ENDOTHELIAL GLYCOCALYX-MEDIATED INTERCELLULAR INTERACTIONS: MECHANISMS AND IMPLICATIONS FOR HEALTH AND DISEASE
A Dissertation Presented
By
Solomon Arko Mensah
To
The Department of Bioengineering
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the field of
Bioengineering
Northeastern University Boston, Massachusetts
October 2019
Northeastern University Graduate School of Engineering Dissertation Signature Page
Dissertation Title: Endothelial Glycocalyx-Mediated Intercellular Interactions: Mechanisms and
Implications for Health and Disease
Author: Solomon Arko Mensah NUID: 001753218
Department: Bioengineering
Approved for Dissertation Requirement for the Doctor of Philosophy Degree
Dissertation Advisor
Dr. Eno. E. Ebong, Associate Professor Print Name, Title Signature Date
Dissertation Committee Member
Dr. Arthur J. Coury, Distinguished Professor Print Name, Title Signature Date
Dissertation Committee Member
Dr. Rebecca L. Carrier, Professor Print Name, Title Signature Date
Dissertation Committee Member
Dr. James Monaghan, Associate Professor Print Name, Title Signature Date
Department Chair
Dr. Lee Makowski, Professor and Chair Print Name, Title Signature Date
Associate Dean of the Graduate School
Dr. Waleed Meleis, Interim Associate Dean Associate Dean for Graduate Education Signature Date
ii ACKNOWLEDGEMENTS
First of all, I will like to thank God for how far he has brought me. I am grateful to you,
God, for sending your son JESUS CHRIST to die for my sins. I do not take this substitutionary death of CHRIST for granted, and I am forever indebted to you for my salvation.
I would like to express my sincerest gratitude to my PI, Dr. Eno Essien Ebong, for the mentorship, leadership and unwaivering guidance through my academic career and personal life.
Dr Ebong, you taught me everything I know about scientific research and communication and I will not be where I am today if not for your leadership. You lead by example and your work ethics is unparalleled. Thank you, from the bottom of my heart.
I will also like to acknowledge my committee members Dr. Arthur Coury, Dr. Rebecca
Carrier and Dr. James Monaghan, for your continual support throughout this project.
Many thanks to project collaborators Dr. Vladimir Torchilin and Dr. Mark Niedre for your contributions to this project.
I will like to acknowledge the Ebong Lab mates both former and current, including Dr.
Homa Homayoni, Dr. Ming Cheng, Ian Harding, Ronodeep Mitra, Nandita Bal and Alina
Nersesyan for your contribution to this project. I also want to sincerely thank all the undergraduate students, including Michelle Zhang, Claire Lee, Paolo Di Pinto and Melis Tirhi for your support.
I am also thankful to Dr. Sheldon Weinbaum, Dr John Tarbell and Dr. Philip Payton all of
City College of New York. I couldn’t have gone this far without your encouragement and support.
Finally, I thank my parents and Auntie Rida Lamptey-Mills (R.I.P), who have selflessly supported me throughout my life journey. To my wife, Mrs. Gillian Arko-Mensah, God bless you for your support and prayers, and to my children, Ethan Papa Ekow Arko-Mensah and Solomon
Papa Arko-Mensah II, I pray for you to do better than daddy.
iii ABSTRACT
The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system.
Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Intercellular interactions are physiologically relevant activities that ensure proper EC function. Various intercellular interactions including those mediated by gap junction proteins, like connexin, for maintaining cell to cell communication, and adhesion molecules, like those mediated by E-selectin and integrins for regulating cell to cell contact between ECs and leukocytes, cancer cells, or other circulating cells. To-date, limited progress has been made to best understand the role of the GCX in intercellular interactions. Previous work demonstrated that GCX degradation disrupts EC gap junction connexin (Cx) proteins, likely blocking interendothelial communication that maintains EC and vascular tissue homeostasis to resist disease. Other reports suggest that the ability of immune cells to interact with EC could be a model for the way cancer cells interact with EC, and these interactions are modulated by the GCX.
We hypothesize that the GCX controls the opening and closing of Cx containing gap junction proteins for regulating communication and also controls accessibility to receptors on the surface of the endothelium for regulating intercellular interactions.
To test our hypothesis, we performed multiple EC experiments to investigate the role of
GCX in intercellular interactions. To understand GCX involvement in gap junction regulation we tested the effect of different GCX conditions on the expression of Cx isotype 43 (Cx43) containing gap junctions. Expression of Cx43 at EC borders was characterized immunocytochemically, and the function of Cx-containing gap junctions were assessed by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. We further examined the activities of the gap
iv junctions and Cx43 after applying various regeneration techniques for GCX. GCX regeneration was achieved via treatment with exogenous heparan sulfate (HS), a major component of GCX. HS was applied with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P).
With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication.
This work validates the importance of GCX in Cx activities.
We also investigated the importance of GCX in concealing or uncovering receptors that mediate cancer-endothelial cell interactions. While it is known that cancer cell interactions with vascular EC drive metastatic cancer cell extravasation from blood vessels into secondary tumor sites, the mechanisms of action are still poorly understood. It is known that GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This dissertation presents evidence that disturbed flow (DF) induces GCX degradation, and leads to circulating tumor cells
(CTC) homing to the endothelium, a first step in secondary tumor formation. To understand the role of GCX in cancer-EC interactions, a two-pronged experiment was performed. First, we tested the hypothesis that neuraminidase-induced degradation of EC GCX, particularly the sialic acid
(SA) residue components of the GCX, will substantially increase metastatic cancer cell attachment to ECs. To our knowledge, our study is the first to isolate the role of GCX SA residues in cancer cell attachment to the endothelium, which were found to be differentially affected by the presence of neuraminidase and to indeed regulate metastatic cancer cell homing to ECs. Second, we
v investigated the effect of DF on cancer-EC interactions. A 2-fold greater attachment of CTCs to human ECs was found to occur in DF conditions, compared to uniform flow (UF) conditions.
These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA) labeled components of the GCX in DF conditions, versus UF conditions. E-selectin receptor expression was similar in DF and UF conditions. Neuraminidase induced degradation of WGA- labeled GCX in UF cell culture conditions or in Balb/C mice led to an over 2-fold increase in CTC attachment to ECs or to Balb/C mouse lungs, respectively, compared to non-enzymatic conditions.
These experiments confirm that GCX degradation enables CTC attachment, providing new insight into a possible GCX-mediated pathway to secondary tumor formation.
Collectively, we have shown that GCX indeed plays a significant role in gap junction mediated intercellular communication and adhesion molecules-mediated intercellular interactions.
We reported that the expression and functionality of Cx43 protein and gap junctions depend on the structural stability of GCX, specifically the HS component. In addition, we also demonstrated that destabilizing subcomponents of the GCX specifically SA, results in increased receptor- mediated cancer-EC interactions. Finally, we showed that DF-induced GCX degradation, or, alternatively, enzymatic degradation of GCX, results in increased attachment, clustering and migration of cancer cells through the endothelium.
vi PREFACE
The work described in this thesis includes published material by the author. Below are the publications:
Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity. Mensah SA, Nersesyan A, Harding IC, Xuefei Tan, Mitra R, Lee C, Niedre MJ, Torchilin VP, Ebong EE. Submitted to FASEB, revision after review by FASEB. 2019
Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation. Mensah SA, Harding IC, Zhang M, Jaeggli MP, Torchilin VP, Niedre MJ, Ebong EE. AIChE J. 2019 Aug;65(8). pii: e16634. doi: 10.1002/aic.16634. Epub 2019 May 9.
Endothelial barrier reinforcement relies on flow-regulated glycocalyx, a potential therapeutic target. Harding IC, Mitra R, Mensah SA, Nersesyan A, Bal NN, Ebong EE. Biorheology. 2019 Mar 29. doi: 10.3233/BIR-180205
Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation. Harding IC, Mitra R, Mensah SA, Herman IM, Ebong EE. J Transl Med. 2018 Dec 18;16(1):364. doi: 10.1186/s12967-018-1721-2
Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target. Mitra R, O'Neil GL, Harding IC, Cheng MJ, Mensah SA, Ebong EE. Curr Atheroscler Rep. 2017 Nov 10;19(12):63. doi: 10.1007/s11883-017-0691-9. Review.
Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter- endothelial communication. Mensah SA, Cheng MJ, Homayoni H, Plouffe BD, Coury AJ, Ebong EE. PLoS One. 2017 Oct 12;12(10): e0186116. doi: 10.1371/journal.pone.0186116. eCollection 2017.
Endothelial glycocalyx, apoptosis and inflammation in an atherosclerotic mouse model. Cancel LM, Ebong EE, Mensah S, Hirschberg C, Tarbell JM. Atherosclerosis. 2016 Sep; 252:136-146. doi: 10.1016/j.atherosclerosis.2016.07.930. Epub 2016
vii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………………………...iii
ABSTRACT………………………………………………………………………………...vi
PREFACE…………………………………………………………………………………. vii
LIST OF TABLES………………………………………………………………………….x
LIST OF FIGURES………………………………………………………………………...xi
LIST OF SYMBOLS AND ABBREVIATIONS…………………………………………. xiv
1. CHAPTER ONE: Introduction……...………………………………………………………………….1
1.1 Problem Statement……………………………………………………………...1
1.2 Atherosclerosis and Breast Cancer Metastasis………………………………….2
1.3 Blood Vessels in Health, Atherosclerosis, and Cancer…………………………3
1.4 Intercellular Interactions in Normal physiology and Disease………………….9
1.4a Intercellular Interactions Involving Gap Junction Proteins…………...10
1.4b Intercellular Interactions Involving Adhesion Molecules…………...... 12
1.5 The Endothelial Glycocalyx: Its Structure………………………………………13
1.6 Endothelial Glycocalyx: Its Function and Implications for Gap Junction and Adhesion Molecule Mediated Intercellular Interactions……………………………………….15
1.7 Hypothesis and Aims…………………………………………………………...16
2. CHAPTER TWO: REGENERATION OF GLYCOCALYX BY HEPARAN SULFATE AND SPHINGOSINE 1-PHOSPHATE RESTORES INTER-ENDOTHELIAL COMMUNICATION……………………………………………………….19
2.1 Introduction……………………………………………………………………..20
2.2 Materials and Methods………………………………………………………….23
viii 2.3 Results…………………………………………………………………………29
2.4 Discussion……………………………………………………………………..38
3. CHAPTER THREE: METASTATIC CANCER CELL ATTACHMENT TO ENDOTHELIUM IS PROMOTED BY ENDOTHELIAL GLYCOCALYX SIALIC ACID DEGRADATION………………………………………………………………………47
3.1 Introduction…………………………………………………………………….48
3.2 Materials and Methods…………………………………………………………54
3.3 Results and Discussion…………………………………………………………58
3.4 Conclusion……………………………………………………………………...69
4. CHAPTER FOUR: FLOW-REGULATED ENDOTHELIAL GLYCOCALYX DETERMINES METASTATIC CANCER CELL ACTIVITY……………………………...71
4.1 Introduction……………………………………………………………………...72
4.2 Materials and Methods…………………………………………………………..76
4.3 Results……………………………………………………………………………86
4.4 Discussion………………………………………………………………………..99
5. CHAPTER FIVE: CONCLUSION AND FUTURE PERSPECTIVES………………….107
APPENDIX A…………………………………………………………………………….....110
Curriculum Vitae………………………………………………………………….....110
APPENDIX B………………………………………………………………………………..117
Engineering for the Less Fortunate…………………………………………………..117
REFERENCES……………………………………………………………………………….121
ix LIST OF TABLES
Chapter 1
Table 1.1: Endothelial cell function in healthy and disease conditions……………….8
Table 1.2: Endothelial cell receptors and their corresponding tumor ligands…………12
Chapter 2
Table 2.1. Summary of experimental design…………………………………………..23
Chapter 3
Table 3.1: Representative reports of the role of the glycocalyx in blocking or enabling endothelial cell adhesiveness to cells in the blood circulation ………………………..51
Table 3:2: Table indicate the lectins (purchased from Vector Labs) used in labeling glycocalyx……………………………………………………………………………..55
x
LIST OF FIGURES Chapter 1
Figure 1.1A: Structure of a blood vessel………………………………………5
Figure 1.1B: Atherosclerosis progression……………………………………...5
Figure 1.2A: Sequence of events leading to cancer metastasis………………...7
Figure 1.2B: Cancer-endothelial cell attachment mediated by adhesion molecules……7
Figure 1.4: Connexin gap junction protein……………………………………1
Figure 1.5: Glycocalyx structure………………………………………………14
Chapter 2
Figure 2.1: Connexin and Lucifer yellow dye transfer quantification model…………………………...... 28
Figure 2.2: Scanning electron micrographs of glycocalyx ……………… 30
Figure 2.3: Image and quantification of heparan sulfate expression in glycocalyx…………………………………………………………………. 32
Figure 2.4: Negative control images for connexin and heparan sulfate…….33
Figure 2.5: Images and quantification of Connexin 43 …………………….34
Figure 2.6: Image and quantification of Lucifer yellow dye transfer………37
Figure 2.7: Proposed pathway for translation of glycocalyx recovery techniques to the clinic………………………………………………………………………...39
Figure 2.8: Dose dependent test on the effect of Cytochalasin D on rat fat pad endothelial cells actin filaments………………………………41
Figure 2.9: Effect of Cytochalasin D on Lucifer yellow dye transfer……….42
Figure 2.10: Conceptual hypothesis of the role of glycocalyx in cell-to-cell communication……………………………………………………………....45
xi
Chapter 3
Figure 3.1: Conceptual hypothesis of the role glycocalyx in endothelial-cancer cell interactions…………………………………………………………………. 49
Figure 3.2: Images show the effect of exposure to various concentrations of Neur enzyme on the GCX, endothelium integrity, and cancer cell attachment to ECs……………59
Figure 3.3: Comparing the coverage and thickness of glycocalyx and sialic acid components………………………………………………….60
Figure 3.4: Comparison of glycocalyx expression and cancer attachment….64
Figure 3.5 Comparison of alpha 2,6 sialic acid and cancer attachment…….65
Figure 3.6: Comparison of alpha 2,3 sialic acid and cancer attachment…….66
Figure 3.7: Preliminary data comparing the glycocalyx expression of human umbilical vein endothelial cells with rat fat pad endothelial cells…...68
Chapter 4
Figure 4.1: Conceptual hypothesis…………………………………………...73
Figure 4.2: Effect of disturbed flow and uniform flow on attachment, clustering and migration of cancer cells…………………………………………………………………...88
Figure 4.3: Computer aided simulation, showing changes in flow patterns after vertical step………………………………………………………………….78
Figure 4.4: Dose response experiment to establish appropriate enzyme concentration for human umbilical vein endothelial cell glycocalyx degradation……………………...79
Figure 4.5: Diffusive in vivo Flow Cytometry (DiFC) system used to track circulation of 4T1 breast cancer cells in BALB/C mice……………………………...... 85
Figure 4.6: Control static experiments for glycocalyx expression, cancer attachment and E- selectin expression…………………………………………………………..87
Figure 4.7: Flow conditioned human umbilical vein endothelial cells exposed to 4T1 or MCF7 cells in static conditions and not in circulation……………………………..104
Figure 4.8: Effect of disturbed flow and uniform flow on endothelial glycocalyx and E-selectin expression……………………………………………………………………91
xii Figure 4.9: Effect of the presence of Neuraminidase on cancer attachment, glycocalyx and E- selectin expression…………………………………………………………………….92
Figure 4.10: Expression of glycocalyx in the abdominal aorta of Balb/c mice………96
Figure 4.11: En face confirmation of intact endothelium after treatment with 5 U/mL of Neuraminidase……………………………………………………….99
Figure 4.12: In-vivo attachment of 4T1 breast cancer cells to the lungs of BALB/C mice……………………………………………………………………………………97
Chapter 5
Figure 5.1: Cartoon depicting intercellular interactions mediated by the glycocalyx…109
xiii LIST OF SYMBOLS AND ABBREVIATIONS
BSA: Bovine Serum Albumin
Cx: Connexin
CS: Chondroitin Sulfate
CTC: Circulating Cancer Cells
CVD: Cardiovascular Disease
DMEM: Dulbecco’s Modified Eagle Medium
DAPI: 4’6-diamidino-2-phenylindole
ECs: Endothelial Cells
EMEM: Eagle’s Minimum Essential Medium eNOS: Endothelial Nitric Oxide Synthase
FBS: Fetal Bovine Serum
GCX: Endothelial Glycocalyx
GAGs: Glycosaminoglycans
HS: Heparan Sulfate
Hep III: Heparinase III
HA: Hyaluronic Acid
LSM: Laser Scanning Microscope
Lu-ECAM: Lung EC Adhesion Molecule-1
MAL II: Maakia Amurensis Lectin II
MCF7: Human Breast Cancer Cells
Neur: Neuraminidase
NDST: N-deacetylase/N-sulfotransferase
xiv
PFA: Paraformaldehyde
PS: Penicillin-Streptomycin
PECAM-1: Platelet Endothelial Cell Adhesion Molecule-1
RFPEC: Rat Fat Pad Endothelial Cells
RT: Room Temperature
RFPEC: Rat Fat Pad Endothelial Cell
SA: Sialic Acid
SNA: Sambucus Nigra (Elderberry Bark) Lectin
S1P: Sphingosine 1-Phosphate
TNF-alpha: Tumor Necrosis Factor- alpha
VEGF: Vascular Endothelial Growth Factor
WGA: Wheat Germ Agglutinin Lectin
ZO-1: Zona Occludin 1
4T1: Stage IV Metastatic Breast Cancer Cells
xv
1. CHAPTER ONE: INTRODUCTION
1.1 PROBLEM STATEMENT
Cardiovascular disease (CVD) and cancer metastasis, combined, are responsible for approximately 50% of all deaths globally [1], making them the top two leading causes of death
[2]. Atherosclerosis is an underlying cause of CVD, and breast cancer is a common form of cancer
[3, 4]. Recent reports by Suzuki et al. and others suggest that patients with atherosclerotic plaques could be at a higher risk for developing cancer [5-7]. Other studies also suggest that CVD and cancer demonstrate similar pathophysiological symptoms such as inflammation [8, 9], neovascularization [10, 11] and epigenetics in the form of DNA methylation and chromatin remodeling [12, 13]. These two diseases, although different, share some common risk factors which include obesity and hypertension, suggesting that they could develop via common cellular and molecular pathways [6].
One common cellular and molecular pathway is thought to involve endothelial cells (ECs) and their glycocalyx (GCX). The GCX is a hydrated sugar-rich layer coating the ECs, making it a lining for the inside of blood vessels, which is dysfunctional during the onset and progression of both atherosclerosis and metastatic cancer [14]. GCX dysfunction results in the lack of proper control of intercellular interaction between EC with neighboring EC and with circulating cells, including inflammatory cells and circulating tumor cells (CTCs), leading to disease progression
[15]. The modes of intercellular interactions are many. However, those mediated by connexin proteins that form gap junction channels and adhesion molecules that form receptor-ligand bonds will be the focus of this thesis. These structures are of particular interest based on their physical
1 proximity to the GCX and their implications in the onset and progression of atherosclerosis and metastatic cancer. Furthermore, the role of the GCX in mediating EC interactions with its neighboring cells and with CTCs has not been fully clarified. It is important that the role of the
GCX in regulating intercellular interactions be clarified to strengthen our understanding of the GCX-mediated mechanisms that contribute to either healthy or disease conditions, and to eventually lead to the development of novel GCX-targeted drugs to prevent GCX degradation and disease.
1.2 ATHEROSCLEROSIS AND BREAST CANCER METASTASIS
Atherosclerosis and breast cancer metastasis are characterized by modifications in the host blood vessels, due to inflammation and change in intercellular interactions. Atherosclerosis is distinguished by plaque formation as a result of accumulation of cholesterol and other cellular debris within the vascular wall, which leads to vessel damage [16]. On the other hand, cancer metastasis is distinguished by the formation of secondary tumors away from the location of the primary tumor. The process of secondary tumor formation involves CTC survival in the bulk flow followed by extravasation and re-growth in the microenvironment of the secondary organ[17].
Atherosclerosis and cancer metastasis may be connected. Recent reports suggest that the progression of breast cancer leads to induction of vascular pathology in patients, leading to increased CVD risks. Other reports indicate that long-term cardiovascular risk attenuates breast cancer survival rate and efficacy of early breast cancer therapy [18, 19], and it is known that CVD contributes to a majority of the deaths amongst breast cancer patients [20].
2 1.3 BLOOD VESSELS IN HEALTH, ATHEROSCLEROSIS, AND CANCER
Progression of these diseases is enhanced by dysfunction of the blood vessel, characterized by a weakened barrier at the interface between the blood compartment and the blood vessel wall tissue.
In health, the blood compartment is responsible for transporting oxygen and nutrients to various parts of the body (Fig. 1.1A). Blood contains cells such as red blood cells (Fig. 1.1A) that have hemoglobin, which helps carry oxygen [22]. The blood also carries white blood cells to specific locations in the body to fight off infections and diseases. The most common type of white blood cells are neutrophils and lymphocytes [23]. In addition to red and white blood cells, there are platelets which help blood to clot by gathering at the site of injury [24](Fig. 1.1A). Blood also and carries waste products to the kidneys and liver and regulates body temperature [21].
The blood vessel wall comes in different types, namely arteries and veins which have specific functions. Arteries carry oxygenated blood away from the heart and veins take deoxygenated blood back to the heart. These specific functions dictate the unique structures of the vessel walls. Generally, the normal anatomy of blood vessel wall includes multiple layers: tunica intima, tunica media, and tunica externa, as depicted in Figure 1.1A. Each layer plays a distinctive role in ensuring proper functioning of the blood vessel wall.
The tunica intima layer (Fig 1.1A) is particularly interesting to us due to its unique position at the blood-tissue interface [25]. The tunica intima layer provides a frictionless pathway for the flow of blood and it is the thinnest layer of the vascular wall, made up of a single layer of ECs with well-defined basement membrane and extracellular matrix [26]. ECs present an anti- thrombotic surface, the GCX, for the easy movement of plasma and its cellular components throughout the blood compartment [27]. The ECs of the tunica intima layer interact and
3 communicate with each other and with the cells in the blood compartment (like inflammatory cells and platelets) and with cells and extracellular matrix in the wall (tunica media layer and in the tunica externa layer) to maintain vascular homeostasis. For example, ECs generate compounds that activate smooth muscle cells to induce vasodilation or constriction in order to adjust blood vessel diameter. Blood pressure is decreased in response to the influx of blood pumped from the heart, by EC release of vasorelaxation compounds including nitric oxide and prostaglandin F2. In order to increase blood pressure when the heart is not pumping, at rest, and refilling with blood,
ECs release compounds involved in vasoconstriction including angiotensin ll and endothelin [28].
ECs also extensively interact with vascular smooth muscle cells via gap junction proteins to adjust blood vessel diameters to manage blood pressure. As another example, the ECs are gatekeepers between the blood compartment and vessel wall to regulate transport of oxygen, nutrients like lipids, and other molecules from the blood into tissues and organs [29, 30]. In addition, ECs play very significant roles in immune cells trafficking to sites of immune surveillance for wound healing (Table 1). With this, the ECs coordinate collaboration between the blood and vessel wall on physiological functions such as inflammation, wound healing and vascular tone [29, 30].
In contrast to the tunica intima layer, the tunica media layer and tunica externa layer have different cellular composition. The tunica media layer is made up of smooth muscle cells and they are contractile cells that play a key role in maintaining vascular tone by regulating constriction and dilation (Fig 1.1A) [31, 32]. The tunica externa layer is the outer layer of the blood vessel and gives structural support to the media and intima (Fig 1.1A) [33]. The cellular composition of the tunica externa includes fibroblasts, myofibroblasts and macrophages [33].
4
A
Smooth Muscle
Externa
Lipids
Endothelium
Figure adapted from Vector-Medical Education Chart of Biology for blood vessel, Image ID: 79651710. B
Endothelial cell layer Smooth muscle cell layer Figure adapted from John A. Rumberger, Cardiac CT. Princeton Longevity center. Feb, 2019 Fig 1.1 A: Structure of blood vessel, showing the three main layers. The tunica intima is the inner most part of the vessel and it comprises of ECs that line the entire cardiovascular network. The tunica media is composed of smooth muscle cells that give structural support to the endothelium. The tunica externa cellular composition includes fibroblast, myofibroblast and macrophages. The tunica externa gives structural support to both the tunica media and intima. The blood component shows the various cells that make up the blood. This include white and red blood cells. The blood component also consists of platelets and nutrients, for example, lipids. B. Cross-section of an artistic rendering of a blood vessel, revealing atherosclerosis plaque progression. Onset of plaque, characterized by fatty streak formation as a result of increased lipid accumulation within the vessel, which triggers white blood cell infiltration leading to the formation of foam cells. The formation of an intermediate lesion composed of intracellular accumulation of lipid and small extracellular lipid pools, forming a lipid rich plaque. The plaque becomes calcified, initiating the formation of scars encapsulating the necrotic core of the plaque, which contains macrophages, foam cells and cellular debris. Progression of the necrotic core and the formation of a fibrotic or calcific layer around the necrotic core. The plaque develops surface 5 defects, possible hemorrhage, and thrombosis, which eventually lead to cardiovascular disease. In atherosclerosis, the blood compartment and vascular wall layers undergo structural and functional modifications. For instance, in the formation of atherosclerotic plaques the tunica intima endothelium becomes dysfunctional. There is loss of gap junction mediated cell-to-cell communication [40] (Table 1) between ECs and smooth muscle cells, which affects vascular tone and blood pressure regulation [41]. ECs lose gatekeeping capability leading to increased permeability to and deposition of lipids in the intima (Fig 1.1B). ECs become overactive and involved in excessive immune cell recruitment [34]. Immune cells respond to heal the situation.
