A Dissertation

entitled

Alpha7 Nicotinic Acetylcholine : Novel Role in Survival and Murine Atherosclerosis

by

Robert H. Lee

Submitted to the Graduate Faculty as partial fulfillment of the requirements for

the

Doctor of Philosophy Degree in Biomedical Sciences

______Guillermo Vazquez, PhD, Committee Chair

______David Giovannucci, PhD, Committee Member

______Joseph Margiotta, PhD, Committee Member

______Sandrine Pierre, PhD, Committee Member

______R. Mark Wooten, PhD, Committee Member

______Patricia R. Komuniecki, PhD, Dean College of Graduate Studies

The University of Toledo

December 2014

Copyright 2014, Robert Hugh Lee

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Alpha7 Nicotinic Acetylcholine Receptor: Novel Role in Macrophage Survival and Murine Atherosclerosis

by

Robert H. Lee

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences

The University of Toledo

December 2014

Atherosclerosis is a chronic inflammatory disease, characterized by infiltration and accumulation of leukocytes within the vascular wall.

Macrophages play a particularly crucial role in all stages of atherosclerotic lesion development. These cells react to their local environment and can alter their function accordingly, resulting in polarization of to divergent phenotypes. Notably, of lesional macrophages undergoing chronic endoplasmic reticulum (ER) stress coupled with impaired clearance of dying cells promotes formation of necrotic cores, a preamble for plaque rupture and thromboembolic events and clinical manifestation as myocardial infarction or stroke. The alpha7 nicotinic acetylcholine receptor (α7nAChR) is recognized as an important regulator of macrophage function in , and activation of this receptor in other cell types can protect against apoptosis. We hypothesized that activation of α7nAChR could protect macrophages against

iii apoptosis, and therefore play an anti-atherogenic role in protecting against apoptosis and necrosis. We investigated the potential role of α7nAChR in macrophage survival in bone marrow derived macrophages (BMDMs) polarized to the M1 or M2 phenotype, and found that α7nAChR stimulation preferentially protected M2 BMDMs from ER stress-induced apoptosis. We then investigated the impact of macrophage α7nAChR deficiency on lesion development by utilizing a bone marrow transplantation model using low density lipoprotein receptor knockout (LDLR-/-) mice. Bone marrow deficiency of α7nAChR had no effect on early plaques, but at the advanced stage α7nAChR deficiency resulted in reduced lesion area and macrophage content, and this was accompanied by reduction lesional cell proliferation. These studies represent novel findings in the role of α7nAChR in macrophage survival in vitro and in the development of murine atherosclerotic lesions, and set a foundation for future studies to further investigate the therapeutic potential of macrophage α7nAChR in the progression of atherosclerosis.

iv

This dissertation is dedicated to my Grandma and Grandpa Lee, and my late

Grandma and Grandpa Caverly. You have given me so much love and support and I am lucky to have grown up with grandparents who were so involved in my life. I love you all very much.

Acknowledgements

I first want to thank my mentor, Dr. Guillermo Vazquez, for giving me the opportunity to earn my Ph.D. under his guidance. I have learned so much more than I ever expected to beyond just the basics of doing scientific research, and I want to thank you for your support, your trust in me, and your tolerance of my excessive use of commas in scientific writing. I hope our scientific and personal relationship continues for a long time to come. To my parents, thank you for your help in the hardest times and in the little moments too. You helped me to become the honest, hardworking and reasonable person I am today, love you both. To Dr. Rande Worth, thank you for your friendship and your sincere interest in my success, I always appreciate your honesty and good advice, and carrying our team when I shoot a bad round. To my current and past labmates and all my other “science friends”, thank you for your friendship and best of luck with everything ahead of you. And to my siblings, cousins, aunts, uncles and all the rest of my family, I am so blessed to have grown up in a fun, loving and close-knit family and I appreciate all the love and support over the years.

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Table of Contents

Abstract...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... ix

List of Abbreviations ...... xii

List of Symbols ...... xiv

1 Introduction ...... 1

1.1 Atherosclerosis: disease overview ...... 1

1.2 Early atherosclerosis: role of the /macrophage……………. ..3

1.3 Advanced atherosclerosis and macrophage apoptosis…………… ...... 8

1.4 Macrophage differentiation and polarization…………………………..13

1.5 α7 nicotinic acetylcholine receptor……………………………… ...... 17

1.6 Summary and hypothesis ...... 20

2 Materials and Methods ...... 22

2.1 Experimental Animals ...... 22

2.2 Preparation of bone marrow derived macrophages ...... 22

vi

2.3 Macrophage polarization into M1 and M2 phenotypes ...... 23

2.4 In vitro TUNEL assay ...... 24

2.5 Cell lysis and immunoblotting ...... 24

2.6 Real Time PCR (RT-PCR) ...... 25

2.7 Fura-2-based real time fluorescence ...... 26

2.8 Bone marrow transplantation (BMT) ...... 27

2.9 Determination of plasma cholesterol and triglycerides ...... 27

2.10 Aortic root sectioning ...... 28

2.11 Immunohistochemistry ...... 28

2.12 In situ immunostaining for M1 and M2 macrophages ...... 30

2.13 In situ TUNEL ...... 30

2.14 Necrotic core evaluation and cap thickness ...... 31

2.15 Statistical analysis ...... 32

3 Results ...... 33

3.1 α7nAChR is expressed in polarized bone marrow derived

macrophages ...... 33

3.2 α7nAChR mediated Ca2+ influx is undetectable in BMDMs ...... 38

3.3 α7nAChR stimulation promotes activation of survival signaling

pathways ...... 39

3.4 α7nAChR stimulation reduces ER stress-induced apoptosis in M2

BMDMs ...... 48

3.5 α7nAChR stimulation is associated with upregulation of Bcl-2 ...... 52 vii

3.6 Role of macrophage α7nAChR in development of murine

atherosclerosis ...... 55

3.7 Impact of bone marrow deficiency of α7nAChR on early

atherosclerotic lesions ...... 56

3.8 Impact of bone marrow deficiency of α7nAChR on advanced

atherosclerotic lesions ...... 61

4 Discussion and Conclusions ...... 72

References ...... 85

A Publications and select presentations ...... 105

viii

List of Figures

3-1 Expression of polarization markers in M1 and M2 BMDMs from wild-type

and α7nAChR-deficient mice ...... 36

3-2 Expression of α7nAChR in polarized BMDMS ...... 37

3-3 Phosphorylation of p38 and ERK1/2 in nicotine-treated α7+/+

M1 and M2 BMDMs ...... 42

3-4 Phosphorylation of STAT3 in nicotine-treated α7+/+ M1 and M2

BMDMs ...... 43

3-5 Phosphorylation of STAT3 in nicotine-treated α7-/- M1 and M2

BMDMs ...... 44

3-6 Phosphorylation of STAT3 in nicotine-treated α7+/+ M1 and M2

BMDMS pretreated with αBT ...... 45

ix

3-7 Phosphorylation of STAT3 in PNU-282987-treated α7+/+ and

α7-/- M1 and M2 BMDMs ...... 46

3-8 Phosphorylation of STAT3 in nicotine or PNU-282987-treated

α7+/+ BMDMs pretreated with AG-490 ...... 47

3-9 Analysis of thapsigargin-induced apoptosis in α7+/+ and α7-/-

M2 BMDMs treated with nicotine or PNU-282987 ...... 50

3-10 Analysis of thapsigargin-induced apoptosis in α7+/+ M2

BMDMs with α7 agonists and STAT3 inhibitor ...... 51

3-11 Expression of Bcl-2 in α7+/+ and α7-/- M2 BMDMs treated

with nicotine or PNU-282987 during chronic ER stress ...... 54

3-12 H&E and ORO sections, lesion area 8 weeks HFD ...... 59

3-13 Moma-2 IHC sections, macrophage abundance 8 weeks HFD ...... 60

3-14 H&E sections, lesion area 14 weeks HFD ...... 65

3-15 Moma-2 IHC sections, macrophage content 14 weeks HFD ...... 66 x

3-16 M1 macrophage co-localization 14 weeks HFD ...... 67

3-17 M2 macrophage co-localization and quantification 14 weeks HFD ...... 68

3-18 Gomori’s Trichrome sections, necrotic core area 14 weeks HFD ...... 69

3-19 In situ TUNEL sections, apoptotic cells and apoptotic

macrophages 14 weeks HFD ...... 70

3-20 Ki67 IF, proliferating cells and proliferating macrophages

14 weeks HFD ...... 71

xi

List of Abbreviations

α7nAChR ...... Alpha 7 Nicotinic Acetylcholine Receptor αBT ...... Alpha Bugarotoxin µm ...... Micrometer µM ...... Micromolar

ACAT ...... Acyl-CoA:Cholesterol Acyltransferase ApoE ...... Apolipoprotein E ArgI ...... Arginase I

Bcl-2 ...... B Cell Lymphoma 2 BMDM ...... Bone Marrow Derived Macrophage BMT ...... Bone Marrow Transplantation

CAD ...... Coronary Artery Disease cDNA ...... Complementary DNA CHOP ...... CCAAT-enhancer-binding Homologous Protein

ER ...... Endoplasmic Reticulum ERK1/2 ...... Extracellular Signal-Regulated Kinase ½

GAPDH...... Glyceraldehyde 3-Phosphate Dehydrogenase

H&E ...... Hematoxylin and Eosin HFD ...... High Fat Diet

IFNγ ...... Interferon-gamma IgG ...... Immunoglobulin G IL-4 ...... Interleukin 4 iNOS ...... Inducible Nitric Oxide Synthase

JAK2 ...... Janus Kinase 2

LDL...... Low Density Lipoprotein xii

LDLR ...... Low Density Lipoprotein Receptor

MAPK ...... Mitogen-Activated Protein Kinase MR ...... Mannose Receptor

OCT ...... Optimum Cutting Temperature ORO ...... Oil Red O oxLDL ...... Oxidized LDL qRT-PCR ...... Quantitative Real Time Polymerase Chain Reaction

SERCA ...... Sarcoendoplasmic Reticulum Calcium ATPase STAT3 ...... Signal Transducer and Activator of Transcription 3

TNFα ...... Tumor Necrosis Factor Alpha TUNEL ...... Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling

xiii

List of Symbols

α ...... Alpha β ...... Beta γ…………………….Gamma δ…………………….Delta ε……………………..Epsilon

º………...... Degrees

xiv

Chapter 1

Literature Review

1.1 Atherosclerosis: disease overview

Atherosclerosis is a devastating disease which is common in western industrialized society, and is the underlying condition responsible for coronary artery disease (CAD), the leading cause of death in the United States. According to the American Heart Association CAD is the cause of 1 in 6 deaths, and the economic burden of cardiovascular disease and stroke exceeded 300 billion U.S. dollars in 2010 (Go, Mozaffarian et al. 2013), making this disease more costly than all cancers combined. This progressive inflammatory disease affects large and medium sized arteries including the coronary and carotid arteries and multiple locations throughout the aorta, and the associated clinical events depend on which artery is affected. or occlusion of the coronary arteries can lead to angina pectoris or myocardial infarction, occlusion of the carotid artery leads to ischemic stroke, and disease of the abdominal aorta can cause aortic aneurism. Evidence of atherosclerotic lesions has been seen in humans

1

dating back thousands of years (Thompson, Allam et al. 2013), but we have only begun to understand the intricacies of lesion pathogenesis over the last few decades. Atherosclerosis was first thought to be primarily a disorder of lipid imbalance and cholesterol accumulation, but we now know that lesions form as a result of a complex interplay between metabolic and inflammatory processes.

This is even further complicated by a variety of genetic and environmental risk factors including hypertension, smoking and metabolic syndrome among many others. Advances in genetic and molecular biology research have allowed researchers to identify many of the cellular and molecular events crucial to lesion formation and progression; however, major gaps in knowledge continue to exist.

The current standard of treatment for preventing initial and secondary coronary events is lipid lowering therapy. The most common class of cholesterol lowering medications is statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and interfere with cholesterol biosynthesis.

This new era of cardiovascular disease management saw a massive reduction in cardiovascular events (LaRosa, He et al. 1999, Heart Protection Study

Collaborative 2002). However, acute coronary syndromes continue to represent the major cause of death in industrialized society (Libby 2005 J Am Coll Cardiol) therefore potential combinatory therapies for disease prevention or reduction are still in critical demand. In humans, acute coronary events most commonly occur as a consequence of luminal arterial occlusion due to thrombus formation that

2

often follows plaque rupture. Although reduction of the arterial lumen can also occur in ischemic events – i.e., critical stenosis, as seen in patients with stable angina pectoris- outward remodeling of the affected artery can compensate to maintain lumen reduction below critical stenosis and therefore preventing – or minimizing- ischemia (Glagov, Weisenberg et al. 1987). Along those lines, it has also been shown that plaque composition and not size is the main indicator in predicting risk of rupture. The so-called “vulnerable plaque” has been defined in humans as having a large necrotic core covered by a thin fibrous cap (<65 μM)

(Stary, Chandler et al. 1995). A number of factors including inflammation, smooth muscle cell death and matrix metalloproteinase production are detrimental to lesion stability and cap thickness and promote development of rupture-prone lesions (Hansson 2005, Virmani, Burke et al. 2006). Understanding the underlying mechanisms causing lesion progression to the dangerous

“vulnerable plaque” stage is the major focus of current research in the pathogenesis of atherosclerosis.

1.2 Early atherosclerosis: role of the monocyte/macrophage

The initiating events in the formation of atherosclerotic lesions are not completely understood and remain somewhat controversial, but the “response- to-retention” theory has been generally accepted as explaining what we observe over the course of the disease (Williams and Tabas 1995). This theory proposes 3

that the initial stages of lesion formation occur as a consequence of the retention of low density lipoprotein (LDL) within the tunica intima, the innermost layer of the arterial wall. One of the crucial early responses to lipoprotein retention is the activation of overlying endothelial cells. Although lipoproteins circulate throughout the entire vascular system, LDL retention and endothelial activation is site specific, generally occurring to a greater extent at arterial branch points or other sites of disrupted flow (Topper, Cai et al. 1996). There is a “chicken and egg” conundrum over which occurs first, LDL retention or endothelial activation.

