Calcium Homeostasis Modulator (CALHM1/2) in Pulmonary Arterial Hypertension

Item Type text; Electronic Thesis

Authors Rodriguez, Marisela

Citation Rodriguez, Marisela. (2020). Calcium Homeostasis Modulator (CALHM1/2) in Pulmonary Arterial Hypertension (Master's thesis, University of Arizona, Tucson, USA).

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Download date 27/09/2021 03:13:25

Link to Item http://hdl.handle.net/10150/648659 CALCIUM HOMEOSTASIS MODULATOR (CALHM1/2) IN PULMONARY ARTERIAL HYPERTENSION

By

Marisela Rodriguez

Copyright © Marisela Rodriguez 2020

A Thesis Submitted to the Faculty of the

COLLEGE OF MEDICINE

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

WITH A MAJOR IN CLINICAL TRANSLATIONAL SCIENCES

In the Graduate College

THE UNIVERSITY OF ARIZONA

2020

- 2 -

Acknowledgements

I would like to acknowledge the members of the Yuan Lab throughout my undergraduate years who inspired me to join research, taught me what it means to be a part of the scientific community, and always showed support for a young student as myself. It has led me to pursue a graduate education in this field and continue as a part of the scientific investigatory community. I would like to thank my thesis committee for their advising and support. Dr. Jiyuan Chen (Era) for contributing and expanding this project with me. I would also like to thank my mentor, Professor

Jason Yuan for his patience and teachings, never having a direct way of saying things but allowing for the message to be said in parables and applicable to everything in life. Also, for always reminding me that there are three factors to my work- heart, brain, and hands.

- 3 -

Dedication

This work is dedicated to my family and friends for their unconditional support, encouragement, and belief in me; especially my father for always setting standards and my mother for advising on hard work ethic and both reminding me to implement my all into everything I do.

- 4 -

TABLE OF CONTENTS

LIST OF FIGURES ...... - 6 - LIST OF TABLES ...... - 7 - LIST OF ABBREVIATIONS AND ACRONYMS: ...... - 8 - ABSTRACT ...... - 11 - SIGNIFICANCE AND INTRODUCTION ...... - 15 - PHENOTYPICAL SWITCH AND PAH ...... - 27 - ION CHANNELS AND PULMONARY HYPERTENSION ...... - 33 - CALCIUM HOMEOSTASIS MODULATOR ...... - 38 - MATERIALS AND METHODS ...... - 47 - Pulmonary Artery Isolation ...... - 47 - Isolation and preparation of rat PASMC ...... - 47 - Western Blotting ...... - 54 - Polymerase Chain Reaction ...... - 60 - Statistical Analysis ...... - 65 - RESULTS ...... - 66 - CONCLUSION ...... - 83 - REFERENCES ...... - 86 -

- 5 -

LIST OF FIGURES

Figure 1 – Treatment algorithm of pulmonary arterial hypertension ……………………. 21

Figure 2 – Immunoflourescent staining of human SMCs………………………………... 27

Figure 3 – PASMC Phenotypic Plasticity………………………………………………… 29

Figure 4 – Mechanisms and signaling cascade of Ca2+ in PAH………………………...... 34

Figure 5 – Crystal structure images of human CALHM2 dimer…………………………. 36

Figure 6 – Transmembrane topology of CALHM channel………………………………. 38

Figure 7 – CALHM 1-3 mRNA expression level………………………………………... 42

Figure 8 – Freshly isolated PA branches from rat………………………………………... 49

Figure 9 – Primary PASMC Isolation Steps……………………………………………... 50

Figure 10 – Contractile vs proliferative phenotype markers…………………………….. 64

Figure 11 – Upregulated CALHM1 and 2……………………………………………….. 66

Figure 12 – Upregulated AKT/mTOR signaling proteins……………………………….. 68

Figure 13 – CaSR and Notch upregulated in phenotypical transition……………………. 71

Figure 14 – Pulmonary vascular remodeling in chronic hypoxia………………………… 74

Figure 15 – Rapamycin downregulates CALHM1 and 2………………………………… 77

Figure 16 – Upregulated CALHM1 and 2 in MCT………………………………………. 79

Figure 17 – Upregulated CALHM1 in IPAH hPASMCs………………………………… 79

- 6 -

LIST OF TABLES

Table 1 – Hemodynamic definition of pulmonary hypertension (PH)…………………… 13

Table 2 – Clinical classification of pulmonary hypertension (PH)………………………. 15

Table 3 – Female to male ratios in major PAH registries………………………………… 18

Table 4 – Drugs approved by FDA to treat PAH…………………………………………. 20

Table 5 – Contractile and proliferative SMC markers…………………………………… 26

Table 6 – CALHM 1-3 PCR primer sequences………………………………………….. 43

- 7 -

LIST OF ABBREVIATIONS AND ACRONYMS:

ACTA2 Smooth muscle cell actin ANG-1 Angiopoietin-1 AVD Apoptotic volume decrease 2+ 2+ [Ca ]cyt Cytosolic free Ca concentration CALHM Calcium homeostasis modulator Came calmodulin CaSR Calcium-sensing receptor CCE Capacitive Ca2+ entry CO Cardiac Output

CO2 Carbon dioxide CpcPH Combined pre-and post-capillary PH CREB cAMP response element binding protein CTEPH Chronic thromboembolic pulmonary hypertension DAG Diacylglycerol EC-coupling Excitation-contraction coupling ECM Extracellular matrix ER Endoplasmic reticulum FBS Fetal bovine serum FGFR Fibroblast growth factor receptor GPCR G protein-coupled receptor HIF-1α Hypoxia-inducible factor 1α HIV Human immunodeficiency virus HPV Hypoxic pulmonary vasoconstriction IH Intimal hyperplasia inh. Inhalation

IP3 Inositol 1,4,5-triphosphate IpcPH Isolated post-capillary PH i.v. Intravenous injection LVEF Left ventricle ejection fraction

- 8 -

MAPK Mitogen-activated protein kinase pathway MHC11 Myosin heavy chain-11 MLCK Myosin light chain kinase mPAP Mean pulmonary arterial pressure mmHg Millimeters mercury NFAT Nuclear factor of activated T-cells NO Nitric oxide NYHA-FC New York Heart Association Functional Classification PA Pulmonary artery PASMC Pulmonary artery smooth muscle cell PAP Pulmonary arterial pressure PAH Pulmonary arterial hypertension PAWP Pulmonary arterial wedge pressure PCH Pulmonary capillary hemangiomatosis PDGF Platelet-derived growth factor

PGI2 Prostacyclin PH Pulmonary hypertension PM Plasma membrane p.o. Oral administration PVOD Pulmonary vino-occlusive disease PVR Pulmonary vascular resistance REVEAL Registry to Evaluate Early and Long-term PAH disease management RHC Right heart catheterization ROCC Receptor-operated Ca2+ channel ROCE Receptor-operated Ca2+ entry ROS Reactive oxygen species RTK Receptor tyrosine kinase RV Right ventricle SMC Smooth muscle cell SOCC Store-operated Ca2+ channel SOCE Store-operated Ca2+ entry s.q. Subcutaneous injection - 9 -

SR Sarcoplasmic reticulum STIM Stromal interaction molecule TKR Tyrosine kinase receptor TRPC transient receptor potential channel VDCC Voltage-dependent Ca2+ channel VSMC Vascular smooth muscle cell WHO-FC World health organization functional classification WSPH World Symposium on Pulmonary Hypertension WU Wood Units

- 10 -

ABSTRACT

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. The increased pulmonary arterial pressure (PAP) in patients with

PAH is mainly generated by increased pulmonary vascular resistance (PVR) (18, 70, 103).

Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular wall stiffness are the major causes for the elevated

PVR and PAP in patients with PAH. Concentric pulmonary vascular remodeling is among one of the major causes for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP), also one the major causes for increasing afterload of right ventricle (RV) and inducing right heart failure leading to death if untreated (103). Excessive pulmonary artery smooth muscle cell (PASMC) proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in patients with PAH and animals with severe experimental pulmonary hypertension (PH).