This causes inflammation at the tunica intima, since ECs are the initial site for immune cell interaction [35]. Immune cell response is not successful, leading to the formation of foam cells, which are immune cells filled with lipids. Smooth muscle cells in the tunica media excessively proliferate. Extracellular matrix, elastin and collagen, remodel and some degradation occurs, which is mediated by matrix metalloproteinase enzymes. These events eventually lead to the formation of advanced plaques filled with lipid, inflammatory cells, smooth muscle cells, collagen and cores of dead tissues, events which are exacerbated in hypertensive individuals (Fig 1.1B)
[36]. In hypertensive individuals, there is also an increase in the production of contracting factors such as endothelin-1 and angiotensin ll, which result in vasoconstriction, sympathetic nervous system stimulation, increased aldosterone biosynthesis and renal action, which leads to increased hypertension [37-39] (Table 1). Calcification of the plaque can occur. Initiation of a fibrous capsule can also occur, which stabilizes the plaque. Surface defects causing plaque hemorrhage and thrombosis is another end point for atherosclerotic plaques and this substantially contributes to the progression of CVD (Fig 1.1B).
In cancer metastasis, tumor cells leave the primary site, enter the blood, and the abnormal presence of tumor cells in the blood compartment leads to imbalance in the cellular and chemical
6 composition of blood [42] (Fig 1.2A). The chemical composition is increased with respect to reaction oxygen species, matrix metalloproteinases, interleukin-8 (IL-8), IL-18, tumor necrosis factor-alpha and other cytokines. This chemical composition can degrade the EC GCX and other components of the blood vessel wall, resulting in damage to the endothelium as well as whole vessel complications. Degraded GCX and damaged endothelium lead to expression of adhesion molecules like E-selectin and integrins (Fig 1.2B), which help the cancer cells firmly attach to the endothelium and extravasate the vessel to initiate and grow secondary tumors (Fig 1.2A).
Fig 1.2: A. Cancer metastasis: Cells from the primary tumor gain migratory properties and detach from the primary tumor. They intravasate by crossing through the blood vessel and through blood flow move to distant locations within the host body. The presence of tumor creates inflammation resulting in
endothelium activation causing an increase in adhesion receptor expression. This enhances circulating tumor cell attachment to the endothelium. This is followed by extravasation between the ECs, leading to the formation of secondary tumor. B. Interaction between EC and cancer cell mediated by receptor- ligand complexes. The ligands on the cancer cell align with the receptors on the surface of the EC for attachment.
7 Endothelial Cell Functions
Healthy condition Disease condition
Atherosclerosis Metastatic Cancer
Barrier • ECs ensure limited • Inflammatory • Increased chemokines or migration of leukocytes conditions triggered by chemoattractant, such as protection across the oxidized lipids result in interleukin-8 and monocyte and endothelium[43]. EC activation[43]. chemoattractant protein-1, permeability • EC receptors like E- • ECs express more around the secondary tumor site selectin and integrins are adhesion molecules, [49, 50]. responsible for leukocyte like E-selectin and • Increased expression of adhesion trafficking[44]. Receptor integrins[44, 47]. molecules, like E-selectin on the expression is low in • Increased trafficking of EC surface and also the ligands healthy conditions. leukocytes across the like CD44 on the cancer cell EC layer[44]. surface[51, 52]. • EC GCX is known to • During atherosclerosis, • Cancer cell trafficking to regulate endothelium dysfunctions in the secondary tumor sites is barrier. The complex endothelium lead to the enhanced[52]. mesh of GAGs and influx of lipid • Matrix glycoproteins prevents deposition in the vessel metalloproteinases(MMPs) vascular infiltration by wall[48]. released by tumors dissolve lipids and leukocytes[45]. • EC GCX is degraded interendothelial tight junctions, • Signaling pathways decreasing barrier disrupt GCX and enable the initiated with ECs by protection. intravasation of CTCs[53]. binding of vasoactive factors regulate junctional protein permeability[46].
Vascular • ECs release nitric oxide • During atherosclerosis • The lack of smooth muscle layer and enothelin-1 for apoptotic EC layer leads in most tumors enhances tumor tone regulating smooth muscle to deletion of ligands invasion and metastasis. EC activities including associated with smooth activation of caspase-3 initiates proliferation control[54]. muscle cell smooth muscle cell • Connexin protein differentiation[59-61]. apoptosis[63]. between endothelial cells • Communication via gap • EC-to-EC, EC-to-fibroblast and and smooth muscle cells junction proteins is EC-to-CTC communication is enhance intercellular impaired[55] due to enhanced in cancer[64, 65]. communication[55]. change in the type of • CTC release of interleukins and • ECs release chemicals like connexin protein tumor necrosis factor alpha in nitric oxide, bradykinin, expressed. the tumor environment increase and epinephrine that • Excessive endothein-1 EC dependent release of promote vasodilation, and released by ECs causes endothelin-1, which is a norepinephrine, serotonin, smooth muscle vasoconstrictor[66]. endothelin etc. that proliferation, which • Enhanced nitric oxide signaling promote increases intima-media by ECs results in increased vasoconstriction[56, 57]. thickness, which leads vasorelaxation and cancer • Connexin containing gap to the progression of metastasis [67, 68]. junctions allow the atherosclerosis[54, 62]. transfer of molecules like • EC dysfunction results calcium and acetylcholine in the increased that promote expression of connexin vasomotion[58]. 43, which is indicative of atherosclerosis progression[58]. Table 1: EC function in both health and in disease. EC plays significant role in endothelium barrier integrity, interaction between neighboring EC and with circulating cells, as well as regulating smooth muscle cell activities.
8 1.4 INTERCEULLAR INTERACTIONS IN NORMAL PHYSIOLOGY AND IN DISEASE
In both disease cases, atherosclerosis and cancer, dysfunctions in the endothelium play a primary role in the progression of disease. In atherosclerosis, leukocytes interact with ECs during immune surveillance through EC surface adhesion molecules like E-selectin and integrins [69]
(Fig 1.1B). There is also an increase in the expression of Cx43 containing gaps through which ECs communicate with each other in the plaque environment [70, 71]. In cancer, CTCs interact with
ECs during metastatic extravasation or intravasation via EC surface adhesion molecules [72] [73]
(Fig 1.2A).
Generally, endothelium interactions with neighboring cells is mediated in different forms which include direct contact with other cells mediated by integrins [74, 75], junctional proteins
[76, 77], adhesion molecules [78, 79], extracellular vesicles [80, 81] or the secretion of proteins
[82] and cytokines [83] in the extracellular space. Integrins are transmembrane proteins that function as receptors for extracellular ligands, and play significant roles in vascular development and vascular health [84]. Integrins function as signal transduction molecules that can control intracellular pathways to regulate cellular activities [85].The clustering of integrins results in the formation of focal adhesion complexes which form mechanical connections between intracellular cytoskeleton and extracellular substrates [86]. Junctional proteins mediate the adhesion and interaction between neighboring ECs, and these junctional proteins include tight junctions, adherence junctions and gap junctions [87] (Fig 1.3). The expression of these junctional proteins depend on the tissue type and the communication requirement between the cells [87]. ECs also interact with themselves and others through adhesion molecules on the EC surface which are essential for cellular activities with EC surrounding [88]. Interactions between ECs and CTCs or immune cells are initiated via expression of adhesion molecules on the EC surface. An example of
9 EC adhesion molecules include the family of selectins [89]. Other adhesion molecules such as
CD11a/CD18(LFA-1) etc. interact with intercellular adhesion molecule-1 on the endothelial surface to enhance CTC or immune cells migration through the endothelium [90]. Through extracellular vesicles, ECs are able to send small lipid-enclosed particles to distant cells to effect physiological changes, locally or systemically. Extracellular vesicles are a relevant intercellular signaling mechanism that enables transfer of molecules between cells [91, 92].
All of these forms of intercellular interactions have been extensively reviewed, but for the purpose of exploring the role of the GCX in the mediation of intercellular interactions, we will herein focus on connexin containing gap junction proteins and adhesion molecules, especially those formed by the endothelial cellular receptor E-selectin.
1.4a INTERCELLULAR INTERACTIONS INVOLVING GAP JUNCTION PROTEINS
Gap junction proteins provide channels through cell membranes, creating a semi permeable pathway for the diffusion of ions and small molecules between neighboring cells [93] (Fig 1.4).
These channels are made up of transmembrane proteins called connexins(Cx) [94]. Six Cx from each neighboring cell membrane form a connexon (Fig. 1.4). Two connexons contributed by neighboring cells come together to construct a cylindrical channel, called a gap junction, that becomes a mode of communication between the cells [95] (Fig 1.4). Several types of Cx combinations may assemble to form the gap junctions between cells, and because of their short- life span they are renewed daily [96]. In relation to ECs, three different Cx types are described, and the expression of these Cx types are dependent on the vessel type [97, 98] and the disease condition[99]. Connexin 37(Cx37), Cx40 and Cx43 are mostly expressed by ECs [70, 100, 101].
The expression of the different types of Cx in healthy and diseased conditions have been
10 investigated and reviewed [102]. For example, Cxs are noted to be responsible for vasomotor responses and tone in smooth muscle cells [99] (Table 1), Cx40 specifically is known to be very
important in the regulation of
blood pressure [103]. Cxs are also
known to play significant roles in
cell migration and proliferations
[104, 105].
Healthy ECs mostly express
Cx37 and Cx40. During the
initiation of atherosclerotic
lesions, Cx43 begins to be
expressed in addition to Cx37 and
Cx40[96]. During the late stage of
the disease only Cx43 is expressed
indicating the drastic change in the
Cx makeup of the vessel [106]. In other diseases like cancer, there is growing evidence to suggest that expressed Cx regulates tumor growth [107, 108]. This regulation is known to happen at the transcription [109, 110], post transcription [111] and the protein synthesis levels [112]. In cancer, the study of Cxs is more complicated because CTCs may express more than three Cx proteins
(there are over twenty Cx proteins in the body), although ECs only express three Cxs.
1.4b INTERCELLULAR INTERACTIONS INVOLVING ADHESION MOLECULES
Interactions via adhesion molecules (Fig 1.2B) are usually complicated and may involve multiple steps in a sequence to ensure intercellular binding. In general, three separate steps
11 characterize the formation of adhesion molecule interactions, which are mediated by receptors and ligands. First, there is the primary recognition stage where receptors on one cell are recognized by corresponding ligands on another cell via electrostatic forces. Second, structural conformational changes and proper orientation occur to match ligands to their binding sites on receptors. Third, physical contact and binding are achieved, which creates a receptor-ligand complex between different cell types [113, 114] (Fig. 1.2B).
ECs have
several surface
receptors that initiate
interactions with
Table 2. EC receptors and their corresponding ligands on tumor cells. other cell types such During tumor invasion of the endothelium, these tumor ligands locate their respective receptors on the endothelium to initiate EC-Tumor as leukocytes [120], interactions. These interactions result in firm attachment of tumor cells to ECs and also initiate biochemical changes with ECs and tumor cells. The cancer cells, and activation of receptors or ligands have been used as markers for determining the aggressive nature of cancers. other cell types
(Table 2). These receptors include E-selectin, intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and integrins etc. [121] (Fig. 1.2B, Table 2).
Of these adhesion receptors, E-selectin is of particular interest because it is the first EC adhesion molecule to interact with CTCs during cancer or leukocyte invasion in atherosclerosis.
E-selectin belongs to a group of selectins which are glycoproteins that mediate circulating blood cell attachment to the endothelium [122] (Fig. 1.2B). E-selectin is mostly expressed by ECs via endothelial activation during inflammation or in the presence of cytokines [122]. As a
12 consequence, E-selectin can promote atherosclerosis and cancer metastasis progression leading to poor prognosis of disease [124, 125]. Gakhar et al reported that in cancer, CTCs isolated from men with castration-resistant prostate cancer exhibited significant physical interactions through tethering and firm adhesion with E-selectin-coated surfaces [126]. These interactions were diminished by anti-E-selectin antibodies [126].
1.5 THE ENDOTHELIAL GLYCOCALYX: ITS STRUCTURE
The gap junction proteins and adhesion molecules both interface with and are embedded in the GCX on the EC surface (Fig 1.3). The GCX is a sugar-rich layer that encapsulates ECs. It is connected to EC membrane through several backbone molecules [127], mainly proteoglycans and glycoproteins like syndecans and glypicans, as indicated in Figure 1.3. These backbone molecules have sugar chains covalently or loosely linked to them. The glycosaminoglycan (GAG) sugar chains, characterized by distinct disaccharide unit repeats, include hyaluronic acid (HA), heparan sulphate (HS), and chondroitin sulphate (CS). HA are long GAG chains attached to EC membrane bound receptors, such as CD44 (Fig 1.3), and are presumed to intertwine through GCX and provide a scaffold for the GCX [45]. The HS GAG is the dominant constituent of GCX (Fig
1.3). HS is a linear sulfated polysaccharide chain and anchored to the syndecan and glypican core proteins [128]. The CS GAG is also an abundant GAG and is bound to syndecan alongside HS.
CS is covalently linked to its core protein via the GAG-protein linkage [129]. The ratio of HS to
CS is reported to be in the order of 4:1. The combination of HS and CS plays a very critical role in the structural stability of GCX [130, 131]. In addition to the GAGs, a sialoglycoprotein, sialic acid (SA) (Fig 1.3), also commonly associates with the EC GCX. SA consists of complex sugar units and is mostly located at the innermost part of GAGs (Fig 1.3). Given its location in proximity to the EC surface adhesion molecules, SA should play a significant role in maintaining the barrier
13 integrity of GCX [132]. SA has the added advantage of being negatively charged and so engages
in the repulsion of unwanted intercellular and molecular interactions from components of the blood
circulation [133]. The functions of GCX are dependent on the way its components are arranged
[134].
Figure 1.5: Physical structure and components of the EC GCX. Image depicts the various GCX components like HS, SA etc. The GCX extends from the endothelial cell membrane to the lumen of blood vessel interacting with circulating cells in the blood. The image also depicts the GCX extending between neighboring cells and interacting with the junctional proteins like gap junctions and tight junctions. The image also shows the height disparity between adhesion receptors like E-selectin and integrin and the GCX. 1.6 ENDOTHELIAL GLYCOCALYX: ITS FUNCTIONS AND IMPLICATIONS FOR GAP
JUNCTION AND ADHESION MOLECULE MEDIATED INTERCEULLAR
INTERACTIONS
Several reports have highlighted the following functions of the GCX: barrier protection
[136] and permeability [137], mechanotransduction [14], and other functions. The barrier and
permeability properties of GCX are usually deduced from the rate at which small molecules like
14 dextran and other tracers as well as leukocytes and CTCs move into the GCX and across the endothelial layer [45]. Regarding mechanotransduction, due to the transmembrane nature of GCX core proteins, it is noted to transduce shear and stretch forces into biomolecular responses of ECs
[138]. It has been shown that GCX is anchored to a scaffold of cytoskeleton actin filaments that form an actin cortical web [139]. Since this cytoskeleton actin web is in close proximity to and, in some cases, linked to transmembrane adhesion molecules and gap junction proteins, it is presumed that GCX is an important factor in intercellular interactions. Researchers studied the transmission of fluid shear stress through the GCX to the actin cortical web of the cytoskeleton and discovered that with intact GCX F-actin is distributed mostly at the cell borders [140] where junctional proteins like Cx are located. Using a GCX digesting enzyme that specifically targets the HS GAG of GCX, it was shown that the absence of HS GAG results in disorganization of the actin filaments and loss of gap junction proteins. Conversely, reinforcing the HS component of GCX by adding fetal bovine serum (FBS) and albumin to the culture medium in addition to prescribed shear stresses resulted in dramatic enhancement of the actin cortical web [140, 141]. Due to the GCX- actin-gap junction protein relationship, it was proposed that functional performance of Cx containing gap junction channels would also be enhanced and lead to active cell-to-cell communication between ECs.
In addition to GCX’s suspected role in regulating gap junction proteins, we speculate that the GCX is relevant in regulating adhesion molecules on the surface of the endothelium thereby increasing blood circulating cell accessibility to the endothelial surface. Interaction between ECs and white blood cells, for example, is adhesion molecule mediated and governed by the availability of receptors, such as E-selectin, which bind to the ligands on white blood cells. It has been reported that the ability of immune cells and other cells to penetrate the GCX layer to access the adhesion
15 receptors is dependent on the porosity and stiffness of the GCX [142]. GCX thickness relative to
the length (thickness) of the receptors on the endothelial surface is also important in determining
if receptors are shielded from ligands on circulating cells [143] [45, 144].
Enzymatic degradation of GCX could be a possible mechanism through which EC
receptors are exposed to circulating cell ligands for the formation of intercellular connections. In
support of this idea, it has been shown that by suppressing the activities of matrix metalloproteases,
an enzyme reported to degrade GCX, leukocyte-EC interactions can be inhibited [145].
Recent work has shown that in addition to enzymatic degradation of GCX, hemodynamic
factors could also result in the degradation of GCX. In areas of the vasculature where disturbed
flows exist, the GCX is degraded significantly compared to uniform flow areas of the vasculature
[146]. Therefore, flow-induced degradation of the GCX could be another mechanism through
which EC surface adhesion molecules are exposed.
1.7 HYPOTHESIS AND AIMS
Relevant to understanding the underlying cellular and molecular causes of atherosclerosis and cancer, to date, limited efforts have been made to explain the role of the GCX in intercellular interactions. The evidence provided above draws an incomplete correlation between gap junction functionality due to GCX health. It provides limited evidence that GCX regulates adhesion molecule interactions between ECs and blood circulating cells. Therefore, our goal is to elucidate the role played by GCX in intercellular interactions, specifically related to how GCX mediates EC-to-EC communication and EC-to-CTC attachment in atherosclerosis and cancer. We hypothesize that
GCX dysfunction results in impairment of gap junctions as well as leads to adhesion molecule accessibility, for disrupted interendothelial communication and increase in CTC attachment,
16 clustering and migration through the endothelium. To test this hypothesis, there were three specific aims for this research.
First, we investigated the role of GCX and its HS component in regulating interendothelial communication (Aim 1). We studied the role of GCX HS on Cx43 gap junction protein functionality.
HS was degraded by enzymes. The effect of HS degradation on the expression and functionality of
CX43 was assessed through immunostaining, Lucifer Yellow dye transfer technique, and microscopic interrogations. Different GCX recovery techniques were employed in an attempt to regenerate lost
HS and to further investigate the importance of HS for Cx43 functionality. This study shed light on the role of GCX in enabling EC-to-EC communication function, which is lost during the progression of certain cardiovascular related pathologies including cancer and atherosclerosis. This work is reported in a peer-review paper entitled, “Regeneration of Glycocalyx by Heparan Sulphate and
Sphingosine 1-Phosphate Restores Inter-Endothelial Communication.”
Second, we investigated the role of GCX sialic acids in the attachment of 4T1 breast cancer cells to the endothelium (Aim 2). We showed the effect of dose dependent administration of neuraminidase (enzyme specific to SA) on rat fat pad endothelial cells (RFPEC). After administering the enzyme, immunostaining and confocal microscopy were used to investigate structural and morphological changes in the GCX. In addition, we investigated the effect of the presence of the enzyme on the attachment of 4T1 breast cancer cells to the endothelium. Understanding the effect of
GCX degradation on the attachment of cancer cells to the endothelium is important to creating therapeutic measures that will combat the spread of cancer via strengthening of GCX against GAG degrading cytokines released by cancer tumors. This work is finalized as a peer-reviewed publication entitled, “Metastatic Cancer Cell Attachment of Endothelial is Promoted by Endothelial
Glycocalyx Sialic Acid Degradation.”
17 Finally, we investigated the effect of disturbed flow patterns on the attachment of CTC to the endothelium (Aim 3). We studied the effect of different flow patterns on EC and breast cancer cell interactions, using a customized flow chamber to introduce disturbed and uniform flow patterns to ECs mimicking in-vivo conditions. The effect of different flow patterns on the changes in GCX structure and morphology was assessed with immunostaining and confocal microscopy. This was followed by EC-4T1 and EC-MCF7 attachment experiments to determine the effect of different flow patterns on the attachment, clustering and migration of cancer cells to the endothelium. Experiments were repeated in a mouse model to validate the in vitro results. This study has increased our understanding of the role played by vascular geometry and flow patterns on GCX structure and morphology as well as the effect of such flow patterns on GCX-mediated EC-cancer cell interactions.
This work has been reported in manuscript form and is currently in the second review after revisions for peer-reviewed publication. The title of the paper is “Flow-Regulated Endothelial Glycocalyx
Determines Metastatic Cancer Cell Activity.”
18 CHAPTER TWO: REGENERATION OF GLYCOCALYX BY HEPARAN SULFATE AND SPHINGOSINE 1-PHOSPHATE RESTORES INTER-ENDOTHELIAL COMMUNICATION
PUBLISHED IN PLOS ONE JOURNAL, 2017
Solomon A. Mensah, B.S.1, Ming J. Cheng, B.S.2, Homa Homayoni, Ph.D.2, Brian D. Plouffe, Ph.D.2, †, Arthur J. Coury, Ph.D.2, and Eno Essien Ebong, Ph.D.1,2,3,‡
1Department of Bioengineering, Northeastern University, Boston, MA 2Department of Chemical Engineering, Northeastern University, Boston, MA 3Department of Neuroscience, Albert Einstein College of Medicine, New York, NY
19 2.1 INTRODUCTION
The vasculoprotective endothelial cells (ECs) exhibit a number of behaviors that include regulation of vascular permeability and inflammation along with control of vascular tone [147].
An important contributor to these cell functions is the EC membrane-anchored mesh-like extracellular matrix a sugar coated known as the glycocalyx (GCX) [148, 149].
The location and anchoring of the GCX enables EC sensitivity to extracellular microenvironment conditions [150], which ECs transduce into specific biological behaviors in a temporal and spatial manner [151, 152]. GCX composition consists largely of integrated and functional heparan sulfate (HS), chondroitin sulfate, hyaluronic acid, and sialic acid glycosaminoglycans (GAGs) [144, 148]. Proteoglycan and glycoprotein core proteins tether the
GAGs and give the GCX a structurally stabilizing backbone [144, 153]. ECs express syndecan and glypican core proteins, and they primarily bind HS [128]. Syndecan is a major EC GCX stabilizer, because it is a trans-membrane protein rooted in the cell body. Plasma proteins also integrate into the GCX, extend its thickness, and stabilize it. Bovine serum albumin (BSA), for example, prevents GCX collapse and its known for delivering sphingosine 1- phosphate (S1P) to the cells to inactivate matrix metalloprotease degradation of the GCX and promote synthesis [154-157].
GCX composition is actively regulated by EC through continuous shedding and synthesis
[158-163]. At healthy vascular sites, shedding and synthesis are balanced and the intact GCX can transmit signals into the cell to trigger vasculoprotective cell function [150, 164]. At diseased vascular sites, shedding exceeds synthesis leading to increased GCX permeability and/or reduced thickness [15, 165, 166]. Consequently, cell signal transmission becomes dysfunctional [150] in a manner that is permissive of disease. Regeneration of shed GCX to reverse this dysfunctional cell
20 signaling and prevent disease was the focus of the study described herein. Specifically, we sought to functionally regenerate the most abundant GCX constituent, HS.
Proposed HS regeneration approaches include its replacement, structural stabilization, competitive binding, and synthesis enhancement [167]. Previously, our group and others successfully replaced shed HS with commercial HS [168] or sulodexide [169, 170], a compound containing both heparin and dermatan sulfate. In other studies, a non-animal heparan sulfate-like polysaccharide, called rhamnan sulfate, was used as a vascular EC HS mimetic [167, 171-173].
For non-vascular applications, semi-synthetic and heparin-like pentosane polysulfate was used as an HS substitute [174]. Compared to HS replacement strategies, structural stabilization of HS is less common, and was only recently achieved through employment of S1P [154, 155]. For competitive binding of HS, wheat germ agglutinin lectin was previously employed to bind to sialic acid and N-acetyl-D-glucosamine, which is a residue and cleavage site of HS and hyaluronic acid, blocking access to cleavage enzymes [175]. HS synthesis enzymes, N-deacetylase/N- sulfotransferase-1 and -2 were previously overexpressed to regulate HS elongation and sulfation in human embryonic kidney cells [176, 177] but this has never been done in ECs to our knowledge.
We considered the previous HS regeneration studies, and focused our approach by regenerating
HS using exogenous HS. We did not combine exogenous HS alone and not in combination with dermatan sulfate as it exists in sulodexide, because dermatan sulfate is not among the GAGs that are naturally present in vascular EC GCX [144, 148]. GCX susceptibility to collapse, shedding, or other damage in the presence of destabilizing chemical and mechanical factors[144] was taken into consideration. To minimize GCX instability, we combined HS with S1P to prevent GCX degradation during the regeneration process [154, 155, 178]. In summary, we examined EC with
21 [i] intact HS; [ii] enzymatically degraded HS; and [iii] HS that was artificially regenerated after enzyme degradation.