However, there is evidence that endothelium exposed to non-laminar flow produce modified extracellular matrix which is more conducive to LDL binding

(Hoff and Wagner 1986). Once retained within the subintima, LDL can be modified by enzymatic and non-enzymatic processes and to different extents from minimally modified (mmLDL) to extensively oxidized (oxLDL) (Steinberg,

Parthasarathy et al. 1989, Navab, Berliner et al. 1996). The current dogma is that lesional macrophages arise from circulating which are recruited to sites of activated endothelium and enter the subintima. Although we know this to be true in the setting of the early lesion, in advanced lesions macrophage proliferation may replace continuous monocyte recruitment as the dominant mechanism regulating continued macrophage occupancy of the lesion (Robbins,

Hilgendorf et al. 2013). Activation of arterial endothelial cells induces production of involved in the recruitment and migration of monocytes including

4

the chemokines monocyte chemoattractant protein (MCP-1) and fractalkine, and the adhesion molecule members of the immunoglobulin (Ig) superfamily vascular cell adhesion molecule (VCAM-1) and intercellular cell adhesion molecule (ICAM-1) (Cybulsky and Gimbrone 1991, Yla-Herttuala, Lipton et al.

1991, Nakashima, Raines et al. 1998, Stolla, Pelisek et al. 2012). Additional sources of chemokines in atheroprone regions include deposition of the platelet- derived factor regulated on activation, normal T cell expressed and secreted

(RANTES) (von Hundelshausen, Weber et al. 2001). The importance of these early chemotactic proteins is shown by the remarkable abrogation of lesion formation with the combined inhibition of MCP-1 and receptors for fractalkine

(chemokine (C-X3-C motif) receptor 1 (CX3CR1)) and RANTES (C-C chemokine receptor type 5 (CCR5)) (Combadiere, Potteaux et al. 2008).

As recruited monocytes differentiate into tissue macrophages, they upregulate scavenger receptors which recognize and internalize oxLDL including scavenger receptor A (SR-A), cluster of differentiation 36 (CD36) and

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), among others (Kunjathoor, Febbraio et al. 2002, Mehta 2004). Lipoprotein internalization can also occur through other non-receptor mediated pathways such as macropinocytosis (Jones, Reagan et al. 2000) or phagocytosis (Tabas, Li et al.

1993). The net result of this massive lipoprotein (LP) uptake is intracellular accumulation of LP-derived cholesterol within lipid droplets (Maxfield and

5

Tabas 2005), giving macrophages a foamy appearance which led to the initial description of “foam cells” in fatty streaks (Gerrity 1981). In early atherosclerotic lesions, these foam cells dominate the cellular environment of the lesion. Besides macrophage foam cells, evidence continues to accumulate for involvement of several other cell types including T and B lymphocytes, and eosinophils, although these are in far fewer numbers than macrophages (Song,

Leung et al. 2001, van Leeuwen, Gijbels et al. 2008).

In macrophages, LDL-derived cholesterol is trafficked through the endosomal pathway, and after leaving the late endosome is shuttled to the endoplasmic reticulum (ER) membrane where it is re-esterified by the ER enzyme acyl-CoA:cholesterol acyltransferase (ACAT) and stored in lipid droplets

(Brown, Ho et al. 1980). Several cholesterol efflux pathways exist in macrophages, including transfer from ATP-binding cassette transporter (ABCA1) to lipid-poor apolipoprotein A (ApoA1) and from ATP-binding cassette sub- family G member 1 (ABCG1) to mature high density lipoprotein (HDL) particles among others (Oram, Lawn et al. 2000, Kennedy, Barrera et al. 2005).

Macrophages undergoing transformation into foam cells are still able to perform various functions despite massive intracellular lipid accumulation including production, promoting further monocyte recruitment and chronic inflammation. However, they are also able to efficiently recognize and uptake lesional apoptotic cells, an anti-inflammatory process (Chung, Kim et al. 2006).

6

The balance between these inflammatory and reparative signals within the lesion can determine the extent of disease progression.

In normal inflammatory processes, there is a general timeline of disease progression which includes initial immune cell recruitment, attack and clearance of the insulting agent, and a final resolution phase which includes emigration of immune cells and tissue repair. Atherosclerosis is a disease of chronic inflammation, where the initial recruitment phase becomes extended and the resolution phase is impaired (Tabas 2010). Macrophages within atherosclerotic lesions are generally unable, due to mechanisms yet unknown, to clear from the lesion site and therefore are chronically exposed to pro-inflammatory and pro- apoptotic factors and begin to undergo apoptosis (Kockx 1998). Macrophage apoptosis is recognized to play a crucial role in lesion progression and necrotic core formation, especially in advanced atherosclerosis (discussed later).

However, macrophage apoptosis in the early lesion stage is detected infrequently, due to efficient clearance of apoptotic cells by local phagocytes, a process known as efferocytosis (Poon, Lucas et al. 2014). In fact, several studies have shown that inducing macrophage apoptosis in early atherosclerosis can have a beneficial impact by reducing lesion cellularity due to rapid clearance of apoptotic cells, while inhibiting macrophage apoptosis can enhance atherogenesis (Arai, Shelton et al. 2005, Liu, Thewke et al. 2005, Wang, Liu et al.

2008). Disrupting efferocytosis via loss of the efferocytic mediator complement

7

C1q also promotes plaque growth at the early lesion stage (Bhatia, Yun et al.

2007). Overall, the early stages of atherosclerosis are relatively benign and reversal of hypercholesterolemia can halt or even induce regression in these lesions (Feig, Parathath et al. 2011). However, in the absence of dramatic intervention early on, most atherosclerotic lesions continue to progress to a more complex state of disease.

1.3 Advanced atherosclerosis and macrophage apoptosis

As atherosclerotic lesions progress, sustained cholesterol uptake begins to have detrimental effects. Macrophages are unable to regulate lipoprotein uptake through scavenger receptors, which unlike the low density lipoprotein receptor

(LDLR) are not negatively regulated by lipoprotein binding and uptake (Brown and Goldstein 1997). Therefore, even when completely engorged in cholesterol, macrophages continue to uptake modified lipoproteins but can no longer store this massive amount of lipoprotein-derived cholesterol in lipid droplets.

Lipoprotein-derived cholesterol is normally re-esterified in the ER membrane by the ACAT enzyme, but in advanced lesions the enzyme’s capacity is overwhelmed and there is an accumulation of unesterified free cholesterol within the ER membrane (Feng, Yao et al. 2003). Normally fluid, the ER membrane becomes stiff and impairs the function of ER membrane proteins, such as inhibition of channel function of the sarcoendoplasmic reticulum calcium 8

ATPase (SERCA) (Li, Ge et al. 2004). Disruption of ion flux and depletion of Ca2+ stores, for example, lead to ER stress. Besides free cholesterol accumulation, other ER stressors in the lesion setting include toxic lipids such as 7- ketocholesterol, oxidative stress and homocysteine (Scull and Tabas 2011).

Importantly, chronic and unresolved ER stress is known to trigger the unfolded protein response (UPR). UPR activation occurs in both physiological and pathological settings, and acts to restore the normal function of the endoplasmic reticulum, an organelle important for proper protein folding, protein modifications and Ca2+ storage (Xu, Bailly-Maitre et al. 2005, Wu and

Kaufman 2006). ER stress triggers signaling through three conserved branches of the UPR, inositol requiring 1 (IRE1), activating 6 (ATF6), and

RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK)

(Szegezdi, Logue et al. 2006). Activation of these signaling pathways promotes synthesis of chaperone proteins, degradation of unfolded proteins, and reduction in protein translation (Kozutsumi, Segal et al. 1988, Travers, Patil et al. 2000,

Harding, Calfon et al. 2002). However, chronic activation of the UPR can eventually trigger apoptosis, and this is known to occur in macrophages treated with atherorelevant ER stressors (Feng, Yao et al. 2003, Seimon, Wang et al.

2009). Persistent activation of UPR signaling pathways can upregulate expression of the transcription factor CEBP-homologous protein (CHOP) and its accumulation in the nucleus (Ron and Habener 1992). Evidence exists for ER

9

stress and UPR activation in lesional macrophages in both mice and humans. In apolipoprotein E knockout (ApoE-/-) mice, UPR activation increases as a function of lesion stage, and both UPR proteins and CHOP co-localize in areas of TUNEL

(terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive apoptotic macrophages (Zhou, Lhotak et al. 2005). In human coronary artery lesion sections, CHOP staining was higher in thin cap atheromas and ruptured plaques compared to stable fibrous plaques (Myoishi, Hao et al. 2007). Co- staining of CHOP and TUNEL in ruptured plaque specimens, along with detection of 7-ketocholesterol near the lipid core, further supports the link between chronic UPR activation, CHOP induction and apoptosis (Myoishi, Hao et al. 2007). It is clear from experimental evidence that lesional macrophages undergo chronic ER stress and UPR activation, and this is a critical factor in triggering macrophage apoptosis in advanced atherosclerotic lesions.

While in early atherosclerotic lesions macrophage apoptosis can be beneficial by reducing cellularity, in advanced lesions apoptosis has a clear detrimental effect on lesion progression. Proof of concept for this theory comes from a number of studies in genetically modified mice. In a study performed by

Gautier et al., the effect of inhibiting macrophage apoptosis via B-cell lymphoma

2 (Bcl-2) overexpression was observed at both the early and late lesion stage

(Gautier, Huby et al. 2009). While early lesions in transgenic mice showed increased size, advanced lesions were smaller with decreased macrophage

10

abundance. At the same time, induction of macrophage apoptosis led to enhanced lesion area at the advanced stage (Gautier, Huby et al. 2009). Mice harboring a myeloid-specific deficiency of p38α MAPK, an important compensatory survival signaling molecule in macrophages undergoing ER stress, develop lesions with increased apoptosis and necrosis at the advanced stage

(Seimon, Wang et al. 2009). Mouse models with macrophage deficiency of the pro-survival Bcl-2 or Bcl-xL show a correlative increase in lesional macrophage apoptosis and necrosis in advanced atherosclerotic lesions (Thorp,

Li et al. 2009, Shearn, Deswaerte et al. 2012). Contrarily, bone marrow deficiency of signal transducer and activator of transcription 1 (STAT1), a critical pro- apoptotic mediator in macrophages, leads to decreased lesional apoptotic cells and necrotic core area (Lim, Timmins et al. 2008). A recent study from our lab examined the effects of bone marrow deficiency of transient receptor potential canonical 3 (TRPC3) channel on progression of atherosclerotic lesions in ApoE-/- mice. At the advanced lesion stage, mice with TRPC3-deficient bone marrow showed reduced macrophage apoptosis and less necrosis in the absence of a change in lesion size (Tano, Solanki et al. 2014). Reduction in necrosis independent of changes in lesion area or macrophage content has also been seen in studies from other laboratories (Lim, Timmins et al. 2008, Seimon, Wang et al.

2009) Genetic-causation studies have also confirmed the involvement of ER stress-induced apoptosis in advanced atherosclerosis. Mice with heterozygosity

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of the cholesterol trafficking protein Niemann-Pick C (Npc1) are protected from free cholesterol accumulation in the ER membrane, and in the setting of atherosclerosis these mice show reduced plaque necrosis (Feng, Zhang et al.

2003). Separate labs reported a decrease in apoptotic lesional macrophages in

ApoE-/- mice with deficiency of CHOP (Thorp, Li et al. 2009, Tsukano, Gotoh et al. 2010). In one report, both global and bone marrow deficiency of CHOP reduce susceptibility for plaque rupture in a cuff ligation model (Tsukano, Gotoh et al.

2010). Finally, as a more general proof of principle, pharmacological inhibition of macrophage ER stress by administration of a chemical chaperone protects against lesion progression (Erbay, Babaev et al. 2009).

It is important to note that increased apoptosis alone does not drive lesion necrosis. The process of apoptotic cell clearance by local phagocytes, called efferocytosis, is an efficient process which functions in all healthy tissues to rapidly uptake free apoptotic cells (Poon, Lucas et al. 2014). This is an anti- inflammatory process, as uptake of apoptotic cells by macrophages induces secretion of interleukin-10 (IL-10) and transforming growth factor β (TGF-β)

(Fadok, Bratton et al. 1998, Chung, Kim et al. 2006). In early atherosclerotic lesions, efficient efferocytosis minimizes apoptotic cell accumulation within the lesion. However, the ability of local phagocytes to take up apoptotic cells becomes impaired in advanced atherosclerosis. Why efferocytosis becomes impaired in advanced lesions is not completely understood, however some

12

potential mechanisms have been described including competition by oxidized lipids for receptors recognizing apoptotic cells (Aprahamian, Rifkin et al. 2004), shedding of apoptotic cell receptors (Thorp, Vaisar et al. 2011), or inhibition of efferocytosis by pro-inflammatory molecules (Friggeri, Yang et al. 2010).

However this impairment occurs, the net result is an increase in free apoptotic cells within the tissue, leading to secondary necrosis. The accumulation of free apoptotic cells due to impaired efferocytosis was observed in human atherosclerotic plaques whereas in the tonsil, where efferocytic mechanisms are intact, free apoptotic cells are infrequent (Schrijvers, De Meyer et al. 2005). As discussed earlier, plaques with large necrotic lipid cores become high risk for rupture and thromboembolic events manifesting in the clinical scenario associated with atherosclerosis such as myocardial infarction and stroke.

Molecular targets which could inhibit macrophage apoptosis and necrotic core formation in advanced atherosclerosis have thus become a significant focus of research in the field.