2+ 2+ An increase in cytosolic free Ca concentration ([Ca ]cyt) in PASMCs is not only a trigger for PASMC contraction and pulmonary vasoconstriction, but also an important stimulus for

2+ PASMC proliferation, migration, and pulmonary vascular remodeling (16, 83, 85). [Ca ]cyt in

PASMCs is increased by Ca2+ influx through Ca2+-permeable cation channels in the plasma membrane (PM) and Ca2+ release or mobilization from the intracellular Ca2+ stores, mainly the sarcoplasmic (SR) or endoplasmic (ER) reticulum. There are at least three classes of Ca2+- permeable cation channels identified in human and animal PASMCs that are responsible for Ca2+ influx associated with excitation-concentration coupling (EC-coupling) and Ca2+-mediated

PASMC proliferation and migration: (i) voltage-dependent Ca2+ channels (VDCC), (ii) receptor- operated Ca2+ channels (ROCC), and (iii) store-operated Ca2+ channels (SOCC) (50, 57). VDCC are opened or activated by membrane depolarization due to, for example, decreased activity or

- 11 - downregulated K+ channels (57) while ROCC is opened or activated by ligand-mediated binding to membrane receptors including G protein-coupled receptors (GPCR) and tyrosine kinase receptors (TKR). Activation of GPCR or TKR upon binding to respective ligands increases production of diacylglycerol (DAG) and inosital 1,4,5-triphosphate (IP3), two important intracellular second messengers. DAG then activates ROCC and introduces receptor-operated Ca2+

2+ entry (ROCE), while IP3 activates IP3 receptors, also referred to as Ca release channels, in the

SR/ER membrane and induces Ca2+ release from the intracellular stores to the cytosol contributing

2+ 2+ 2+ to increasing [Ca ]cyt. Depletion or significant reduction of Ca levels in the ER/SR due to Ca mobilization or release leads to Ca2+ influx through SOCC, commonly referred to as store-operated

Ca2+ entry (SOCE). Active depletion of intracellularly stored Ca2+ in the SR/ER then also results in the dimerization and translocation of STIM1 (and/or STIM2) in the SR/ER membrane and forms

STIM protein puncta close to the SR/ER-plasma membrane junctions. Then the multimer STIM1/2 proteins in the ER-PM recruit Orai proteins in the plasma membrane to form SOCC responsible for SOCE (16). It has been shown that transient receptor potential (TRP) channels are involved in forming ROCC in PASMCs and can be activated directly by DAG. In our previous publishing, we have determined that the proliferative phenotype of PASMCs employs SOCC leading to increased expression levels of STIM2, not STIM1, and also Orai2 and TRPC6 expressions from IPAH patients- altogether providing an underlying mechanism for enhanced SOCE. TRP channels are also reported to participate in the regulation of SOCE in many cell types (21). In addition to TRP channels, many other types of Ca2+-permeable cation channels may also participate in ROCE and

SOCE.

Calcium homeostasis modulators (CALHM) including CALHM1 and CALHM2, have been identified as a family of physiologically important plasma membrane ion channels that are permeable to both cations and anions. These channels are allosterically regulated by membrane

- 12 - voltage (or membrane potential) and extracellular Ca2+; CALHM1 and CALHM2 channels are closed at the resting membrane potential but can be opened by strong membrane depolarization.

Reduction of extracellular [Ca2+] increases the probability for CALHM channels to open, which allow the channels to be activated at a negative potential. Ultimately, it is widely known that the

2+ 2+ increased [Ca ]cyt due to upregulated and activated Ca -permeable cation channels contribute to pulmonary vasoconstriction and excessive proliferation of PASMCs (and other cell types, for example fibroblasts and myofibroblasts) in patients with PAH, eventually this leads to concentric pulmonary vascular remodeling (17). Therefore, a rise in intracellular [Ca2+] and activated Ca2+ in

PASMC via upregulated and/or activated Ca2+-permeable cation channels play a major role.

CALHM1 and CALHM2 have a significant impact on the pathogenesis that lead to the development and progression of PAH.

As discussed earlier, sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling comprise of two major causes for the elevated PVR and PAP in patients with

PAH and animals with experimental PH (50). Pulmonary vasoconstriction is certainly a major cause for increasing PVR and PAP at the early stage of disease development, while concentric pulmonary vascular remodeling and obliterative intima and plexiform lesions are made up of the late state pathological changes that contribute to maintaining high PVR and PAP (57).

The transition from the contractile or differentiated phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling (62) in which we aimed at investigating. In this study, I hypothesized that CALHM1 and/or CALHM2 are involved in PASMC phenotypical transition from the contractile or differentiated phenotype to the synthetic or proliferative phenotype, while CALHM1/2 are upregulated in PASMC from patients with PAH and animals with experimental PH.

To test the hypothesis, I proposed the following specific aims in my thesis study:

- 13 -

Aim 1: To study whether CALHM1 and/or CALHM2 are involved in the phenotypical transition of PASMC. We identified contractile markers and proliferative markers that were used to represent a contractile phenotype of healthy smooth muscle cells from freshly isolated PA tissue

(with the endothelium removed) and a proliferative or synthetic phenotype as in primary cultured

PASMC prepared from PA tissue, together from the same animal; rat. This way we obtain a model that shows the ability of cells to change drastically towards the disease phenotype to assimilate smooth muscle cell diversity in PAH.

Aim 2: To study whether CALHM1 and CALHM2 are upregulated in highly proliferative

PASMC from patients with PAH and animals with experimental PH. Utilizing our proliferative and contractile phenotype model in smooth muscle cells, we measured and compared the expression of CALHM1 and CALHM2 in freshly isolated PA tissue (representative of the contractile phenotype) and primary cultured PASMCs from the same rat (representative of the proliferative phenotype) to see that the displaced environment could shift the cells' characterization markers to match disease protein expression.

Aim 3: To determine pharmacological interventions that target the proliferative phenotypical transition of PASMC in our proliferative PASMC model. Treatment of primary cultured PASMC with media containing drug rapamycin was used to observe proliferation measured by downregulated proliferative cell markers and increased expression of contractile protein markers. We examined whether inhibition of cell proliferation by inhibiting mTORC1 and mTORC2 activity using rapamycin affects CALHM1 and CALHM2 expression in PASMC.

- 14 -

SIGNIFICANCE AND INTRODUCTION

The pulmonary circulation is a circulatory system that delivers deoxygenated venous blood from the right ventricle (RV) through the pulmonary artery to the lungs to get the venous blood oxygenated in the lung capillaries and bring the oxygenated arterial blood through the pulmonary vein back to left atria. The oxygenated blood is then pumped to the whole body and all organs via left ventricle through the systemic circulatory system. Pulmonary hypertension (PH) is a condition in which pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) are increased due to genetic, epigenetic, and environmental causes as well as other diseases in other organs (e.g., congenital heart disease, chronic obstructive pulmonary diseases, sickle cell anemia)

(29, 36, 57). The elevated blood pressure in the lungs or the pulmonary circulation system, along with the elevated PVR, increases RV afterload and if not treated, causes right heart failure and death. The Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL

Registry) is an observational registry of the demographics, disease course, and management of patients with PAH in the United States. In spite of the recent progress and advancement of new therapeutic approaches, the 5-year survival of patients with advanced PAH remains poor (15), while the New York Heart Association Functional Classification (NYHA-FC) is an important predictor of future survival. These observations reinforce the importance of continuous monitoring of NYHA-FC in PAH patients and continuous developing of novel therapies that improve hemodynamics and RV function in PAH patients.

The normal value of mean PAP in healthy control subjects is 14.0±3.3 millimeters mercury

(mmHg) based on recent publications (77). The clinical criteria for diagnosis of PH has been updated recently; PH is now defined as mean PAP > 20 mmHg at rest, measured by right heart catheterization (RHC) (77). The hemodynamic definition of PH is reproduced and modified from reference 77 as shown in Table 1.