In previous studies, described above, efficacy of HS regeneration was tested by examining transendothelial permeability and vascular inflammation as markers of HS-dependent cell function [155, 168-170, 175]. Few other important EC functions were tested [170]. To help advance efforts to therapeutically rebuild the GCX in a functional manner, we assessed the efficacy of HS regeneration by probing gap junctions, which are of great interest due to their complexity and mediation of many vasculoprotective EC functions [41, 179, 180]. Gap junctions in EC are formed by connexin (Cx) proteins of various isoforms. Cx43 is the most abundantly expressed connexin in cultured EC [181, 182] making it the focus of many previous studies, as well as the present investigation, via immunocytochemical characterization of Cx43 expression at EC borders.
Connexins chemically adapt to form multi-protein oligomers called connexons, the half channels of gap junctions [183]. Each of two neighboring EC contributes a connexon [183], which couple to form a full gap junction [184] with a central pore through which neighboring ECs can exchange ions, metabolites, and other small molecules (< 1 kDa) [185]. Cx-containing gap junctions are adaptive, and opening and closure of intercellular communication are dynamic events [186]. Based on prior work by Thi et al [150], we expect to find that HS regeneration will stabilize Cx expression, supporting open gap junction communication. Thi and colleagues noted that HS-bound syndecan and F-actin are connected, and F-actin is linked to the intercellular junction complex via zona occludin 1 (ZO-1) [187], which plays a role in docking the Cx gap junction proteins at the cell membrane [181, 188, 189]. Thi also noted coordinated F-actin stress fiber formation and ZO-
1 and Cx43 reorganization in response to flow stimuli, only when HS is intact and not when it is degraded [150]. This prior work [150] suggests that reinforcing HS to neutralize the effect of its
22 shedding from the GCX will maintain Cx43 expression and open gap junctional communication.
To confirm this, our study determined HS regeneration efficacy by measuring EC-to-EC spread of gap junction permeable Lucifer Yellow dye as an indicator of the activity level of Cx-containing gap junctions.
Shedding of GCX and its component HS, as well as alterations in gap junctional communication, have all been connected to the onset and the progression of many vascular diseases including atherosclerosis [190-194]. Therefore, the results of this study will enhance our understanding of vascular disease mechanisms and may introduce a new approach to restoring vascular health.
2.2 MATERIALS AND METHODS
General Methods
The general design of this investigation is summarized in Table 2.1.
Table 2.1: Summary of experimental design. Abbreviations: Hep III is heparinase III; HS is heparan sulfate; S1P is sphingosine 1-phosphate; DMEM is Dulbecco’s Modified Eagle Medium; P/S is penicillin/streptomycin; FBS is fetal bovine serum; BSA is bovine serum albumin; IU is international units; Cx43 is connexin isoform 43.
23 Cell Culture
Rat fat pad ECs (RFPEC) [195] at passages 20 to 39 were offspring of cells isolated from rat epididymal fat pad [195] and provided by Dr Mia Thi of Albert Einstein College of Medicine
(Bronx, NY). They were used because they exhibit abundant glycocalyx compared to other cell types and also respond to shear stress like other endothelial cells [140, 195, 196]. The RFPEC are immortalized, making it possible for us to use the late passages. RFPEC were seeded on 12 - 14 mm diameter and 0.13 - 0.17 mm thick glass coverslips at a seeding density of 15,000 - 20,000 cells/cm2. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, USA) with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS, Gibco Life
Technologies). Cells were maintained in humidity at 37°C and 5% CO2. RFPEC reached full confluence in 3 days. Cultured RFPEC produce thick and robust GCX in this time period, which is an advantage. At 3 days post-seeding the RFPEC culture medium was supplemented with 1%
BSA in place of FBS, to stabilize the GCX during preservation and immunolabeling (described below). For enzyme treatment experiments, 25 micro-international units per milliliter (IU/ml) of heparinase III (Hep III; IBEX, Canada) were added to BSA-containing culture medium for 2 hours to degrade HS from the GCX [52]. HepIII was washed out, followed by 16-hour incubation of
RFPEC in regular BSA-containing medium. In self-recovery experiments, RFPEC GCX was left to recover for 24 hours after enzyme degradation. The time frame was chosen in accordance with the 20-hour time period that is required for HS restoration to occur on the surface of ECs [128].
For HS recovery experiments, exogenous HS and/or S1P were applied for a critical 16-hour time frame to accommodate our communication test as an assessment of functional glycocalyx recovery. Our group previously showed that, upon external stimulation, induced communication
(transfer of gap junction permeable Lucifer Yellow dye) between neighboring endothelial cells is
24 time-dependent exhibiting a substantial increase in communication in 16 hours compared to a low level of communication in times limited to 5 hours[152]. After HS was degraded with HepIII it was replaced by a 16-hour feeding of RFPEC with 59 µg/ml exogenous porcine mucosal HS
(Celsus, Cincinnati, OH), based on published serum concentration of HS [50]. In Sphingosine 1- phosphate experiments, culture medium containing enzyme was substituted for media containing either 10µM of S1P (Sigma-Aldrich) or a combination of 59 µg/ml of HS and 10µM of S1P for
16 hours after enzyme degradation. Below we describe how GCX structure, connexin expression, and gap junctional coupling were characterized in RFPEC with intact GCX, HS degraded GCX, or different modes of GCX repair.
Scanning Electron Microscopy
RFPECs, still adherent on glass, were fixed for 1 hour in a mixture of 2% paraformaldehyde, 2% glutaraldehyde, and 0.1 M cacodylate buffer, with or without 0.15% ruthenium red, at a pH of 7.4.
After fixation, RFPEC were washed three times at 5 to 10 minute intervals, prior to being incubated for 1 hour in 0.15 M cacodylate buffer containing 1% osmium tetroxide. Following another cacodylate buffer wash cycle, RFPEC were dehydrated using a graded series of ethanol concentrations that included 30%, 50%, 70%, 85%, and three times at 100%, each for 5 minutes at a time. The dehydration series was followed by critical point drying from CO2. The RFPEC on glass were then attached to sample mounts using double-sided carbon adhesive and coated with 5 nm platinum using a Cressington 208 HR sputter coater. Imaging was performed using a Hitachi
S-4800 scanning electron microscope at low accelerating voltage of 3 kV and a magnification of
3000x.
25 Immunostaining
RFPEC have high affinity to most antibodies that are available for performing immunocytochemistry [52]. For heparan sulfate (HS), RFPEC monolayers treated with 2% paraformaldehyde/1% glutaraldehyde in phosphate buffered saline (PBS) for fixation and treated with 2% goat serum in PBS to block non-specific ligands, were stained for three nights at 4°C with a 1:100 10E4-epitope HS antibody (Amsbio). As specified by manufacturer, the HS antibody reacts with many types of heparan sulfate. The reactivity of the HS antibody is abolished after treatment with Hep III, indicating antibody specificity [197, 198]. For secondary detection of HS,
Alexa Flour 488 conjugated goat anti-mouse IgG/IgM (H + L; Life Technologies) secondary antibody was used. RFPEC to be processed for connexin 43 (Cx43) were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 (Fisher), and blocked in 5% goat serum combined with 0.2% Triton X-100. Connexin 43 staining was performed overnight at 4°C with
1:100 mouse monoclonal Cx43 antibody (Millipore). The manufacturer indicates that anti-Cx43 corresponds to amino acids 131-142 of human Cx43 and is homologous with rat Cx43 (Millipore).
Secondary Cx43 detection was done with Alexa Flour 488 conjugated goat anti-mouse IgG.
Control RFPEC samples in which anti-HS or anti-Cx antibodies were omitted prior to the application of secondary antibodies did not exhibit immunofluorescence. These controls confirmed that the staining observed in the RFPEC is not artefactual.
Confocal Immunofluorescence Microscopy Imaging and Analysis
Alexa Fluor 488 fluorescent RFPEC were mounted with Vectashield containing DAPI (Vector
Labs) and imaged with a Zeiss LSM 700 laser scanning confocal microscope. HS and Cx43 were imaged at 63X magnification (oil objective) and 40X magnification (oil objective), respectively.
Lasers with excitation wavelengths of 490 nm (for HS or Cx43) and 350 nm (for DAPI) were used
26 to obtain XY-plane slices. Laser gain and transmission were kept below fluorophore saturation levels. Confocal slice intervals were 0.2 µm for HS and 0.7 µm for Cx43. Slices were Z-projected using NIH ImageJ software. For further HS analysis, from the en face view of the 490 nm channel
Z –projection, ImageJ measured and divided the area of fluorescence by the area of the total field of view to obtain a percent value. This percentage represented the amount of RFPEC that was covered by GCX. Cross-sectional images were qualitatively assessed to ensure HS presence on the
RFPEC surface and not inside the cells. To complete our analysis of Cx43, images of multiple fields were collected to form tiles capturing a 10,0000 µm2 field of view. From the en face view of the 490 nm channel Z –projection, ImageJ randomly selected nine cells (Fig. 2.1A). The ImageJ freehand tool was used to outline the border of each of the nine cells, and to measure each cell perimeter. The length of the perimeter portion that showed Cx43 fluorescence was also measured.
The Cx43 length was divided by the perimeter length to determine the percentage of Cx43 distribution along the perimeter of the cell.
Dye Transfer Assay, Fluorescence Microscopy, & Image Analysis
Gap junction functionality was assessed by loading Lucifer Yellow in RFPEC, using a scratch technique[199], and observing the extent of Lucifer Yellow transfer from loaded cells to neighboring cells (Fig. 2.1B). Lucifer Yellow has a molecular weight of 457.3 and can only enter cells via broken membranes or via gap junctions. The scratch loading technique involved pre- incubation of RFPEC (for 1 min) with calcium and magnesium free Hanks’ Balanced Salt Solution
(HBSS; Life Technology) containing 5 mg/ml Lucifer Yellow dye (Life Technology). We then used a 5µm diameter tip scribe (Ted Pella, Inc, USA) to carefully create a straight scratch in the
27 Figure 2.1: A. To quantify Cx43 coverage of RFPEC monolayer ImageJ automatically selected nine locations, as marked with red crosses, to randomize the cells that were quantified. B. To quantify gap junction mediated cell communication, cells were scratch- loaded (red-labeled cells only) with Lucifer Yellow dye, the dye spread to neighbors, and the neighboring cells that contained Lucifer yellow dye (green-labeled cells) were counted along lines perpendicular to the scratch.
RFPEC monolayer and to allow the dye to enter the scratched cells.
After the dye was loaded, it spread via open gap junctions to
adjacent intact cells, with the extent of Lucifer Yellow spread
reaching a plateau by 10 minutes after loading. At that point, excess
dye was washed out and RFPEC were imaged at 10X magnification
(dry objective) using a Zeiss Observer Z1 fluorescence microscope.
From recorded images of dye transfer after scratch loading, ImageJ randomly selected dye spread locations that would be quantified along the scratch. At those random locations, lines perpendicular to the scratch axis were drawn along the perpendicular lines, the intact fluorescent cells (scratched cells excluded) were counted. The resultant values were taken to represent the extent of Lucifer
Yellow spread across the cell monolayer.
Statistics
Data were obtained from 3 – 5 separate experiments. Per condition, duplicate samples were examined. For HS samples we obtained 5 data points, for Cx43 9 data points, and for Lucifer
Yellow 20 data points. Data was reduced to mean ± SEM. GraphPad Prism software was used to analyze the data via one-way ANOVA and Bartlett’s statistical correction test, which is sensitive to deviation from normality. Differences in means were statistically significant at p < 0.05.
Statistical significance of differences in means is specified in the Figures and captions.
28 2.3 RESULTS
HS Expression in Intact GCX, Enzyme Degraded GCX, and Repaired GCX Conditions
We examined the membrane-attached extracellular matrix of RFPEC cultured at passages 20 to 39 on 0.13 - 0.17 mm thick and 12 - 14 mm diameter round glass. Expression of the GCX and its HS component on the surface of these RFPEC was confirmed using scanning electron microscopy [200] and immunofluorescence confocal microscopy [144, 196], as utilized in previous studies.
At baseline conditions (untreated control), where RFPEC were fully confluent and had not yet undergone any treatment, scanning electron micrographs distinctly showed long, thin, extracellular microvilli structures extending from the surface of the RFPEC plasma membrane (Arrows shown in Fig. 2.2A). Ruthenium red staining revealed a large portion of other extracellular structures consisting of GAGs, localized on the apical surface as well as at the junction between cells (Arrow shown in Fig. 2.2B). The GCX collapses during the dehydration process that is required for scanning electron microscopy, complicating quantification of GCX structure [144]. Our scanning electron microscopy experiments revealed a number of intercellular gaps (Arrowheads shown in
Fig. 2.2), which we suspected were due to dehydration and which reinforced that this approach would be useful only for GCX localization and structure but not for precise quantification. A correlative microscopy approach that requires less detrimental cell preparation, such as immunofluorescence confocal microscopy, would be required [144].
29
Figure 2.2: Scanning electron micrographs show brush-like protrusion from the cell surface. A. No GCX-specific contrast agent was used. Long arrows point to long, thin extracellular fibrillar structures extending from the surface of the RFPEC plasma membrane. Arrowheads point to intercellular gaps. B. Ruthenium red was used as contrasting agent. Ruthenium red specifically binds GAGs and revealed the strong presence of GAGs at the apical surface and at borders of the cell body. Medium length arrows point to ruthenium red at cell borders. Arrowhead points to intercellular gap. Bar = 10 µm.
Confocal micrographs illustrated that one of the GAGs on the apical EC surface included HS, as expected (Fig. 2.3). For baseline conditions in which RFPEC GCX was left intact and untreated,
71.85 ± 1.90% of cell surfaces were covered with HS as indicated in immunofluorescence labelling
(Fig. 2.3A and 2.3G). Control RFPEC samples in which HS antibody were omitted prior to the application of secondary antibodies did not exhibit immunofluorescence (Fig. 2.4A). These controls confirmed that the staining observed in the RFPEC is not artefactual. The effect of enzymatic removal of HS from RFPEC GCX using Hep III is shown in Fig. 2.3B and quantified in Fig. 2.3G. RFPEC treated with a 25 IU/ml concentration of Hep III for 2 hours, and probed at
16 hours post-enzyme treatment, showed HS coverage of 46.4 ± 1.20% which is statistically significantly less than in untreated control samples (Fig. 2.3G). As a positive control, after enzyme degradation the RFPEC were allowed to recover for 24 hours and self-regenerate their HS
30 coverage.
Figure 2.3: HS expression in intact GCX, enzyme degraded GCX, and repaired GCX conditions. A. Untreated (control) cells show intact HS at baseline conditions (green is HS with blue DAPI staining the cell nucleus). B. With 25 μIU/ml of Hep III, HS is degraded. C. Degraded HS samples were left for 24hrs to allow cells to regenerate HS. D. With the addition of HS at a 59 μg/ml concentration, there is a significant recovery of HS back to baseline conditions. Bar = 20 µm, and applies to A through F. E. Adding 10µM S1P to enzyme treated samples significantly recovered the expression of HS in GCX, back to baseline conditions. F. Combined treatment of exogenous HS and S1P effected HS expression HS by restoring baseline conditions. G. Quantification of HS in control, Hep III-treated, self-recovery, and artificial recovery conditions. ANOVA and Tukey post hoc test showed statistical significance as noted in the plot and summarized in the table. The results of various treatment conditions were compared to either control or enzyme (Hep III) conditions.
31 The 24-hour time period for self-regeneration was selected because it is established that HS restoration on the surface of ECs requires 20 hours in static conditions [201]. This cellular self- regeneration resulted in restoration of HS coverage to 69.79± 3.92% (Fig. 2.3C and 2.3G), which was statistically similar to baseline conditions and statistically greater than enzyme treatment conditions (Fig. 2.3C and 2.3G). We found that restoration of HS by artificial regeneration could be achieved in a shorter time period (16 hours) than what was required for restoration of HS by cellular self-regeneration. For artificial regeneration of HS, to counteract the effect of Hep III, immediately after Hep III treatment we exposed RFPEC to 59 µg/ml of exogenous HS during the
16-hour period following enzyme treatment. This caused an increase in the coverage of HS to
65.58 ± 2.26% (Fig. 2.3D and 2.3G). The added HS was also found to be non-toxic for RFPEC
(data not shown) and showed up on the cell surface and was not internalized by the cells (Fig. 2.3D orthogonal view). Treatment for 16-hr with 10µM of S1P to neutralize Hep III yielded HS coverage of 71.97 ± 5.02% (Fig. 2.3E and 2.3G). Delivering 10µM S1P in combination with 59
µg/ml exogenous HS for 16 hours resulted in HS coverage of 60.97 ± 4.59%. Statistical analysis confirmed that artificially recovered HS, via the addition of exogenous HS and/or S1P, was significantly more than HS in enzyme treated conditions and similar to HS in baseline conditions
(Fig. 2.3F and 2.3G).
Cx43 in Intact GCX, Enzyme Degraded GCX, and Repaired GCX Conditions
To study the effect of GCX HS conditions on Cx-containing gap junctions, we first analyzed the distribution of Cx43 at cell borders in samples of EC with intact GCX HS, degraded GCX HS, and
GCX HS recovered by various strategies. Our confocal microscopy studies confirmed that Cx43 is abundantly expressed by RFPEC and localized primarily at the cell borders where functional gap junctional communication takes place. The initial Cx43 distribution in untreated control
32 RFPEC samples was 59.61 ± 3.20% along the perimeter of the cells (Fig. 2.5A and 2.5G).
Specificity of Cx43 immunolabeling was confirmed by omission of Cx43 antibody and incubation of control RFPEC samples with secondary antibody only, which did not result in any detectable immunofluorescence (Fig. 2.4B).
Figure 2.4: A. Negative Control for HS: primary antibody targeting HS was omitted to confirm the specificity of the antibody. No HS was stained as observed both in the en face image and the orthogonal view (blue stain indicates DAPI staining for cell nucleus). B. Negative control for Cx43: Primary antibody specific to Cx43 was omitted in the immunostaining protocol to confirm the specificity of the antibody. No Cx43 was stained (blue stain indicates DAPI which stains the cell nucleus).
Enzymatic removal of HS by using 25 IU/ml of Hep III resulted
in a statistically significant decrease (p<0.05) in the percentage of
Cx43 distribution, to 30.40 ± 4.65% (Fig. 2.5B and 2.5G) which
is approximately 30% less than in untreated control samples (Fig.
2.5A and 2.5G). In RFPEC that were allowed to self-regenerate their HS coverage after enzymatic degradation, Cx43 appeared along 53.89 ± 3.44% of cell borders, which was statistically equivalent to control conditions and statistically greater than enzyme treatment conditions (Fig. 2.5C and 2.5G). Addition of 59µg/ml of HS to counteract enzymatic degradation resulted in Cx43 increase to 46.45 ± 4.21%, representing a statistically significant recovery compared to enzyme treated samples and statistical similarity to control conditions (Fig. 2.5D and 2.5G). Samples with S1P treatment after enzyme degradation presented a Cx43 distribution of 34.81 ± 5.16%, which, to our surprise was not statistically different than enzyme treated samples and statistically less than baseline levels at control conditions (Fig. 2.5E
33 and 2.5G). Addition of both HS and S1P resulted in an increase in Cx43 distribution to 56.21 ±
6.32%, a statistically significant recovery from enzyme treatment conditions and statistically close to baseline levels (Fig. 2.5F and 2.5G). This HS recovery due to combined HS and S1P can be attributed primarily to exogenous HS, since S1P alone could not induce HS recovery.
Figure 2.5: Images and quantification of Cx43 after various modes of GCX recovery: A - F. Representative images of Cx43 expression at the monolayer level, with insets clarifying Cx43 distribution at cell borders. A. Untreated sample. B. After Hep III treatment Cx43 distribution was reduced. C. After enzyme treatment, samples were left to self-recover their Cx43 over 24 hours. D. Cx43 distribution was partially restored after addition of 59 μg/ml exogenous HS to counteract enzyme treatment. Bar = 100 µm in low magnification image and bar = 2.5 µm in high
34 magnification image, and both apply to A through F. E. Addition of 10µM S1P alone did not show a significant restoration of Cx43 expression in comparison of enzyme treated samples. F. Combined treatment of HS and S1P after Hep III treatment resulted in expression of Cx43 that matched controls and was greater than in enzyme treated samples. G. Quantification of Cx43 distribution at cell borders of RFPEC for control, Hep III-treated, self-recovery, and artificial recovery conditions. ANOVA and Tukey post hoc test showed statistical significance as noted in the plot and summarized in the table. The results of various treatment conditions were compared to either control or enzyme (Hep III) conditions.
Gap Junction Communication is Lost with GCX Degradation and Recovery Depends on
Mode of GCX Repair
Having confirmed Cx expression levels per GCX condition, we measured the extent of functional communication via Cx-expressing gap junctions. The opening and closing of the gap junctions was assessed by end-point quantification of the extent of movement of gap junction permeable scratch-loaded Lucifer Yellow dye between neighboring cells, in monolayers of RFPEC with intact GCX and in comparison to monolayers of RFPEC with degraded GCX or after different modes of GCX recovery. In cultured RFPEC monolayers, dye spread (intercellular communication) from the scratch to relatively few neighboring cells was taken to suggest weak communication. Spreading of dye among a relatively large number of cells indicated stronger communication. Dye spread was never observed to extend throughout the entire cell monolayer despite the fact that most RFPEC were expressing Cx43.
As indicated in Fig. 2.6A and 2.6G, for every cell along the scratch, uploaded Lucifer Yellow dye transferred to 2.88 ± 0.09 neighboring RFPEC in untreated control conditions. Following Hep III removal of HS from RFPEC, we observed a statistically significant decrease in Lucifer Yellow spread, with 1.87 ± 0.06 cells receiving the dye from an adjacent scratch-loaded cell (Fig. 2.6B and 2.6G). Self-regeneration of HS after enzyme degradation yielded Lucifer Yellow spread of
2.64 ± 0.07 cells which was statistically similar to control conditions and statistically significant
35 recovery from enzyme conditions, as expected (Fig. 2.6C and 2.6G). In RFPEC that had been exposed to HS degradation enzyme, artificial replacement of HS with the exogenous GAG did not yield any recovery of Lucifer Yellow dye-coupling. We were surprised to discover that, under conditions of HS recovery with exogenous HS, scratch-loaded dye was only transferred to 1.03 ±
0.07 cells (Fig. 2.6D and 2.6G). This was statistically less than dye transfer in both baseline and
Hep III conditions (Fig. 2.6D and 2.6G). Artificial recovery with only S1P added after enzyme degradation showed a Lucifer Yellow dye spread of 2.06 ± 0.08 cells (Fig. 2.6E and 2.6G), statistically similar to the dye transfer level under Hep III treatment conditions. We were pleased to find that, after the treatment of RFPEC with combined HS and S1P, 3.96 ± 0.23 cells received
Lucifer Yellow dye. This cell number is statistically significantly high when compared to cell number in both untreated control and Hep III-treated conditions (Fig. 2.6F and 2.6G). This communication data, taken together with the Cx43 data, demonstrates that treatment of ECs with exogenous HS combined with S1P is the best artificial HS regeneration approach for simultaneous recovery of both Cx43 protein and gap junction function.
36
Figure 2.6: Quantification of Lucifer Yellow dye transfer after various modes of GCX recovery: A. Control B. With 25 μIU/ml Hep III treatment, the movement of Lucifer Yellow dye across cell junctions was impaired. C. Self-recovery of HS, after enzymatic degradation, allowed dye transfer across cell junctions at near baseline levels D. Not much Lucifer Yellow dye transfer improvement was observed after recovery of lost HS by addition of 59 μg/ml HS. Bar = 100 µm and applies to A through F. E. Addition of 10µM S1P also did not improve cell-to-cell communication. F. A combined treatment of RFPEC with both HS and S1P resulted in increase in Lucifer Yellow dye transfer; reversing the effects of enzyme treatment. G. Quantification of Lucifer Yellow flux across connexin-containing gap junctions in RFPEC in control, Hep III-treated, self-recovery, and artificial recovery conditions. ANOVA and TUKEY post hoc test showed statistical significance as noted in the plot and summarized in the table. The results of various treatment conditions were compared to either control or enzyme (Hep III) conditions.
37 2.4 DISCUSSION
We commenced our study by creating cell culture models of degraded and regenerated GCX, using
RFPEC as an ideal experimental cell culture system because of the inherent RFPEC expression of abundant GCX that contains substantial HS (Fig. 2.3). We created a model of degraded GCX via
Hep III-induced HS degradation (Fig. 2.3B and 2.3G), an approach that is consistent with previous work [191, 196, 201]. This enzymatic degradation model mimics diseased conditions for which release of GAGs from the endothelium surface and into the bloodstream have been reported [194,
202, 203]. Our GCX regeneration model, on the other hand, is a new contribution to the GCX research field. To our knowledge, this is the first demonstration that endothelial GCX can be considerably restored in vitro by addition of exogenous HS (Fig. 2.3D).
We took advantage of the cell surface GCX capacity to recognize and bind to extracellular
GAGs. The concentrations of supplemental HS, 59 µg/ml, was chosen to align with values for the amount of HS found in the arterial blood of patients suffering from global ischemia, reported by
Rehm et al. [194]. We anticipated a potential pitfall, in that RFPEC may not incorporate the exogenous HS in a functional manner. Our alternative GCX regeneration strategy was to use S1P to both regenerate and stabilize the GCX [154, 155] after enzymatic degradation and regeneration.
It was encouraging to find that feeding RFPEC with the prescribed concentration of exogenous
HS alone, S1P alone, or HS together with S1P led to renewed HS expression in the RFPEC surface
GCX, at a status that matched control conditions. Although using the rat ECs was ideal because of the abundant GCX that could be easily manipulated, this study is limited by the possibility that the results could be different if other cells were used. For example, using ECs from different vascular beds with potentially different GCX expression patterns could affect the outcomes of this study.