1.4 Macrophage differentiation and polarization

Macrophages are involved in a variety of crucial processes in the body, limited not only to their role in the but also function in development, tissue homeostasis and remodeling (Mosser and Edwards 2008).

Macrophages are able to respond to environmental cues and adapt their 13

repertoire of expressed genes depending on what they detect in the local environment, giving rise to a wide spectrum of phenotypes. Among these, some of the best described are two types with characteristics that place them in opposite extremes of such spectrum, and thus often called polarized macrophages. These are the classically activated “M1” macrophages and the alternatively activated “M2” macrophages, originally named so for their alignment with Th1 and Th2-mediated responses (Mills, Kincaid et al. 2000).

Although evidence for these precise phenotypes in vivo is not definitive, activating factors and subsequent expression profiles have been described in in vitro systems. Polarization to the M1 phenotype occurs by macrophage exposure to interferon-gamma (IFNγ) alone or IFNγ in the presence of

(LPS). These macrophages gain enhanced anti-microbial activity, increased (ROS) production via upregulation of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokine production including tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) (Adams 1989,

Dalton, Pitts-Meek et al. 1993, Martinez, Sica et al. 2008). Programming of macrophages to the alternatively activated M2 phenotype most notably occurs through interleukin-4 (IL-4) or IL-13 signaling, although subsets of the M2 type have been described which differentiate in response to glucocorticoids or immune complexes (Mantovani, Sica et al. 2004). IL-4 induced M2 macrophages upregulate mannose receptor (MR) expression and show suppressed

14

inflammatory cytokine and ROS production (Stein, Keshav et al. 1992, Gordon

2003). Functionally, M1 macrophages are microbicidal, have increased antigen presentation capabilities, and greater respiratory burst, while M2 macrophages are canonically involved in parasite killing and tissue remodeling (Sica and

Mantovani 2012).

Although these distinct, divergent macrophage phenotypes have not been confirmed in vivo, there is accumulating evidence for macrophage polarization in numerous pathological settings. In healthy adipose tissue, macrophages acquire an M2 phenotype and regulate insulin sensitivity in adipocytes (Lumeng, Bodzin et al. 2007, Odegaard and Chawla 2013). Excess caloric uptake and development of obesity induces a phenotype switch to M1 macrophages which produce inflammatory and promote insulin resistance in adipose tissue

(Weisberg, McCann et al. 2003, Lumeng, Bodzin et al. 2007). In myocardial infarction, the initial injury phase and the secondary phase of inflammatory resolution and fibrosis is regulated by infiltration of distinct monocyte subsets

(Nahrendorf, Swirski et al. 2007).

Cytokines driving macrophage polarization, including both IFNγ and IL-

4, are expressed in atherosclerotic lesions (Gupta, Pablo et al. 1997, Zhou,

Paulsson et al. 1998) and polarized macrophage subtypes have been identified in developing and advanced lesions, although the function of different macrophage types in atherosclerotic lesions is not completely understood. Based on

15

expression of arginase isoforms, where M1 macrophages express arginase II

(ArgII) and M2 macrophages express ArgI, M2 macrophages are observed in greater number in early atherosclerotic lesions, while the ratio of M1 to M2 macrophages increases as lesions progress to advanced stage (Khallou-Laschet,

Varthaman et al. 2010). In a mouse model of lesion regression where hypercholesterolemia is induced and then later reversed to extremely low levels

(<80 mg/dl), regression is associated with reduced macrophage and lipid content, increased collagen deposition and upregulated markers of M2 macrophage polarization (Feig, Parathath et al. 2011). In hyperlipoproteinemic

ApoE2.Ki mice, administration of the oxidative-stress limiting protein thioredoxin-1 skews macrophage polarization toward the M2 phenotype and reduces aortic lesion area (El Hadri, Mahmood et al. 2012), while administration of its truncated form thioredoxin-80 enhances M1 polarization in vivo and increases lesion area (Mahmood, Abderrazak et al. 2013). An association between enhanced M1 polarization and increased lesion area is also seen in mice with bone marrow deficiency of subfamily 4A1 (NR4A1/Nur77)

(Hanna, Shaked et al. 2012), although these findings were not consistently observed in studies from other laboratories (Hamers, Vos et al. 2012, Chao, Soto et al. 2013).

In human atherosclerotic lesions, markers of M1 macrophages are localized to a greater extent in unstable carotid plaques and in areas of lipid

16

accumulation, while M2 markers were increased in more stable femoral artery plaques and in areas of calcification (Shaikh, Brittenden et al. 2012).

Additionally, M1 macrophages are the dominant macrophage type in rupture- prone shoulder areas of advanced human plaques (Stoger, Gijbels et al. 2012), although microarray analysis demonstrated upregulation of both M1 and M2 genes during lesion progression from stable to rupture-prone plaques (Stoger,

Gijbels et al. 2012).

1.5 α7 nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChR) are a member of the cys-loop receptor family, which also includes gamma-aminobutyric acid (GABA) and serotonin receptors, and are classified as ligand-gated ion channel receptors

(Albuquerque, Pereira et al. 2009). In humans and mice, there are nine alpha subunits (α1-7, 9, 10), four beta subunits (β1-4), and a gamma (γ), delta (δ) and epsilon (ε) subunit, and these subunits form a multitude of hetero- and homopentamers depending on the cell or tissue type and expression system. Of these receptors, one of the most extensively studied is the α7nAChR (α7nAChR) homopentamer, comprised of five α7 subunits. The α7nAChR gene, located on 15q14 in humans and 7c7 in mice, includes 10 exons coding for a

502 amino acid, ~56 kDa protein (Chini, Raimond et al. 1994, Orr-Urtreger,

Seldin et al. 1995). The subunit structure includes a ligand-binding domain 17

located in the extracellular N-terminus region, four transmembrane (TM) domains, and an intracellular domain located between TM3 and TM4

(Albuquerque, Pereira et al. 2009). When complexed together into a functional homopentamer, the pore region of the receptor becomes a cation-permeable channel which is lined mostly by the TM2 domains of each subunit (Miyazawa,

Fujiyoshi et al. 2003). The extra- and intracellular domains also include regulatory sites for palmitoylation (Alexander, Govind et al. 2010), putative glycosylation sites (Schoepfer, Conroy et al. 1990, Seguela, Wadiche et al. 1993,

Chen, Dang et al. 1998), and sites for interaction with intracellular signaling proteins including Src-family kinases, which can regulate receptor phosphorylation in (Charpantier, Wiesner et al. 2005, Jones,

Buckingham et al. 2010). Proper receptor processing and trafficking seems to require the molecular chaperone resistance to inhibitors of cholinesterase 3 (RIC-

3) (Williams, Burton et al. 2005, Valles, Roccamo et al. 2009). The α7nAChR is a unique nicotinic receptor characterized by sensitivity to binding by the snake toxin α-bungarotoxin (αBT) (Chen and Patrick 1997, Drisdel and Green 2000) and a high permeability to Ca2+ compared to other nicotinic receptors (Fucile

2004).

The α7nAChR was first discovered in mammalian brain, and has typically been recognized and studied as a neuronal receptor (Leiser, Bowlby et al. 2009).

More recently, expression of α7nAChR has been identified in many non-

18

neuronal cell types where it is involved in a variety of cellular functions. These non-neuronal α7-expressing cells include endothelial cells (Li, Liu et al. 2010,

Smedlund, Tano et al. 2011), keratinocytes (Arredondo, Nguyen et al. 2002), microglia (De Simone, Ajmone-Cat et al. 2005), lymphocytes (Razani-Boroujerdi,

Boyd et al. 2007) and monocytes/macrophages (Wang, Yu et al. 2003), among others. In non-macrophage cell types including neurons of various origin and endothelial cells, α7nAChR activation can protect against apoptosis and cell death (Messi, Renganathan et al. 1997, Pugh and Margiotta 2000, Akaike,

Takada-Takatori et al. 2010, Smedlund, Tano et al. 2011). Expression of α7nAChR has been seen in different macrophage populations from both humans -primary in vitro differentiated macrophages- (Wang, Yu et al. 2003) and mice -peritoneal, alveolar- (de Jonge, van der Zanden et al. 2005, Kawashima, Yoshikawa et al.

2007, Su, Lee et al. 2007) at both the transcript and protein level. An important role for α7nAChR beyond the central nervous system was first described in the setting of acute . An endogenous anti-inflammatory circuit was described where afferent vagal nerve stimulation by endotoxin or cytokines triggers efferent vagal activation and release of acetylcholine, which in turn dampens the inflammatory response during sepsis (Borovikova, Ivanova et al. 2000). Termed the “cholinergic anti-inflammatory reflex” or CAIR, subsequent studies defined macrophage α7nAChR activation in the spleen as the central mediator of the anti-inflammatory effects (Wang, Yu et al. 2003, Huston, Ochani et al. 2006,

19

Olofsson, Katz et al. 2012). In vitro, α7nAChR stimulation reduces lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and toll-like receptor 4 (TLR4) expression in human monocytes and macrophages

(Wang, Yu et al. 2003, Hamano, Takahashi et al. 2006, Rosas-Ballina, Goldstein et al. 2009). In the context of atherosclerosis, peritoneal macrophages loaded with oxLDL show greater oxLDL internalization and higher intracellular cholesterol mass in α7nAChR-deficient macrophages, along with diminished anti-oxidant capacity (Wilund, Rosenblat et al. 2009). In a model of atherosclerosis where lesion formation was induced in ApoE-/- mice by a combination of high fat diet feeding and angiotensin II infusion, in vivo administration of an α7nAChR agonist over the course of the diet significantly decreased plaque burden and reduced aortic inflammation (Hashimoto, Ichiki et al. 2014). However, the specific cellular targets of the α7nAChR agonist and whether these included lesional α7nAChR-expressing macrophages was not established. Evidence for the involvement of α7nAChR in regulating macrophage functions potentially related to atherogenesis warrants further investigation into the role of this receptor in disease progression.

1.6 Summary and hypothesis

Macrophages are involved in all stages of atherosclerotic lesion development, and lesional macrophage apoptosis contributes to formation of 20

necrotic cores in advanced atherosclerotic plaques. The α7nAChR is expressed in several macrophage types, and in other non-macrophage cell types activation of

α7nAChR promotes cell survival. Based on this knowledge, we sought to investigate the potential role of α7nAChR in survival of macrophages by examining survival signaling pathways and apoptosis in M1- and M2-polarized bone marrow derived macrophages in vitro. We further examined the potential role of macrophage α7nAChR in atherogenesis using low density receptor knockout (LDLR-/-) mice, a mouse model of atherosclerosis. Mice were made chimeric by bone marrow transplantation of wild-type or α7nAChR-deficient bone marrow and fed a high fat diet for 8 or 14 weeks to examine both early and advanced atherosclerotic lesions. Based on evidence that α7nAChR is expressed in macrophages and can promote cell survival in other cell types, we hypothesize that activation of α7nAChR will protect polarized macrophages from chronic ER stress- induced apoptosis, and by virtue of this anti-apoptotic effect, LDLR-/- mice with bone marrow deficiency of α7nAChR will have increased lesional macrophage apoptosis and larger necrotic cores.

21

Chapter 2

Materials and Methods

2.1 Experimental animals

All studies involving animals described in this work conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National

Institutes of Health, and have been approved by University of Toledo IACUC.

tm1Bay/J C57BL/6 mice, the α7nAChRKO (B6.129S7-Chrna7 ) mice and LDLRKO

(B6.129S7-Ldlrtm1Her/J) mice were obtained from Jackson Labs (Jackson Labs, ME) and colonies were maintained in our animal facility. Euthanasia was performed by intraperitoneal injection of sodium pentobarbital (150 mg/kg) added to an anticoagulant (heparin, 10 U/mL).

2.2 Preparation of bone marrow‐derived macrophages

Bone marrow‐derived macrophages were obtained essentially as described by Wooten and colleagues (Lazarus, Meadows et al. 2006, Lazarus,

22

Kay et al. 2008). Briefly, femurs and tibias were flushed with sterile RPMI (1% penicillin/streptomycin) and cells were plated with L929‐conditioned medium for 7 days (37°C, 5% CO2 atmosphere); after that, cells were replated in 6‐well

(immunoblotting) and 96‐well (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)) plates for experiments. At this point, and before proceeding with the macrophage polarization protocol (see below), the macrophage phenotype of the cells was confirmed (>99%) by their cobblestone appearance, positive immunostaining with F4/80 antibody and acetylated‐LDL uptake, as we previously described in (Tano and Vazquez 2011). L929 cells

(ATCC, CCl‐1, mouse fibroblastic cell line) were grown in RPMI + 10% fetal bovine serum + 1% penicillin/streptomycin during 7 days; the supernatant

(cell‐free) was collected, filtered (0.22 μm pore), and added to the macrophage differentiation media at a final concentration of 30%.

2.3 Macrophage polarization into M1 and M2 phenotypes

Bone marrow‐derived macrophages were recovered with ice‐cold PBS, collected by centrifugation (380×g), and plated in complete medium (RPMI +

10% FBS + 1% penicillin/streptomycin) containing either 10 ng/mL interferon‐γ

(IFNγ; M1 polarization) or 5 ng/mL interleukin-4 (IL‐4; M2 polarization, predominantly M2a) for 24 h. After 24 h, macrophages were treated for experiments as noted. Polarization to the M1 or M2 phenotype was confirmed by 23

qRT‐PCR with primers for markers of M1 (iNOS, inducible nitric oxide synthase;

TNFα, tumor necrosis factor α) and M2 macrophages (ArgI, mannose receptor

(MR)). IFNγ and IL‐4 were from Millipore (Billerica, MA).

2.4 In vitro TUNEL assay

Apoptosis was assayed by using the in situ cell death detection kit, TMR red (Roche, Indianapolis, IN). Following treatment, media was removed from wells and cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Cells were lysed for 5 mins on ice in PBS containing 0.1% TritonX-

100, washed in cold PBS, and then incubated in TUNEL enzyme solution for 1 hr at 37º C. Negative control was performed by omitting enzyme from the labeling solution. Cells from 4-5 fields per well were counted and expressed as a percent of DAPI-positive nuclei.