- 15 -

Table 1. Hemodynamic definition of pulmonary hypertension (PH)

Definition Characteristics Clinical Groups

mPAP > 20 mmHg Pre-capillary pulmonary PAWP ≤ 15 mmHg Groups 1,3,4 and 5 hypertension (PH) PVR > 5 WU

mPAP > 20 mmHg Isolated post-capillary PH PAWP >15 mmHg Groups 2 and 5 (IpcPH) PVR < 3 WU

mPAP > 20 mmHg Combined pre- and post- PAWP > 15 mmHg Groups 2 and 5 capillary PH (CpcPH) PVR > 3 WU

Note: mPAP, mean pulmonary arterial pressure; PAWP, pulmonary arterial wedge pressure; PVR, pulmonary vascular resistance; WU, Wood units; Group 1, PAH; Group 2, PH due to left heart disease; Group 3, PH due to lung disease and/or hypoxia; Group 4, PH due to pulmonary artery obstructions; Group 5, PH with unclear and /or multifactorial mechanisms (see Table 2).

- 16 -

PH is clinically classified into five groups based on pathogenic mechanisms, clinical manifestations, hemodynamic characteristics and therapeutic approaches at the World Symposium on Pulmonary Hypertension (WSPH) (77). The new classification of PH has also recently been published with a guideline for clinical management and translational research in the field of pulmonary vascular disease, Table 2 has been modified and reproduced from reference 77.

- 17 -

Table 2. Clinical classification of pulmonary hypertension (PH)

Group 1: 1.1 Idiopathic PAH Pulmonary arterial 1.2 Heritable PAH hypertension (PAH) 1.3 Drug- and toxin-induced PAH 1.4 PAH associated with: 1.4.1 Connective tissue disease 1.4.2 HIV infection 1.4.3 Portal hypertension 1.4.4 Congenital heart disease 1.4.5 Schistosomiosis 1.5 PAH long-term responders to calcium channel blockers 1.6 PAH with overt features of venous/capillaries (PVOD/PCH) involvement 1.7 Persistent PH of the newborn syndrome

Group 2: 2.1 PH due to heart failure with preserved left ventricular ejection fraction (LVEF) PH due to left heart disease 2.2 PH due to heart failure with reduced LVEF 2.3 Valvular heart disease 2.4 Congenital/acquired cardiovascular conditions leading to post- capillary PH

Group 3: 3.1 Obstructive lung disease PH due to lung diseases 3.2 Restrictive lung disease and/or hypoxia 3.3 Other lung disease with mixed restrictive/obstructive pattern 3.4 Hypoxia without lung disease 3.5 Developmental lung disorders

Group 4: 4.1 Chronic thromboembolic pulmonary hypertension (CTEPH) PH due to pulmonary artery 4.2 Other pulmonary artery obstructions obstructions

Group 5: 5.1 Hematological disorders PH with unclear and/or 5.2 Systemic and metabolic disorders multifactorial mechanisms 5.3 Others 5.4 Complex congenital heart disease

Note: PVOD, pulmonary veno-occlusive disease; PCH, pulmonary capillary hemangiomatosis

- 18 -

The lungs are the only organ that receives the entire cardiac output (CO), which should be maintained at a high flow, low resistance and low pressure system (50). When the pulmonary circulation fails to preserve this system there is a disruption in the transfer of blood carried into the pulmonary capillaries where the venous blood takes up oxygen and unloads excess carbon dioxide (CO2). This fatal hemodynamic abnormality in the lungs is often characterized as pulmonary hypertension. Pulmonary hypertension can be idiopathic (spontaneous or of unknown etiology), heritable, or secondary to other diseases, see Table 2.

When patients are in suspicion of a diagnosis towards pulmonary hypertension, they are evaluated and undergo extensive diagnostic testing to confirm PH exists. PH is defined by an elevated mean pulmonary arterial pressure (mPAP) greater than 20 mmHg, measured by a performed right-heart catheterization (RHC). Patients with PH are then classified into five groups based upon the mechanisms behind the disease as well as etiology (Table 2). All patients in Group

1 are considered to have pulmonary arterial hypertension (PAH) which include heritable primary causes and drugs, as well as connective tissue disease. Patients in the classification of Group 2 are diagnosed with PH due to left-sided heart disease. Patients within group 3 are diagnosed due to chronic lung disorders and hypoxemia. Group 4 classification is if the disease is induced by pulmonary artery obstructions. Finally, Group 5 is classified to those who are considered to have

PH due to unidentified mechanisms (18, 77). The term PH is then used when the five groups are described collectively.

Currently, the diagnostic algorithms commence with a collection of symptoms, signs and historical features. A list of identified symptoms defined by the Mayo Clinic include shortness of breath (dyspnea), initially while exercising and eventually while at rest, fatigue, dizziness or fainting spells (syncope), chest pressure or pain along with edema, especially noticeable in the ankles, legs, and eventually in the abdomen. Unfortunately, signs and symptoms of PH can be

- 19 - nonspecific as well and progress in present dyspnea and fatigue to eventually severe PH with right ventricular failure in place (8). A recent study has found that 1 in 5 patients in the REVEAL

Registry who were diagnosed with PAH take more than 2 years of reported symptoms before their disease is recognized (9). Another unfortunate feature of PH, includes its predominance found/diagnosed in women- where registry data worldwide demonstrate a predominance for PAH of 2-4 over men (4) which has led to studies that investigate gender differences within the discriminating disease, more so having to do with X chromosome expressing gene functions

(estrogen receptor) that amplify disease features (reference Table 3, modified and reproduced from reference 15, for breakdown of ratios in registries across various countries) (4).

- 20 -

Table 3. Female to male ratio in major PAH registries

Time Period Registry Number Median Age Overall F:M (years) Ratio 1981-1985 USA/NIH 187 36 1.7:1

1982-2006 USA 578 48 3.3:1

1986-2001 Scotland 374 51 2.3:1

1999-2004 China 72 36 2.4:1

2000-2010 Czech Republic 191 52 1.9:1

2001-2009 UK/Ireland 482 50 2.3:1

2002-2003 France 674 50 1.9:1

2006-2007 USA/REVEAL 2,535 50 3.9:1

2007-2008 Spain 866 45 2.5:1

2007-2013 Europe/COMPERA 1,283 68 1.8:1

Note: Female to male registries across the world15.

- 21 -

Current therapies and strategies for PAH suggest induced relaxation of the pulmonary arteries, diuretics to help with edema, oral anticoagulants, or feeding supplemental oxygen. Others include the correction of increased ratio of circulating vasoconstrictors, such as endothelin, to vasodilators that include prostacyclin (PGI2) and nitric oxide (NO) by blocking endothelin receptors. The list grows by treating with increasing circulating PGI2 or orally in-taking PGI2 analogues, inhaling nitric oxide (NO), stimulating soluble guanylate cyclase or inhibiting phosphodiesterase, but they are not robustly aimed at the vascular remodeling that is a main characteristic of PAH (35, 85). Although these therapies do relieve or improve some of the symptoms that patients often experience- they do not rid the patient of progressing disease unfortunately, this only dissipates ongoing progression. Approximately 15% of patients die within a year of medical follow up despite having had treatment (51). Despite some improvements drug interventions have had on the symptoms of patients and even improved survival, most become resistant to therapy and yield to disease (57). Table 4 shows a list of drugs approved by FDA to treat PAH during the last 20 years and Figure 1, modified and reproduced from reference 51, depicts the treatment algorithm for PAH or Group 1 PH.