Also, primary cell cultures could give different results. Reports of the patterns of HS expression
38 in bovine- and human-derived primary ECs, for example, differ from the RFPEC HS pattern and are regulated by external mechanical stimulation [204]. We are conducting other investigations with primary ECs originating from human vessels and a variety of vascular beds. The other investigations are outside the scope of the study described in this report, and will be published in a separate research article. With this being said, RFPEC are ideal, because they enable translation of this research to preclinical in vivo studies in the future (Fig. 2.7). Rat animal models have commonly been used for preclinical translational research to test the ability of new drug treatments to mitigate pro-atherosclerotic factors and attenuate atherogenesis in vivo [205]. The RFPEC cultures used in this study are meant to be a species match for the rat animal pre-clinical trials that will be conducted in the future.
Figure 2.7: Proposed pathway for eventual translation of functional glycocalyx regeneration to the clinic: According to this pathway, the work presented in this report represents the first step toward clinical translation. This step involves basic cell culture experiments for initial determination of the efficacy of our proposed glycocalyx replacement and stabilization strategy. We used rat endothelial cells to match rat animal experiments that are common for atherosclerosis pre-clinical studies, because the cell culture work will be followed by a series of rat and other animal experiments to test in-vivo performance of the HS/S1P treatment. These cell culture and animal studies are prerequisites for phase I and phase II clinical trials on human volunteers.
39 GCX importance in key physiological events is well documented in numerous reports on studies of how characteristic cell functions are gained or lost as a consequence of GCX subcomponent degradation [131, 196, 206, 207]. To date, our colleagues Thi et al have been the only group to study GCX regulation of intercellular gap junctional communication, in a very elegant study [150]. They removed the principal GCX component, HS, and demonstrated gap junction protein desensitization to externally applied flow stimuli that would normally displace the protein. In our study, by successfully degrading and subsequently regenerating HS, we created a tool with which to substantially enhance the body of knowledge about GCX role in inter- endothelial gap junction functionality. Similar to previous findings [150], enzymatic degradation of HS reduced Cx43 at the RFPEC perimeter where cell-to-cell communication activity takes placed, by ~50% compared to baseline conditions. Our study is the first to observe significant
Cx43 recovery at cell borders, to ~80% of baseline conditions, as a result of simple replacement of HS to counteract enzymatic HS degradation. Combined treatment of RFPEC with HS and S1P resulted in further enhanced Cx43 recovery at cell borders, to ~95% of baseline conditions. These results emphasize the importance of the GCX as a regulator of cell membrane expression of gap junction protein. The expectation is that similar findings can be obtained for active cell communication, which directly depends on Cx expression and mediates other key EC functions that are prominent in vascular health and disease [180, 208].
In contrast to our Cx43 results, changes in active communication (assessed by Lucifer Yellow dye transfer) did not parallel the experimentally induced changes in HS and the GCX. As we expected, removal of HS from RFPEC reduced the spread of dye from scratch-loaded to neighboring cells by ~50%, compared to untreated RFPEC. This reduction was attributed to HS-induced decrease in membrane Cx43 and an associated decline in the number of open gap junction channels. This result
40 further confirmed the fundamental relationship between the GCX and gap junction function.
However, with HS regeneration via exogenous HS, gap junction communication remained closed.
Figure 2.8: Dose dependent test on the effect of Cytochalasin D on RFPEC actin filaments. A1. Control sample (blue is DAPI stain for cell nucleus and red stain is phalloidin 647 labelling actin filaments). A2. Phalloidin 647 stain only showing intact actin filaments. B1. Adding 50nM of Cytochalasin D initiates the process of arresting actin filament polymerization, resulting in the gradual deterioration of cytoskeletal stability. B2. Phalloidin 647 stain only. C1. Increasing the concentration of Cytochalasin D to 100nM totally arrests actin filament polymerization, deforming the cytoskeleton of the cells and causing a rounded cell morphology.C2. Phalloidin 647 stain only. (Scale bar is 20µm and confocal magnification is 63x. Data was not quantitatively analyzed)
It is unclear why exogenous HS restoration did not induce gap junction channels to re-open along with renewal of Cx43 expression at cell borders. It is plausible that GCX restoration by exogenous
HS does not translate to full repair of the plasma membrane, which is critical for gap junction proteins to be expressed at the membrane [94] at a level that is sufficient for gap junction formation.
41
Figure 2.9: A1. Bright-field image of control RFPEC. A2. Lucifer yellow dye transfer to neighboring cells in control samples. B1. Bright-field image of enzyme (Hep lll) only treated RFPEC. B2. Lucifer yellow dye transfer of enzyme treated sample: Lucifer yellow dye is bared from moving through gap junctions to neighboring cells.C1. Bright-field image of artificially recovered RFPEC GCX: Artificial recovery includes the addition of exogenous HS and S1P after Hep lll treatment for 16 hours.C2. Lucifer yellow dye transfer to neighboring cells, recovery sample exhibited significant dye transfer across neighboring cells in comparison with both control and enzyme treated samples. D1. Bright-field image of RFPEC after adding 50nM of Cytochalasin D to artificially recovered samples. D2. Adding 50nM of Cytochalasin D for the last 30 minutes during artificial recovery of RFPEC GCX reduces Lucifer yellow dye transfer in comparison with artificial recovered samples only. E1. Bright-field image of RFPEC after adding 100nM of Cytochalasin D to artificially recovered RFPEC. E2. Adding 100nM of Cytochalasin D for the last 30 minutes during artificial recovery bars Lucifer yellow dye transfer to neighboring cells similar to enzyme treated samples. F1. Bright-field image of Dimethyl sulfoxide(DMSO) only samples: This was necessary to determine if DMSO alone had an effect on cell to cell communication because DMSO was used to conjugate the Cytochalasin D powder. F2. Lucifer yellow dye transfer to neighboring cells almost similar to what was observed in control samples, indicating that adding DMSO alone has no significant effect on cell to cell communication. This implies that the reduction in cell to cell communication in the presence of Cytochalasin D could essentially be attributed to arrest of actin cytoskeleton by Cytochalasin D. (Note: Data is not quantified quantitatively. Scale bar is 100µm, magnification is 10x)
The most probable explanation is that the experimentally restored GCX may have a configuration of low stability that exerts reduced tension [209] on the trans-membrane core proteins and, consequently, reduced tension on the cytoskeleton. Downstream, we speculate, this
42 low tension may destabilize the cytoskeleton link with the intercellular junction complex. This weak link could result in improper Cx43 alignment and keep connexins from combining to form connexons, block connexon docking to form gap junctions, or prevent gap junction gates from opening. This hypothesis is supported by the results of preliminary studies that actin disruption by
Cytochalasin D bars cell-to-cell movement of Lucifer Yellow dye and other ions and molecules
(Fig. 2.8 and Fig. 2.9).
The bioactive agent S1P has been reported to preserve GCX [155, 178, 210], and was recently shown to induce recovery of the HS GCX component in the absence of HS at baseline conditions
[178]. The detailed mechanism underlying S1P’s role in GCX preservation and growth is still under investigation.
In addition, S1P has been shown to enhance the strength of the intercellular junction complex, in a study that showed S1P causes redistribution of ZO-1 to lamellipodia and cell-to-cell appositions [211] and in another study that demonstrated that S1P enhances the role of Cx in vasculoprotection [212]. Through these reported mechanisms, and possibly others, S1P maintains vascular functions such as regulation of transendothelial permeability [155, 211]. Surprisingly, the use of S1P to regenerate degraded HS did not improve the expression of Cx43 or the level of active communication. Instead, S1P turned out to be an important co-factor for exogenous HS.
Regeneration of the GCX via the combination of S1P with exogenous HS resulted in both increased Cx43 at cell borders and greater movement of Lucifer Yellow dye across cells, indicating a recovery of structure and function of both GCX and gap junctions. In fact, HS/S1P-treatment over-recovered gap junctional dye spread, achieving a 1.4-fold increase compared to pre- degradation baseline conditions. Arguably, this gap junction hyperactivity (above baseline level) may signify some form of abnormality [213] or pathology. However, in a recent report by Jiang et
43 al, it was demonstrated that high levels of Cx43-mediated gap junctional communication attenuate the degree of malignancy in multiple cancer cell lines [214]. Considering that cancer and vascular diseases share common pathology progression pathways [215], the results of the study conducted by Jiang et al can be extrapolated to suggest that over-recovery of interendothelial gap junction- mediated dye spread is not necessarily pathological and may actually be physiological beneficial.
In future studies, a conscious effort must be made to identify and treat ECs with levels of S1P that ensure the stability of HS while at the same time confirming that restored interendothelial gap junction activity is within physiological limits. The mechanism of action of HS/S1P-treatment should also be clarified. For example, recovery of gap junction activity via HS/ S1P may involve non-Cx43 gap junction proteins (Cx40 and Cx37) as well as other junctional proteins (ZO-1, cadherins, and catenins, etc.) and other GAGs (chondroitin sulfate, sialic acid, and hyaluronic acid). This work is outside the scope of this study but of great relevance for fully understanding the relationship between GCX regeneration, Cx protein expression, and gap junction function.
The findings of this work add fundamental knowledge to this understudied area and are encouraging for future therapeutics. As depicted in Fig. 2.10, the key novel and compelling findings are: (i) removal of GCX-associated HS not only has the ability to alter organization of gap junction proteins, namely Cx43, but it also shuts down gap junction channel activity, and (ii)
GCX repair by treating cells with exogenous HS and S1P restores gap junction protein placement which translates to the reactivation of gap junction channel activity.
44
Figure 2.10: Conceptual Hypothesis: A. Inter-endothelial gap junction is fully functional due to intact GCX (green arrows show Lucifer Yellow dye flux) in control conditions, when cells self-recover HS, or when HS is artificially recovered by treatment of cells with combination of exogenous HS and S1P. B. Degraded HS from GCX will cause the dislocation of Cx43 and malfunction of gap junction channels thereby preventing the movement of Lucifer Yellow dye across inter-endothelial gap junctions (red line indicates the improper functioning or blockage of gap junctions). C. Treatment of enzyme- treated cells with exogenous HS or S1P does not fully reset CX43 and/or recover intercellular gap junctional communication.
To our knowledge, we are the first
group to achieve functional GCX
regeneration in ECs in a manner that
effectively restores vasculoprotective
EC communication within a short time frame. This current finding could translate to therapy for preventing atherosclerosis and motivates future development of new therapies targeted at the GCX to treat vascular disease. The ‘Holy
Grail’ would be to discover innovative methods for regulating EC GCX enzymatic degradation and synthesis with subcellular Golgi and endoplasmic reticulum specificity and at vascular tree locations that are most relevant. In the meantime, in the absence of widely adopted approaches to reinforce the vascular GCX, implementation of our innovative approach in basic cell culture
45 studies, pre-clinical in vivo studies, and, potentially, in clinical settings (Fig. 2.7) will be highly significant to advance GCX knowledge and to better address endothelial-dependent disease processes.
46
CHAPTER THREE: METASTATIC CANCER CELL ATTACHMENT TO ENDOTHELIUM IS PROMOTED BY ENDOTHELIAL GLYCOCALYX SIALIC ACID DEGRADATION
PUBLISHED IN AIChE JOURNAL, 2019
Solomon A. Mensah1, Ian C. Harding1, Michelle Zhang2, Michael P. Jaeggli1, Vladimir P. Torchilin3, Mark J. Niedre1, 4 Eno E. Ebong1,2,5, ‡
1. Bioengineering Department, Northeastern University, Boston, MA. 2. Chemical Engineering Department, Northeastern University, Boston, MA. 3. Pharmaceutical Sciences Department, Northeastern University, Boston, MA. 4. Electrical and Computer Engineering Department, Northeastern University, Boston, MA. 5. Neuroscience Department, Albert Einstein College of Medicine, New York, NY
47 3.1 INTRODUCTION
Cancer metastasis is one of the major causes of cancer-related deaths 1,2. During metastasis, primary tumor cells migrate from the parent tumor into neighboring tissues to form secondary tumors 3. This occurs when cells from the primary tumor migrate to and intravasate nearby blood vessels, travel through these vessels, and eventually extravasate the vessels at distant tissues where secondary tumors can form 4. The migration of cancer cells out of blood vessels, requires initial homing to and crossing of the adhesive endothelium, and it has been established that this occurs due to dysfunction of the endothelium’s glycocalyx (GCX) coating (Fig. 3.1A) 5,6.
GCX covers most mammalian cells. For endothelial cells (ECs), a healthy GCX forms a selective barrier between ECs and their neighboring environment, by blocking adhesion receptors on the endothelium from binding to ligands on cells and certain molecules from the environment
(Fig. 3.1A) 7-12. The EC GCX is composed of a variety of sugar chains, glycoproteins, and soluble proteins. For example, the GCX includes core proteins on which heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) glycosaminoglycan (GAG) sugar chains are attached 13,14.
HS is the most abundant GCX component. The core proteins are also attached to sialic acid (SA) on their terminal ends 15. Degradation of these key components of GCX is associated with certain conditions and diseases including inflammation, sepsis, atherosclerosis, and cancer 16-21.
To date, only a few cell mechanism studies have explored how HS, CS, HA, and SA regulate homing of cells, particularly white blood cells and cancer cells, from vessel circulation to the endothelium surface 6,22-30. Selected reports are summarized in Table 3.1, which show that the majority of investigations have been performed in the context of white blood cells and inflammation 22-25,27,31-33, more work is required in the cancer milieu in order to address metastasis
6,28-30. The white blood cell studies inform our understanding of GCX-mediated EC adhesiveness
48 to cancer cells, because cancer cells utilize inflammation mechanisms to metastasize 21,34-36 and inflammatory agonists are known to cause endothelial GCX shedding 37 that can expose endothelium to circulating cell adhesion.
Figure 3.1 – (A) Drawing shows endothelium with intact GCX. As metastatic cancer cells move with blood flow, the healthy GCX blocks the adhesion ligands on the cancer cells from attaching to the adhesion receptors on the endothelium lining of the blood vessel wall. We hypothesize that cancer cell attachment to the endothelium is caused by endothelial GCX degradation. (B, C) These drawings illustrate the conclusions of the findings reported herein. Our observations provide evidence that the systemic increase in Neur (B), which coincides with metastatic cancer conditions, degrades the GCX as a whole and as applied to its ∝-2,6- linked and ∝-2,3-linked SA residues (C). This GCX degradation leads to increased cancer cell attachment to ECs, and we speculate that this is mediated by exposure of adhesion receptors on the endothelium which become accessible to adhesion ligands on cancer cells (C).
49 With respect to GCX component-specific shedding, research has largely been focused on studying the role of HS loss in cancer and inflammatory cell adhesion, due to its abundance in the
GCX (Table 3.1) 6,23,25,26,38. Some work has gone into investigating how losses in HS and CS, combined, contribute to endothelium adhesiveness 24,27. Experiments conducted to isolate and elucidate the mechanisms of EC adhesiveness involving HA are rare 29, and studies isolating SA- mediated EC adhesiveness have not been performed at all, to our knowledge (Table 3.1).
Relevant to endothelium adhesiveness to cells in the blood vessels, SA is of particular interest because it is a major contributor to cellular and molecular recognition 39-41. This is due to the diversity of SA residues, which are differentiated through post-transcriptional sialytransferase activity [22, 27, 28]. On ECs, several SA residues are expressed, but the most notable include the
∝-2,6-linked, ∝-2,3-linked, and ∝-2,8-linked SA residues. These residues recognize and interact with cells and molecules through lectins, which are proteins that recognize and bind to SA and other sugars 40,42,43. In addition, SA residues are uniquely composed of amino acids and carboxyl groups that make them negatively charged, further enhancing SA’s ability to facilitate recognition and binding or repulsion to other cells or molecules. SA’s terminal location, diversity, composition, and charge, taken together, are important determinants of EC GCX-mediated endothelial barrier function. Yet, while the role of SA in the cancer cell GCX has been extensively studied and found as a marker of oncogenesis and tumor survival 44-46, the role of SA in the endothelial GCX during oncogenesis has yet to be studied.
Another reason for our interest in SA stems from reports that the SA-degrading enzyme, neuraminidase (Neur) 47, is strongly associated with cancer metastasis and other pathologies.
Specifically, Neur upregulation has been reported in hepatocellular carcinoma and ovarian cancer, and Neur has been noted to be an oncogene that enhances proliferation and migration of the
50 metastatic cancer cells 38,48. Based on these reports, it is surprising that not much effort has been made to elucidate the impact of excessive Neur on ECs, which are likely to respond by shedding their SA residues, leading to cancer cell access to the endothelium.
EC Glycocalyx Components Endothelial Circulating SA Author Type Cell Type Residues Summary/Conclusion HS CS HA α- α- α- 2,8 2,6 2,3 HEVs present themselves as rounded ECs, giving rise to weak endothelium barrier. On these cells, Human post- GCX layer is thick, capillary vein Human T- which likely enhances Anderson high lymphocytes binding sites for and Shaw endothelial (T-cells) soluble factors like venule (HEV) macrophage cells inflammatory protein-
1훃 or interleukin-8
that are critical triggers for T-cell adhesion to the endothelium. HS plays important role in leukocyte Leukocyte extravasation. Upon EC type not Blood Cells White Celie et al type not inflammation, HS specified specified shedding facilitates EC-leukocyte interactions. Blueberry metabolites helps restore cell surface GCX that was Human aortic Human Cutler et lost due to diabetes. endothelial monocytes al HS and CS presence in cells (HAEC) (THP1) the restored GCX was confirmed. GCX restoration coincided
51 with low endothelial binding of monocytes. Enzyme induced HS degradation increases adhesion of fluorescently labeled microspheres to ECs.
However, leukocyte Fluorescently adhesion to ECs Mulivor Rat Venular labeled decreases. It is et al ECs microspheres possible that the & rat enzyme that is applied leukocytes to degrade HS also removes selectins, disabling EC adhesiveness to leukocytes. Inflammation triggers lead to pulmonary Human endothelial GCX Schmidt pulmonary Mouse degradation, mediated et al microvascular neutrophils by heparinase, to ECs induce neutrophil adhesion to the endothelium. Knockout of HS- and CS-bound syndecan-1 Mouse Human Voyvodic results in ECs pulmonary monocytes et al becoming more ECs (THP-1) adhesive to monocytes. Preserving the GCX by orosomucoid, a Human plasma glycoprotein, Rat post malignant decreases Cai et al capillary breast cancer microvascular venule ECs cells (MDA- permeability and MB-231) reduces the tumor cell
Cancer Cells Cancer adhesion. Mouse brain Human Degradation of HS Fan and microvascular malignant increases tumor cell Fu ECs (bEnd3) breast cancer adhesion to ECs.
52 cells (MDA- MB-231) Removal of HA from PHAEC GCX significantly decreases Human the adhesion of cancer pulmonary Malek- Human lung cells to the aorta Zietek et carcinoma endothelium, endothelial al cells (A549) indicating the strong cells importance of HA as a (PHAEC) barrier against PHAEC-cancer cell adhesion. Receptor destroying enzyme degrades endothelial GCX and Neur degrades Mouse endothelial SA. Loss Gasic and Mouse lung ascitic tumor of GCX was found to Gasic and liver ECs cells (TA3) coincide with reduced cancer metastasis, while effect of loss of SA, specifically, was not confirmed.
Table 3.1 – Representative reports of the role of the GCX in blocking or enabling EC adhesiveness to cells in the blood circulation, including white blood cells and cancer cells. Green shading indicates that some progress has been made in understanding the role of a specific GCX components in EC interaction with circulating cells, while red shading indicates that little or no progress has been made for some GCX components. Abbreviations not defined in the table are ECs: endothelial cells; GCX: glycocalyx; HS: heparan sulfate; CS: chondroitin sulfate; HA: hyaluronic acid; and SA: sialic acid.
In the present study, we aimed to test the hypothesis that Neur-induced degradation of EC
GCX, particularly the shedding of SA residues, will result in substantial increase in metastatic cancer cell attachment to the endothelium. To test our hypothesis, we first characterized the expression of the GCX, both generally and with a specific focus on a SA residue. We then investigated the effects of Neur on the GCX. Lastly, we investigated the effect of GCX degradation
53 on the attachment of metastatic cancer cells to the endothelium. Our findings indicate that SA residues are differentially affected by the presence of Neur and confirm that GCX degradation indeed leads to increased cancer cell attachment to the endothelium. We anticipate that this body of work will extend our knowledge on the role played by GCX, specifically regarding SAs, in regulating movement of metastatic cancer cells from blood vessels to secondary metastatic sites.
This will lead to future research to identify innovative GCX-based markers that can be therapeutically targeted to hinder the progression of cancer metastasis.
3.2 MATERIALS AND METHODS Endothelial Cell Culture
GCX-rich, immortalized rat fat pad endothelial cells (RFPECs) 49-51, originally isolated by
Dr. Mia Thi of Albert Einstein College of Medicine (Bronx, NY), were used between passages 24 and 30. RFPECs were seeded onto 12 mm No. 1 glass coverslips (Fisher Scientific) at a seeding density of 15,000 - 20,000 cells/cm2. The cells were grown in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen, USA) with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum
(FBS, Gibco Life Technologies), in a humidity-controlled environment at 37°C and 5% CO2, for
3 days until they reached full confluency. For control experiments, human umbilical vein endothelial cells(HUVEC)(ATCC) were used at passages between 2 to 8. HUVEC were seeded on
22mm X 40mm rectangular coverslips with thickness of 0.17mm at a seeding density of 15,000 –
20,000 cells/cm2. HUVEC were cultured with Vascular Cell Basal Medium (ATCC® PCS-100-
030™) supplemented with Endothelial Cell Growth Kit-VEGF (ATCC® PCS-100-041™).
Neur Enzyme Degradation of the Endothelial GCX
In order to model endothelial GCX degradation, RFPECs were cultured for 2 hours in
DMEM/1% PS/10% FBS with 0 (untreated control), 15, 135, 1215, or 3645 mU/mL of the SA- degrading enzyme, Neur from Clostridium perfringens (Sigma) 52. After the 2-hour period ended,
54 the medium containing enzyme were exchanged with
Primary
Labels enzyme-free culture media before further
biotin - experimentation.
Endothelial GCX Lectin Staining, Confocal
Microscopy, and Image Analysis
Fluorescent staining was performed to assess
488 Conjugated Anti Conjugated 488 -
GCX expression on untreated or Neur-treated RFPECs.
Biotinylated Wheat Germ Germ Wheat Biotinylated (WGA) Agglutinin Sambucus Nigra Biotinylated Bark) Lectin (Elderberry (SNA) Maakia Biotinylated (MAL II Lectin Amurensis AF II) DAPI
∝-2,6 This was achieved using lectins, proteins that recognize X x X X linked and bind to sugars 40,42,43. After the Neur treatment period, ∝-2,3 X x X X linked RFPEC monolayers were quickly washed with 2% bovine
acetylneuraminic acetylneuraminic ∝-2,8
- X X X serum albumin (BSA) and fixed with 2% acid (SA) residues: acid N linked paraformaldehyde/0.1% glutaraldehyde for 30 minutes at HS X X X room temperature.
Table 3.2 – This table indicates the lectins (purchased
HA X x x from Vector Labs) that were used in labeling GCX. As
component of: component acetlyglucosamine acetlyglucosamine
- shown, WGA is a general lectin for GCX, labeling three
N SA residues along with N-acetlyglucosamine, a component of HS and HA. SNA and MAL II are specific to only ∝-2,6 linked and ∝-2,6 linked residues of SA, respectively 26,69. All lectins were biotinylated, enabling fluorescence labeling with biotin antibody conjugated to AF-488. Once lectins were applied, followed by the secondary antibody, cell nuclei were all labeled with DAPI.
The cells were then blocked for 30 minutes at room temperature with 3% BSA and subsequently incubated with various GCX labeling-lectins (Table 3.2) for 1 hour at 4⁰C. After lectin incubation, cells were incubated with Alexa Fluor 488 (AF-488) conjugated streptavidin
(Table 3.2) for an hour at 4⁰C. Fluorescently stained samples were then mounted with Vectashield anti-fade media containing 4’6-diamidino-2-phenylindole (DAPI) (from Vector Labs; indicated in
55 Table 3.2) to stain the cell nuclei. HUVEC were exposed to the same WGA staining protocol as described above and outlined in table 1 for control purposes. HUVEC were also stained for heparan sulfate as previously described (refer to Ming’s paper here).
RFPECs and HUVEC were then imaged using a Zeiss Confocal Laser Scanning
Microscope (LSM) 700 with a 63X magnification objective (oil). Lasers with excitation wavelengths of 490 nm (to detect AF-488 labeled lectins) and 350 nm (to detect DAPI labeled cell nuclei) were used to obtain images of XY-plane slices, with inter-slice spacing of 0.2 µm. The slices were then combined to create a 3D Z-projection. To determine GCX thickness and the extent of RFPEC coverage, the GCX fluorescence signal of Z-projection images were analyzed using
NIH ImageJ software as previously published 53.
Cancer Cell Culture, Attachment Assay, Fluorescence Microscopy, and Image Analysis
Stage IV metastatic mouse breast cancer cells (4T1) (ATCC) were used between passages
5 and 10. The 4T1 cells were cultured in DMEM/1% PS/10% FBS and maintained in a humidity- controlled environment at 37°C and 5% CO2. Passaging or experimental usage occurred at 80% confluency.