2.5 Cell lysis and immunoblotting

Cell lysis and immunoblotting was performed essentially as we described in (Tano and Vazquez 2011, Tano, Smedlund et al. 2011). Briefly, cells were lysed in lysis buffer containing: 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-

40, 0.2% sodium deoxycholate and 1 mM NaF. Solubilized proteins were then separated in 10% acrylamide gels, electrotransferred to PVDF membranes and

24

immunoblotted with the indicated primary antibody. After incubation with appropriate HRP‐conjugated secondary antibodies, immunoreactive bands were visualized by ECL (Amersham, Pittsburgh, PA). Primary antibodies used were: phospho‐AKT (Ser473, clone 587F11), total AKT, phospho‐p38 MAPK

(Thr180/Tyr182, clone D3F9), total p38MAPK, phospho‐STAT3 (Tyr705, clone

3E2), total STAT3, phospho‐ERK1/2 (Thr202/Tyr204 of ERK1, Thr185/Tyr187 of

ERK2), and total ERK1/2, and were all obtained from Cell Signaling (MA).

2.6 Real‐time PCR (RT-PCR)

Total RNA was prepared from BMDMs using PerfectPure RNA Tissue kit

(5Prime, Gaithersburg, MD) according to manufacturer's instructions. cDNA was synthesized with random primers and reverse transcriptase (Applied Biosystems high‐capacity cDNA RT kit; Applied Biosystems, Grand Island, NY) using 1 μg of total RNA. cDNA was evaluated with semi-quantitative real‐time PCR

(qRT‐PCR) using TrueAmp SYBR green qPCR supermix (Applied Biosystems).

The relative amount of mRNA (RQ) was calculated by comparison to the corresponding controls and normalized relative to GAPDH. Results are expressed as mean ± SEM relative to controls. Sequences of primers used are as follows: Arginase I (F: CAGAAGAATGGAAGAGTCAG; R: CAGATATGCAGG

GAGTCACC), iNOS (F: TGCATGGACCAGTATAAGGCAAGC; R: GCTTCTGG

25

TCGATGTCATGAGCAA), TNFα (F: CAGGCGGTGCCTATGTCTC; R: CGATCA

CCCCGAAGTTCAGTAG), MR (F: CTCTGTTCAGCTATTGGACGC; R: CGGAA

TTTCTGGGATTCAGCTTC), α7nAChR (F: AATTGGTGTGCATGGTTTCT; R:

AGCCAATGTAGAGCAGGTTG), Bcl‐2 (F: ATGCCTTTGTGGAACTATATGGC;

R: GGTATGCACCCAGAGTGATGC), GAPDH (F‐ AGGTCGGTGTGAACGGAT

TTG; R‐ TGTAGACCATGTAGTTGAGGTCA. Primerbank (Spandidos, Wang et al. 2010, Wang, Spandidos et al. 2012) identification numbers: TNFα:

133892368c1; MR: 224967061c1; Bcl‐2: 6753168a1; GAPDH: 6679937a1. Primers for

Arginase I and iNOS were as in (Khallou-Laschet, Varthaman et al. 2010).

Primers for α7nAChR were ordered from Realtimeprimers.com (#VMPS‐1143).

The specificity of all primers used in qRT‐PCR was evaluated from melting curve analysis.

2.7 Fura-2-based Real Time Fluorescence

Coverslip-plated cells loaded with the Ca2+-sensitive dye Fura-2 were used to monitor real-time fluorescence changes of intracellular Ca2+ on multiple cells with a charge-coupled device (CCD) camera-based imaging system

(Intracellular Imaging Inc) as previously described. Measurements were performed at room temperature and treatment conditions were in HEPES- buffered saline solution (HBSS) containing (in mmol/L): 140 NaCl, 4.7 KCl, 1

MgCl2, 10 glucose, 10 HEPES pH 7.4, 2 CaCl2. 26

2.8 Bone marrow transplantation (BMT)

BMT was performed essentially as we described in detail in ref. (Tano,

Solanki et al. 2014). Briefly, recipient mice (LDLRKO females, 6-week-old,

C57BL/6 background) were irradiated (10 Gy, 3 min; 137Cs-Gammacell 40

Exactor, Nordion Int. Inc.) and 4 hours later injected via tail vein with bone marrow cells (~5×106 cells) from C57BL/6 (B6) or α7nAChRKO (α7KO, on

C57BL/6 background) mice. Preparation of bone marrow cells from donors is described in detail in ref. (Tano, Solanki et al. 2014). To determine chimerism,

PCR was performed on cDNA from peripheral blood cells for α7nAChR (for

α7KOLDLRKO mice) and on gDNA from peripheral blood cells for wild-type

LDLR (for B6LDLR mice). Primers used for α7nAChR were: F (5'-->3'): AAT

TGG TGT GCA TGG TTT CT; R (5'-->3'): AGC CAA TGT AGA GCA GGT TG

(Realtimeprimers.com, accession NM_007390). PCR conditions were as we described in (Lee and Vazquez 2013). For LDLR, primer sequences and PCR cycling conditions were as recommended by Jackson Labs (Jackson Labs, ME).

2.9 Determination of plasma cholesterol and triglycerides

After a 12 h fasting period blood was collected by submandibular vein puncture. Total plasma cholesterol and triglyceride concentrations were

27

determined using Cholesterol-E and L-Type Triglyceride-M (Wako Chemicals

USA, Inc.) following manufacturer’s instructions.

2.10 Aortic root sectioning

Aortic root sections were prepared as we described in refs (Smedlund,

Tano et al. 2010, Tano, Solanki et al. 2014). Briefly, euthanized mice were perfused through the left ventricle with 4% paraformaldehyde followed by PBS.

The heart was cut so that all three aortic valves were in the same geometric plane. The upper portion of the heart was embedded in optimal cutting tempterature medium (OCT), frozen in the Peltier stage of the cryostat (Thermo

Scientific R. Allan HM550 Cryostat) and processed for sectioning. Sections (10

µm) were collected onto Fisher Superfrost Plus-coated slides, starting from where aorta exits the ventricle and moving towards the aortic sinus over ~650-700 μm.

Additional sections were collected at the end to be used as controls in immunostaining procedures. Lesion analysis and Oil Red O (ORO), hematoxylin and eosin (H&E) or trichrome stainings were as we described in ref. (Tano,

Solanki et al. 2014).

2.11 Immunohistochemistry

28

Immunohistochemistry (IHC) was essentially as we described in refs.

(Smedlund, Tano et al. 2010, Tano, Solanki et al. 2014). Briefly, sections were fixed in acetone and processed for immunostaining for MOMA-2 (#sc-59332,

Santa Cruz Biotechnology, TX) followed by incubation with biotinylated rabbit anti-rat antibody (Dako). After treatment with secondary antibodies sections were incubated with alkaline phosphatase-conjugated streptavidin (Dako).

Counterstaining was with hematoxylin. Negative controls were performed by substituting the primary antibody with non-immune IgG from the same species and at the same concentration. Under these conditions, nonspecific immunostaining was not detected. Stained areas were captured (Micropublisher

3.3 Megapixel Cooled CCD Color Digital Camera) and measured (NIS Elements

D). Either MOMA-2 or the rabbit polyclonal antibody to AIA31240 (Accurate

Chemical and Science Corp.) was used to identify macrophages in co-localization immunofluorescence staining. Secondary antibody was Alexa Fluor-488 goat anti-rabbit (#A11008, Invitrogen, for AIA31240) or Alexa Fluor-488 anti-rat

(#4416, Cell Signaling, for MOMA-2). Immunostaining for the Ki67 antigen was performed by incubating acetone-fixed frozen sections with an anti-Ki67 antibody (#ab66155, Abcam; 1:100 dilution) overnight at 4oC followed by anti- rabbit IgG Alexa Fluor-555 (#4413, Cell Signaling) at 1:1,000 dilution for 1 h.

29

2.12 In situ immunostaining for M1 and M2 macrophages

M1 and M2 macrophages in lesions were identified by co-staining for iNOS (Abcam, #ab15323) or mannose receptor (#HM1049, Hycult Biotech), respectively, and macrophage (Accurate Chemical, #AIA31240), as follows: frozen sections were fixed in acetone (15 min, 4°C) followed by incubation with iNOS antibody (1:100 dilution) overnight at 4oC and then anti-rabbit IgG Alexa

Fluor-555 (#4413, Cell Signaling) at 1:1,000 dilution for 1 h, or with mannose receptor antibody (1:100 dilution) overnight at 4oC and then anti-rat IgG Alexa

Fluor-555 (#4417, Cell Signaling) at 1:1,000 dilution for 1 h. Next, AIA31240 antibody was used at a 1:100 dilution followed by staining with anti-rabbit IgG

Alexa Fluor-488 (#A11008, Invitrogen) at 1:1,000 dilution for 1 h. Slides were mounted using the Prolong Gold Antifade Reagent with DAPI (Cell Signaling,

#8961). All antibody dilutions were done using the IHC TEK antibody diluent

(IW-1,000) from IHC World.

2.13 In situ TUNEL

Staining of sections for in situ TUNEL was performed using an in situ cell death detection kit (Roche, IN) and incorporating adjustments from the stringent method by Kockx et al. (Kockx, Muhring et al. 1998) to minimize non-specific staining. In addition, negative controls for TUNEL staining were included in

30

which TdT (enzyme solution) was omitted from the labeling mixture. TUNEL positive cells that co-localized with the nuclei (DAPI staining) were counted by two independent operators blinded to study groups and total number was normalized by the corresponding lesion area as determined by hematoxylin and eosin staining.

2.14 Necrotic core evaluation

To evaluate lesion necrotic core (NC), we used the combination of criteria used by Seimon et al. and Thim et al. (Seimon, Wang et al. 2009, Thim, Hagensen et al. 2010). NC was defined as those areas within a lesion that were negative for hematoxylin-positive nuclei (i.e., acellular/anuclear) and Gomori’s trichrome staining (i.e., lack of extracellular matrix, collagen). Boundary lines were delineated around those regions and the area was measured by image analysis software as described above. Based on the criterion by Seimon et al., areas <3,000

μm2 were not counted as they likely do not constitute substantial areas of necrosis. Measurements were performed by two independent observers blinded to study group. Relative NC area was calculated by dividing total NC area by total lesion area. Evaluation of cap thickness was performed based on the protocol described in (Seimon, Wang et al. 2009) with modifications. Cap thickness was assessed from the largest necrotic cores from at least duplicate sections stained with Gomori’s trichrome and measuring the thinnest section of 31

the cap as determined by the distance between the outer edge of the cap and the

NC border. Buried caps in deep necrosis areas, when present, were not considered for measurements.

2.15 Statistical analysis

Values are shown as mean ± SEM. Comparison of mean values between groups was performed with a two‐tailed Student's t test for in vitro data analysis, or by

Mann-Whitney U-test for analysis of lesion parameters, using Prism Graph Pad version 6 for Windows 2007 (San Diego, CA). P values below 0.05 were considered significant.

32

Chapter 3

Results

3.1 α7nAChR is expressed in polarized bone marrow derived macrophages

In order to study polarized macrophages in vitro, we use bone marrow- derived macrophages which are differentiated with L929 fibroblast-derived macrophage colony stimulating factor (mCSF), and then further polarized to the

“M1” classically-activated phenotype with interferon-gamma (IFNγ, 10 U/ml) or the “M2” alternatively-activated phenotype with interleukin 4 (IL-4, 5 ng/ml).

This model of in vitro polarized macrophages is accepted as the standard model to study distinct, divergent macrophage types (Murray, Allen et al. 2014).

Activation to each phenotype was confirmed by measuring expression levels of markers typical of each phenotype by quantitative real time PCR (RT-PCR). M1 macrophages upregulate inducible nitric oxide synthase (iNOS or NOS-2) and tumor necrosis factor alpha (TNFα) (Khallou-Laschet, Varthaman et al. 2010,

Murray, Allen et al. 2014), while M2 macrophages upregulate mannose receptor

33

(MR) arginase I (ArgI) and Ym1/2 (Khallou-Laschet, Varthaman et al. 2010,

Murray, Allen et al. 2014). Expression of each marker was measured in M1 and

M2 macrophages from wild-type C57BL/6 (“BL6” or “α7+/+”) or α7nAChR- deficient (“α7KO” or “α7-/-”) mice. M1 and M2 macrophages from both mouse genotypes showed expected upregulation of phenotypic markers, and no significant difference in expression of M1 or M2 markers was observed between genotypes (Fig. 3-1). We were then interested in determining if polarization to either phenotype had an effect on expression levels of α7nAChR. Standard PCR was performed on RNA-derived cDNA from BL6 and α7KO M1 and M2

BMDMs, and the PCR products were run on a 1.5% agarose gel. Brain cDNA served as a control for each group. We observed an amplicon of the expected size

(157 bp) in BL6 M1 and M2 macrophages, with a more intense band observed in the brain control (Fig. 3-2). Neither BMDMs nor brain from α7KO mice expressed the expected amplicon for α7nAChR, confirming the knockout phenotype at the mRNA level. qRT-PCR was then used to compare expression level of α7nAChR between BL6 M1 and M2 BMDMs, and there was no significant difference in expression between macrophage types (Fig. 3-2). As expected, when we compared mRNA expression level in these macrophage types versus brain, macrophage α7nAChR expression was significantly reduced compared to expression in brain (on average, 5-10 fold greater expression in brain than

BMDMs; data not shown). We are able to conclude that α7nAChR is expressed at

34

similar levels in both M1 and M2 bone marrow-derived macrophages, and that

α7nAChR-deficiency does not impair polarization to either the M1 or M2 phenotype.