- 22 -

Table 4

Table 4. Drugs approved by FDA to treat PAH

Commercial Name Drug action and Drug (FDA approval Pharmacological Action mechanisms time)

Epoprostenol (i.v.) Flolan (1995) Prostacyclin (PGI2) Activate PGI2 IP receptor to increase cGMP/PGK Epoprostenol (i.v.) Veletri (2010

Bosentan (p.o.) Tracleer (2001) Dual ET receptor blocker Block endothelin receptors A (ETA) and B (ETB)

Treprostinil (s.q.) Remodulin (2002) PGI2 analogue Treprostinil (i.v.) Remodulin (2005) Treprostinil (inh.) Tyvaso (2009) Treprostinil (p.o.) Orenitram (2013

Iloprost (inh.) Ventavis (2004) PGI2 analogue Activate IP receptor to increase cGMP/PKG

Sildenafil (p.o.) Revatio (2005) Phosphodiesterase (PDE) Inhibit PDE4 to enhance inhibitor endogenous NO

Ambrisentan (p.o.) Letairis (2007) Selective ETA blocker Selectively block ETA

Tadalafil (p.o.) Adcirca (2009) PGI2 analogue

Riociguat (p.o.) Adempas (2013) Soluble guanylate cyclase Activates sGC to increase (sGC) stimulator cGMP and activate PKG

Macitentan (p.o.) Opsumit (2013) PGI2 analogue

Salexipag (p.o.) Uptravi (2015) PGI2 analogue PGI2 IP receptor agonist

Note: i.v., intravenous injection; inh., inhalation; p.o., oral administration; s.q., subcutaneous injection

- 23 -

Figure 1 - Treatment algorithm of pulmonary arterial hypertension

Figure 1. Treatment algorithm of pulmonary arterial hypertension (PAH) or Group 1 pulmonary hypertension (PH) (51). CCB, calcium channel blocker; WHO FC, World Health Organization Functional Classification.

- 24 -

Pulmonary arterial hypertension is a progressive disease where increased pulmonary arterial pressure (PAP) is generated by increased pulmonary vascular resistance (PVR) (70, 103).

PVR and PAP are caused by the intraluminal obliterations and occlusions of small pulmonary arteries and arterioles, and the neointimal and plexiform lesions in the pulmonary vasculature- all play critical roles in the progression of pulmonary hypertension (17, 103). Elevated PVR in patients is caused by sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. Once at a late stage with PAH, patients experience a gradual transition from sustained vasoconstriction to vascular remodeling characterized by adventitial, medial, and intimal hypertrophy of the pulmonary arteries (81). Pulmonary vascular remodeling occurs in the forms of pulmonary vascular wall thickening, small vessel obliteration, and formation of plexiform lesions of which in turn leads to elevated right ventricular afterload (58, 96). If left untreated, PAP imposes a sustained pressure load on the right ventricle resulting in its hypertrophy and can advance to right heart failure and lead to death (41). A loss or decrease on the compliance of the pulmonary arterial wall, for example, when the pulmonary vascular wall stiffens due to pulmonary vascular remodeling leads to progressive pulmonary hypertension (50, 52, 57). As mentioned previously, the lungs are the only organ in the body to receive the entire CO; therefore, it is of high importance to maintain a high-flow, low resistance, low pressure system within the pulmonary circulation. CO together with PVR are factors of the resulting pulmonary artery pressure (PAP),

Formula 1: PAP= CO × PVR

Formula 2: PAP = CO × [PVRarteries + PVRcapillaries + PVRveins] (50)

- 25 -

Given these equations, PAP measured in millimeters mercury (mmHg), can be increased by an increase in CO while also having PVR contributed by arteries, capillaries, and veins which are all governed by physical laws that can predict a very small change in the diameter of the pulmonary vasculature can significantly increase PVR (50). Anytime a liquid flows, such as blood, through a cylindrical structure, in this case a vessel, the resistance of such tube is inversely proportional to the fourth power of the radius of lumen of the vessel, this is demonstrated by physicist Poiseuille's formula known as Poiseuille's Law (50, 63). It states that for laminar flow of a fluid (liquid or gas) along a pipe:

Formula 3: ��� = !"# $%!

where L is the length of the vessel, η is the coefficient of viscosity of blood, and r is the inner radius of the vessel.

Elevated pulmonary arterial pressure PAP in patients with PAH is mainly caused by an increase in PVR. Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH (16, 57). Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype (16). Although there is crosstalk between endothelium and vascular smooth muscle in vasoconstriction, in particular, we focus on the role of SMCs within

PAH.

- 26 -

PHENOTYPICAL SWITCH AND PAH

Vascular smooth muscle cells (VSMCs) in pulmonary arterial hypertension (PAH) have been a recent interest due to their phenotypic diversity which can perform both contractile and synthetic functions. The resulting phenotypic diversity of SMCs has been studied as a function of collective gene programming, biochemical factors, extracellular matrix components and physical factors such as stretch and shear stress (68). Another way of defining this change is cellular differentiation, which is defined as the process by which multipotential cells in the developing organism acquire cell-specific characteristics that distinguish them from other cell types. Three major regulatory components have been suggested in VSMCs multifunctional development: a) selective activation of the subset of genes required for the cell’s differentiated functions, b) the coordinate control of expression of cell selective and/or specific genes at precise times and stochiometries and lastly c) there is the continuous regulation of gene expression through effects of local environmental cues on the genetic program that determines cell lineage, which includes the control of chromatin structure or epigenetic programming that can influence the ability of transcription factors to access regulatory regions of genes (61).

In mature animals, VSMCs are specialized cells that contract and regulate blood vessel tone, diameter, blood pressure, and blood flow distribution (61). Differentiated SMCs in adult blood vessels proliferate at an extremely low rate, exhibit low synthetic activity, and express a collection of contractile proteins, ion channels, and signaling molecules required for the cell’s contractile function (1, 60, 82). They produce the extracellular matrix (ECM) during development which provides the arterial wall with the capacitance for high pressure and to maintain a healthy contractile tone (peripheral resistance to flow). This is mainly dependent on sympathetic nervous system signaling that is also responsible for the redistribution of blood flow in relation to organ

- 27 - specific metabolic demand which entails the cardiac output generated by the heart (42). VSMCs present within the arterial wall are highly sensitive to the mechanical stress (42).

A phenotypic switch of PASMC from a contractile to proliferative phenotype is inevitable for any pathological and physiological vascular remodeling process to occur. In healthy arteries,

SMCs are surrounded by a basement membrane composed of laminin, collagen type IV, and heparan sulfate proteoglycan (38). In the normal arterial media, VSMCs express a range of 'SMC markers' which include Smooth muscle cell myosin heavy chain (MHC11 in our hands),

SM22α/TAGLN22, Smooth muscle cell actin (ACTA2), smoothelin and others (7, 10) (reference

Table 5 modified and reproduced from reference 68, for a complete list), all of which are important factors of VSMCs' ECM integrity. During the development of intimal hyperplasia (IH), for example, and other vascular pathologies, such as restenosis and atherosclerosis, SMCs lose their contractile proteins and cellular quiescence and increase their proliferation, migration, and production of ECM proteins (6). These processes define a shift from normal, contractile SMC phenotype toward a gradient shift towards a synthetic or proliferative phenotype. Figure 2 shows an example of Immunoflourescent staining of human coronary artery SMCs in culture that have been treated to re-induce contractile phenotype (5, 6).

- 28 -

Table 5

Marker Protein Phenotype Subcellular Function specificity localization a-smooth muscle C > S Contractile filaments Contraction actin/ACTA2 Smooth muscle- C Contractile filaments Contraction myosin heavy chain

SM22a/TAGLN22 C > S Actin-associated Regulation contraction SM-calponin C > S Actin- Regulation associated/cytoskeleton contraction/signal transduction H-caldesmon C Actin-associated Regulation contraction Smoothelin C Actin-associated Regulation contraction Telokin C > S Cytoplasmic/membrane Regulation contraction Meta-vinculin C > S Cytoskeleton Anchoring cell-ECM Desmin C > S Cytoskeleton Structural mechanical integrity CRBP-1 S > C Cytoplasm Retinoid transport and metabolism Smemb S > C Contractile filaments Contraction Collagen I S Cytoskeleton ECM framework Collagenase IV S > C Cytoskeleton ECM framework Connexin43 S > C Cytoskeleton ECM framework Vimentin S > C Cytoplasmic type II Maintains cell shape intermediate filament and integrity of cytoplasm Tropomyosin/TPM4 S > C Actin-associated Contraction S = Synthetic, C = Contractile

- 29 -

Figure 2 – Immunoflourescent staining of human SMCs

Figure 2. Immunoflourescent staining of human coronary artery SMCs in culture that have been treated to re-induce contractile phenotype (5, 6). A: Calponin (green) co-localizes (yellow) with smooth muscle a-actin (SMaA) (red) fibrils in the central region of the cells. B: SM22a (green) co-localizes (yellow) along the SMaA (red) fibrils. Nuclei are counterstained with DAPI (blue), scale bars = 50 µm (6).