For easy identification of 4T1 cells in co-culture with RFPECs, live 4T1 cells were labelled with CellTracker Red CMTPX Dye (Thermo Fisher Scientific). 4T1 cell labeling was accomplished by adding 15 M of the dye to FBS-free DMEM containing a total of ~106 suspended 4T1 cells. The cell suspension was incubated at 37 °C for 20 minutes after which the sample was centrifuged, the supernatant was removed, and the pellet of CellTracker-labeled 4T1 cells was retained. The cells were then re-suspended in regular DMEM/1% PS/10% FBS, and allowed to equilibrate for 45 to 60 minutes before utilization.
56 To study cancer cell attachment to RFPECs and control HUVEC, CellTracker- labelled 4T1 breast cancer cells were co-incubated at a concentration of 103 cells/mL with confluent monolayer of either untreated or enzyme treated RFPECs. After 30 minutes of co- incubation, non-attached 4T1 cells were removed by washing with DMEM/1% PS/10% FBS three times, leaving attached 4T1 cells behind. The attached 4T1 cells and underlying RFPEC layer were then imaged using a Zeiss Z1 Observer fluorescence microscope at 10X magnification, using a dry objective. A red filter (excitation of 558 nm) was used to distinguish and image CellTracker- labelled 4T1 cells. RFPECs were visualized by phase contrast. The total number of attached 4T1 cells and the total number of RFPECs in the field of view were counted using the ImageJ cell counting tool. To quantify the extent of adhesion, the number of attached 4T1 cells was divided by the number of RFPEC cells in the field of view. This quantity was normalized by the value obtained for 4T1 adhesion to untreated RFPECs (control).
Statistics
The number of experimental repeats varies with the experiment type, and details are indicated in the data plots. Data sets were reduced to means ± SEM. When two treatments were compared, after checking for normal distributions using the D'Agostino & Pearson normality test and verifying equality of variance using the two sample F-test, student’s t-tests were used to determine statistical significance. For multiple comparisons, after confirming that the data fit normal distributions, one-way ANOVA analyses and Tukey post-hoc tests were used to determine statistical significance. An alpha value of p < 0.05 was used for both t-tests and ANOVA. All statistical analyses were performed using GraphPad Prism.
57 3.3 RESULTS AND DISCUSSION
The endothelial GCX plays a significant role as a barrier between the endothelium and circulating cells present in the blood stream, including cancer cells 6,22-30. The importance of the endothelial GCX in enabling EC adhesiveness to cancer and other types of circulating cells, primarily when its HS component is shed or in the presence of heparinase enzyme that specifically degrades HS, has been well established (Table 3.1) 6,22-28,30. Regulation of EC adhesiveness by endothelial GCX components such as CS and HA has also been demonstrated (Table 3.1) 6,23-29.
Another important endothelial GCX component, namely SA, its residues, and its degrading enzyme, Neur, remains understudied in the context of EC adhesion to circulating cancer cells
(Table 3.1). The present study aimed to fill this gap, by studying the impact of Neur-induced degradation of EC GCX, particularly the SA component, on metastatic cancer cell attachment to the endothelium.
Neur Elevation in the EC Environment Destabilizes WGA-Labeled GCX
RFPECs were selected as an appropriate cell model for this study because of their ability to produce robust GCX even in static conditions, while other cell culture models require shear stress to stimulate the synthesis of GCX 51. Upon the initial inspection of RFPECs treated with
Neur, we observed that RFPEC monolayers exposed to Neur had preserved morphology
(compared to untreated samples) indicating that only the GCX would be affected by the presence of the enzyme and not the underlying endothelium (Fig. 3.2A-3.2E).
58
Figure 3.2 – Images show the effect of exposure to various concentrations of Neur enzyme on the GCX, endothelium integrity, and cancer cell attachment to ECs. As shown, Neur concentrations include 0, 15, 135, 1215, and 3645 mU/mL. A-O: 5X magnification phase contrast images merged with red fluorescence micrographs, to confirm integrity of the EC layer in all conditions and show cancer cells attached to the endothelium. Scale bar equal to 500 µm is shown. P-T: 63X magnification confocal micrographs of GCX labelled with green fluorescence conjugated to the following lectins: WGA (A-E), SNA (F-J), and MAL II (K-O). The blue is DAPI, which labels EC nuclei. Scale bar equal to 20 µm is shown. U-Y: 5X magnification red fluorescence micrographs clarify the presence of CellTracker Red labeled cancer cells. Scale bar equal to 500 µm is shown.
59 To determine the effects of Neur on GCX integrity, RFPECs were first labeled with WGA (Fig.
3.2F-3.2J). WGA is reported to delineate the overall GCX structure better than other lectins 54,55, and binds to a number of SA residues along with a few other GCX components (Table 3.2).
Figure 3.3 – Comparing the coverage (A) and thickness (B) of GCX components that bind WGA, SNA and MAL II at various Neur concentrations. Compared to untreated baseline conditions as indicated by the dashed line (- - -), Neur concentration of 15mU/mL slightly reduced thickness of and coverage of the endothelium by WGA-labeled GCX, SNA-labeled ∝-2,6-linked SA residue, and MAL II-labeled ∝-2,3-linked SA residue. SNA-labeled ∝-2,6-linked SA residue was reduced most statistically significantly. 135 mU/mL of Neur slightly reduced WGA- labeled GCX but statistically significantly reduced SNA-labeled ∝-2,6-linked SA residue and MAL II-labeled ∝-2,3-linked SA residue. 1215 mU/mL and 3654 mU/mL of Neur further reduced WGA-labeled GCX, SNA-labeled ∝-2,6-linked SA residue, and MAL II-labeled ∝-2,3-linked SA residue. Results are normalized based on 0 mU/ML conditions. Significance differences between groups are denoted as *****P<0.0001, and ‘ns’ denotes non-significance.
We discovered that at baseline
conditions (i.e. in the absence of Neur), WGA- labeled GCX covered 73.87.4 % of RFPEC monolayers (Fig. 3.2F), represented by a normalized value of 1.000.07 (Fig. 3.3A and 3.4A). WGA-labeled GCX thickness was 1.460.02µm, a normalized value of 1.000.02 (Fig. 3.3B, 3.4B). Previously, Reitsma et al. reported a WGA- labeled GCX layer to cover about 90% of the endothelium and to be 2.30.1µm thick 56. The
60 coverage and thickness of the WGA-labeled GCX in our study was less, which could be attributed to differential GCX expression in varying vascular tissue beds 7,57,58. Additionally, our in vitro
GCX is expected to be thinner than that found in vivo due to the absence of BSA in the experimental media and also the collapse of the GCX that was previously reported to occur ex- vivo and in-vitro 56,59,60.
After 2-hour treatment of RFPEC monolayers with 15, 135, 1215, and 3645mU/mL of
Neur, the normalized WGA-labeled GCX coverage of ECs decreased to 0.80.1, 0.740.08,
0.620.07, and 0.590.07, respectively (Fig. 3.3A and 3.4A). Normalized thicknesses of WGA- labeled GCX for the same sequence of enzyme treatments decreased to 0.960.03, 0.650.02,
0.590.02, and 0.540.02, respectively (Fig. 3.3B and 3.4B).
In summary, WGA-labeled GCX was resistant to the initial enzyme dose of 15 mU/mL.
However, as WGA-labeled GCX degraded, statistically (p-value) significant reduction in thickness, but not in coverage, was observed starting at an enzyme concentration of 135mU/mL.
Statistically (p-value) significant reductions in both coverage and thickness required an enzyme concentration of 1215mU/mL. At this 1215mU/mL concentration, in the orthogonal views we also started to see discontinuity in the overall appearance of the GCX layer (Fig. 3.2I, orthogonal view), which may expose the underlying endothelial cell membranes. With the highest dose of 3645 mU/mL, we noticed an ~50% decrease in both the coverage and thickness of WGA-labeled GCX.
This reduction in coverage and thickness matches pathological conditions, such as sepsis and ischemia/reperfusion and some cancers, in the degree of GCX degradation 61,62.
Increased Neur in the EC Environment Leads to Degradation of ∝-2,6-linked SA Residue.
∝-2,6-linked SA residues exhibited a patchy morphology when compared to WGA-linked
GCX (Fig. 3.2K). The discontinuity in expression of ∝-2,6-linked SA residue was confirmed using
61 orthogonal views of RFPEC monolayers (Fig. 3.2K) and has also been reported by other studies
63. When we quantified our observation, coverage of the ECs by the ∝-2,6-linked SA residue at baseline conditions (i.e. in the absence of enzyme treatment) was 25.85.9% of the RFPEC surface
(Fig. 3.2K), represented by a normalized value of 1.00.23 (Fig. 3.3A, 3.5A). SNA-labeled SA thickness was 1.190.04µm, a normalized value of 1.00.1 (Fig. 3.3B, 3.5B).
We found that 15mU/mL of Neur enzyme did not significantly degrade the coverage of
RFPECs by ∝-2,6 SA residues (Fig. 3.3A, 3.5A). Conversely, at the same enzyme concentration
∝-2,6-linked SA exhibited a significant decrease in normalized thickness from 1.00.2 to
0.400.08 (Fig. 3.3B, 3.5B), representing an ~60% decrease from baseline conditions. Recall, at the same enzyme concentration of 15mU/mL, WGA-linked GCX did not exhibit significant decrease in thickness (Fig. 3.3B, 3.4B). Taken together, these results suggest that ∝-2,6 SA residues could possibly be the first GCX component degraded by Neur.
Further increases in enzyme concentration lead to an exponential decrease in ∝-2,6-linked
SA residue coverage and thickness on RFPECs. After 2-hour treatment of confluent RFPECs with
15, 135, 1215, and 3645mU/mL of Neur, the resulting coverage of ECs by ∝-2,6 SA residue decreased to 0.70.1, 0.090.05, 0.040.02, and 0.0100.001, respectively (Fig. 3.3A and 3.5A).
The corresponding effect on the thickness of ∝-2,6-linked sialic acid for the same Neur doses was observed to decrease 0.400.08, 0.180.002, 0.060.01, and 0.030.01, respectively (Fig. 3.3B,
5B). Clearly, as Neur concentration increases, the underlying endothelium membrane is exposed.
Neur Increase Degrades ∝-2,3-linked Residue of GCX SA
33.72.4% of the untreated RFPEC surface was covered by ∝-2,3-linked SA. This result was normalized to 1.000.08 (Fig. 3.6A). The corresponding thickness was 1.830.06 µm, also normalized to a value of 1.00.08 (Fig. 3.6B). Taking these results into consideration clarifies that
62 ∝-2,3-linked SA residue provided less baseline coverage than WGA-labeled GCX and higher baseline RFPEC coverage than ∝-2,6-linked SA residue (Fig. 3.2F-3.2T). In addition, comparing
∝-2,3-linked SA residue to ∝-2,6-linked SA residue, it is worth mentioning that ∝-2,3-linked SA residue expression is less patchy and discontinuous than ∝-2,6-linked SA residue (Fig. 3.2K-3.2T).
These observations reveal differential expression of these SA residues and may also indicate that different ECs could have different presentations of these SA residues 64. In fact, Cioffi et al. reported that ∝-2,3-linked SA residues, specifically, are more abundantly expressed in microvessels compared to other SA residues 64.
RFPEC coverage by ∝-2,3-linked SA residue was drastically affected starting from the initial stages of enzymatic treatment (Fig. 3.3A, 3.6A). After the treatment of RFPECs with 15,
135, 1215, and 3645 mU/mL of Neur, the normalized coverage of RFPEC by ∝-2,3-linked SA was decreased to 0.980.03, 0.0070.001, 0.0080.005, and 0.0070.004, respectively (Fig. 3.3A and
3.6A). The corresponding normalized thicknesses of ∝-2,3-linked SA after enzyme treatment were decreased to 0.90.2, 0190.09, 0.20.1, and 0.080.03, respectively (Fig. 3.3B and 3.6B).
Neur-Induced GCX Loss Leads to Increased Cancer Cell Attachment to ECs
After establishing a degradation profile for GCX and its SA components we co-incubated
Neur treated and non-treated RFPEC monolayers with labeled 4T1 breast cancer cells to determine the effect of Neur on cancer attachment to the endothelium. Before the addition of the enzyme to
ECs, there was a baseline attachment of cancer cells to ECs (Fig. 3.2U), normalized to a value of
1.000.22 (Fig. 3.4, 3.5, 3.6). This could be attributed to the barrier created by the terminal positions of sugar chains within the GCX matrix 65,66. These GCX structures found on the apical surface of the GCX structure mediate intercellular recognition and binding 65.
63
Figure 3.4 – The extent of EC coverage by GCX and the thickness of GCX, as assessed by quantifying WGA labeled GCX, are inversely proportional to the number of cancer cells that attach to endothelium. Results are normalized to 0 mU/mL baseline conditions, which are indicated by the dashed lines (- - -). Significance is denoted as *P<0.05, **P<0.01, and ****P<0.0001. A: Compared to 0 mU/mL Neur conditions, WGA-labeled GCX coverage of ECs only becomes statistically low at high Neur doses of 1215 mU/mL and 3645 mU/mL. N = 3, and representative en face images are shown in Fig. 2A-2E. B: Compared to 0 mU/mL Neur conditions, WGA-labeled GCX thickness is statistically significantly affected by Neur doses of 135 mU/mL, 1215 mU/mL and 3645 mU/mL. N = 3, and representative cross- section images used for the data are shown in Fig. 2A-2E. A - B: Exponential increase in cancer attachment was observed with the increasing Neur concentration. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1215 mU/mL, N = 9; and at 3645 mU/mL, N = 9, and representative images used for this data are shown in Fig. 2U – 2Y.
Adding 15mU/mL of Neur resulted in attachment of 1.20.3-fold more 4T1 breast cancer cells to ECs, a 20% increase (Fig. 3.2V, 3.4, 3.5, 3.6). At this enzyme concentration, WGA-labelled
GCX and ∝-2,3-linked SA decrease in coverage and thickness was not as significant as the decrease in ∝-2,6-linked SA. (Fig. 3.3, 3.4, 3.5,3.6). Therefore, 4T1 attachment to ECs could likely be related to the degradation of ∝-2,6-linked SA, which may have exposed receptor sites on the surface of the endothelium to facilitate attachment.
64 Increasing the enzyme dosage to 135mU/mL resulted in 1.40.5-fold increase in cancer cell attachment to ECs (Fig. 3.2W, 3.4, 3.5, 3.6).
Figure 3.5 – The extent of EC coverage by SA and the thickness of SA, as assessed by quantifying SNA- labeled ∝-2,6-linked SA residue, are compared to the number of cancer cells that attach to endothelium. 0 mU/mL baseline conditions are indicated by the dashed lines (- - -). Significance is denoted as *P<0.05, **P<0.01, and ***P<0.001. A: Compared to 0 mU/mL Neur conditions, SNA-labeled ∝-2,6-linked SA residue coverage of ECs becomes statistically low at Neur doses of 135 mU/mL, 1215 mU/mL, and 3645 mU/mL. N = 3, and representative en face images are shown in Fig. 2F-2J. B: Compared to 0 mU/mL Neur conditions, SNA- labeled ∝-2,6-linked SA residue thickness is statistically significantly affected by Neur doses of 15 mU/mL, 135 mU/mL, 1215 mU/mL and 3645 mU/mL. N = 3, and representative cross-section images used for this data are shown in Fig. 2F-2J. A - B: The observed Neur-induced increase in cancer attachment as shown in Fig. 3 is shown again, for comparison to expression of SNA-labeled ∝-2,6- linked SA residue. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1215 mU/mL, N = 9; and at 3645 mU/mL, N = 9, and representative images used for this data are shown in Fig. 2U – 2Y.
Further increasing the enzyme dose to 1215 mU/mL resulted in a 1.40.3-fold increase in cancer cell attachment compared to control conditions (Fig. 3.2X, 3.4, 3.5, 3.6) a result that was not significantly different from those at the 135mU/mL concertation. This can be explained by the
65 fact that at both of these Neur concentrations, ∝-2,6-linked and ∝-2,3-linked SA residues were not visible and WGA-labeled GCX became clearly discontinuous (Fig. 3.2H, 3.2L-N, 3.2R, 3.2S).
Figure 3.6 – The extent of EC coverage by SA and the thickness of SA, as assessed by quantifying MAL II-labeled ∝-2,3-linked SA residue, are compared to the number of cancer cells that attach to endothelium. Results are normalized to 0 mU/mL baseline conditions, which are indicated by the dashed lines (- - -). Significance is denoted as *P<0.05, **P<0.01, and ****P<0.0001. A: Compared to 0 mU/mL Neur conditions, MAL II- labeled ∝-2,3-linked SA residue coverage of ECs becomes statistically low at Neur doses of 135 mU/mL, 1215 mU/mL, and 3645 mU/mL. N = 3, and representative en face images are shown in Fig. 2K-2O. B: Similarly, compared to 0 mU/mL Neur conditions, SNA-labeled ∝-2,6-linked SA residue thickness is statistically significantly affected by Neur doses of 135 mU/mL, 1215 mU/mL and 3645 mU/mL. N = 3, and representative cross-section images used for this data are shown in Fig. 2K-2O. A - B: The observed Neur-induced increase in cancer attachment as shown in Fig. 3 and 4 is shown again, for comparison to expression of MAL II-labeled ∝- 2,3-linked SA residue. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1215 mU/mL, N = 9; and at 3645 mU/mL, N = 9, and representative images used for this data are shown in Fig. 2U – 2Y.
These results indicate a point at which loss of both ∝-2,6-linked and ∝-2,3-linked SA permitted 4T1-EC attachment. Furthermore, due to statistical insignificance between 4T1
66 attachment at 135 mU/mL and 1215 mU/mL, we believe that at enzyme concentration of 135 mU/mL cancer cell saturation of exposed adhesion receptors occurred. Thus, additional cancer cells were prevented from binding. The reverse of this phenomena was observed by Zhu et al. who reported blocked mouse melanoma cell (B16) binding to lung EC adhesion molecule-1 (Lu-
ECAM-1) that was competitively bound to monoclonal antibody (6D3) against Lu-ECAM-1 67.
A final Neur dose of 3645 mU/mL resulted in a coverage and thickness decrease of WGA- labeled GCX to 40% and 50%, respectively, in comparison to baseline conditions (Fig. 3.4). The
∝-2,6-linked and ∝-2,3-linked SA residues were completely removed (Fig. 3.5, 3.6). The high level of Neur enzyme concentration and subsequent GCX and SA degradation lead to a 2-fold increase in attachment of 4T1 breast cancer cells to RFPECs, in comparison with control (Fig.
3.2Y, 3.4, 3.5, 3.6). A similar result was reported by Gasic et al., who treated 8-week old CAF1/J mice with Neur and discovered that there was a significant increase in metastasis formation in mice treated with Neur in comparison with untreated mice 30.
Our investigation and the study reported by Gasic 30 reveal the important role played by the presence of Neur in the spread of cancer. Recall our finding that intact GCX components extend
1.2 to 1.8 µm into the extracellular space of the ECs. The shedding of these GCX components presents an opportunity for exposure of EC surface adhesion molecules such as E-selectin 32, which are membrane-attached, less than 100 nm in height, and typically covered by the thick GCX. The disparity between GCX thickness and adhesion molecule height could prevent 4T1 breast cancer cells from attaching to the surface of the endothelium 39,68 in conditions where the GCX is not damaged. Conversely, GCX degradation makes the adhesion molecules more accessible to cancer cells, thus facilitating the high level of 4T1-EC attachment (Fig. 3.1B and 3.1C).
67
Figure 3.7 – Preliminary data were collected regarding human ECs (HUVEC), their GCX, and the extent of their recruitment of 4T breast cancer cells in comparison with RFPEC controls. This human EC data were used to confirm and validate the rat EC data that was the focus of this report. A. Phase image shows that an untreated HUVEC layer is healthy. B. WGA-labeled untreated HUVEC reveals the presence of GCX even in the absence of physiological flow stimulation, which is usually required for in vitro human EC studies. C. Low expression of HS is observed when these HUVEC, which lack flow stimulation, are labeled with HS antibody. The limited HS is presumably insufficient to expose the EC surface adhesion molecules to 4T1 breast cancer cells, because WGA is abundant enough to compensate and provide adequate coverage. D. As expected, the level of attachment of 4T1 breast cancer cells to untreated HUVEC is low, similar to what was observed in untreated RFPEC. E. Phase image shows healthy untreated RFPEC monolayer. F. WGA- labeled untreated RFPEC monolayer showing intact GCX. G. HS is abundantly expressed in in RFPEC without flow stimulation. H. Low attachment of 4T1 breast cancer cells to RFPEC monolayers. I. Data quantification for RFPEC and HUVEC controls, there was no statistical significance between RFPEC and HUVEC controls in regards to 4T1 breast cancer cell attachment.
68 Non differential attachment of 4T1 breast cancer cells to HUVEC controls compared to
RFPEC controls.
Untreated HUVEC (Fig. 3.7A) after staining revealed that some glycocalyx components like those targeted by wheat germ agglutinin (WGA) (Fig. 3.7B) show high expression while other glycocalyx components like heparan sulfate (HS) (Fig. 3.7C) show low expression, particularly in the absence of flow stimulus. Co-incubation of untreated HUVEC with labeled 4T1 breast cancer cells (Fig. 3.7D) showed similar results as untreated RFPEC cells. These results indicate that
HUVEC could not be a relevant control model for this study due to the requirement of flow induced
GCX regulation, unless all enzyme treatment protocols that we applied to RFPEC were to be repeated for HUVEC in a flow dependent manner.
3.4 CONCLUSIONS
The goal of this study was to clarify how the GCX, specifically the understudied SA component of the GCX, contributes to GCX-mediated protection of the endothelium against cancer cell adhesion. Towards this end, we have confirmed differential expression of WGA-labeled GCX,
∝-2,6-linked SA, and ∝-2,3-linked SA on the endothelium. We found that the presence of Neur in the EC environment sheds these components and enables ECs to become adhesive to floating cancer cells (Fig. 3.1B and 3.1C). Graded Neur dosing and systematic GCX degradation revealed that there is a necessary threshold of degradation of both GCX coverage and thickness on ECs at which the attachment of 4T1 breast cancer cells to the endothelium is substantially enhanced in a manner that could promote metastasis.
Further understanding of endothelial GCX mechanisms of cancer metastasis is needed.
Ongoing work in our lab includes studies of human EC interaction with human cancer cells. We expect the human cell studies will confirm and build upon the rodent cell results reported herein.
69 In addition, we are incorporating flow perfusion into our EC culture environment. This approach will more completely replicate the natural environment in which both the circulating cancer cells and the endothelial cells are conditioned by flow, which tends to affect the morphology, biochemical activities, and other aspects of the cells. For example, as indicated by our preliminary data, the endothelial GCX can become anti-metastatic in a healthy flow environment while the endothelial GCX may become pro-metastatic, not simply due to enzyme conditions, but due to unhealthy flow conditions. Finally, in our human cell studies, under flow conditions, we are also comparing the role of SA to the role of EC surface adhesion molecules. In the future, we look forward to interrogating additional GCX components (Table 3.1). The aim is to eventually clarify how the various GCX components and the adhesion molecules synergistically contribute to cancer cell attachment to the endothelium. Studying the role of the EC GCX as compared to the role of the EC adhesion molecules from a perspective that has a human-context, considers both enzyme and flow conditions, and looks at the multiple GCX components, we will expand upon our pre- existing knowledge of the relationship between endothelial GCX degradation and cancer attachment. Our long-term goal is to apply this knowledge to the development of therapeutic means to prevent the endothelial GCX degradation in a hope of hindering or even preventing cancer metastasis.
70 CHAPTER FOUR: FLOW-REGULATED ENDOTHELIAL GLYCOCALYX DETERMINES METASTATIC CANCER CELL ACTIVITY
SUBMITTED TO FASEB, 2019
Solomon A. Mensah1, Alina A. Nersesyan,1 Ian C. Harding1, Claire I. Lee1, Xuefei Tan3, Mark J. Niedre1,3, Vladimir P. Torchilin4, Eno E. Ebong1,2,5‡
1. Department of Bioengineering, Northeastern University, Boston, MA 2. Department of Chemical Engineering, Northeastern University, Boston, MA 3. Department of Electrical and Computer Engineering, Northeastern University, Boston, MA 4. Department of Pharmaceutical Science, Northeastern University, Boston, MA 5. Neuroscience Dept., Albert Einstein College of Medicine, New York, NY
71
4.1 INTRODUCTION
The metastatic spread of cancer is the leading cause of cancer related deaths globally [216,
217]. A common therapeutic approach to addressing this problem has involved targeting the leaky blood vessel network of primary tumors to prevent tumor cells from escaping into the circulation
[218, 219]. New therapies to treat cancer metastasis and invasiveness will need to mitigate secondary tumor formation once tumor cells have already entered the circulation. However, the mechanisms leading to the initiation of secondary tumors by metastatic and invasive cancer remain elusive.