35

TNFα (M1)

** *

ns

Figure 3-1: Bone marrow derived macrophages from C57BL/6 (α7+/+) or α7nAChR-deficient (α7-/-) were polarized to the M1 or M2 phenotype with IFNγ or IL-4, respectively. RNA was then isolated, reverse transcribed to cDNA, and qRT-PCR performed for markers of M1 (iNOS, TNFα) or M2 (ArgI, MR) macrophages and expressed as relative quantification (RQ) compared to α7+/+ M1 (for iNOS and TNFα) or α7+/+ M2 (for ArgI and MR). of GAPDH was used as endogenous control. *P = 0.04 for the difference between α7+/+-M1 and α7+/+-M2; **P = 0.002 for the difference between α7-/--M1 and α7-/-- M2. ns: not statistically significant for the difference between α7-/--M2 and α7+/+- M2. Modified from (Lee and Vazquez 2013).

36

bp 300 Non specific 200 α7 (157 bp) 100

ns

Figure 3-2: cDNA from wild-type or α7nAChR-deficient BMDMs polarized to the M1 or M2 phenotype was analyzed for α7nAChR expression by normal PCR (top panel) or qRT-PCR (lower bar graph). Expected amplicon size for α7 is 157 bp. Bars in B are an average (mean ± SEM) of 5 independent experiments, each performed in triplicate. ns: not significant. Modified from (Lee and Vazquez 2013).

37

3.2 α7nAChR mediated Ca2+ influx is undetectable in BMDMs

In neurons and other cell types α7nAChR agonist binding induces cation influx (primarily Ca2+ and also Na+) which initiates cellular effects including activation of signaling pathways and changes in membrane potential (Nai,

McIntosh et al. 2003, Shen and Yakel 2009). We were therefore curious whether activation of α7nAChR in the macrophage functions as an ion channel and thus mediates Ca2+ influx. To explore this, we first examined changes in intracellular

Ca2+ using the fluorescence-based Fura-2 method, where the ratio of Ca2+-bound

Fura-2 (340 nm) and Ca2+-free Fura-2 (380 nm) gives an indication of increases in cytosolic Ca2+. BMDMs were plated on glass coverslips overnight, loaded with 2

μM Fura-2 AM for 20-30 minutes and then washed in Fura-2 AM-free buffer and allowed to rest briefly before agonist treatment. We first examined if the pan- nAChR agonist nicotine (10 μM) could induce Ca2+ influx; in the presence of either 2 mM or 10 mM Ca2+, no change in Fura-2 ratio was detected in either M1 or M2 BMDMs from B6 or α7KO mice. BMDMs were also treated with the

α7nAChR-specific agonist PNU-282987 (1 μM), and again no Ca2+ influx was detected in any macrophage type. To confirm efficiency of Fura-2 loading in

BMDMs, several positive controls were used including treatment with ATP, which induces Ca2+ influx through purinergic receptors (Naumov, Kaznacheyeva et al. 1995) and thapsigargin, which induces Ca2+ release from ER stores into the

38

cytoplasm (Tran, Watanabe et al. 2001). Treatment with both ATP and thapsigargin induced significant changes in Fura-2 ratio (not shown), indicating that under our experimental conditions changes in intracellular Ca2+ levels can be detected. Overall, we were unable to detect α7nAChR-mediated Ca2+ influx into either M1 or M2 BMDMs.

3.3 α7nAChR stimulation promotes activation of survival signaling pathways

There is substantial evidence in multiple cell types that activation of nicotinic acetylcholine receptors can promote cell survival and inhibit apoptosis

(Messi, Renganathan et al. 1997, Pugh and Margiotta 2000, Marrero and

Bencherif 2009, Smedlund, Tano et al. 2011). We performed a preliminary screen of typical survival signaling pathways in macrophages, including AKT, p38

Mitogen-Activated Protein Kinase (MAPK), Extracellular signal-Regulated

Kinase (ERK)1/2 MAPK, and Signal Transducer and Activator of Transcription 3

(STAT3), upon nicotinic acetylcholine receptor activation. Cells were treated for

0-60 minutes with the pan nicotinic acetylcholine receptor agonist nicotine (10

μM) or the α7nAChR-specific agonist PNU-282987 (1 μM), and lysates were collected and proteins subsequently separated by SDS-PAGE and analyzed by western blot to determine their phosphorylation status. Assessing phosphorylation status of phospho-sites known to contribute to regulation of

39

protein activity is an indirect way to determine the activation state of these signaling proteins. Nicotine induced phosphorylation of p38 MAPK

(Thr180/Tyr182), ERK1/2 MAPK (ERK1, Thr202/Tyr204; ERK2, Thr185/Tyr187)

(Fig. 3-3) in both M1 and M2 α7+/+ BMDMs, while AKT (Ser473) phosphorylation levels were unchanged in all macrophage groups following treatment with either nicotine or PNU-282987 (not shown). Phosphorylation of p38 and ERK1/2 were similar in BL6 M1 and M2 BMDMs, and no significant difference was seen in corresponding α7KO BMDMs (not shown). This suggests that nicotine-mediated phosphorylation of p38 and ERK1/2 may be regulated by nicotinic receptors other than α7nAChR. When we examined STAT3 phosphorylation (Tyr705) we observed that nicotine was able to induce phosphorylation in BL6 M1 and M2

BMDMs but in a more robust manner in M2s (2.58 ± 0.25 vs. 1.25 ± 0.08, fold induction over control for M2 and M1, respectively, P = 0.003, Fig. 3-4). In α7KO

BMDMs, nicotine-induced phosphorylation was slightly but significantly reduced (Fig. 3-5). As a secondary method to examine the role of α7nAChR in this signaling pathway, we pre-treated cells with α-bungarotoxin (αBT), an irreversible inhibitory toxin with high affinity for α7nAChR, prior to agonist treatment. We observed that αBT pre-treatment was able to significantly reduce

STAT3 phosphorylation (~40% at 5–10 min, Fig. 3-6). To examine activation of survival signaling with specific stimulation of α7nAChR we used the α7-specific agonist PNU-282987. Treatment of macrophages with PNU-282987 resulted in

40

STAT3 phosphorylation only in BL6 M2 BMDMs (Fig 3-7) and was not able to induce STAT3 phosphorylation in either α7KO M1 or M2 BMDMs (Fig 3-7).

These findings suggest that stimulation of nicotinic receptors can induce STAT3 phosphorylation, and a significant amount of this effect was contributed by.

Notably, the α7nAChR-specific phosphorylation of STAT3 was seen only in the

M2 BMDM phenotype.

STAT proteins are commonly the targets of the nonreceptor tyrosine kinase Janus Kinase proteins (JAKs) (Rawlings, Rosler et al. 2004). STAT3 is a target for JAK2, and this signaling axis is activated by nicotine treatment in peritoneal macrophages (de Jonge, van der Zanden et al. 2005). To determine if

JAK2 was required for STAT3 phosphorylation also in polarized BMDMs, we pre-treated cells with the JAK2 inhibitor AG-490 (10 μM) prior to agonist treatment. Blocking JAK2 prevented nicotine and PNU-282987 from inducing

STAT3 phosphorylation (Fig. 3-8), suggesting operation of a JAK2/STAT3 axis.

41

M1 BMDMs (α7+/+) M2 BMDMs (α7+/+) +Nicotine +Nicotine pERK1/2 pERK1/2

ERK1/2 ERK1/2

M1 BMDMs (α7+/+) M2 BMDMs (α7+/+) +Nicotine +Nicotine

pP38 pP38

P38 P38

Figure 3-3: M1 and M2 BMDMs from α7+/+ mice were treated with nicotine (10 μM) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P-ERK1/2 and total ERK1/2 MAPK (top panels) or P-p38 and total p38 MAPK (bottom panels). Blots are representative of 3 independent experiments. Modified from (Lee and Vazquez 2013).

42

M1 BMDMs (α7+/+) M2 BMDMs (α7+/+) +Nicotine +Nicotine

pSTAT3 pSTAT3

STAT3 STAT3

* ** *** ****

*

pSTAT3/STAT3

pSTAT3/STAT3

(fold change over control) over change (fold (fold change over control) over change (fold

Figure 3-4: M1 and M2 BMDMs from α7+/+ mice were treated with nicotine (10 μM) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P-STAT3 and total STAT3 to control for protein loading. Band intensity was measured by densitometry (ImageJ) and normalized values from 3 independent experiments are shown. For M1 BMDMs, *P = 0.024; for M2 BMDMs, *P = 0.0001, **P = 0.002, ***P = 0.032, ****P = 0.03. Modified from (Lee and Vazquez 2013).

43

M1 BMDMs (α7-/-) M2 BMDMs (α7-/-) +Nicotine +Nicotine

pSTAT3 pSTAT3

STAT3 STAT3

* **

***

pSTAT3/STAT3

pSTAT3/STAT3

(fold change over control) over change (fold (fold change over control) over change (fold

Figure 3-5: M1 and M2 BMDMs from α7-/- mice were treated with nicotine (10 μM) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P-STAT3 and total STAT3 to control for protein loading. Band intensity was measured by densitometry (ImageJ) and normalized values from 3 independent experiments are shown. For M2 BMDMs, *P = 0.001, **P = 0.006, ***P = 0.02. Modified from (Lee and Vazquez 2013).

44

M1 BMDMs (α7+/+) M2 BMDMs (α7+/+) + α Bgt + α Bgt +Nicotine +Nicotine

pSTAT3 pSTAT3

STAT3 STAT3

Figure 3-6: M1 and M2 BMDMs from α7+/+ mice were treated with nicotine (10 μM) in the presence of α-bungarotoxin (“αBgT”, 100 ng/ml, 15 min preincubation) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P- STAT3 and total STAT3. Blots are representative of 3 independent experiments. Modified from (Lee and Vazquez 2013).

45

M1 BMDMs (α7+/+) M2 BMDMs (α7+/+) +PNU-282987 +PNU-282987

pSTAT3 pSTAT3

STAT3 STAT3

* **

pSTAT3/STAT3

pSTAT3/STAT3

(fold change over control) over change (fold (fold change over control) over change (fold

M1 BMDMs (α7-/-) M2 BMDMs (α7-/-) +PNU-282987 +PNU-282987

pSTAT3 pSTAT3

STAT3 STAT3

Figure 3-7: M1 and M2 BMDMs from α7+/+ (top panels) or α7-/- (bottom panels) mice were treated with PNU-282987 (1 μM) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P-STAT3 and total STAT3. Blots are representative of 3 independent experiments. For α7+/+ blots, band intensity was measured by densitometry (ImageJ) and normalized values from 3 independent experiments are shown. For M2 BMDMs, *P = 0.023, **P = 0.016. Modified from (Lee and Vazquez 2013).

46

M2 BMDMs (α7+/+) +AG490 + PNU-282987

pSTAT3

STAT3

Figure 3-8: M1 and M2 BMDMs from α7+/+ mice were treated with nicotine (10 μM, top panels) or PNU-282987 (1 μM, bottom panel) in the presence of AG-490 (10 μM, 15 min preincubation) in serum-free RPMI for 0-60 minutes, and subsequently lysed and processed for immunoblot. Membranes were then probed with antibodies for P-STAT3 and total STAT3. Blots are representative of 3 independent experiments. Modified from (Lee and Vazquez 2013).

47

3.4 α7nAChR stimulation reduces ER stress-induced apoptosis in M2 BMDMs

We next wanted to evaluate if the α7nAChR-STAT3 pathway had any effect on apoptosis in BMDMs. Chronic ER stress and UPR activation can induce apoptosis in macrophages both in vitro and in the setting of the lesion

(Rivadeneira, Grobmyer et al. 2001), therefore BMDM apoptosis was induced by chronic treatment with the ER stressor thapsigargin. An irreversible inhibitor of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump, thapsigargin induces chronic ER stress by inhibiting Ca2+ reuptake into the ER, causing rapid release of Ca2+ from ER stores and disrupting ER Ca2+ homeostasis. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. When we treated BL6 M1 and M2 BMDMs with thapsigargin, we observed an increase in TUNEL-positive cells compared to serum-free RPMI alone. In M1 BMDMs, co-treatment with nicotine and thapsigargin gave inconsistent results between experiments, while PNU-282987 had no effect on apoptosis (not shown). However, in M2 BMDMs both nicotine and PNU-282987 were able to significantly reduce the number of TUNEL- positive apoptotic BMDMs when cells were co-treated with either agonist during thapsigargin treatment (Fig. 3-9). Because we saw no significant effects on apoptosis in BL6 M1 BMDMs, from here on only M2 BMDMs were studied.

Next, we wanted to determine if the effects seen in M2 BMDMs were lost when

48

α7nAChR is absent. When α7KO M2 BMDMs were treated with thapsigargin, we again observed a significant increase in TUNEL-positive cells, with no significant difference compared to thapsigargin-treated BL6 M2 BMDMs (Fig. 3-9.). This suggests that α7nAChR deficiency alone does not seem to impact the sensitivity of BMDMs to ER stress-induced apoptosis. Interestingly, the pro-survival effects of nicotine and PNU-282987 were lost in α7KO M2 BMDMs, although there was a trend toward a protective effect with nicotine (p=0.07). However, the complete loss of PNU-282987 mediated protection in the α7KO M2 BMDMs suggests that the pro-survival effect of nicotinic receptor activation is mediated primarily by

α7nAChR.

We have observed that α7nAChR agonists can induce survival signaling and protect BL6 M2 BMDMs from ER stress-induced apoptosis, however the question remained whether STAT3 contributed to the mechanism underlying the pro-survival effect of α7nAChR activation. To determine if STAT3 activation is involved, we pre-treated cells with a cell permeable STAT3 inhibitor (STAT3i, 10

μM) to block STAT3 activation. Pre-treatment with the STAT3i alone did not induce apoptosis, and had no effect on thapsigargin-induced apoptosis.