- 30 -

In contrast, SMC activations following arterial injury is associated with the disappearance of laminin and other basement membrane structures and the appearance of abundant deposits of proliferative cells in the media and intima (38). The types of intima thickening are varied. They can be briefly summarized as based on the predominance of collagen and mucin, fibroblastic-like cells, or endothelial cells (97). Hypertension and the excessive strain on the ECM (mainly elastin in physiological conditions) stretches the VSMCs, which transduces intracellular signals of tensegrity (37, 42). The ECM integrin-cytoskeleton interactions play a role in mechanosensing, which enables VSMCs to respond and react to changes in intraluminal pressure (42). This then allows hypertrophic inward remodeling of resistance arteries characterized by reduction in lumen diameter and in an increased media to lumen ratio. The transformation of SMCs from the contractile to the synthetic proliferative state is an important indicator towards the progression of a pathological state. In vascular diseases such as atherosclerosis, this switch has also described increases in lipid phagocytosis of SMCs and their transfer of intima of the arteries which then are characterized as foam cells due to their proliferative properties (24, 107). The list of factors that can activate or inhibit them continues to grow. A key component of this pathological mechanism, more specifically, the proliferation of VSMCs holds PDGF with a key role within this cell population in PAH (4, 73). PDGF drives hypertrophy and proliferation as it is significantly increased in lung tissue from PAH patient in comparison to control lungs, like many others (TGF-

β1, TLR-4, IL-1β, IL-18, etc.) (4, 66, 74). PDGF mediated SMC proliferation involves an increase

2+ in cytosolic free calcium concentration [Ca ]cyt which is also a key stimulus for this phenotypic transition and our motive of investigating this induced signaling cascade that ties together and encompasses most proliferating contributing factors (4, 57).

- 31 -

Figure 3 - PASMC phenotypic plasticity

A PASMC Phenotypic Plasticity

§ PDGF-BB results in SMC phenotypical transition from contractile to proliferative phenotype, causing PA remodeling. § Rapamycin induces SMC differentiation from proliferative to contractile phenotype, by inhibiting Akt/mTOR signaling pathway. § Prostacyclin induces SMC differentiation, reversing PA remodeling B Synthetic phenotype Contractile phenotype

α1 integrin, β1 integrin, α7 integrin, Myocardin, N/-T-cadherin, SMA-α1, Desmin, SM22α, ACLP SM-calponin, h-caldesmon, meta-vinculin, CRBP-1, Smemb Y-SMA, APEG-1, CRP-2

PDGF-A, I-caldesmon, Osteopontin, SM-MHC, Smoothelin ICAM-1, MGP

Collagen I, Collagenase IV, Conexin43, MMP isoforms, Syndecan-1/4, Moesin

Figure 3. Schematic diagram showing changes or different markers during the phenotypical transition between the contractile (or differentiated) phenotype and the synthetic (or proliferative) phenotype of smooth muscle cells. PDGF, platelet-derived growth factor; SMA-α1, smooth muscle α1-actin; SM22α, transgelin (or TAGLN); SM-MHC, smooth muscle myosin heavy chain (or MHC11); PA, pulmonary artery.

- 32 -

ION CHANNELS AND PULMONARY HYPERTENSION

2+ 2+ A rise in cytosolic Ca concentration ([Ca ]cyt) in PASMCs triggers pulmonary vasoconstriction and is a key stimulus for PASMC proliferation and migration, which subsequently

2+ results in pulmonary vascular remodeling (16, 85). In PASMCs, [Ca ]cyt is an important determinant of contraction, migration, and proliferation that contributes to the development and progression of concentric pulmonary vascular remodeling and arteriole muscularization (57). A

2+ rise of [Ca ]cyt in PASMC can activate transcription factors and further signal transduction of

2+ proteins that are necessary for cell proliferation and cell migration (85). [Ca ]cyt in PASMCs can be increased by:

• Ca2+ influx through voltage-dependent Ca2+ channels, receptor-operated Ca2+ channels

(ROC), and store-operated Ca2+ channels (SOCC), and

• Ca2+ release from intracellular stores such as the sarcoplasmic reticulum (SR) via Ca2+

release channels (IP3 receptors and ryanodine receptors are examples) (57).

2+ 2+ 2+ [Ca ]cyt in PASMCs can also be increased by inward transport of Ca via Ca transporters in the plasma membrane, such as the reverse mode of Na+/Ca2+ exchanger, which is considered an

2+ 2+ important pathway for increasing [Ca ]cyt (57). In opposition, [Ca ]cyt in PASMCs can be decreased by:

• Ca2+ ejection by the Ca2+-Mg2+ ATPase, Ca2+ pump; by the forward mode of Na+/Ca2+

exchanger in the plasma membrane, and

• Ca2+ sequestration by the Ca2+-Mg2+ ATPase in the SR (also known as SERCA) (57)

Similarly, a decreased expression of K+ channels leads to membrane depolarization and

2+ contributes to sustained elevation of [Ca ]cyt. This is accomplished by facilitating the production

- 33 -

2+ 2+ of IP3, which stimulates the release of SR Ca into the cytoplasm and promoting Ca entry via the reverse mode of Na+/Ca2+ exchange.

2+ 2+ 2+ A rise in cytoplasmic [Ca ] also rapidly increases the nuclear [Ca ] ([Ca ]n) due to the

2+ 2+ fact of the nuclear pore envelope being highly permeable to Ca (88). An increase in [Ca ]cyt and

2+ 2+ [Ca ]n stimulates cell proliferation by activating Ca -dependent kinases such as CaMK and transcription factors that include c-Fos, nuclear factor of activated T-cells (NFAT), and cAMP response element binding protein (CREB) that are necessary for cell proliferation (40, 88, 100).

Ca2+ can also affect gene expression by interacting with protein kinase C (PKC) and calmodulin

(Came) as well as by activating proteins involved in the cell cycle (cyclins and cyclin dependent kinases) (19, 39). Similarly aside from gene expression, calmodulin is activated when Ca2+ rises in the cytosol, which leads to the activated Ca2+/calmodulin (CaM) increased phosphorylation of myosin light chain kinase (MLCK) (94). This subsequently phosphorylates myosin heavy chain allowing myosin interaction with actin and finally causes smooth muscle contraction and pulmonary vasoconstriction. In addition to stimulating quiescent cells to enter the cell cycle (G0 to

2+ G1 transition), Ca /CaM is also required for the transition from G1 to S (DNA synthesis) phase and from G2 phase to mitosis, and the progression of mitosis (67).

2+ In vascular smooth muscle cells, an increase in [Ca ]cyt is also associated with a rise in

2+ 2+ 2+ 2+ 2+ [Ca ] in the SR ([Ca ]SR) (32) and the nuclei ([Ca ]n); all increases in [Ca ]cyt , [Ca ]SR and

2+ [Ca ]n are essential for cell proliferation. In the presence of serum and growth factors, removal of extracellular Ca2+ and depletion of intracellularly stored Ca2+ in the SR both significantly inhibit

PASMC proliferation, further establishing the role of Ca2+ for the cell cycle progression and cell proliferation (44, 85). Removal of extracellular Ca2+ abolishes agonist-induced pulmonary vasoconstriction and also significantly inhibits PASMC proliferation in culture (89). Inhibition of canonical TRP (TRPC) channel expression with antisense oligonucleotides also markedly

- 34 - decreases the amplitude of capacitative Ca2+ entry (CCE), or store-operated Ca2+ entry (SOCE), and significantly inhibits PASMC proliferation (57). We have previously reported that the resting

2+ [Ca ]cyt in PAH-PASMCs is significantly higher than in normal PASMCs, while ROCE and store- operated Ca2+ entry SOCE are both enhanced in PAH-PASMCs compared to normal PASMCs

(25). Thus upregulation of TRPC channels is also a significant mechanism in the induction of

PASMC proliferation; as enhanced CCE, possibly via this upregulation may represent a critical mechanism involved in the development of severe PAH (16, 57).