In late stage cancer, circulating tumor cells (CTCs), having escaped from primary tumors
(Fig. 4.1A), can be detected in peripheral blood flow of human patients [220, 221]. This indicates that CTCs are able to survive in the bulk blood flow environment and maneuver their way through complex geometries within the vasculature of human hosts [222]. In the vascular system, abrupt changes in vessel geometry have been implicated in the progression of disease [223]. These geometric irregularities or non-uniformities affect blood flow patterns and result in the formation of disturbed flow (DF) regions (Fig. 4.1C and 4.1E) within blood vessels. DF erodes the blood vessel by causing inflammation and damage to the endothelial cells (ECs) that line the blood vessel wall, leading to pathogenic conditions [224, 225]. We hypothesize that DF conditions can promote
CTC attachment to EC-covered blood vessel walls, followed by migration into surrounding tissue and leading to secondary tumor formation (Fig. 4.1B) [222]. We also hypothesize that CTC clustering can be enhanced by DF conditions. CTC clustering is a very important phenomenon that enhances their survival in the circulation. In addition, as shown by human pathology studies, CTCs invade host tissues as strands, cords and clusters [226], which ensures proliferation into secondary
72 tumors. DF-induced CTC clustering and subsequent migration through the endothelium could be a possible pathway leading to cancer progression [227, 228].
Figure 4.1: A schematic showing the effect of DF patterns on endothelial GCX and circulating cancer cell attachment to the endothelium. A. Cancer cells within the primary tumor gain migratory properties and leave the primary tumor, intravasate through a nearby blood vessel, enter the bulk flow, and B. form secondary tumor sites in distant organs including the lungs. C. Geometric changes within the blood vessel results in different flow patterns. D. UF regions of blood vessels are known to have intact GCX resulting in the inability of CTC to attach to the endothelium. E. Branched areas will produce DF; we hypothesize that this DF will result in degradation of the endothelial GCX enhancing attachment of CTC to the endothelium.
ECs are covered by glycocalyx (GCX), which is a complex sugar coat. Due to its unique position, the GCX lines the luminal side of blood vessels and plays an essential role as a
73 vasculoprotective barrier [210, 229, 230]. While there have been numerous studies focusing on
DF-induced endothelial dysfunction, only a few studies have paid attention to DF regulation of the
GCX structure and function [146, 159, 231, 232] despite its importance in vascular homeostasis.
Recently, Harding et al labeled heparan sulfate, a major component of GCX, to determine the effects of DF on GCX structure and function compared to the effects of uniform flow (UF) conditions [146]. It was found that DF destabilizes the GCX and consequently disrupts the expression and localization of caveolin-1, potentially leading to blockage of endothelial nitric oxide synthase (eNOS) activation [146].
Relevant to CTC attachment to EC-covered blood vessel walls, other major GCX components like sialic acid (SA) are of great importance because of their role in cell and molecular recognition [233]. To our knowledge, studies investigating the activities of CTCs in relation to degradation of the GCX as a whole, or in relation to degradation of its SA sub-component, are limited. Recently, we performed a study in which ECs were treated with GCX degradative enzyme,
Neuraminidase (Neur), to cleave various SA residues of the GCX and to determine their correlation to enhanced cancer attachment to the endothelium [234]. We concluded that the ∝-2,6-linked residue of SA is the first to be degraded in the presence of Neur, resulting in vulnerability of the
GCX layer, which is sufficient to initiate CTC attachment to the endothelium [234]. Whether DF will have the same effects as Neur, by degrading SA and leading to enhanced attachment of CTCs to the endothelium, is unknown. Answering this question will be of great clinical importance.
Adhesion molecules on the endothelium play a significant role in CTC migration across the endothelium [235, 236]. They enhance CTC binding during attachment, consistent with how leukocytes attach to the endothelium [237]. GCX and these adhesion molecules, especially E- selectin, are similarly positioned as transmembrane glycoproteins within the endothelium [216].
74 EC GCX thickness ranges from 0.02 to 10 μm depending on species size, vascular bed, the microenvironment niche, in vivo versus in vitro conditions, and the GCX preservation and visualization approach used [144]. The E-selectin adhesion molecule extends to 0.03 to 0.07 μm on cell surfaces [238]. The height differences between healthy GCX and E-selectin suggest that
CTC ligands will find it difficult to access E-selectin adhesion receptors on the endothelium (Fig.
4.1D and 1E) [118]. By regulating the expression of GCX through DF or enzyme activity, we hope to learn more about the mechanism of interaction between ECs and CTCs.
Here we report for the first time the effects of DF-induced endothelial GCX degradation, versus E-selectin expression, on the attachment of cancerous cells to the endothelium. Our results bring to light a GCX-mediated pathway to secondary tumor formation. Furthermore, this work suggests that the creation of endothelial GCX reinforcing drugs, specifically targeted to the DF areas of the vasculature, may reduce the metastatic potential of CTCs.
75 4.2 MATERIALS AND METHODS
Cell Culture
Human umbilical vein ECs (HUVEC), purchased from ATCC (Manassas, VA), were used at passages 2 to 8. HUVEC were seeded at a density of 15,000 – 20,000 cells/cm2 on 0.17 mm- thick 22 mm x 40 mm rectangular coverslips coated with 60µg/ml of fibronectin. HUVEC were cultured with Vascular Cell Basal Medium. The medium was supplemented with recombinant human vascular endothelial growth factor (rh VEGF: 5 ng/mL), epidermal growth factor (rh EGF:
5 ng/mL), basic fibroblast growth factor (rh FGF basic: 5 ng/mL), and insulin-like growth factor-
1 (rh IGF-1: 15 ng/mL). Additional supplements included L-glutamine (10 mM), heparin sulfate
(0.75 Units/mL), hydrocortisone (1 µg/mL), ascorbic acid (50 µg/mL), and fetal bovine serum
(FBS: 2%). The medium and supplements were all purchased from ATCC. HUVEC reached 100% confluency at 3 to 4 days after seeding.
Two different types of CTCs were used. 4T1 mouse metastatic breast cancer cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (purchased from Invitrogen) with 1% penicillin-streptomycin and 10% FBS (Gibco Life Technologies). MCF-7 human metastatic breast cancer cells (ATCC) were cultured in Eagle’s Minimum Essential Medium (purchased from
ATCC) with 0.01 mg/ml of human recombinant insulin (Gibco) and 10% FBS.
All cultured ECs and CTCs were maintained in a humidity-controlled environment at 37°C with 5% CO2.
Application of Flow Conditions in Cell Culture Studies
To introduce DF and UF to HUVEC, a vertical step flow chamber (Fig. 4.2A) was designed and manufactured in our lab [146]. HUVEC monolayers cultured on glass slides as
76 described above were inserted into a recess in the bottom chamber downstream from the step
(Fig. 4.2A).
The DF flow region, through SolidWorks simulations, was previously confirmed to consist of a reversal in flow direction, a high shear stress gradient, and shear stress ranging from -8 dynes/cm2 to 12 dynes/cm2 [146]. UF flow conditions consists of a shear stress of 12 dynes/cm2.
The flow setup was placed in a humidity-controlled environment at 37°C and 5% CO2. HUVEC monolayers were exposed to flow for 4 hours using the supplemented Vascular Cell Basal
Medium, with the addition of 0.5% of bovine serum albumin (BSA), to stabilize the GCX, and 0.4 ng/ml of tumor necrosis factor-α (TNF-α), to activate E-selectin.
When CTCs were added to the flow stream, for the purposes of enabling CTC attachment to ECs, shear stress was reduced to 1 dyne/cm2 for 1 hour after the 4-hour UF and DF pre- conditioning period was completed. This reduced flow condition best mimics flow conditions that are relevant where CTCs approach the vessel wall in vivo. Moreover, it minimizes the recirculation region so that CTC residence time over DF-conditioned ECs matches CTC residence time over
UF-conditioned ECs (Fig. 4.3).
77
Figure 4.3: Computer aided simulation, showing changes in flow patterns after the introduction of a vertical step in the flow path. The DF region shows eddy current formation immediately after the step, which is characterized by flow detachment, circulation and reattachment sections. This is immediately followed by a transition region where the flow gradually changes until it become uniform. The transition region is followed by the UF region where the flow is void of any disturbances. A. Flow patterns generated when UF is 12 dynes/cm2. This was the setting in which HUVEC were flow conditioned prior to exposure to 4T1 or MCF7 cells. B. Flow patterns generated when UF is 1 dyne/cm2. These were the conditions used for co-incubation of HUVEC with circulating 4T1 or MCF7 cells.
Neuraminidase Enzyme Treatment in Cell Culture Studies
Neuraminidase (Neur) enzyme from clostridium perfringens was used to target the N- acetyl-neuraminic components of GCX in order to degrade all the forms of SA present in GCX.
To achieve this, regular growth media from cultured HUVEC was replaced with media containing
Neur enzyme at a concentration of 15 mU/ml. This concentration of Neur, was selected after a careful pilot study of the effects of 0 mU/ml, 15 mU/ml, 135 mU/ml, 1215 mU/ml and 3645 mU/ml of Neur, to determine the appropriate enzyme concentration (Fig. 4.4). 15mU/ml of Neur was
78 chosen because it was found to best maintain cell viability during flow experiments while significantly degrading SA. The Neur-modified culture media was used during UF experiments only.
Figure 4.4: Dose response experiment to establish appropriate enzyme concentration for HUVEC GCX degradation. A. Control sample shows intact GCX as labeled with WGA for both coverage and thickness. B. After treating HUVEC with 15mU/mL of Neur, we observed changes in the coverage of GCX but not the thickness. C. Adding 135mU/mL of Neur resulted in a significant decrease in both the coverage and thickness of GCX. D. After treating HUVEC with 1215mU/mL of Neur, we observed an exponential decrease in the coverage and thickness of GCX on HUVEC monolayers. E. Finally, by adding an increased enzyme concentration of 3645mU/ml, we noticed a total degradation of GCX on the surface of HUVEC monolayers. F and G. Data quantification for the coverage and thickness of GCX after dose response treatment with Neur enzyme respectively. One-way ANOVA was used to determine differences between groups, and the sample size was N=3. Statistical significance between groups is denoted as *P<0.05, ****P<0.0001, or not significant (ns).
79 In Vitro Cancer Cell Labeling, Attachment Assay, Imaging, and Quantification
CTCs (4T1 or MCF7) were cultured as described above. Prior to the performance of attachment assays, 103/ml of suspended CTCs were labeled with 15µM CellTracker Red CMTPX
Dye (Thermo Fisher Scientific) for easy identification during co-culture experiments. The
CellTracker-labeled CTCs were then injected into the parallel plate flow circulation for co- incubation with HUVEC, as mentioned above. When Neur was present in the flow circulation, effort was made to ensure that Neur impacted only ECs and not CTCs. To achieve this, HUVEC were removed from the flow/Neur environment and the CellTracker-labeled CTCs were co- incubated with Neur- and flow-conditioned HUVEC under static conditions for 1 hour in Neur- free media. After HUVEC and CTC co-incubation, whether under flow or static conditions, non- attached cells were removed by washing with growth media.
A Zeiss Z1 Axio Observer fluorescence microscope with a 10x magnification objective was used to capture CTC and EC interactions. For each attachment experiment, 6 different fields of view were captured with an excitation wavelength of 558 nm to locate red CellTracker-labeled
CTCs. Brightfield images were also captured to determine the health of HUVEC. NIH Image J and a cell counter plug-in was used to count the number of attached CTCs. In addition, CTC clusters were quantified, by counting the number of groups of two or more cells together. Lastly, the total number of CTCs that migrated through the endothelium was determined by using the working distance of the microscope, marking the HUVEC level as the baseline and considering only 4T1 or MCF7 cells that were beneath that baseline as migrated cells. In other words, cells that were migrated were considered as cells that were out of focus compared with HUVEC. CTC attachment, clustering, and migration results were all normalized with data obtained from conditions of UF in the absence of Neur.
80 GCX and E-Selectin Labeling, Imaging, and Quantification for Cell Culture Studies
After various treatment conditions, we assessed structural changes in GCX caused by conditions of UF, DF, or UF with Neur. First, HUVEC monolayers were fixed with 2% paraformaldehyde (PFA) combined with 0.1% glutaraldehyde in phosphate buffered saline (PBS), for 30 minutes at room temperature (RT). To label the GCX, HUVEC were incubated for an hour at 4⁰C with 5-20 µg/ml biotinylated wheat germ agglutinin (WGA; Vector Labs) lectin in PBS with 3% BSA to block nonspecific binding. WGA largely tags the SA component of the GCX, although it has affinity for other components [127, 239-241]. A concentration of 1:1000 of Alexa flour 488-conjugated anti-biotin was used for secondary detection.
For E-selectin staining, HUVEC monolayers were fixed with 4% paraformaldehyde in
PBS, for 20 minutes at RT. Fixed cells were incubated overnight at 4°C with human E-selectin antibody (R&D Systems) at a concentration of 10 µg/ml in a solution of 10% goat serum in PBS to block non-specific staining. Secondary labeling was performed by incubation with 1:1000 concentrated biotinylated goat anti mouse IgG (H+L) highly cross adsorbed secondary antibody, for 1 hour at RT. Signal amplification was performed via tertiary labeling with 1:1000 concentrated
Alexa Flour 488-conjugated anti-biotin, for 1 hour at RT.
GCX and E-Selectin labeled samples were covered with Vectashield anti-fade mounting medium (Vector Labs), containing 4’,6-diamidino-2-phenylindole (DAPI) to label cell nuclei.
Samples were then sealed in preparation for imaging and quantification.
Confocal microscopy provided enhanced resolution and allowed for the capturing of different planes within our cell monolayers, which were later reconstructed into a three dimensional (3D) image. For this process a Zeiss LSM 700 confocal microscope with a 63x oil objective with excitation wavelengths of 490 nm (for WGA or E-selectin) and 350 nm (for DAPI)
81 was utilized to obtain XY-plane slices. Intervals for slices were 0.2 µm for both WGA and E- selectin. Using NIH Image J software, these slices were reconstructed into complete 3D images.
WGA or E-selectin coverage of the surface of endothelial monolayers was quantified from the z- projected en face view of the 3D image. The total area of WGA or E-selectin fluorescence was measured, and this measurement was divided by the total area of the field of view. WGA thickness was also quantified. In cross-sectional views derived from the 3D images, lines were drawn perpendicular to the cell surface, from the top of the WGA fluorescence to its base. The length of the lines was measured to represent the thickness of WGA on the surface of HUVEC monolayers.
Overview of Animal Studies.
All animal studies were done in accordance with the regulations of the Institutional Animal
Care and Use Committee (IACUC) at Northeastern University, under protocol number 18-0725R.
Eight male 6-8 week old BALB/C mice were obtained from Jackson Laboratories. The animals were fed a regular chow diet for 1 week before commencing the experiments. Four mice were studied for control experiments and four mice were studied for Neur treatment experiments, to determine the in vivo effect of enzyme treatment on GCX and CTC attachment at locations prone to secondary tumor formation.
Neuraminidase Enzyme Treatment in Animals
Animals were weighed to calculate the volume of blood. The volume of blood informed the volume of enzyme to inject in order to achieve the desired concentration. Control animals were intravenously injected with saline. Neur-treated mice were intravenously injected with 5U/mL of
Neur. Animals were allowed 14 hours to recover before further experimentation.
Cancer Cell Injection and Enumeration in Animals
After 14 hours, both control and Neur-treated mice were intravenously injected with 106
82 CTCs in 200µL of phenol free media via the tail vein. 4T1 CTCs were chosen to match the species of the animal. These cells were labeled with far-red CellTrace (Thermo Fisher Scientific), which is similar to the red CellTracker that was used for the cell culture studies but easier to measure in vivo using the Diffuse diffusive in vivo Flow Cytometer (DiFC) [242-244] technique.
We performed DiFC (Fig. 4.5A), which is a new technique to detects rare fluorescently labeled cells moving in circulation in highly scattering biological tissue, to investigate the in vivo clearance kinetic of the CellTrace labeled 4T1 CTCs. After the mice were injected with the cells they were scanned by DiFC for 15 minutes at multiple time points (10, 30 and 60 minutes) after the injection. During each scan, the injected mouse was kept anesthetized under isoflurane and the tail was secured with a custom-made holder (Fig. 4.5C).
Figure 4.5: Diffusive in vivo Flow Cytometry (DiFC) system used to track circulation of 4T1 breast cancer cells in BALB/C mice. A. The DiFC system configuration is shown. BP: band pass filter; ND: neutral density filter; M: mirror; BS: beam splitter; FC: fiber coupler; PMT: photomultiplier tube; PA: pre-amplifier; DAQ: data acquisition board; PC: personal computer. Subscripts, x: excitation; m: emission. This image was previously published and is being used with permission [243]. B. Example of a peak which corresponds to a ‘detected’ cell on DiFC. C. During the measurement, fiber probes were placed in firm contact with the surface of a mouse tail. DiFC can identify cells moving in the arteriosus (red arrows) or venous (blue arrows) direction.
83 We placed the DiFC probe on the ventral surface of the tail about 3 cm from the mouse body, approximately above the ventral caudal vascular bundle (Fig. 4.5C). DiFC uses two custom designed groupings of optical fibers consisting of a central ‘source’ fiber and eight fluorescence collection fibers, as well as integrated optical filters and collection lens [244] (Fig. 4.5A). The
DiFC probe was placed in firm contact with the skin, to prevent motion artifacts during scanning
(Fig. 4.5). A heating pad was also placed over the exposed area of the mouse tail during the scanning to maintain blood flow. As each fluorescently labeled CTC passed through the field of view of the probe, a transient fluorescence pulses were detected as in our previous work (Fig.
4.5B). Using a custom-developed signal-processing algorithm [243], we analyzed the DiFC data to determine the rate of CTCs in circulation (counts per minute) and the average peak amplitude
(linearly related to the brightness of individual cells) during scanning. DiFC distinguishes cells moving in arterial venous directions (forward and reverse) which mitigates double counting of
CTCs (Fig. 4.5). A noticeable decrease in CTCs in circulation was taken to indicate CTC clearance from the blood circulation. After 60 minutes, the mice were euthanized.
Animal Euthanization, Tissue Dissection, and Fluorescent Staining
Each mouse was injected with ketamine and xylazine anesthetics based on their weight.
The hind limbs were pinched to ensure that each mouse was completely under anesthesia. A surgical incision was made from the abdominal wall to the thoracic wall of the mouse to expose the heart. Using a small needle inserted into the heart, the vessels of each mouse were pressure perfused with 2% BSA in PBS to euthanize by exsanguination while preventing the GCX from collapsing. Once exsanguination was complete, 4% PFA in PBS was pressure perfused to fix the vessels. The lungs and abdominal aorta were excised.
The lungs, a well-established site of secondary tumor formation, were refrigerated in PBS.
84 Thanks to the auto fluorescence of the lung tissue and prior fluorescent labeling of the CTCs, no further staining was required. The lungs could be immediately imaged to count the number of attached fluorescent CTCs in the absence and presence of Neur.
Some segments of excised abdominal aortas were refrigerated in PBS. Later, the aortas were longitudinally cut to expose the luminal vessel wall en face and to verify the integrity of the endothelium in the absence and presence of Neur. On the luminal vessel wall, endothelium markers were immunolabeled using antibodies against eNOS (Fisher), platelet endothelial cell adhesion molecule-1 (PECAM-1; Novus Biologicals), and E-selectin (Fisher). Immunostaining was done as previously described [146]. Secondary detection was achieved by incubating samples in Alexa
Flour 488-conjugated goat anti-rabbit secondary antibody. The stained en face tissue was covered with Vectashield anti-fade medium containing DAPI and sealed before imaging.
Remaining excised abdominal aortas were frozen in optimum cutting temperature media
(OCT) and stored at -80oC. A Leica cryostat was used to create 6-µm thin axial sections of the frozen aortas. The thin sections were mounted and allowed to dry out on positively charged glass slides (Fisher Scientific). The aorta sections were later stained and imaged to quantify GCX expression in the presence and absence of Neur. As before, WGA was used as a marker of GCX.
Sections were then incubated in 1:100 biotinylated WGA in 5% BSA for 2 nights. Secondary detection of WGA-labeled GCX was achieved by incubating sections in 1:500 HRP-conjugated streptavidin (Thermo Scientific) for 1 hour, which was amplified using 1:100 of TSA Plus Cyanin
3 (Perkin Elmer) for 7 minutes. DAPI and Vectashield anti-fade mounting medium were applied before imaging.
Imaging and Analysis in Animal Studies
WGA-labeled GCX in aorta sections were visualized using a Zeiss LSM 710 Confocal
85 Laser Scanning microscope at 63X/oil magnification. GCX coverage and thickness were quantified as previously described [15]. PECAM-1, E-selection, and eNOS labeled en face aorta tissue samples were also visualized by confocal microscopy, using a 40x/water objective [146].
Whole excised lungs after experimentation were mounted on glass sides without mounting media and imaged using a Zeiss LSM 710 Confocal Laser Scanning microscope at 40X/water objective.
A red filter of excitation of 558 nm was used to identify Cell Trace labeled 4T1 cells attached to the lungs.
Statistics. Data is expressed as means ± SEM. Graph Pad Prism was used for all statistical analysis and determination of statistical significance of differences between means. Additional details of the statistical tests are described in Figure captions.
4.3. RESULTS
Interactions between CTCs and the endothelium are increased in DF conditions
HUVEC exposed to both UF and DF patterns (Fig. 4.2A) did not show any morphological changes after 4 to 5 hours of conditioning with flow (Fig. 4.2B and 4.2C) when compared to HUVEC in static conditions (Fig. 4.6A, Fig. 4.7). In static conditions, when
CellTracker-labeled 4T1 cells were co-incubated with HUVEC, approximately 11 4T1 CTCs were attached per every thousand ECs (Fig. 4.6B, Fig. 4.7). CellTracker-labeled 4T1 CTCs were also co-incubated with flow-conditioned
86
Figure 4.6: Control static experiments for GCX expression, cancer attachment and E-selectin expression before the introduction of both DF and UF patterns to HUVEC. A. Phase contrast image showing healthy endothelium. B. 4T1 breast cancer cell attachment to the endothelium. C. Staining for GCX with WGA shows intact GCX on the surface of the HUVEC monolayer (green is WGA-labeled GCX and blue is DAPI-labeled nuclei). D. E- selectin expression in static conditions (green is E-selectin and blue is DAPI-labeled nuclei).
HUVEC monolayers, for 1 hour at a reduced flow rate of 1 dynes/cm2. Under UF conditions we observed an attachment of approximately 6 4T1 cells for every thousand ECs. Normalized, 4T1 CTC attachment to the endothelium was at a level of 1.00 ± 0.06 (Fig. 4.2D and 4.2F). In the DF region of the flow chamber, a normalized number of 2.35±0.29 cells were attached per field of view (Fig. 4.2D and
4.2F). This represents a statistically significant 2-fold increase in the attachment of 4T1 CTCs to
HUVEC in DF conditions compared to UF conditions. We investigated the rate of clustering of
4T1 CTCs after co-incubation with HUVEC monolayers in circulation. The normalized data, under
UF conditions, was 1.00 ± 0.09 clusters of 4T1 CTCs on HUVEC (Fig. 4.2D and 4.2G). With introduction of HUVEC to DF conditions, the clustering of 4T1 CTCs was at 3.19 ± 0.63 (Fig.
4.2D and 4.2G), representing a significant 3-fold increase with DF compared to UF conditions.
Migration of cancer cells through the endothelium is important for secondary tumor formation.
We therefore quantified the extent of migration of 4T1 breast cancer cells through the endothelium.
87 In UF conditions the extent of migration was normalized to 1.00±0.09 (Fig. 4.2D and 4.2H). The presence of DF on HUVEC monolayers resulted in a significant increase in the migration of 4T1 breast cancer cells through the endothelium, with the normalized value of migration reaching 3.66
± 1.22 (Fig. 4.2D and 4.2H).
Figure 4.2: A. A glass slide covered with ECs was placed at the bottom of the flow chamber. The ECs were exposed to a dynamic flow pattern that was generated by the introduction of a vertical step in the flow path. This computer aided simulation shows some of the flow pattern features. Here, the DF region is exaggerated for illustration purposes. It can be seen that eddy currents form immediately after the step, which is characterized by flow detachment, circulation and reattachment sections. In addition, the DF region includes a transition in which the flow gradually adapts until it becomes UF. The transition region is followed by the UF region where the flow is void of any disturbances. Additional details can be found in SI Fig. 1 and its caption. B and C. Phase contrast images indicating healthy HUVEC monolayer after the introduction of both DF and UF. D and E. Attachment of 4T1 breast cancer cells (D; red dots) and MCF7 cells (E; red dots) to the endothelium respectively. As expected cancer cells preferred to attach to the DF area than the UF area. F. Number of 4T1 and MCF7 breast cancer cells attached to the DF- conditioned endothelium. The dotted line represents normalized UF data. Significant increase in the attachment of cancer cells in the DF region compared to UF region. G. Number of cancer cell clusters formed in the DF region. The dotted line represents normalized UF data. We observed a significant increase in the clustering of 4T1 and MCF7 breast cancer cells to the endothelium in comparison with UF regions. H. Migration of 4T1 and MCF7 breast cancer cells through the DF- conditioned endothelium. The dotted line represents normalized UF data. Compared to UF regions, we observed a significant increase in the migration of cancer cells through the DF region,
88 compared to UF areas. All data “Normalized with UF”. Student t test was used to compare DF vs. UF. Sample sizes: 4T1 attachment N=9, MCF7 attachment N=5. Significance is compared to the UF condition and denoted as *P<0.05, **P<0.01, ***P>0.001, or not significant (ns).