However, in the presence of STAT3i there was no significant reduction in apoptosis following treatment with either nicotine or PNU-282987 in BL6 M2

BMDMs (Fig. 3-10).

49

α7+/+ α7-/- * ** # ## ns ns

Figure 3-9: M2 BMDMs from α7+/+ (black bars) or α7-/- (white bars) mice were plated in 96-well plates and treated for 12 hours in serum-free RPMI or thapsigargin (“Thap”, 1 μM) in the presence or absence of nicotine (“Nic”, 10 μM) or PNU-282987 (“PNU”, 1 μM). Following treatments, cells were evaluated for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cells were counted by fluorescence microscopy, and results shown as percentage of TUNEL-positive nuclei relative to total DAPI-positive cells. Shown are means ± SEM, n = 6. *P = 0.001 and **P = 0.002, compared to RPMI; #P = 0.026 and ##P = 0.005, compared to thapsigargin alone for α7+/+; ns: not statistically different compared to thapsigargin alone for α7-/-. Modified from (Lee and Vazquez 2013).

50

ns *

ns

Figure 3-10: M2 BMDMs from α7+/+ mice were plated in 96-well plates and treated for 12 hours in serum-free RPMI or thapsigargin (“Thap”, 1 μM) in the presence or absence of nicotine (“Nic”, 10 μM) or PNU-282987 (“PNU”, 1 μM), all in the presence of a STAT3 inhibitor peptide (“S3i”, 25 μM, 15 min pretreatment) Following treatments, cells were evaluated for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cells were counted by fluorescence microscopy, and results shown as percentage of TUNEL-positive nuclei relative to total DAPI-positive cells. Shown are means ± SEM, n = 6. *P = 0.0005 compared to RPMI+S3i; ns: not statistically different from Thap+S3i. Modified from (Lee and Vazquez 2013).

51

3.5 α7nAChR stimulation is associated with upregulation of Bcl-2

The anti-apoptotic effect of α7nAChR activation in BL6 M2 BMDMs seems to be mediated, at least in part, by activation of STAT3, but the mechanism by which the α7nAChR-STAT3 pathway can inhibit apoptosis remained undefined.

STAT3 is an oncogene which promotes cell survival in many types of cancer, generally by upregulation of Bcl-2 family genes including Bcl-2 and Bcl-Xl (Real,

Sierra et al. 2002, Lebedeva, Sarkar et al. 2003), and these genes are important for macrophage survival in atherosclerosis (Thorp, Li et al. 2009, Shearn, Deswaerte et al. 2012). We therefore were interested in evaluating the potential regulation of

Bcl-2 genes downstream activation of the α7nAChR-STAT3 pathway. RT-PCR was performed on cDNA derived from BL6 and α7KO M2 BMDMs to evaluate expression of Bcl-2, Bcl-XL and Mcl-1 after thapsigargin treatment in the presence or absence of nicotine or PNU-282987. In both BL6 and α7KO M2

BMDMs, thapsigargin treatment alone induced upregulation of Bcl-2 (Fig 3-11) and Bcl-XL (not shown). Although it may seem unexpected at first that a pro- apoptotic compound would upregulate pro-survival genes, this could be a compensatory upregulation in response to thapsigargin treatment, an effect we have previously seen in TNF-treated THP-1-derived human macrophages (Tano and Vazquez 2011). However, co-treatment with nicotine further upregulated

Bcl-2 and Bcl-XL, and although not statistically significant compared to

52

thapsigargin alone, this effect was completely absent in α7KO M2 BMDMs.

Specific activation of α7nAChR with PNU-282987 also induced further upregulation of Bcl-2 compared to thapsigargin alone, and again this effect was completely absent in α7KO M2 BMDMs (Fig. 3-11.). These findings suggest that the anti-apoptotic effect of α7nAChR in M2 BMDMs may be partially mediated by regulating the expression of pro-survival Bcl-2 genes.

53

Bcl-2 α7+/+ α7-/-

* **

Bcl-2 α7+/+ α7-/-

*** ***

Figure 3-11: M2 BMDMs from α7+/+ (black bars) or α7-/- (white bars) mice were incubated for 12 h in serum-free RPMI alone or containing thapsigargin (“Thap”, 1 μM) in the presence or absence of nicotine (“Nic”, 10 μM, top graph) or PNU- 282987 (“PNU”, 1 μM, bottom graph), as indicated. Following treatments, cells were processed for RNA isolation and preparation of cDNA. Expression levels of Bcl-2 were measured by qRT-PCR. Graphs represent data (means ± SEM) of 4 independent experiments performed in triplicate wells. Gene expression was normalized with GAPDH as an endogenous control. *P = 0.016, **P = 0.023, ***P = 0.05 compared to respective RPMI controls. Modified from (Lee and Vazquez 2013).

54

3.6 Role of macrophage α7nAChR in development of murine atherosclerosis

We have observed that α7nAChR activation can protect M2 bone marrow- derived macrophages against ER stress-induced apoptosis in vitro. Chronic ER stress is known to induce apoptosis in macrophages within atherosclerotic lesions (Tabas 2010), therefore it was of importance to determine if macrophage

α7nAChR has a potential impact in lesion development. Until very recently

(Hernandez, Cortez et al. 2014), there was no available conditional knockout mouse model for α7nAChR. Therefore we turned to bone marrow transplantation (BMT), a method used by our lab and many others to study the role of macrophage-specific genes in murine atherosclerosis (Aparicio-Vergara,

Shiri-Sverdlov et al. 2012, Tano, Solanki et al. 2014). Inbred mouse strains such as

C57BL/6 are relatively resistant to development of atherosclerotic lesions unless fed an extreme diet containing high fat, cholesterol and cholic acid (Paigen,

Morrow et al. 1987). Therefore we chose to perform BMT in low density lipoprotein receptor knockout (LDLR-/-) mice. This mouse model mimics the human condition of familial hypercholesterolemia whereby the loss of function of LDLR, primarily in the liver, leads to unusually high plasma concentrations of

LDL and VLDL (Ishibashi, Brown et al. 1993). Hypercholesterolemia can be further exaggerated by placing the mice on a high fat “Western” diet. For our studies, LDLR-/- mice were subjected to myeloablatory irradiation and BMT was

55

performed using bone marrow cells isolated from C57BL/6 (BL6LDLRKO) or

α7nAChR-deficient mice (α7KOLDLRKO). After four weeks of recovery, bone marrow conversion to the donor’s phenotype was confirmed by PCR genotyping of peripheral blood cells (not shown). For B6LDLRKO mice, genomic DNA was isolated and PCR was performed for expression of wild-type LDLR gene; for

α7KOLDLRKO mice, RNA was isolated from peripheral blood, reverse transcribed to complementary DNA (cDNA) and PCR performed for expression of mutant α7nAChR. Upon confirmation of bone marrow conversion, mice were placed on a Western diet (HFD, 42% kcal from fat, 0.2% cholesterol) ad libitum for eight or fourteen weeks to promote development of early stage (8 weeks HFD) and advanced stage (14 weeks HFD) atherosclerotic lesions.

3.7 Impact of bone marrow deficiency of α7nAChR on early atherosclerotic lesions

Prior to sacrifice at the end of the diet time point, mice were fasted overnight and blood was collected for analysis of plasma lipids. After 8 weeks of

HFD feeding both groups of mice were hypercholesterolemic, however the

α7KOLDLRKO mice had reduced total cholesterol levels compared to

B6LDLRKO controls (746 ± 94 mg/dl (n=7) vs. 953 ± 211 mg/dl (n=6) respectively; p=0.035). Analysis of plasma triglycerides showed comparable levels between groups (103 ± 25 mg/dl (n=6) vs. 151 ± 106 mg/dl (n=7),

56

B6LDLRKO vs. α7KOLDLRKO; p=0.90). There was no difference in the average body weight between groups (21.0 ± 2.9 g (n=13) vs. 21.2 ± 1.6 g (n=15),

B6LDLRKO vs. α7KOLDLRKO; p=0.84).

For analysis of atherosclerotic lesion development, the hearts were isolated and frozen cross sections were collected throughout the aortic root. First, lesion area was determined by morphometric analysis of hematoxylin-eosin

(H&E) stained sections; both groups showed similar total lesion area (227,219 ±

24166 μm2 vs. 246,792 ± 25201 μm2, B6LDLRKO vs. α7KOLDLRKO; p=0.59,

Fig. 3-12). Early atherosclerotic lesions are dominated by foam cells and lipid accumulation. Sections were stained with Oil Red-O (ORO) for neutral lipid content; ORO-positive areas were not different between groups (217,816 ± 24166

μm2 vs. 229,553 ± 31578 μm2, B6LDLRKO vs. α7KOLDLRKO; p=0.88, Fig. 3-

12). Sections stained using the anti-macrophage Moma-2 antibody showed no difference in macrophage area (151,345 ± 19,266 μm2 vs. 117,110 ± 20518 μm2,

B6LDLRKO vs. α7KOLDLRKO; p=0.86, Fig. 3-13). Although it is well established that apoptotic cells are efficiently cleared in early atherosclerotic lesions (Tabas 2007), they can still be detected (Zhou, Lhotak et al. 2005, Yancey,

Blakemore et al. 2010). Staining for apoptotic cells by in situ TUNEL showed no difference in the number of TUNEL-positive cells per mm2 lesion area (not shown). Taken together, these data indicate that bone marrow deficiency of

57

α7nAChR does not affect the development of early stage aortic root lesions in mice.

58

B6LDLR α7-/-LDLR

H&E ORO

8 w e e k s H F D

5 0 0 0 0 0 p = 0 .5 9 p = 0 .8 8

) 2

m 4 0 0 0 0 0

u (

a

e 3 0 0 0 0 0 r

A

n 2 0 0 0 0 0 o

i s

e 1 0 0 0 0 0 L

0 O O O O K K K K R R R R L L L L D D D D L L L L > -> > -> ------6 O 6 O B K B K 7 7   H & E O R O

Figure 3-12: Aortic root sections from B6LDLRKO or α7KOLDLRKO mice on an 8 week high fat diet were stained with hematoxylin-eosin (H&E, closed circles) or Oil-Red-O (ORO, open circles) to evaluate lesion area and neutral lipid content, respectively. Quantitation of mean stained areas is shown. Mean values and corresponding standard errors and n numbers are provided in the text. P values were determined using the Mann-Whitney U-test.

59

B6LDLR α7-/-LDLR

8 w e e k s H F D )

2 3 0 0 0 0 0 p = 0 .8 7

m

(

a

e r

a 2 0 0 0 0 0

e

v

i

t

i

s o

p 1 0 0 0 0 0

2

-

a

m o

M 0

O O K K R R L L D D L L -> > - -- 6 O B K 7 

Figure 3-13: Atherosclerotic lesions in aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) that were maintained on a 8 week high fat diet, were stained with MOMA-2 antibody to evaluate macrophage content. Shown are representative sections of MOMA-2 staining and corresponding quantitations of the mean stained areas. P values were determined using the Mann-Whitney U- test.

60

3.8 Impact of bone marrow deficiency of α7nAChR on advanced atherosclerotic lesions

In order to evaluate the impact of macrophage α7nAChR deficiency at the advanced lesion stage, we allowed a second set of mice from each group to remain on HFD for an additional 6 weeks for a total of 14 weeks on the atherogenic diet. There was no difference in average weight between groups at this time. When we analyzed plasma lipid levels, we observed comparable total cholesterol levels between groups, however there was a significant increase in plasma triglycerides in the KOLDLR mice compared to controls. Peripheral blood was also used to analyze circulating monocyte subsets by flow cytometry.

Monocytes were first gated by CD11b expression and then analyzed for expression of marker Ly6C, where Ly6Chigh monocytes represent the inflammatory subset and Ly6Clow monocytes are considered the patrolling subset

(Ley, Miller et al. 2011). Comparable ratios of Ly6Chigh:Ly6Clow monocytes were observed in both groups of mice (not shown).

We then analyzed the aortic root atherosclerotic lesions of these mice.

After 14 weeks on HFD we observed a significant decrease in total lesion area in

α7KOLDLRKO mice compared to the control group (354,672 ± 18,246 μm2 vs.

469,927 ± 34028 μm2, respectively; p=0.015, Fig. 3-14). In line with the notion that cellularity contributes to lesion size, and in correlation with lesions at this stage being smaller than in control animals KOLDLRKO mice also had significantly

61

decreased lesional macrophage content compared to the control group as determined by Moma-2 staining (10.6 ± 0.3% vs. 14.4 ± 2.0%, respectively; p=0.03,

Fig. 3-15). The relative abundance of lesional macrophage phenotypes change depending on lesion stage (Khallou-Laschet, Varthaman et al. 2010). We next wanted to determine if α7nAChR deficiency influenced the abundance of M1 and

M2 macrophages in the lesions of these mice. We performed co-immunostaining using markers for M1 (iNOS) and M2 (mannose receptor, MR) macrophage subsets combined with a pan-macrophage antibody (AIA31240), and co- localization within the lesion areas was visualized by fluorescence microscopy.

Staining for M1 macrophages showed abundant co-localization in lesions from both groups with no observable differences (Fig. 3-16). Co-staining for M2 macrophages was less abundant, and no significant difference in the relative number of M2 macrophages was observed between groups (50 ± 14 cells/mm2 vs. 71 ± 20 cells/mm2, B6LDLRKO vs. α7KOLDLRKO; p=0.39, Fig. 3-17).

Next, it was necessary to determine if macrophage α7nAChR deficiency had an effect on macrophage apoptosis and necrosis at the advanced lesion stage.

Based on our in vitro findings, we hypothesized that α7nAChR deficiency could potentially increase macrophage apoptosis which, at the advanced lesion stage, would result in enlargement of necrotic cores (Tabas, Seimon et al. 2009).