As shown in Figure 4, reproduced from reference 57, there are multiple potential

2+ pathogenic “hits” or molecular causes involved in development of PAH. A rise in [Ca ]cyt in

PASMCs (due to decreased Kv channel activity (1) and membrane depolarization, which opens voltage-dependent Ca2+ channels (VDCC); upregulated TRPC channels that participate in forming receptor- (ROC) and store-operated (SOC) Ca2+ channels (2); and upregulated membrane receptors [e.g., serotonin, endothelin and/or leukotriene receptors (3) and their downstream signaling cascades] causes pulmonary vasoconstriction, stimulates PASMC proliferation, and inhibits the BMP-signaling pathway that leads to antiproliferative and proapoptotic effects on

PASMCs. Dysfunction of BMP signaling due to BMPR2 mutation and BMP-RII/BMP-RI downregulation (4) and inhibition of Kv channel function and expression (1) attenuate PASMC apoptosis and promote PASMC proliferation. Increased angiopoietin-1 (Ang-1) synthesis and release (5) from PASMCs enhance 5-HT production and downregulate BMP-RIa in PAEC and further enhance PASMC contraction and proliferation, whereas inhibited nitric oxide and prostacyclin (PGI2) synthesis (6) in PAECs would attenuate the endothelium-derived relaxing effect on pulmonary arteries and promote sustained vasoconstriction and PASMC proliferation.

Increased activity and expression of the 5-HT transporter (5-HTT) (7) would serve as an additional pathway to stimulate PASMC growth via the mitogen-activated protein kinase (MAPK) pathway.

- 35 -

Furthermore, exogenous viral and bacterial infection and inflammation (8) may contribute to vasoconstriction and vascular medial hypertrophy in patients with mutations in multiple genes or with “susceptible predispositions” in these pathways. In addition, a variety of splicing factors, transcription factors, protein kinases, extracellular metalloproteinases, and circulating growth factors would serve as the so-called “hits” or facilitators to mediate the phenotypical transition of normal cells to contractive or hypertrophied cells and to maintain the progression of PAH. SR indicates sarcoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PLC, phospholipase C; PKC, protein kinase C; GPCR, G protein coupled receptor; RTK, receptor tyrosine kinase, PDGF, platelet-derived growth factor; ROS, reactive oxygen species; and AVD, apoptotic volume decrease.

- 36 -

Figure 4 – Mechanisms and signaling cascade of Ca2+ in PAH

Figure 4. Proposed pathogenic mechanisms and Ca2+ signaling cascades in pulmonary vascular smooth muscle and endothelial cells that are involved in the development and progression of PAH (57).

- 37 -

CALCIUM HOMEOSTASIS MODULATOR

The calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C (48), has been identified as a physiologically important plasma membrane ion channel that is permeable to both cations and anions, regulated by membrane voltage and extracellular Ca2+ concentration.

Through convergent evolution, CALHM has structural features that mimic that of pannexin and innexins’ gene families that include four transmembrane helices with cytoplasmic amino and carboxyl termini, based on membrane topology prediction algorithms (Figs. 5 and 6). Human

CALHM1 is predicted to be a membrane protein with 346 amino acids. The CALHM1 channel is a hexamer of CALHM1 monomers containing a functional pore diameter that is ~14 Å in size (48)

(Fig. 5). As mentioned previously, CALHM1 enables both cations and anions due to its inability to discriminate between the two charged molecules, consequently both Ca2+ and ATP can permeate as signaling molecules through its pore. In the presence of physiological Ca2+ (~1.5 nM), CALHM is closed at membrane resting potentials but can be opened by strong depolarizations. Thus, reducing extracellular Ca2+ increases channel opening probability, enabling channel activation at negative membrane potentials (48) (Fig. 6).

- 38 -

Figure 5

Figure 5. Structure of a dimer of undecameric human CALHM2, active open state. RCSB PDB ID: 6UIV

- 39 -

In humans, there have been five homologs of CALHM identified. The CALHM gene family, CALHM1, and its homologues were collectively identified as the FAM26 gene family, where six genes are in two clusters on two chromosomes. CALHM1/FAM26C is clustered on chromosome 10 with FAM26A and FAM26B genes, which were designated as CALHM3 and

CALHM2, respectively. FAM26D, FAM26E, and FAM26F genes, of which CALHM names have not been assigned, are located in a cluster on chromosome 6. All of the CALHM genes and previously named FAM26 genes, are present throughout vertebrates and conserved across more than 20 species including mouse, human, and C. elegans which are suggested of biological importance in disease processes (48).

- 40 -

Figure 6

2+ Figure 6. Voltage- and Ca o-gated CALHM channels. a) Transmembrane topology of CALHM channel. Asn72, Pro86, Asp121, and Asn140 are indicated for human CALHM1. ECL1 and ECL2 are extracellular loops 1 and 2, respectively. N amino terminus, C carboxyl terminus, TM transmembrane domain. b Normalized conductance-voltage (G-V) relations for human CALHM1 in 5 mM Ca2+ and 0 Ca2+. Solid lines are Boltzmann function fits to the experimental data. c CALHM1 channel contains a voltage sensor and a Ca2+ sensor that detects extracellular Ca2+ concentration. The CALHM channel 2+ gate is closed by physiological [Ca ]o and hyperpolarized voltages (left). Depolarization and/or 2+ + 2+ lowering [Ca ]o can open the gate, enabling Na and Ca influx and ATP efflux (right). Reproduced from Ma et al., Pflugers Arch. 468:395–403, 2016 (48).

- 41 -

CALHM expression though still lacking full clarity in its functional features and gating mechanisms. In recent studies, whole-cell patch clamp electrophysiology of CALHM1 expressed in mammalian cell cultures, revealed the presence of a new Gd3+-sensitive and Ca2+ permeable outward rectifying current (12). Interestingly enough, when extracellular Ca2+ was re-introduced

2+ 2+ to cells who had been lacking Ca in the media- there was a sustained rise in [Ca ]cyt possibly due to artificial responses caused by leaked extracellular Ca2+, multiple studies have coincided with similar results (20, 47, 76). In conclusion, the apparent increased Ca2+ permeability in response to Ca2+ removal and re-introduction is insensitive to both genetic and pharmacologic inhibition of store-operated Ca2+ entry (SOCE) (48) and so the magnitude of CALHM1- dependent

2+ SOCE and [Ca ]cyt signals is solely additive (12, 47).

In hippocampal HT-22 cells, CALHM1 expression led to a robust and relatively selective activation of the Ca2+-sensing kinases-ERK1/2. CALHM1 also triggered activation of MEK1/2, the upstream ERK1/2-activating kinases, and of RSK1/2/3 and MSK1, two downstream effectors of ERK1/2 signaling. CALHM1-mediated activation of ERK1/2 signaling was controlled by the small GTPase, Ras. Rho (Ras homologous) GTP-binding proteins regulate many cellular processes, including gene transcription, differentiation, proliferation, hypertrophy, apoptosis, phagocytosis, adhesion, migration, contraction, and are recognized as GTPases in endothelial dysfunction (57).

In PAH, ERK1/2 signaling is activated in response to growth factors regulating vascular endothelium (VEGFR), fibroblast growth factor (FGFR), and PDGF receptor (2). The signaling pathways that include ERK, c-Jun-N-terminal kinase, p38 mitogen activated protein kinases, Akt,

Rho/Rho-kinase, and calcineurin/calmodulin kinases have been associated with cofactors and transcription factors that are in part mediated by transcription repression in phenotypic modulation of vascular smooth muscle cells (VSMC) as well (42, 61). In addition, pharmacological inhibition

- 42 - of CALHM1 permeability using Ruthenium Red, Zn2+, and Gd3+, or expression of the CALHM1

N140A and W114A mutants, which are deficient in mediating Ca2+ influx, prevented the effect of

CALHM1 on the MEK, extracellular signal-regulated kinase (ERK), RSK and MSK signaling cascade, demonstrating that CALHM1 controlled this pathway via its channel properties which is also an implication towards its significant role in SMC proliferation in PAH (13).