Similar results were observed when we matched the endothelium species with the CTC species, by utilizing MCF7 CTCs in place of 4T1 CTCs. In static conditions, when CellTracker- labeled MCF7 cells were co-incubated with HUVEC, approximately 20 MCF7 CTCs were attached per every thousand ECs. Under UF conditions we observed an attachment of approximately 11 MCF7 cells for every thousand ECs. The normalized data for MCF7 CTC attachment to the endothelium under UF conditions was 1.00 ± 0.24 (Fig. 4.2E and 4.2F). Exposure to DF resulted in a significant 2.3-fold increase in the attachment of MCF7 cells to HUVEC, with the normalized value of attachment increasing to 2.34 ± 0.42 (Fig. 4.2E and 4.2F). The level of clustering of MCF7 CTCs on HUVEC was at 1.00 ± 0.41 for UF (Fig. 4.2E and 4.2G. After
HUVEC exposure to DF, MCF7 CTC clustering significantly increased to a normalized value of
2.49 ± 0.44 (Fig. 4.2E and 4.2G. Unlike 4T1 CTCs, when the migration of MCF7 CTCs across the endothelial barrier in UF conditions was compared to DF conditions the difference was statistically insignificant. The normalized level for UF conditions was at 1.00 ± 0.16 (Fig. 4.2E and 4.2H) and for DF conditions was at 1.66 ± 0.39 (Fig. 4.2E and 4.2H).
We verified that the flow circulation could have no effect on attachment, clustering and migration results. This was accomplished by co-incubating CTCs with flow-conditioned ECs under static conditions not in 1 dyne/cm2 flow conditions (Fig. 4.7).
The high levels of CTC interactions with DF-condition endothelium coincide with endothelial
GCX degradation but no change in E-selectin expression
To determine whether increased cancer cell attachment to the endothelium could occur as a result of GCX changes, the GCX was visualized by labeling it with fluorescently-tagged WGA,
89 which labels primarily the SA component of the GCX [127, 239]. The GCX was found to be intact on the surface of HUVEC monolayers even in static (no flow) conditions (Fig. 4.6C). Under UF conditions, WGA-labeled GCX covered 88.65 ± 4.35% of HUVEC monolayers, represented by a normalized value of 1.00 ± 0.07 (Fig. 4.8A, 4.8C, and 4.8F). The WGA-labeled GCX thickness was 1.57 ± 0.15µm, represented by a normalized value of 1.00 ± 0.14 (Fig. 4.8A, 4.8C, and 4.8G).
After introducing HUVEC monolayers to DF patterns, the normalized GCX coverage for the DF area decreased to 0.44 ± 0.01 (Fig. 4.8A, 4.8B, and 4.8F). Normalized thickness at the DF area was also 0.41 ± 0.08 (Fig. 4.8A, 4.8B, and 4.8G). This is a statistically significant difference in
GCX coverage and thickness in the DF conditions, compared to the UF condition.
To determine whether cancer cell attachment to the endothelium could be due to increased adhesion receptor expression, we analyzed the expression of E-selectin in static, DF and UF conditions. In static conditions, E-selectin was barely expressed (Fig. 4.6D). The normalized E- selectin expression in the UF region was 1.00±0.19 (Fig. 4.8A, 4.8E, and 4.8H). After introducing
DF to HUVEC monolayers, normalized E-selectin expression was 1.29±0.29 (Fig. 4.8A, 4.8D, and 4.8H). representing a nonsignificant change in expression of E-selectin adhesion molecules between DF and UF regions.
90
Figure 4.8: Effect of DF and UF patterns on the GCX and the expression of E-selectin. A. Computer aided simulation of DF formation immediately after the step (refer to Fig. 2A for more details). B. The coverage and thickness of GCX is significantly low within the DF region (green is WGA-labeled GCX and blue is DAPI-labeled nuclei). C. In the UF region the GCX is shown to be abundantly expressed both in coverage and thickness. D and E. Expression of the endothelial surface adhesion molecule E-selectin. The introduction of different flow patterns did not affect the expression E-selectin. F, G and H. Data quantification for GCX and E-selectin expression on the surface of HUVEC cells. F. GCX coverage in UF region compared to DF region. G. GCX thickness in UF region compared to DF region. F. E-selectin expression in UF region compared to DF region. All data “Normalized with UF”. Student t test was used to compare DF vs. UF. Sample sizes: WGA-labeled GCX expression N=3, E-selectin expression N=9. Significance is denoted as *P<0.05, **P<0.01, or not significant (ns).
Introducing GCX-degrading Neur into the UF environment results in an increase in CTC interactions with the endothelium; this indicates that the low level of CTC-EC interactions in UF can be attributed to the abundance of the protective GCX.
We investigated the effects of Neur-induced GCX degradation on the attachment of CTCs to the endothelium. Based on a dose response experiment in static conditions (Fig. 4.4) and a
91
Figure 4.9: Effect of the presence of Neur on cancer attachment, GCX and E-selectin expression. A and F. Phase contrast images revealing intact and healthy HUVEC monolayers in untreated conditions and after treatment with Neur. B WGA-labeled GCX in UF regions is abundant. G. Addition of Neur enzyme to the UF environment abolishes WGA-labeled GCX. C and H. Expression of E-selectin in conditions of isolated UF versus conditions of UF together with Neur enzyme. D and I. Attachment of 4T1 cells to HUVEC in UF region, prior to or after the introduction of 15mU/mL of Neur. E and J. Attachment of MCF7 cells to HUVEC in UF region, prior to or after the introduction of 15mU/mL of Neur. K and L. The quantification of coverage and thickness of GCX labeled by WGA, respectively. M. Expression of E-selectin in UF with enzyme treatment, compared to UF conditions. N, O, and P. Data quantification of the attachment, clustering and the migration of 4T1 and MCF7 breast cancer cells to HUVEC monolayers, respectively. All data are normalized with UF results. Student’s t test was used to compare “UF” vs. “UF + Neur”. Sample sizes are as follows: GCX expression N=3, E-selectin expression N=6,
92 4T1 data N=7, MCF7 data N=4. Significance is denoted as *P<0.05, **P<0.01, ***P<0.001, or not significant (ns).
confirmatory experiment in UF experiments, it was determined that a Neur concentration of 15mU/mL was the best concentration of enzyme to keep HUVEC layers intact, stable, and firmly attached to their substrate (Fig. 4.9A and 4.9F) so that they would be able to withstand the force of UF conditions. In static conditions, this 15mU/mL concentration was effective to statistically significantly reduce the coverage of the ECs by the WGA-labeled GCX, which is primarily the SA component of the GCX (Fig. 4.4B). In static conditions 15 mU/mL of Neur did not affect WGA-labeled GCX thickness (Fig. 4.4G). However, the presence of 15 mU/mL of Neur in UF conditions, compared to untreated UF conditions, was effective to reduce WGA-labeled
GCX with respect to both its coverage of the ECs and its thickness on top of the ECs (Fig. 4.9B and 4.9G). In UF without Neur, the normalized GCX coverage was 1.00±1.11 (Fig. 4.9B and
4.9K) and coverage for GCX after supplementing UF with 15mU/mL of enzyme was 0.21±0.01
(Fig. 4.9G and 4.9K). The corresponding normalized thickness of GCX with UF in the absence of
Neur was at 1.00±0.14 (Fig. 4.9B orthogonal view and Fig. 4.9L). In UF with Neur the normalized
GCX thickness was at 0.37±0.01(Fig. 4.9G orthogonal view and Fig. 4.9L). These results indicate the fact that, in UF conditions, the presence of GCX degrading enzyme is very detrimental to the health of GCX.
The presence of 15 mU/mL of Neur in UF conditions not only degraded the GCX but also decreased E-selectin expression. It was found that E-selectin is more significantly expressed in isolated UF conditions than in UF conditions where Neur is present at a concentration of
15mU/mL. Exposure of ECs to isolation UF resulted in E-selectin expression at a normalized level of 1.00±0.23 (Fig. 4.9C and 4.9M). After the addition of Neur to UF, we saw a statistically
93 significant reduction of E-selectin expression to 0.21±0.01(Fig. 4.9H and 4.9M), an approximate
80 percent decrease in the expression of E-selectin. Since Neur treatment degraded GCX while also removing E-selectin, it was not clear whether we would observe increased CTC-EC interactions due to GCX loss or whether we would find unchanged or decreased CTC-EC interactions due to E-selectin deficiency.
As previously mentioned, UF conditions in the absence of Neur yielded 4T1 cells to attach to HUVEC at a normalized count of 1.00±0.13 (Fig. 4.9D and 4.9N). Adding 15mU/mL of Neur to UF resulted in a statistically significant increase in 4T1-to-HUVEC attachment to 2.60±0.34
(Fig. 4.9I and 4.9N). We observed a statistically significant increase in migration of 4T1 cells through the endothelium, when conditions of UF with Neur were compared to conditions of isolated UF. Specifically, a normalized 1.00±0.24 (Fig. 4.9D and 4.9P) count of 4T1 cells migrated through samples of HUVEC layers which had been exposed to only UF conditions, while a normalized 6.50±1.72 (Fig. 4.9I and 4.9P) count of 4T1 cells migrated through the endothelium that had been exposed to UF and 15mU/mL of Neur simultaneously. Clustering of 4T1 cells was
1.00±0.21 (Fig. 4.9D and 4.9O) for UF-only conditions and the clustering level increased to
1.90±0.59 (Fig. 4.9I and 4.9O) in conditions of UF with Neur, which was statistically insignificant.
When we matched the endothelium species with the CTC species, by utilizing MCF7
CTCs in place of 4T1 CTCs, we observed a normalized number of MCF7-to-HUVEC attachments of 1.00±0.19 (Fig. 4.9E and 4.9N). Adding 15mU/mL of Neur to the UF condition resulted in a statistically significant increase in the attachment of MCF7 cells to HUVEC, at the normalized level of 3.8±0.55 (Fig. 4.9J and 4.9N). We also observed a statistically significant change in MCF7 cell clustering. In isolated UF conditions, the normalized value of MCF7 clustering was at
1.00±0.22 (Fig. 4.9E and 4.9O). After addition of Neur to the UF environment, clustering
94 significantly increased to the normalized value of 6.10±1.48 (Fig. 4.9J and 4.9O). There was also a change in the migration of MCF7 cells across layers of ECs. Without Neur enzyme treatment,
UF stimuli led to the migration of MCF7 cells at a normalized level of 1.00 ±0.24 (Fig. 4.9E and
4.9P). With Neur enzyme treatment, we observed a statically significant increase in MCF7 trans- endothelial migration to a normalized level of 3.10±0.25 (Fig. 4.9J and 4.9P). The 4T1 and MCF7
CTC attachment, clustering, and migration results were consistent with what would be expected due to GCX loss and inconsistent with what would be expected due to E-selectin deficiency.
In an in vivo setting, the presence of GCX-degrading Neur in the circulating blood results in an increase in CTC homing to the lung, an organ where secondary tumors are commonly found
This in vivo experiment was carried out in Balb/c mice to confirm our observed in vitro findings. We confirmed that the coverage and thickness of GCX lining the vascular walls of the
Balb/c mice were statistically significantly decreased after treating Balb/c mice with 5U/mL of
Neuraminidase. For non-treated mice the normalized expression of GCX coverage was at 1.00±
0.01 (Fig. 4.10A and 4.10C) while the GCX coverage for Neur-treated mice was 0.85±0.04 (Fig.
4.10B and 4.10C). In the case of GCX thickness, the normalized data for non-treated mice was
1.00±0.09 (Fig. 4.10A and 4.10D) and for enzyme-treated samples the normalized thickness was
0.71±0.03 (Fig. 4.10B and 4.10D). We also confirmed that the endothelium integrity was maintained in both control and Neur-treated mice by staining for the following EC markers: eNOS,
E-selectin and PECAM-1 (Fig. 4.11A through 4.11F). Since we were interested in comparing GCX levels to E-selectin levels, we quantified E-selectin expression. The normalized level of E-selectin in untreated mice was 1.00±0.57 (Fig. 4.11C and 4.11G) and for enzyme-treated mice the
95 normalized E-selectin level was 0.60±0.28 (Fig. 4.11D and 4.11G). The E-selectin levels are not statistically significantly different.
Figure 4.10: Expression of GCX in the abdominal aorta of Balb/c mice. A. Control mice showing a uniform layer of GCX within the lumen of the abdominal aorta. B. After treatment of Balb/c mice with 5 U/mL of Neur we observed a decrease in the expression of GCX, with the layer showing discontinuity in coverage across the lumen of the abdominal aorta. C. Data quantification for coverage of GCX. D. Data quantification for the thickness of GCX. The sample size (N) is 4 for WGA-labeled GCX, data was statistically analyzed using the student t-test, and the statistical significance between the two groups is denoted as *P<0.05.
4T1 CTCs were introduced into the blood of Balb/c mice that were not treated or those that were treated with 5U/mL of Neur. With DiFC, the 4T1 cells were observed to significantly clear
96 the arterial and venous blood circulation over the course of 1 hour (Fig. 4.12A and 4.12B). At 10,
30, and 60 minutes after 4T1 cells were introduced, DiFC detected circulating 4T1 at a rate of
8.51, 3.69, and 3.1 cells per minute in the Balb/C arteries and the veins (Fig. 4.12A and 4.12B).
Figure 4.12: In-vivo attachment of 4T1 breast cancer cells to the lungs of BALB/C mice. A and B. In vivo data detected on Diffuse in vivo Flow Cytometry (DiFC) system. (A) 120- seconds data examples on DiFC, showing the number of cells in circulation is decreasing over time after injection. (B) The average CTC count rates detected in ventral caudal artery and ventral caudal vein. The average numbers were calculated in 15-minutes intervals. C. Untreated control mice showing a limited attachment of 4T1 cancer cells to the lungs (green is the lung tissue and red dots are 4T1 breast cancer cells). D. We observed an increase in the attachment of 4T1 breast
97 cancer cells to the lungs of BALB/C mice treated with 5U/mL of Neur. E. Data quantification showing a statistically significant increase in the attachment of 4T1 breast cancer cells to the lungs of BALB/C mice after treatment with 5U/mL of Neur. The sample size (N) is 4, data was statistically analyzed using the student t-test, and the statistical significance between the two groups is denoted as ***P<0.001.
The decreasing rate, which served as an indicator of substantial 4T1 clearance from the blood stream, was expected to correspond to 4T1 attachment to the vascular wall or clearance from the blood circulation system altogether. Therefore, at the 60-minute time point we determined if
4T1 cells were attached by examining the Balb/c lungs. We found that for Balb/c mice with intact
GCX (lack of Neur treatment), the normalized number of 4T1 attachment to the vessel walls in the lungs was 1.00±0.14 (Fig. 4.12C and 4.12E). We observed a statistically significant 2.2-fold increase in the attachment of 4T1 cells to the GCX-deficient (due to Neur treatment) vessel walls of the Balb/c lungs (Fig. 4.12D and 4.12E).
98
Figure 4.11: En face confirmation of intact endothelium after treatment with 5 U/mL of Neur. A and B. Expression of eNOS before and after the treatment with Neur enzyme. Images show that eNOS is expressed around the nucleus of ECs. C and D. E-selectin expression before and after the treatment with enzyme. E-selectin is expression across the surface of the endothelium. E and F. PECAM-1 staining before and after the treatment of enzyme. Visual inspection shows the presence of PECAM-1 across the entire surface of the endothelium. G. Data quantification for E- selectin showing a non-significant difference in expression of E-selectin on the endothelium between control and enzyme treated mice. All data are normalized with UF results. Student’s t test was used to compare endothelium from untreated mice to endothelium from Neur-treated mice. The sample size (N) is 4. NS denotes “not significant”.
4.4 DISCUSSION
In this study, we demonstrated the importance of EC GCX regulation of cancer cell attachment to the endothelium. We also showed that the increase in cancer cell attachment to the endothelium, which results from decreased GCX expression, can more preferentially occur in areas
99 of the vasculature with DF patterns, in comparison with UF areas. Activation of the adhesion molecule E-selectin in different flow conditions was not the primary contributor to increased cancer attachment to the endothelium. On the other hand, the absence of a healthy, robust GCX appeared to be extensively and primarily involved in the enhancement of cancer cell attachment to the endothelium. Our results provide new insights into the possible pathways leading to secondary tumor formations during cancer progression.
The mechanisms underlying secondary tumor formation remain an active area of ongoing research and recent studies suggest that interaction between CTCs and the endothelium is an important driver of cancer cell migration from the primary tumor site to secondary tumor sites
[245, 246]. Until now, there have been few studies conducted to examine the role of the endothelial
GCX in mediating these CTC-to-endothelium intercellular interactions [247]. Here, we have provided evidence that suggests that the GCX is pivotal in mediating CTC-EC interactions leading to secondary tumor formation.
We began our study by using a previously developed parallel plate flow chamber model
[146] that recreates flow patterns (UF and DF) similar to what is observed in vivo [248, 249]. We tested the hypothesis that DF regions of the vascular system could enhance the attachment of cancer cells to the endothelium. After introducing DF and UF conditions to HUVEC, we co- incubated HUVEC monolayers with either 4T1 or MCF7 breast cancer cells. As predicted, we observed a significant increase in the attachment and immobilization of 4T1 or MCF7 breast cancer cells to the endothelium in DF regions compared with UF regions (Fig. 4.2D, 4.2E and
4.2F). Considering the fact that 4T1 cells originate from mice and MCF7 cells originate from humans, our finding shows that CTC attachment to the DF area is not species specific. However, it is important to mention that we observed an increase in the rate MCF7 attachment to HUVEC
100 compared to 4T1 attachment to HUVEC, indicating that proper species match is very important.
Any other cancer cell type that has affinity to receptors on the endothelial surface should produce the same attachment results. This confirms an earlier report by Yamaguchi et al., that cancer cells are more likely to adhere to bifurcated regions in blood vessels where DF exits, and less likely to adhere in other locations of the vascular tree where UF exist [250].
Breast cancer cell clustering has been reported to have a significant effect on the metastatic potential of the tumor [251-253]. Specifically, Nicola et al reported that CTC clusters, although rare to find in circulation, have a 23-fold to 50-fold increased likelihood of resulting in metastasis
[251]. Guided by the prior reports, in the present study we investigated the potential for DF patterns to regulate cancer cell clustering that could enhance secondary tumor formation. We observed substantial increases in the rate of clustering of 4T1 and MCF7 breast cancer cells (Fig. 4.2D, 4.2E and 4.2G) in regions of DF, compared to regions of UF. It was previously suggested that metastatic cancer cells are more likely to break away from the primary tumor in clusters and travel in groupings to the secondary tumor site [251]. It was also suggested that metastatic cancer cells are less likely to break away from the primary tumor as individual cells, a situation in which cells would form clusters somewhere along the vessel pathway or at the vessel wall where the secondary tumor will form [251]. From our study, we cannot determine which situation is the case. However, if the former is true, then our results show that clustered CTCs prefer to land at sites of DF while individual CTCs prefer to land at sites of UF. If the latter is true, then our results show that CTCs more readily aggregate in the presence of DF patterns than they do in the presence of UF patterns.
CTC migration from the apical side of the endothelium, across the endothelium, and into the underlying tissue are the last steps in the process of secondary tumor initiation [254, 255].
Therefore, we investigated the potential of attached 4T1 or MCF7 breast cancer cells to migrate
101 through the endothelium after successful attachment to the endothelium. Both cell types exhibited the same trend of higher transendothelial migration in DF versus UF conditions. 4T1 breast cancer cells were observed to migrate across the endothelium at a statistically significant high rate in the
DF region, in comparison to the rate of migration in UF areas (Fig. 4.2H). For MCF7 cells, on the other hand, exhibited statistically nonsignificant increase in the rate of migration across the endothelium in DF regions compared to UF regions (Fig. 4.2H). This may be due to 4T1 breast cancer cells possessing a higher degree of activated invadopodia than MCF7 breast cancer cells
[256, 257].
We sought to identify a mechanism that could enable DF-induced CTC interactions with
ECs. It has been reported that DF initiates EC dysfunction [249], which leads to the progression of disease [225]. Previous reports rarely consider that DF is a contributing factor to GCX degradation [258-261]. On average, components of the GCX in both humans and animals degrade and regenerate on a daily basis and in a balanced manner [262, 263]. However, as our group and others [146, 264-266] have highlighted, DF has a negative effect on the endothelial GCX [15, 267].
Based on this prior research, we believe that degraded GCX could play a direct role in regulating
DF-induced CTC interactions with ECs. Therefore, we characterized the expression of HUVEC
GCX in DF and UF regions of the flow chamber (Fig. 4.8A, 4.8B, and 4.8C). Compared with UF conditions, we observed a more than 2-fold decrease in both the coverage and thickness of GCX in DF regions (Fig. 4.8B, 4.8F, and 4.8G). This is similar to what we previously observed in rat
ECs [146], suggesting that the effects of DF on GCX is consistent across EC types from different species. It is clear from our observations that GCX degradation outweighs GCX regeneration in the DF regions of the vascular system (Fig. 4.8A-4.8C, 4.8F, and 4.8G). As reported by numerous
102 studies [15, 128, 159, 268], including the present investigation, UF is protective of the endothelium and results in a healthy, robust GCX [231, 269, 270].
In UF regions, healthy GCX can shield receptors on the endothelium surface and block binding of CTC to the endothelium. Degraded GCX in DF regions of the blood vessels could expose receptor sites on the surface of the endothelium for easy binding of CTCs to the endothelium surface (Fig. 4.1) [271, 272] because these adhesion molecules lie beneath the endothelial GCX surface and are attached to the cell membrane. Another plausible explanation is that DF could be pro-inflammatory and could enhance the expression of adhesion receptors. Once the GCX layer is removed and the adhesion molecule receptor sites are exposed in DF, or once adhesion receptors are overexpressed in DF, CTC-to-EC intercellular interactions can become enhanced. It follows that CTCs would likely prefer to undergo transendothelial migration at DF regions of the vasculature, due to the proven decrease in GCX expression.
We examined whether the attachment of cancer cells to the endothelium in either DF or
UF was due to degraded endothelial GCX or overexpressed endothelial surface adhesion receptors, namely activated E-Selectin although there are other adhesion molecules that should be explored in future studies. It was important for us to verify whether decreased GCX expression in DF regions directly causes increased cell attachment, clustering and migration, or whether CTC-to-
EC intercellular interactions are caused by E-Selectin activity in DF conditions. To this end, we first investigated the expression of E-selectin in the different flow conditions. We did not observe any statistically significant difference between E-selectin expression in DF and UF conditions (Fig.
4.8D, 4.8E, and 4.8H). Our E-Selectin findings are unexpected based on prior research publications. For example, Huang et al, showed that UF downregulates E-selectin expression by
103 HUVEC, in a time-dependent manner and to protect
the endothelium against chronic inflammation
[273].
Figure 4.7: Flow conditioned HUVEC were exposed to 4T1 or MCF7 cells in static conditions and not in circulation, to confirm that flow eddy currents do not extend 4T1 or MCF7 residence time which would create artefactual results A. Number of 4T1 and MCF7 breast cancer cells attached to the DF-conditioned endothelium. The dotted line represents normalized UF data. Significant increase in the attachment of cancer cells in the DF region compared to UF region. B. Number of cancer cell clusters formed in the DF region. The dotted line represents normalized UF data. We observed a non- significant change in the clustering of 4T1 and MCF7 breast cancer cells to the endothelium in comparison with UF regions. This lack of clustering could be explained by the fact that we need to study a larger dataset. It could also be due to the reduced DF that we see in the 1 dyne/cm2 setting, which probably promotes aggregation of the cancer cells. Another explanation could be that cancer cells need time to circulate, which could make their surfaces more adhesive to each other. Testing these possibilities is a subject for another and outside the scope of the current project. C. Migration of 4T1 and MCF7 breast cancer cells through the DF- conditioned endothelium. The dotted line represents normalized UF data. Compared to UF regions, we observed a significant increase in the migration of cancer cells through the DF region, compared to UF areas. All data “Normalized with UF”. Student t test was used to compare DF vs. UF. Sample sizes: 4T1 attachment N=7, MCF7 attachment N=5. Significance is compared to the UF condition and denoted as *P<0.05, **P<0.01, or not significant (ns). This prior study and others, unlike ours, compared E-Selectin expression in static versus
UF conditions and did not compare DF versus UF conditions. In addition, our HUVEC exposure time was enough to disrupt GCX expression in the DF region but not enough to have resulted in a significant expression of E-selectin.
We next attempted to investigate the effects of GCX degradation in the presence of stable
E-Selectin expression. It is possible to achieve systemic GCX knockdown by pro-inflammatory
104 cytokines or enzymes that increase reactive oxygen species and matrix metalloproteases activities
[61]. These agents are naturally released in certain disease settings including atherosclerosis, ischemia/reperfusion and cancer [15, 261, 274, 275]. In our study, we mimicked in vivo systemic degradation of GCX in vitro by adding 15 mU/mL of neuraminidase to UF conditions, as previously described above, and we characterized the expression of GCX (Fig. 4.9G). We observed that the enzyme significantly decreased both the coverage and thickness of GCX in UF conditions
(Fig. 4.9B, 4.9G, 4.9K, and 4.9L). This result is similar to the degradation of the endothelial GCX that is a reported effect of systemic enzyme or cytokine release in disease [276-278]. After successful degradation of GCX in UF settings, we would expect a high level of interaction between
CTCs and ECs. However, when we investigated E-selectin expression we unexpectedly found that adding 15 mU/mL of Neur to UF destabilized and decreased the expression of E-Selectin (Fig.
4.9C, 4.9H, and 4.9M). Although, this Neur-induced E-Selectin decrease could hinder CTC-to-EC interactions, we found instead that Neur increased 4T1 and MCF7 breast cancer cell attachment, clustering and migration to ECs to levels that were similar to what we found due to DF stimulation.
This increase in CTC-to-EC interactions can only be attributed to GCX degradation and not to E-
Selectin degradation.