Necrotic cores were measured in sections stained with Gomori’s trichrome, and were characterized by acellular areas lacking extracellular matrix (marked by

62

arrows, Fig. 3-18). When we quantified these areas of necrosis, there was no significant difference in necrotic core area relative to lesion area (27 ± 2% vs. 30 ±

2%, B6LDLRKO vs. α7KOLDLRKO; p=0.37, Fig. 3-18). Similarly, the number of apoptotic cells per mm2 lesion area, as determined by in situ TUNEL, was comparable between groups (53 ± 10 cells/mm2 vs. 66 ± 10 cells/mm2; p=0.34,

Fig. 3-19). When we co-stained sections for TUNEL and an anti-macrophage antibody, no significant differences were found in the number of apoptotic lesional macrophages between groups (23 ± 8 cells/mm2 vs. 33 ± 10,

B6LDLRKO vs. α7KOLDLRKO; p=0.36, Fig. 3-19).

To determine if differences existed in other parameters associated with lesion stability in both murine and human plaques (Schwartz, Galis et al. 2007,

Finn, Nakano et al. 2010) we analyzed lesional collagen content as well as fibrous cap thickness of caps overlying necrotic cores. Despite no statistically significant differences between groups in collagen content or cap thickness, there was a consistent trend for a reduction in these parameters in α7KOLDLRKO compared to B6LDLRKO mice (normalized collagen content: 3.45 ± 0.28 vs.

2.79 ± 0.20, respectively, p=0.12; fibrous cap thickness: 48.9 ± 6.4 μm (n=10) vs.

36.9 ± 3.4 mm (n=8), respectively, p=0.23; not shown).

Altogether, these results indicate that in advanced atherosclerotic lesions of LDLRKO mice, bone marrow deficiency of α7nAChR does not seem to impact lesional macrophage apoptosis or necrotic core development. However,

63

advanced lesions in mice with α7nAChR deficient bone marrow were smaller and with a marked reduction in macrophage content. Recent evidence indicates that the macrophage population of advanced atherosclerotic lesions is maintained mostly through proliferation of lesional macrophages rather than through de novo recruitment of circulating monocytes (Robbins, Hilgendorf et al.

2013). To examine if bone marrow deficiency of α7nAChR had an impact on the proliferation of lesional cells in lesions from mice maintained on 14-week HFD, aortic root sections from B6LDLRKO and α7KOLDLRKO mice were subjected to immunostaining for the nuclear antigen Ki67, a useful marker to detect cells in a proliferative state (Wu, Luo et al. 2000). Interestingly, lesions from mice with α7KO bone marrow showed a marked trend for a reduction in the number of Ki67-positive cells compared to mice from the control group (5.3 ±

4.0 vs. 1.4 ± 0.9 Ki67+ cells/mm2, B6LDLRKO vs. α7KOLDLRKO, respectively; n=6 for both groups, p=0.097, Fig. 3-20). Notably, co-localization of

Ki67-positive cells with macrophage immunoreactivity also exhibited a trend for reduction in mice that received α7nAChRKO bone marrow compared with controls (4.6 ± 1.2 vs. 1.3 ± 0.5 Ki67+ cells/mm2, for B6LDLRKO vs.

α7KOLDLRKO, respectively; n=6 for both groups, p=0.09, Fig. 3-20).

64

B6LDLR α7-/-LDLR

1 4 w e e k s H F D

8 0 0 0 0 0

p = 0 .0 1 5

) 2

m 6 0 0 0 0 0

(

a e

r 4 0 0 0 0 0

a

n

o i

s 2 0 0 0 0 0

e L

0

O O K K R R L L D D L L -> > - -- 6 O B K 7 

Figure 3-14: Atherosclerotic lesions in aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) that were maintained on a 14 week high fat diet were stained with hematoxilin-eosin (H&E) to determine lesion area. Shown are representative sections of H&E staining, and corresponding quantitations of the mean stained areas. P values were determined using the Mann-Whitney U-test.

65

B6LDLR α7-/-LDLR

1 4 w e e k s H F D

2 0

p = 0 .0 3

a

e

r a

1 5

n

o

i

s

e l

+ 1 0

2

-

a m

o 5

M

% 0

O O K K R R L L D D L L -> > - -- 6 O B K 7 

Figure 3-15: Atherosclerotic lesions in aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) that were maintained on a 14 week high fat diet were stained with MOMA-2 antibody to evaluate macrophage content. Shown are representative sections of MOMA-2 staining, and corresponding quantitations of the mean stained areas as a percentage of total lesion area. P values were determined using the Mann-Whitney U-test.

66

B6LDLR α7-/-LDLR

Figure 3-16: Aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) and maintained on a 14 week high fat diet, were stained for macrophages (green, AIA31240), iNOS (M1 marker, red) and DAPI (nuclei). Yellow/orange cells and areas show co-localization of macrophage- and iNOS, suggestive of the presence of M1 type macrophages.

67

B6LDLR α7-/-LDLR

1 4 w e e k s H F D

1 5 0

p = 0 .3 9 2

m 1 0 0

m

/

s

c

a

M

2 5 0 M

0

O O K K R R L L D D L L -> > - -- 6 O B K 7 

Figure 3-17: Aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) and maintained on a 14 week high fat diet, were stained for macrophages (green, AIA31240), mannose receptor (M2 marker, red) and DAPI (nuclei). Yellow cells and areas (arrows) show co-localization of macrophage- and mannose receptor, suggestive of the presence of M2 type macrophages. The number of AIA31240+ + mannose receptor+ cells were counted throughout the aortic sinus and normalized by the total lesion area. Results are shown as mean ± SEM. P values were determined using the Mann-Whitney U-test.

68

B6LDLR α7-/-LDLR

1 4 w e e k s H F D

5 0

p = 0 .3 7

4 0

a

e

r

a

c 3 0

i

t

o r

c 2 0

e

n

% 1 0

0

O O K K R R L L D D L L -> > - -- 6 O B K 7 

Figure 3-18: Atherosclerotic lesions in aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) fed a high fat diet for 14 weeks were stained with Gomori’s trichrome to measure necrosis. Shown are representative sections of trichrome staining, with areas of necrosis noted with arrows, and corresponding quantitations of the mean necrotic area as a % of total lesion area. P values were determined using the Mann-Whitney U-test.

69

1 4 w e e k s H F D 1 4 w e e k s H F D p = 0 .3 6 1 0 0 5 0

p = 0 .3 5

2 2

8 0 4 0

m

m

m

m

a

/

a

/

e

s

e

s

r

l

r c

l 6 0 3 0

a

a

a

e

c

n

n

M

o

o

+

+

i i

L 4 0 2 0

s

s

L

E

e

e

E

l

l

N

N U

2 0 U 1 0

T T

0 0

O O O O K K K K R R R R L L L L D D D D L L L L -> > -> > ------6 O 6 O B K B K 7 7  

Figure 3-19: Aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) that were maintained on a 14 week high fat diet, were stained for in situ TUNEL to detect apoptotic cells, or co-stained for macrophage (AIA31240) and TUNEL to evaluate apoptotic macrophages (as we described in (Tano, Solanki et al. 2014)). The number of TUNEL+ or AIA31240+ TUNEL+ cells were counted throughout the aortic sinus and normalized by the total lesion area. Results are mean ± SEM. P values were determined using the Mann-Whitney U-test.

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B6LDLR α7-/-LDLR

1 4 w e e k s H F D 1 4 w e e k s H F D

8 p = 0 .0 9 8

p = 0 .0 9

2 2

6 6

m

m

m

m

/

/

s

s

l

c

l a

e 4 4

c

M

+

+

7

7

6

6 i

2 i 2

K K

0 0

O O O O K K K K R R R R L L L L D D D D L L L L -> > -> > ------6 O 6 O B K B K 7 7  

Figure 3-20: Aortic root sections from LDLRKO mice transplanted with bone marrow from wild-type (B6LDLRKO) or α7nAChRKO mice (α7KOLDLRKO) and maintained on a 14 week high fat diet, were stained for the Ki67 antigen (red) to detect proliferating cells, and DAPI (nuclei). The arrows show the localization of Ki67 positive cells inside lesion areas. The number of Ki67+ cells or Ki67+ AIA31240+ macrophages (images not shown) were counted throughout the aortic sinus and normalized by the total lesion area. Results are mean ± SEM. P values were determined using the Mann-Whitney U-test.

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Chapter 4

Discussion and Conclusions

Our studies examined for the first time a role for α7nAChR in macrophage apoptosis, and its potential impact on the development of murine atherosclerotic lesions. For our in vitro studies, we used bone marrow derived macrophages

(BMDMs) from wild-type and α7nAChR-deficient mice to specifically examine the impact of both α7nAChR activation or its deficiency on signaling mechanisms related to cell survival, apoptosis, and expression of pro-survival genes. Furthermore, we used macrophages which were polarized to the M1 or

M2 phenotype, in an attempt to study macrophages with characteristics that may better represent those of the macrophage populations found in vivo and in the lesion setting, than non-polarized or peritoneal macrophages. We observed that activation of α7nAChR resulted in activation of the pro-survival JAK2/STAT3 axis in M2 macrophages, but not in M1 cells. When we examined the impact of survival signaling activation in M2 macrophages, we found that α7nAChR activation had a protective effect on this macrophage type regarding their

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susceptibility to ER stress-induced apoptosis and that this occurred, at least to some extent, in a STAT3-dependent manner (Lee and Vazquez 2013).

We found evidence of nicotinic receptor-mediated activation of several macrophage survival signaling pathways in both M1 and M2 macrophages including ERK1/2 MAPK, p38 MAPK and STAT3. While STAT3 activation occurred downstream selective activation of α7nAChR, the activation of ERK1/2 and p38 by nicotine treatment was not altered by blocking α7nAChR with α- bungarotoxin or in α7nAChR-deficent BMDMs. Nicotine is a general agonist for all nicotinic receptors, and we have observed expression of other nicotinic receptors in macrophages by PCR, including α2, 3 and 6 nAChR subunits.

Importantly, we did not detect either the α1 or α9 subunits which are also a target for αBT (Bray, Son et al. 2005, Albuquerque, Pereira et al. 2009), supporting the interpretation that the effects of αBT pretreatment are the result of selective blocking α7nAChR. Because macrophages express other nicotinic receptor subunits, we cannot consider any effects from nicotine, in the absence of receptor blocking or deletion, to be specific for α7nAChR. Contrary to the activation of

ERK1/2 and p38MAPK, nicotine activated STAT3 in a manner that was partially dependent on α7nAChR, as evidenced in both M1 and M2 macrophages by the reduction in STAT3 phosphorylation following αBT pretreatment and by

α7nAChR deficiency. In line with this, the α7nAChR-selective agonist PNU-

282987 induced STAT3 phosphorylation which was much more robust in M2

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than in M1 macrophages. Polarized macrophage populations are commonly identified by their expression profile of certain proteins including signaling proteins, transcriptional factors, components of amino acid metabolism and membrane receptors. Cytokine-induced activation and polarization, for example by IFNγ vs. IL-4, can differentially affect the expression and activity of STAT family proteins (Hoeksema, Stoger et al. 2012). Therefore, it is tempting to speculate that the M2-specific activation of STAT3 downstream α7nAChR stimulation may be due to differences between M1 and M2 macrophages in terms of expression and cellular distribution of STAT3, coupling status of

α7nAChR signaling to STAT3 activation and/or differences in expression of

STAT-regulating proteins (Spence, Fitzsimons et al. 2013).

In peritoneal macrophages, as well as in cell types other than macrophages, nicotine-dependent activation of STAT3 occurs through a

JAK2/STAT3 axis (de Jonge, van der Zanden et al. 2005, Marrero and Bencherif

2009, Marrero, Bencherif et al. 2011, Smedlund, Tano et al. 2011). We also observed operation of a JAK2/STAT3 axis, as evidenced by the significant reduction in nicotine and PNU-282987-induced STAT3 phosphorylation after pretreatment of macrophages with the JAK2 inhibitor AG-490. Interestingly, in peritoneal macrophages α7nAChR and JAK2 co-immunoprecipitate following nicotine treatment, suggesting a direct interaction between them upon receptor activation (de Jonge, van der Zanden et al. 2005). The absence of detectable Ca2+

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influx in bone marrow derived macrophages upon α7nAChR stimulation raises the question whether in macrophages the signaling capacity of α7nAChR is independent of its channel properties, and rather depends on the receptor acting as a signaling platform in which the intracellular domain of α7nAChR facilitates direct interactions with downstream signaling molecules. Proteomic analysis of

αBT-bound protein complexes isolated from the brain of wild-type or α7nAChR- deficient mice identified over 50 proteins interacting with α7nAChR (Paulo,

Brucker et al. 2009). This further supports the idea of α7nAChR, besides its canonical role as a functional ion channel, having the capacity to act as a signaling platform and mediate activation of signaling pathways through protein-protein interactions. Evidence from other laboratories has shown that nicotinic receptor function in hematopoietic cells may not be due to ion permeation, but rather, subsequent to allosteric transitions that occur upon receptor activation and can modulate the activity of downstream effectors

(Hecker, Mikulski et al. 2009, Skok 2009). However, the lack of detectable Ca2+ influx under our experimental conditions does not rule out the possibility that either tiny or rapidly occurring ion fluxes may fall below the detection sensitivity of our system. We have previously shown that both human and murine macrophages are endowed with a very efficient Ca2+ buffering system (Tano and

Vazquez 2011, Tano, Smedlund et al. 2011). Thus, it is possibly that α7nAChR- mediated Ca2+ entry may be rapidly buffered out by endogenous Ca2+ binding

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systems, hindering our ability to detect these changes by the conventional Fura-2 based fluorescence technique. Additional studies will be necessary in order to define the capacity of macrophage α7nAChR to act as a functional ion channel and/or as a signaling platform, and how this impacts any pharmacological maneuver aimed at effectively and selectively activating α7nAChR in these cells.