Since CALHM1 localizes to the ER as well as to the plasma membrane, it has been speculated that CALHM1 might contribute to the increase in the Ca2+ leak from the ER. This possibility is very interesting, as the increased Ca2+ leak would lead to a decrease in the Ca2+

2+ concentration inside the ER ([Ca ]ER) which in turn, could produce ER stress and the unfolded protein response, a reaction that triggers both homoeostatic and pathophysiological mechanisms.

Gallego-Sandin et al. (20) directly address this hypothesis by selectively monitoring Ca2+ transport through the ER membrane using ER- targeted aequorin Ca2+ probes. It is found that CALHM1

2+ 2+ 2+ both increased Ca leak and decreased Ca pumping into the ER. As a result, [Ca ]ER decreases and an unfolded protein response cascade is activated (20).

The current research on CALHM1 is known in the neuroscience field of research, originally was identified as a possible modifier of the age of onset of Alzheimer’s disease and is presently in cerebral-cortical neurons that can also release ATP; as well as signaling from mature taste buds. CALHM1 has been identified as a novel ATP-permeable channel that mediates the action potential-dependent release of ATP from taste cells (TBCs) to the afferent gustatory nerves, therefore CALHM1 is the first example of voltage-gated ATP channels that can mediate action potential-dependent fast purinergic neurotransmission (95). Having known that CALHM is a large enough membrane ion channel that can permeate ATP, CALHM has been linked as a relevant target to attenuate stroke injury (11). It regulates cortical neuronal excitability in response to reduced extracellular Ca2+ concentrations and it is able to forms the endogenous ATP-permeable

- 43 - channel complex in Type II TBCs as wells as large pores in mouse cortical neurons. A recent study investigated the contribution of CALHM1 to post anoxic depolarization and cell death during cerebral ischemia, and found that the pharmacological inhibition or knockdown of CALHM1 provides protection in ischemia (11). Additionally, signaling from mature TBCs shows greater reductions in fat preferences in CALHM1 than CD36 knockout mice (75). The authors focused on the members of the CALHM family, CALHM2 (FAM26B) and CALHM3 (FAM26A), using electrophysiological recording in heterologous expression systems. They found that co-expression of CALHM1 with CALHM3, but not with CALHM2, drastically enhances the activation kinetics of outward currents (presumably induced by ATP release) compared to expression of CALHM1 alone (59, 75). In fact, cells transfected with CALHM1-3 exhibited gating kinetics similar to the kinetics in intact TBCs. Given these results, the authors hypothesized that CALHM1 and

CALHM3 form a complex to mediate rapid taste neurotransmission (59). RT-PCR analyses allowed us to identify transcripts of all CALHM isoforms (CALHM1, CALHM2 and CALHM3) in the lung tissues (Fig. 7).

Intracellular concentration of ATP ([ATP]i) is around mM range, while extracellular ATP concentration is very low. Therefore, ATP released from PASMC and PAEC through CALHM1/2 channels can serve as an important ligand to stimulate PASMC proliferation and migration through activation of purinergic receptors (P2Y and P2X). These elucidating discoveries direct us to hypothesize that CALHM may plan an important role in the development and progression of

2+ pulmonary arterial hypertension due to its important role in regulating [Ca ]cyt in PASMCs.

- 44 -

Figure 7 – CALHM 1-3 mRNA expression level

Figure 7. CALHM 1-3 mRNA expression level in rat brain, lung, PA, and PASMCs (Reference Table 6 for Primers' sequence).

- 45 -

Table 6 – CALHM 1-3 PCR Primer Sequence

Gene PCR Primer Sequence

CALHM1 Fwd: 5-TAG-GCA-ATG-GGA-GCC-TGG-TG-3

Rev: 5-CCC-AGC-GCC-TGA-GAG-ATG-C-3

CALHM2 Fwd: AGT-TTG-TGG-ACC-CCT-CCT-CG-3

Rev: 5-AGC-CAT-CCA-AAG-AGC-TGG-GAC-3

CALHM3 Fwd: 5-AAT-GCC-CTC-TAT-GGC-CTG-GG-3

Rev: 5-AGC-ACA-TGT-ACC-TGA-TGA-TGC-C-3

- 46 -

MATERIALS AND METHODS

Pulmonary Artery Isolation

Protocols involving the use of experimental animals for all experiments were reviewed and approved by the Ethics/Animal Care Committee (IACUC) of the University of California, San

Diego and The University of Arizona, Tucson. Isolation of rat pulmonary artery smooth muscle tissue followed the same protocol as in our recent published article STIM and Orai Proteins in

Calcium Signaling(16). Sprague-Dawley male rats (150–200 g) were decapitated, and the whole lung and heart were removed and placed in warm Hanks’ balanced salt solution (HBSS, Life

Technologies, Carlsbad, CA) supplemented with 10 mM N-2 hydroxyethylpiperazine-N=-2- ethanesulfonic acid (HEPES; Sigma Aldrich, St. Louis, MO). The right and left branches of the main pulmonary as well as the intrapulmonary arteries were first isolated from the whole lung with precise forceps under a dissecting microscope. The fat and connective tissues were then removed gently from the isolated pulmonary artery (PA) under sterile conditions. The isolated PA was incubated in HBSS containing 1.7 mg/ml collagenase type II (Worthington Biochemical;

Lakewood Township, NJ) for 20 min at 37°C. The shortly digested PA ring was rinsed with HBSS to remove residual collagenase and the adventitia of the PA ring was carefully stripped off with forceps, and the endothelium was gently scratched off with dull forceps. The remaining PA smooth muscle tissue was then used to prepare single PASMC and to extract total protein for Western blot experiments.

Isolation and preparation of rat PASMC

Following removal of the adventitia and endothelium, the rat PA was further digested in

HBSS 1.7 mg/ml collagenase type II, 0.5 mg/ml elastase (Sigma), and 1 mg/mL of bovine serum albumin (BSA, Sigma) at 37°C for 50 min. The PA tissue was agitated every 15–20 min to speed

- 47 - digestion. The dispersed PA tissue was then triturated approximately 8–10 times with a Pasteur pipette to further dissociate the cells. Ten milliliters of SMC medium with 5% fetal bovine serum

(FBS, Corning), was then added to the enzymatic solution to stop digestion. The cell suspension was centrifuged for 5 min at 1,200 rpm held at room temperature (22–24°C). The supernatant was aspirated off and the resulting pellet was re-suspended in 2mL of fresh 5% FBS-SMC medium and triturated to separate the cells. For experiments using freshly dissociated PASMC, aliquots of the cell suspension were plated directly onto glass coverslips with 2.5 ml of 5% FBS-SMC medium.

These freshly dissociated rat PASMC were allowed to attach to the coverslips overnight before fresh SMC media was added. The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. The medium was changed 24 h after initial seeding and every 48 h subsequently. When the cells reached 80–90% confluency, they were gently washed with phosphate-buffered saline

(PBS), incubated briefly with 1 ml of 0.025% trypsin-EDTA solution until detachment (3–5 min), and then 9 ml 5% FBS-SMC media was added to the plate. The cell suspension was then transferred to a sterile 15-mL round-bottom tube, centrifuged at room temperature for 5 min at 200 g (1,500 rpm), re-suspended in the appropriate growth media and seeded onto coverslips or petri dishes. The freshly dissociated cells plated directly into the six-well dishes then cultured and transferred directly onto 10-cm petri dishes with 5% FBS-SMC to then be collected as one sample for WB or PCR experiments.