To confirm our cell culture results, we treated Balb/C mice with 5 U/mL of Neur. We quantified the effects of the enzyme on the endothelium of the abdominal aortas of treated and untreated mice. We observed on this endothelium a significant reduction in the expression of GCX
(Fig. 4.10). We also confirmed that only GCX on the endothelial layer was affected by Neur enzyme and that the endothelium was not denuded, by the localizing three EC markers including
E-Selectin (Fig. 4.11A-4.11F). Quantification of E-Selectin revealed that its expression level was not altered by Neur (Fig. 4.6G). Lastly, we investigated the effect of Neur on 4T1 breast cancer
105 cell attachment to mouse lungs. We observed a significant 2-fold increase in the attachment of 4T1 breast cancer cells to the lungs of Neur-treated mice in comparison with untreated samples (Fig.
4.12C-4.12E). These results are consistent with a prior study by Rai et al, in which they observed a significant increase in the attachment of lung cancer cells to the endothelial monolayers after the degradation of the WGA-labeled GCX [216].
In summary, our data indicate that DF areas of the vasculature could represent points of entry for CTCs into tissue sites where they can form secondary tumors and, thereby, advance cancer progression. In addition, DF-induced degradation of endothelial GCX, specifically the SA component, plays a critical role in regulating the interactions of CTCs with the endothelium.
106 CHAPTER FIVE: SUMMARY OF WORK AND FUTURE PERSPECTIVES
Although the role of GCX in regulating intercellular interactions has been understudied, the recent work published by our group proves that there is a correlation between the structural integrity of the GCX and the performance of intercellular interaction proteins like Cx43 and E- selectin[123, 140, 279].
We have shown that the opening and closing of Cx containing gap junctions, especially
Cx43 containing gap junctions, are directly dependent on the health of GCX [279]. In healthy conditions (Fig 5.1B, 5.1D) the GCX is stable and Cx43 is properly aligned to its adjacent connexins, while connexons are also aligned to adjacent connexons, from a neighboring cell, enabling the transport of ions and molecules through gap junctions. However, in diseased conditions, degraded GCX destabilizes Cx43, the connexons and gap junctions, and prevents the transport of ions and molecules (Fig 5.1A, 5.1C,5.1E).
Further studies are necessary to investigate the role of GCX in gap junction mediated intercellular communication. The formation of connexin containing gap junctions includes oligomerization, trafficking, the actual gap junction formation, gating function and internalization
[280-282]. Of these sequences, we have only shown the connection between GCX and the gating function of connexins. Due to reports by Mia Thi et al and others [283, 284] in the involvement of cytoskeleton in Cx expression, could it be possible that the GCX can be implicated in the trafficking and internalization of connexin containing gap junctions? This remains to be clarified.
In addition, the formation of gap junctions could be occurring as a result of the heteromeric combination of different types of connexin proteins [285-289]. While we have only studied GCX regulation of one type, Cx43. It will be very interesting to compare the role of GCX in regulation
107 of homomeric (single or similar subunit of connexin protein) versus heteromeric (different subunits) connexin containing gap junctions. Such an experiment would broaden our understanding of the role played by GCX in modulating cell-to-cell communication.
It has been proposed [290], and we have shown, that accessibility of E-selectin receptors on the surface of the endothelium for easy binding of ligands on circulating cells is GCX dependent
(Fig 5.1A,5.1B) [132]. In disease conditions, degraded GCX enhances the interactions between
ECs and CTCs in a manner that may result in secondary tumor initiation (Fig 5.1A, 5.1C). We further showed that the SA component of the GCX plays a significant role in the process of concealing these receptors from CTCs [123]. It is necessary to study GCX involvement in the attachment of circulating cells at specific stages of the process: slow rolling, adhesion, firm binding, crawling and paracellular and transcellular migration [291, 292]. Each of these stages is mediated by a different form of receptor on the surface of the endothelium (Table 2). E-selectins, which we have studied, are only reported to be important in slow rolling of circulating cells on the endothelium [293]. Future work should investigate the full class of selectins, which in addition to
E-selectin include P-selectin and L-selectin. These selectins could be differentially regulated by
GCX. Another adhesion molecule, integrin, is worth investigating because of the specific role played by integrins in creating firm adhesion complexes during immune and cancer cells interactions with the endothelium.
Lastly, GCX is composed of different GAGs and numerous other components, as previously mentioned. We have shown the importance of SA and HS in regulating intercellular interactions in regards to cell-to-cell communication and cell-to-cell adhesion. Perhaps other components that form an integral part of GCX should be studied to understand their role in intercellular interactions. Investigating the full range of GCX components is particularly important
108 for meeting a two-tiered goal: (1) Better understand the GCX role in intercellular interactions and,
(2) Develop GCX strengthening drugs to prevent unwanted GCX degradation in a component- specific manner, to stop disease progression. Achieving this goal will significantly advance the field.
Fig 5.1: Conceptual depiction of the role played by endothelial glycocalyx in intercellular interactions. A. Vessel showing healthy and diseased conditions. On the left side the vessel is healthy and characterized by intact GCX which ensures proper cell-to-cell communication between neighboring endothelial cells. The healthy GCX also prevents circulating tumor cells from attaching to adhesion receptors on the surface of endothelial cells. The right side shows a diseased condition where glycocalyx is degraded leading to lack of cell-to-cell communication and attachment to circulating tumor cells to the endothelium. B. Zoom-in of healthy endothelial GCX and stable actin cortical web. The GCX prevents ligands on circulating tumor cancer cells from binding to the receptors on the endothelial surface. The actin cortical web is stable, ensuring proper alignment of junctional proteins. C. Zoom-in of diseased GCX and destabilized actin cortical web. The degraded or diseased GCX uncovers the adhesion receptors on the surface of the endothelium for easy binding to ligands on circulating cancer cells. Destabilized actin cortical web leading to misaligned junctional proteins D. Zoom-in of healthy junctional proteins, showing active communication between neighboring endothelial cells. E. Degraded GCX leading to a destabilized actin cortical web, which disrupts junctional protein alignment and prevent intercellular communication between neighboring endothelial cells.
109
APPENDIX A: CURRICULUM VITAE
EDUCATION Ph.D., Bioengineering Northeastern University, Boston, MA, Expected 2019. Concentration: Mechanobiology; GPA: 3.5 Dissertation: Endothelial glycocalyx-mediated intercellular interactions: mechanisms and implications for health and disease Honors and Awards: NSF GRFP, NSF I-Corp and Outstanding graduate research award (College of Engineering) Thesis Advisor: Eno E Ebong, Ph.D.
BSc. Biomedical Engineering, The City College of New York (CCNY) of the City University of New York, NY, 2014. Graduating Honors: Cum laude Special programs: National Institute of Health undergraduate scholar; Louis Stokes Alliance for Minority Participation program Thesis Advisor: John M Tarbell, Ph.D.
Associate Degree, Mechanical Engineering, Takoradi Technical University, Takoradi, Ghana, 2004. Concentration: Industrial Engineering (plant option)
ADDITIONAL TRAINING AND DEVELOPMENT ACTIVITIES • Regulatory Approval Experiential Learning for FDA, Northeastern University XN Program, 2019. • Regulatory Approval Experiential Learning for CE, Northeastern University XN Program, 2018. • Product Development Customer Discovery Course, National Science Foundation Innovation Corp Cohort, 2017. • Entrepreneurship Education, Center for Entrepreneurship Education, Northeastern University, Boston, MA, 2017. • International Association of Healthcare Central Service Material Management Certified Registered Central Service Technician (CRCST), 2009
110 EXPERIENCE Chief Executive Officer Medical Device Development, Therapeutic innovations Inc, Boston, MA (June 2015- January 2019) • Work creatively and analytically in a problem-solving environment, demonstrating teamwork, innovation and excellence. • Develop, communicate project milestones to product development team. • Provide coaching and guidance to all team members. • Identify and procure internal or external resources needed to complete projects. • Evaluate financial goals of the project and ensure project is within financial limits. • Report on project milestone results, metrics, test and management activities. • Monitor staff performance and complete performance reviews for growth. • Understand interdependencies between technology, operations and business needs. • Understand regulatory approval pathways for FDA and CE Mark. Project Outcome: Commercialization of the Bubble CPAP for Low to Middle Income Countries, Therapeutic innovations Inc, Boston, MA, 2019 Laboratory Director Bioengineering, Mechanotransduction Laboratory, Northeastern University, Boston, MA (June, 2017 – August 2019) • Responsible for the overall operation and administration of the laboratory. • Responsible for ensuring laboratory personnel develop and used quality SOPs to ensure accurate and reliable results. • Monitoring defects in laboratory SOPs and taking the necessary corrective actions to prevent recurrence. • Responsible for ensuring that all laboratory personnel adhered to the safety regulations of the university at all times. • Responsible for ensuring laboratory budget related to NSF projects was maintained. • Having mechanisms in place for effective communication between principal investigator and graduate students. • Conducted research progress report meetings to evaluate individual graduate students progress. Graduate Research Assistant Bioengineering, Ebong Laboratory, Northeastern University, Boston, MA (June, 2014 – August 2019) • Conducting interdisciplinary and collaborative cardiovascular and cancer research for the completion of the degree in a timely manner. • Learned theories, practices and research methods of the bioengineering disciple and applied them to research and teaching. • Discovered and pursue unique topics to add to the scientific knowledge and applied this knowledge to existing problems. • Wrote research articles and grants to support research program. • Participated in scientific presentations at conferences to foster collaborative works with other research groups.
111 • Mentored Masters, Undergraduate, and High school students in research programs. • Prepared and submitted NIH, NSF, B-BIC grants to support new research areas. Projects Outcomes: • NSF GRFP, NSF I-CORP, Basis for NIH RO1 in Review. • Flow-Regulated Endothelial Glycocalyx Determines Metastatic Cancer Cell Activity • Metastatic Cancer Cell Attachment to Endothelium is Promoted by Endothelial Glycocalyx Sialic Acid Degradation. • Functional Regeneration of the Endothelial Glycocalyx, Undergraduate Research Assistant Biomedical engineering, Wallace Coulter Cardiovascular laboratory, CCNY, New York. (January 2009- May 2014) • Conducted collaborative cardiovascular research as an undergraduate NIH research fellow • Learned theories, practices and research methods of the bioengineering disciple and applied them to research and teaching. • Participated in scientific presentations at conferences to foster collaborative works with other research groups. • Participated in Laboratory outreach programs on campus to recruit incoming undergraduate students interested in cardiovascular research. Projects Outcomes: • Endothelial glycocalyx, apoptosis and inflammation in an atherosclerotic mouse model, • The development of a stretching device that is compatible with the combined atomic force and confocal microscope, Senior Capstone, CCNY, 2014. • NIH undergraduate research Fellow • Lois Stokes for Minority Participation Scholarship(LSAMP) Fellow
Surgical Instrumentation Specialist, Jacobi Medical Center, NY Certified Registered Central Service Technician (CRCST), (November, 2011 – June 2014) • Performed maintenance on re-usable surgery equipment for surgical specialties including Cardiac, Orthopedic, Neuro and General Surgeries. • Liaison between Central Service Department and the operating room. • Performed high level disinfection on gastrointestinal scopes and Cardiac probes. • Preparation and packaging of surgical instruments. • Performed restocking of orthopedic surgical implant trays. • Performed sterilization techniques for both low and high temperature sterilizations • Organized surgical instruments for both trauma and planned surgeries. Surgical Instrumentation Specialist, Newark Beth Israel Hospital, New Jersey Certified Registered Central Service Technician (CRCST), (January, 2010- November 2010) • Decontamination of surgical equipment • Preparation and packaging of surgical equipment, following all standards and protocols • Sterilization of prepared and packed surgical equipment
112 Surgical Instrumentation Specialist, Mount Saini Hospital, NY Certified Registered Central Service Technician (CRCST) Student, (February, 2009- November 2009) • Assisted certified technicians to decontaminate, prepare and pack re-usual surgical equipment • Fulfilling requirements for certification with IAHCSMM Mechanical Maintenance Engineer Mechanical Maintenance Engineer, Guinness Ghana LTD. Achimota, Accra, Ghana. (January, 2007 – December, 2008) • Performed planned, preventive, shutdown and breakdown maintenance on brewery equipment. • Operated sophisticated brewery machinery including: bottle washers, labelers, fillers and palletizes. • Worked with original equipment manufacturers to install a complete brewery line for Guinness beer. • Lead a team of Junior Engineers to renovate an entire conveyer line to accommodate different shapes and sizes of bottles. • Customized bottle labelers for different brands of beers and Malta Guinness.
HONORS & AWARDS 2019 Outstanding graduate research award, College of Engineering, Northeastern University. 2018 • Research and presentation award, New England Science Symposium. • Nominated for University Outstanding Graduate Student Award for Experiential Learning, Northeastern University, Boston, MA.
2017 National Science Foundation Innovation Corp(I-Corps) Award for Entrepreneurs
2016 • National Science Foundation Graduate Research Fellowship Program (NSF GRFP) Award • Travel Award to attend the Annual Conference of the National Organization for the Professional Advancement of Black Chemist and Chemical Engineers • MassChallenge Entrepreneurship Finalist • Second Runner up Flately Entrepreneurship Challenge • Second Runner up PLUGG Startup Challenge • Sandbox Entrepreneurship Finalist, MIT 2013 • Wallace H. Coulter Award for Outstanding Biomedical Engineering Undergraduate Research (at CUNY CCNY)
113 • Excellence Award in Recognition of an Outstanding Research Project Presented at the 15th Annual Philadelphia AMP Research Symposium and Mentoring Conference 2012 Louis Stokes Alliance for Minority Participation (LSAMP) Scholarship (at CUNY CCNY)
2011 National Institute of Health Scholarship (at CUNY CCNY)
FUNDING • NSF Innovation Corp Grant(I-CORP), $50,000, 12/ 2017- 08/2019 • NSF Graduate research fellowship program(GRFP), $146,499, 08/2016-08/2019 • Sandbox Entrepreneurship Fund, MIT, $5000, 05/2016- 12/2016 • Idea Gap Funding, Northeastern University, $20,000, 08/ 2015- 12/2016
TEACHING ENGAGEMENTS • Project Sponsor, Capstone Course, Bioengineering, Northeastern University, January 2019 • Project Sponsor, Regulatory Affairs Course, Northeastern University, March 2019. • Project Sponsor, Business in Emerging Markets, Northeastern University, April 2019 • Project Sponsor, Regulatory Affairs Course, Northeastern University, November 2018. • Guest Lecturer, Entrepreneurship Education Northeastern University, April 2018. • Guest Lecturer, Design of Biomedical Instrumentation, Northeastern University, June 2017. • Guest Lecturer, Design of Biomedical Instrumentation, Northeastern University, November 2016. MEMBERSHIPS • International Association of HealthCare Central Service Material Management (IAHCSMM) • Biomedical Engineering Society • Founding Member, Biotech Entrepreneurs, Northeastern University • American Hearts Association • Ghana institute of Engineers(Ghana)
PUBLICATIONS Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity. Mensah SA, Nersesyan A, Harding IC, Xuefei Tan, Mitra R, Lee C, Niedre MJ, Torchilin VP, Ebong EE. Submitted to FASEB, revision after review by FASEB. 2019
Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation. Mensah SA, Harding IC, Zhang M, Jaeggli MP, Torchilin VP, Niedre MJ, Ebong EE. AIChE J. 2019 Aug;65(8). pii: e16634. doi: 10.1002/aic.16634. Epub 2019 May 9.
114
Endothelial barrier reinforcement relies on flow-regulated glycocalyx, a potential therapeutic target. Harding IC, Mitra R, Mensah SA, Nersesyan A, Bal NN, Ebong EE. Biorheology. 2019 Mar 29. doi: 10.3233/BIR-180205
Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation. Harding IC, Mitra R, Mensah SA, Herman IM, Ebong EE. J Transl Med. 2018 Dec 18;16(1):364. doi: 10.1186/s12967-018-1721-2
Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target. Mitra R, O'Neil GL, Harding IC, Cheng MJ, Mensah SA, Ebong EE. Curr Atheroscler Rep. 2017 Nov 10;19(12):63. doi: 10.1007/s11883-017-0691-9. Review.
Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter- endothelial communication. Mensah SA, Cheng MJ, Homayoni H, Plouffe BD, Coury AJ, Ebong EE. PLoS One. 2017 Oct 12;12(10):e0186116. doi: 10.1371/journal.pone.0186116. eCollection 2017.
Endothelial glycocalyx, apoptosis and inflammation in an atherosclerotic mouse model. Cancel LM, Ebong EE, Mensah S, Hirschberg C, Tarbell JM. Atherosclerosis. 2016 Sep; 252:136-146. doi: 10.1016/j.atherosclerosis.2016.07.930. Epub 2016
PATENT PENDING • Mensah SA, Cheng MJ, Mitra R, Ebong EE. U.S. Provisional Application No.: 62/534,660 Title: GlycoFix (Structurally and Functionally Repaired Endothelial Glycocalyx). 2017
• Mensah, S., Khadka, N., Peyear, T., Costa, K.D., Cardoso, L., and Wang, S. A Stretching Device that is Compatible with the Combined Atomic Force and Confocal Microscope. 2014
CONFERENCE PRESENTATIONS • Mensah, S., Harding, I., Niedre, M., Torchilin, V., and Ebong, E. “The Role of Glycocalyx and Shear Stress on Endothelial-Cancer Cell Attachment.” American Institute of Chemical Engineers Annual Meeting in San Francisco, CA on November 14, 2016. • Mensah, S. and Ebong, E. “Endothelial Glycocalyx and Cancer Metastasis: The effect of Glycosaminoglycans on 4T1 Breast Cancer Cell Attachment to the Endothelium.” Annual Conference of the National Organization for the Professional Advancement of Black Chemist and Chemical Engineers in Raleigh, NC on November 8-12, 2016. • Mensah, S., Homayoni, H., Cheng, M., Plouffe, B., and Ebong, E. “Glycocalyx and its
115 impact of communication in endothelium.” International Vascular Biology Meeting at Boston, MA on October 31, 2016. Poster Presentation. • Mensah, S., Harding, I., Niedre, M., Torchilin, V., and Ebong, E. “The Role of Glycocalyx of 4T1 Breast Cancer Cell Attachment to the Endothelium.” Annual Biomedical Engineering Society Meeting at Minneapolis, MN on October 7, 2016. • Harding, I., Mensah, S., Ahn, I., Ebong, E. “Disturbed flow, endothelial glycocalyx, and proatherosclerotic oxLDL uptake.” International Vascular Biology Meeting at Boston, MA on October 31, 2016. Poster Presentation. • Cheng M., Mensah S., Harding I., Ebong E. “Glycocalyx in Dynamic Flows and in Atherosclerosis, and Implications for Key Cell Functions.” NIH National Heart, Lung, and Blood Institute K- to-R01 Meeting at Bethesda, MA on August 11-12, 2016. Poster Presentation. • Cheng, M. (presenter), Mensah, S., Ebong, E.E. “Endothelial Extracellular Glycocalyx: Regeneration of Structure & Function.” 2015 North American Vascular Biology Organization Meeting, Hyannis, MA. October 2015. Podium Presentation. • Mensah, S, Cheng, M., and Ebong, E.E. “Role of Glycocalyx Heparan Sulphate in Endothelial Gap Junction Channel Functionality.” Annual Conference of the National Organization for the Professional Advancement of Black Chemist and Chemical Engineers, Orlando, FL. September, 2015. Podium Presentation.
• Mensah S., Cancel, L., Tarbell J.M., Ebong E.E. “The Effect of Flow and Cell Death on the Growth of Atherosclerotic Plaques.” Proceedings of the 2014 IEEE 40th Annual Northeast Bioengineering Conference, 2014 April 25-27. Poster Presentation. • Mensah, S., Tarbell, J.M., Cancel, L.M., and Ebong, E.E. “The role of flow patterns and apoptosis in atherosclerotic plaque formation” presented at the Annual Biomedical Engineering Society Meeting, Seattle, WA, September 2013. Poster Presentation.
116 APPENDIX B:
DESIGN FOR THE LESS FORTUNATE
My passion for product development, coupled with the need to help the less privilege in society, drove me to start a medical device company called Therapeutic Innovations, during my second year as a PhD student. The main goal of Therapeutic Innovations is to redesign medical devices for developing countries. Our first product, which is called the Airbaby, is a respiratory assist device for premature babies suffering from respiratory distress syndrome (RDS).
Vision Statement
The objective of the Airbaby project is to reduce the rate of premature deaths in Ghana.
As reported by the World Health Organization (WHO), Ghana has the highest rate of premature deaths among the nations in the West African sub-Saharan region. We implemented a training program to educate clinicians on the standards of care in regards to premature babies. With the help of the AirBaby device, in addition to the training program, we hope to move Ghana out of the critical zone of 75% neonatal mortality rate. We anticipate improvements within 9 months to a year and thereafter continuous improvement. We will utilize a well-designed tracking and evaluation system and implement revisions along the way to ensure the effectiveness of our treatment strategies.
Executive Summary and Problem Statement
The development and generation of new medical devices tends to be expensive and leads most companies to produce high-priced products and services for high income markets. Low to middle income countries often lack the resources to secure expensive high-priced medical equipment. If they are able to afford these devices, they often lack the necessary training and customer support services to ensure these machines function optimally and are meeting their
117 needs. High infant mortality rates in low to middle countries (LMIC) countries are largely attributed to a lack of training, education, and specialized trained professionals for neonates, such as neonatal intensive care unit(NICU) Registered Respiratory Therapists. There is also a lack of understanding of neonatal medical equipment.
Therapeutic Innovations provides tailored solutions to the health challenges in low to middle income countries. We offer a comprehensive approach to their healthcare issues. This approach involves complementary people, processes and products. We offer training and extensive program development for capacity building as well as design and manufacture low cost, high quality versions of essential medical devices to achieve specific environment and program objectives.
Our combined flagship product is designed to optimize neonatal intensive care unit (NICU) training for staff and provide the infant respiratory breathing system called the AirBaby. The introduction of our AirBaby device will be able to help reduce the infant mortality rate, not just in area hospitals but across the whole nation. RDS is caused by lung immaturity and surfactant deficiency and is common among premature infants globally. Survival and outcomes for infants with RDS has improved within the past 43 years as a result of the development of surfactant, high frequency ventilation, mechanical ventilation, and most recently in the last 30 years, the advent of bubble CPAP (Continuous Positive Airway Pressure). Surfactant reduces mortality and morbidity in premature infants, but requires intubation and mechanical ventilation. Mechanical ventilation increases the risk of bronchopulmonary dysplasia (BPD), a chronic lung disease associated with adverse pulmonary and neurodevelopmental issues. Early treatment with bubble CPAP can preserve an infant’s self-generated surfactant and reduce the need for mechanical ventilation and
118 surfactant administration. Bubble CPAP would be sufficient support for infants born 26 weeks and greater.
The Innovation
Summary: Therapeutic Innovations' core innovation is a customized take of the bubble continuous pressure airway pressure device(CPAP) suitable for low to middle income countries, for treating respiratory distress syndrome(RDS). Therapeutic Innovations device called the
AirBaby will be the first of its kind in the class of bCPAP. Through sponsorship from the NSF I- corp program, the team visited key target markets to conduct customer discovery interviews and collect data regarding the state of current respiratory devices and why they tend to fail, leaving premature infants without the necessary life support. The AirBaby will be innovative in six key areas:
(1) Manufacturing cost reduction: The use of off-the-shelf parts combined
with 3D printing to develop a fully functional bCPAP will result in the reduction of
manufacturing cost, which will ultimately reduce the overall cost of the device.
(2) Ability to interface with external power sources: The lack of continuous
power supply in low to middle income countries will result in the inability of chronic use
medical devices like the bCPAP to support life. The AirBaby will have the ability to
interface with motorcycle batteries to provide continual life support even when external
power supply is not available.
(3) Transportability: Conventional BCPAPs rely exclusively on power
directly from outlets, making it difficult, if not impossible to transport patients who may
need to be continuously hooked to the device during transportation from one hospital to
another. The AirBaby will not only be light weight due to the carefully selected off-the-
119 shelf parts, but will also be able to interface with external batteries to facilitate
transportation. From our customer discovery interviews we noticed that a majority of
premature infant death occurs during their transportation from point of delivery to the
major referral hospitals.
(4) Data collection: The ability of the AirBaby to collect and store vital patient
metadata over the period of care is essential in order to establish standards of care for
patient management. The collection of key data points through device usage will also
empower policy makers to implement changes within their respective hospitals to decrease
neonatal death.
(5) Capacity building training: Our comprehensive clinical and maintenance
training program, which incorporates detailed clinical and engineering training for clinical
staff and clinical engineers in charge of neonatal care, and clinical staff. We hypothesize
that the implementation of a customized Airbaby tailored for the needs of clinicians and
patients in low to middle income countries will drastically reduce the rate of premature
death.
Intellectual Property Strategy
Therapeutic Innovations has taken a proactive approach regarding intellectual property.
We are working with the technology transfer office at Northeastern University, Boston, MA to identify potential areas of novelty in the AirBaby that need protection. Since the AirBaby will be internationally used we are also working with the regulatory affairs clinic (Northeastern University
XN Program) to develop a regulatory affairs strategy for the AirBaby. We plan on pursuing a patent for our designs and algorithms for the AirBaby, with consultation from a legal counsel. We
120 are well aware of the regulatory and intellectual property situation in our target market and are currently in talks with the Ghana Standards Board and the Ghana Food and Drugs Administration to understand their requirements.
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