We have shown that α7nAChR activation can protect M2 BMDMs from

ER-stress induced apoptosis, and that this anti-apoptotic effect is at least partially dependent on activation of STAT3. It remains to be determined whether activation of the α7nAChR-STAT3 axis is actually linked to the observed reduction of apoptosis in M2 macrophages. Bcl-2 family genes have been shown to be a target of STAT3 (Marrero and Bencherif 2009, Cheng, Wang et al. 2012), and we observed that M2 macrophages exposed to nicotine or PNU-282987 under conditions of chronic ER-stress exhibited upregulated mRNA levels of Bcl-

2. It is thus tempting to speculate that upregulation of Bcl-2 occurs downstream activation of the α7nAChR-STAT3 axis. This notion is supported by the finding that M2 BMDMs from α7nAChR-deficient mice completely lacked nicotine or

PNU-282987 induced Bcl-2 upregulation. However, it is unlikely that changes in expression of Bcl-2 alone are responsible for the anti-apoptotic effect that follows

α7nAChR activation, as we observed that the compensatory upregulation of Bcl-

2 that takes place when macrophages are treated with thapsigargin alone is clearly unable to protect the cell from undergoing apoptosis. It is possible that

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the anti-apoptotic effects of the nicotine/PNU mediated Bcl-2 upregulation is accompanied by changes in intracellular localization and binding partners of Bcl-

2, both of which are crucial for its pro-survival effects (Tabas and Ron 2011).

In order to determine the in vivo relevance of these in vitro findings, we examined the impact of bone marrow deficiency of α7nAChR on development and characteristics of atherosclerotic lesions in the aortic sinus of LDLR knockout mice, a well-established animal model of atherosclerosis. After eight weeks on high fat diet, which results in development of early stage atherosclerotic plaques, lesions in mice with α7nAChR-deficient bone marrow were comparable to lesions in control animals. However, in mice maintained for fourteen weeks on high fat diet, which promotes development of advanced atherosclerotic lesions, bone marrow deficiency of α7nAChR had an anti-atherogenic effect in that there was a marked reduction in total lesion size and macrophage content, which corresponded with decreased lesional cell proliferation. Thus, whereas our in vitro findings indicate that activation of α7nAChR protects M2 macrophages- a macrophage phenotype that is present throughout atherosclerotic lesion development- from ER-stress induced apoptosis, our animal studies do not provide evidence of this mechanism operating in vivo and in the setting of atherosclerosis. However, these studies underscored a novel, unexpected role of

α7nAChR in modulating size and cellularity of advanced atherosclerotic lesions.

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Interest in macrophage α7nAChR has increased over the last decade due to accumulating evidence for a role of α7nAChR in regulating cytokine production and macrophage activation in models of acute inflammatory disease such as sepsis (Wang, Yu et al. 2003, Pavlov, Ochani et al. 2007, Rosas-Ballina,

Goldstein et al. 2009). In the so called cholinergic anti-inflammatory circuit, activation of macrophage α7nAChR can suppress pro-inflammatory cytokine production. In sepsis for example, electrical stimulation of the efferent vagus nerve results in reduction of systemic TNFα release and mortality in mice subjected to septic shock (Wang, Yu et al. 2003, Pavlov, Ochani et al. 2007).

Originally thought to operate through direct activation of macrophage α7nAChR by vagus nerve-derived acetylcholine, it was later discovered that the splenic nerve activates a subset of T cells, triggering acetylcholine release which can act in a paracrine fashion to activate macrophage α7nAChR (Rosas-Ballina, Ochani et al. 2008, Rosas-Ballina, Olofsson et al. 2011, Olofsson, Katz et al. 2012). The anti-inflammatory effects of vagal nerve stimulation can also be mimicked by administration of a α7nAChR agonist, suggesting the importance of specific activation of α7nAChR in this setting (Pavlov, Ochani et al. 2007). The discovery of the involvement of macrophage α7nAChR in a physiological anti- inflammatory response led us to speculate on the potential importance of macrophage α7nAChR in atherosclerosis, a disease characterized by a chronic inflammatory component, and the effects of receptor stimulation beyond the

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mere regulation of cytokine production. In our in vitro studies, the findings of a protective action of α7nAChR in M2 macrophages were evident only upon receptor activation, with no obvious differences between wild-type and

α7nAChR-deficient macrophages under basal conditions. Our hypothesis on the potential impact of α7nAChR deficiency on lesion development therefore has implicit the notion that activation of α7nAChR somehow takes places in the lesional macrophage, which requires the existence of an acetylcholine source within or nearby the plaque macrophage. However, the extent of activation of

α7nAChR in lesional macrophages is uncertain. Therefore, future studies should examine whether stimulation in vivo of macrophage α7nAChR reveals functions that may have a physiopathological impact. Potential endogenous sources of ligands for α7nAChR include acetylcholine released directly from local cholinergic synapses, or T cell-derived acetylcholine as occurs in the spleen.

Cholinergic innervation in coronary arteries from human and other mammals is sparse (Kalsner 1989, Kovach, Gottdiener et al. 1995), and it is unlikely that nerve terminal-derived acetylcholine could diffuse through multiple tissue layers and deep enough into atherosclerotic plaques to reach lesional macrophages (as proposed in Figure 4, (Wilund, Rosenblat et al. 2009)). In addition, neither M1 nor M2 macrophages seem to express the enzymatic machinery required to synthesize acetylcholine for autocrine/paracrine receptor activation (Lee and

Vazquez, unpublished observations). All this raises the question of what

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potential sources of acetylcholine could be present in the lesion to modulate macrophage function. T lymphocytes are present in atherosclerotic lesions

(Hansson and Hermansson 2011), however to what extent the phenotype and properties of these T cells resembles that of acetylcholine-releasing lymphocytes in the spleen is unknown. Discovering whether a mechanism similar to that in spleen operates in the lesion microenvironment will require studies on the capacity of lesional cells to produce and locally release effective concentrations of acetylcholine.

In an attempt to examine additional functions of macrophage α7nAChR besides cytokine suppression in the context of atherosclerosis, Wilund et al.

(Wilund, Rosenblat et al. 2009) studied cholesterol accumulation and anti- oxidant function in peritoneal macrophages isolated from α7nAChR knockout or

α7nAChR/ApoE double –global- knockout mice. They found that these cells exhibited increased cholesterol mass and oxidized LDL uptake and decreased anti-oxidant capacity compared to control cells (Wilund, Rosenblat et al. 2009).

Whereas these processes are of significance to the role of macrophages in the development of atherosclerotic lesions, these findings were not corroborated in an in vivo setting. Our results show that early lesions in LDLRKO mice with wild-type or α7nAChR-deficient bone marrow are comparable in size and foam cell content. We observed a significant decrease in plasma cholesterol levels in

α7KOLDLRKO mice compared to control animals, and it could be argued that

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we should expect decreased foam cell formation in those mice. However, although significantly lower than control mice the cholesterol levels in these mice were still at a level of extreme hypercholesterolemia. Furthermore, foam cell development in vivo is a complex process and results obtained from simple in vitro cholesterol uptake assays may not be necessarily reflected in the process of lesion development. Our results in early lesions suggest that macrophage

α7nAChR may not have a major role in regulating lipid accumulation in vivo, but a definitive conclusion requires additional studies.

As mentioned previously, accumulation of free apoptotic macrophages within atherosclerotic lesions is exacerbated in intermediate and advanced stages of disease mostly due to impaired ability of resident phagocytes to efficiently clear apoptotic cells (Tabas 2010, Tano, Lee et al. 2012). In a recent study from our laboratory (Tano, Solanki et al. 2014) we reported that deficiency of the non- selective cation channel Transient Receptor Potential Canonical 3 (TRPC3) in both M1 and M2 macrophages enhances the efferocytic capacity of these cells compared to Trpc3+/+ macrophages. It remains to be determined if α7nAChR deficiency has an impact on efferocytosis in macrophages in vitro and/or in vivo.

If so, this could mask differences in susceptibility of macrophages to apoptosis in the lesion by influencing the rate of apoptotic cell clearance. This warrants further investigation.

81

A novel but unexpected observation from our in vivo studies is that advanced aortic root lesions from mice with α7nAChR deficient bone marrow exhibit a marked reduction in size and cellularity, the latter mostly reflected by a clear decrease in the lesional macrophage content. This is unlikely to be related to differences in lipid metabolism after the fourteen weeks on high fat diet, as both groups were markedly hypercholesterolemic and the knockout group had even greater plasma triglycerides compared with controls. Moreover, any changes in circulating monocyte recruitment or ability to adhere to endothelium overlying lesion sites would have had an impact on lesion size and macrophage infiltration at the early stage, which was not the case. In an attempt to find a potential explanation for the observed decreased size of advanced lesions exhibited by mice with bone marrow deficiency of α7nAChR, we took into consideration the recent observations by Robbins et al. (Robbins, Hilgendorf et al. 2013) which revisited the mechanism of macrophage accumulation during atherosclerotic lesion formation in a mouse model of the disease. As opposed to early lesions, in which cellularity is regulated by continuous monocyte recruitment, these authors found that in advanced plaques proliferation of lesional macrophages accounts for a significant proportion of the macrophage content in the lesion (Robbins,

Hilgendorf et al. 2013). Our immunofluorescence studies on advanced aortic root lesions show a reduced number of total proliferating cells and macrophages in

α7KOLDLRKO mice compared to control animals. Thus, these findings

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suggest that α7nAChR-dependent modulation of the proliferation of lesional macrophages may account, at least in part, for the observed differences in plaque cellularity. A specific mechanism/s for α7nAChR-dependent modulation of macrophage proliferation has yet to be determined.

The human α7nAChR gene has several single nucleotide polymorphisms.

However, clinically associated human variations have not been reported. In expression profile studies using human genomic microarrays and samples from plaques of patients with atherosclerotic lesions in the left anterior descendent coronary artery (Cagnin, Biscuola et al. 2009), circulating mononuclear cells from atherosclerotic patients (Schirmer, Fledderus et al. 2009) or from plaques of patients with advanced carotid artery disease (Ayari and Bricca 2013), levels of

α7nAChR mRNA are not different from those in control patients. It is possible that changes in channel activity and/or its signaling properties, rather than changes in expression level, or alterations in one or more of the signaling partners that complete the α7nAChR signaling pathway, account for differences in α7nAChR functions in physiological vs. disease states.

Despite growing interest in the participation of α7nAChR in mechanisms relating to the pathogenesis of atherosclerosis (Cooke 2007, de Jonge and Ulloa

2007, Wilund, Rosenblat et al. 2009, Smedlund, Tano et al. 2011, Lee and Vazquez

2013), the impact of α7nAChR in either murine or human atherosclerosis, and its effects on the function of different cell types involved in lesion development, has

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remained largely unexplored. In this context, our findings indicating a reduction in size and cellularity of advanced lesions in mice with bone marrow specific deficiency of α7nAChR provide for the first time experimental evidence underscoring a potential role of macrophage α7nAChR in atherogenesis and set a framework for future studies aimed at characterizing the mechanisms coupling

α7nAChR signaling to macrophage function in atherosclerosis.

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Appendix A

Publications and select presentations

Publications

Lee, R.H., Vazquez, G. Reduced size and cellularity of advanced atherosclerotic lesions in mice with bone marrow specific deficiency of alpha 7 nicotinic acetylcholine receptor (manuscript in preparation).

Tano, J.Y., Solanki, S. Lee, R.H., Smedlund, K., Birnbaumer, L., Vazquez, G. Bone marrow deficiency of TRPC3 channel reduces early lesion burden and necrotic core of advanced plaques in a mouse model of atherosclerosis. Cardiovascular Research 101 (2014) 138-144).

Lee, Robert H., Vazquez, Guillermo. Evidence for a prosurvival role of alpha-7 nicotinic acetylcholine receptor in alternatively (M2)-activated macrophages. Physiological Reports 1:7 (2013).

Tano JY, Lee RH, Vazquez G. Involvement of calmodulin and calmodulin kinase II in tumor necrosis factor alpha-induced survival of bone marrow derived macrophages. Biochemical and Biophysical Research Communications 427 (2012) 178-184.

Jean-Yves Tano*, Robert Lee* and Guillermo Vazquez. (*equally contributing authors) Macrophage function in atherosclerosis: Potential roles of TRP channels. Channels 6 (2012) 141-8.

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Guillermo Vazquez, Kathryn Smedlund, Jean-Yves K. Tano and Robert Lee (2012). Molecular and Cellular Aspects of Atherosclerosis: Emerging Roles of TRPC Channels, Coronary Artery Disease - New Insights and Novel Approaches, Dr. Angelo Squieri (Ed.), ISBN: 978-953-51-0344-8, InTech, Available from: http://www.intechopen.com/books/coronary-artery-disease-new- insights-and-novel-approaches/molecular-and-cellular-aspects-of- atherosclerosis-emerging -roles-of-trpc-channels

Tano J.Y., Smedlund K., Lee R., Vazquez G., Abramowitz J., Birnbaumer L. Impairment of Survival Signaling and Efferocytosis in TPRC3 Deficient Macrophages. Biochemical and Biophysical Research Communications 410 (2011) 643-47.

Select Presentations

Robert H. Lee, Guillermo Vazquez. Alpha7 nicotinic acetylcholine receptor and ER stress induced apoptosis in M2 macrophages: potential impact on atherogenesis. 41st Annual Pharmacology Colloquium 2014 (Oral presentation).

Robert Lee, Guillermo Vazquez. Alpha7 nicotinic acetylcholine receptor and ER stress induced apoptosis in M2 macrophages: potential impact on progression of atherosclerotic lesions. Graduate Student Research Forum 2014 (Oral presentation).

R. Lee and Vazquez G. α7 nicotinic acetylcholine receptor protects M2 macrophages against ER stress-induced apoptosis. Experimental Biology 2013 (Poster presentation).

R. Lee and Vazquez G. Alpha7 Nicotinic Acetylcholine Receptor in Macrophage Apoptosis and Efferocytosis. Experimental Biology 2012: Late Breaking Program (Poster presentation).

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