Primary PASMC Isolation from Rat PA Protocol (Figs. 8 and 9) (by Marisela Rodriguez)

Day 1: Pulmonary Artery Isolation (~2 hours total) (Fig. 8)

• Sterilize materials under UV light for 15 minutes

o Microscope

o 10 ml beaker

- 48 -

o 50 ml beaker

o 500 ml beaker

o Gel Petri dish

o One 6-well-plate

o Dissection tools: two forceps, pins, scissors

o Plastic Pasteur pipets

• Warm PASMC media in 37ºC water bath for 30 min (can also use 10% FBS/DMEM)

• Fill 6-well-plate with alternate HBSS- betadine- HBSS- betadine, and leave under hood

• Fill gel Petri dish with HBSS solution and keep near surgical work area

• Sacrifice rat by decapitation. Clean chest area with 70% ethanol, then betadine, and

ethanol once again

• Open chest and abdomen to isolate lungs and heart (Fig. 9)

• Place lungs and heart into Petri dish, swirl and clean from excess blood, and transfer to

the hood workspace

• Take the lung and heart tissue together and dunk the tissue into each of the HBSS-

betadine wells 2Xs times in each

• Clean the tissue from betadine and place back into gel Petri dish

• Pin down the heart, 4 of the largest lung lobes, and cut the smallest lobe away

• Under microscope begin to clean the vessel of fat, tissue, loose adventitia, and blood

while replacing HBSS solution in the petri dish every 15 minutes or as needed

• Measure 1.7 mg of Collagenase enzyme (Worthington Biochem, 229 U/mg) into a 10

mL beaker and add 1 mL of HBSS. Swirl and triturate to dissolve

• Transfer dissolved collagenase to a 5 mL tube and place the PA vessel in the solution

• Incubate the PA vessel in the enzyme solution in cell culture chamber for 20 minutes

- 49 -

• Remove the vessel to a sterile dissecting dish with fresh HBSS. Cut open the PA vessel

longitudinally and clear any blood or tissue still in place

• Gently scrape off the endothelial layer

• Place the PA in a small petri dish containing 3 mL of PASMC media (or 10%

FBS/DMEM) and incubate overnight.

Day 2: Cell Culture of Pulmonary Artery Smooth Muscle Cells (PASMCs) (Fig. 9)

• Sterilize materials under UV light for 15 minutes

o Four 6-well culture dishes

o Coverslips

o 10 ml beaker

o 50 ml beaker

o 15 ml culture tube

o Forceps

o Pasteur Pipettes

• Warm 100 ml HBSS, 10% and PASMC culture media or (10% FBS/DMEM) in

incubator

• Measure the enzymes into a 10 ml beaker

o 1.7 mg collagenase (Worthington Biochem, 229 U/mg)

o 0.5 mg elastase (Sigma, Porcine Type III, 70 U/mg)

o 1.0 mg Bovine Serum Albumin (Sigma)

• Transfer to the hood and add 1 mL HBSS. Swirl and triturate to dissolve

• Gently place the vessel segments into the beaker of enzyme solution. Use a sterile

Pasteur pipette to transfer the vessel and enzyme solution to a sterile 15 ml culture tube

- 50 -

• Incubate for 50-55 min at 37ºC. Gently swirl cells every 10-15 minutes

• While the cells are incubating prepare the coverslips. Place 24 coverslips into a beaker

of 70% alcohol and allow to soak for several minutes

• Gently blot the individual coverslips on sterile gauze and place them on edge in each of

the dish wells to dry. Triturate 10 times with a p-1000 pipet to further dissociate the cells,

the tissue should be cloudy when it is finished digesting

• If it is not dissociated enough continue digestion for a couple more minutes and triturate

again.

• Add 11 ml of PASMC culture media (or 10% FBS/DMEM) to stop digestion and

centrifuge at 1500 RPM for 5 minutes (temp of centrifuge cabinet should be 25 ºC).

• While the cells spin, put 2 ml of PASMC culture media (or 10% FBS/DMEM) into each

of the dish wells and swirl the dishes to get rid of the bubbles under the coverslips

• Remove supernatant and re-suspend the cells in 4 ml of 10% FBS/DMEM

• Add a few drops of cell suspension to the center of each of the coverslips

• Allow the cells to settle for several minutes and then place in the incubator for storage

• Change the media every 48 hours

- 51 -

Figure 8

Figure 8. Diagram showing isolated extrapulmonary and intrapulmonary arterial branches of a rat from which we extracted protein for Western blot analyses on various markers and proteins. The isolated vessels are also used to prepare primary cultured PASMC.

- 52 -

Figure 9

Figure 9. Schematic diagram showing the procedure of preparation of isolated pulmonary arteries (containing mainly contractile PASMC) and primary cultured PASMC (containing mainly synthetic or proliferative PASMC)

- 53 -

Western Blotting

PA smooth muscle tissue was placed in an Eppendorf tube on ice and lysed with cold RIPA lysis buffer (Millipore, Billerica, MA) supplemented with protease inhibitor cocktail (Roche; Basel,

Switzerland) by sonication three times for 30 seconds each time. Cultured rat PASMCs were washed with PBS, scraped, placed into an Eppendorf tube, and centrifuged. The pelleted cells were resuspended in 20–50 µl of RIPA buffer supplemented with protease inhibitor cocktail. Lysed tissues and cells were incubated in lysis buffer for 15 min on ice. The lysates were then centrifuged at 13,300 rpm for 15 min at 4°C. The pellet was discarded and from the supernatant, protein concentration was determined by Bradford Protein Assay (Bio-Rad, Hercules, CA) with BSA as a standard. Proteins (15–25 µg) were mixed and boiled in Laemmli sample buffer supplemented with 2-mercaptoethanol (BME, Sigma) reducing agent. Protein lysates were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.45 µm nitrocellulose membranes (Bio-Rad). Membranes were incubated for 1h at 22–24°C in a blocking buffer [0.1% Tween 20 in TBS (TBST)] containing 5% nonfat dry milk powder. The membranes were then incubated with primary antibodies diluted in TBST containing 5% BSA, with shaking overnight at 4°C. Membranes were washed three times in TBST for 5 min each, followed by incubation in secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature in TBST containing 5% milk. Membranes were washed three times for 5 min each, and peroxidase activity was visualized with enhanced chemiluminescence substrate (Pierce, Rockford, IL).

Primary antibodies included myosin heavy chain (MYH, 1:1,200, Santa Cruz), smooth muscle 22- a (SM22a; 1:1,000, Santa Cruz), proliferating cellular nuclear antigen (PCNA, 1:1,000, Santa

Cruz), and β-actin (1:2,000, Santa Cruz). Band intensity was quantified with ImageJ (National

Institutes of Health, Bethesda, MD), normalized to β-actin control, and is expressed as arbitrary units. - 54 -

Western Blot Protocol by Marisela Rodriguez

Western blotting is a technique that uses antibodies to identify proteins of interest that will be separated based on size by gel electrophoresis. The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane.

The membrane is then processed with antibodies specific for the protein of interest and visualized using secondary antibodies and detection reagents.

Core Process Steps:

1. Sample Preparation

2. Gel electrophoresis

3. Blocking

The following guide discusses the process for producing a western blot experiment from collected pulmonary artery tissue. The same can be done for cells.

Setting up:

Organize and label all samples before beginning experiment with initials, date, and protein.

Research the molecular weight of the protein of interest and calculate how many gels you will run at a time (depending on size of proteins or amount of samples).

Day 1

Sample Preparation (1 hour or more depending on number of samples)

• Always maintain samples at 4º C using ice bucket

• Add 50 microliters (μL) of thawed lysis to the Eppendorf tubes (E-tubes) to denature the

proteins 3 min (always keep lysis on ice). Begin to homogenize the tissue 10 seconds

out of the ice at a time or alternate between samples

- 55 -

• Vortex and centrifuge each sample

Fisher Scientific settings

Temperature: 4º C

Speed: 12 x g

Time: 3-5 minutes

• Leave in ice for 15 min

• Prepare new and labeled sterile set of E-tubes

• Transfer all the liquid from recently centrifuged tubes (supernatant) into new sterile E-

tubes leaving the tissue at the bottom. Centrifuge again to make sure no tissue is left in

supernatant.

Fisher Scientific settings

Temperature: 4º C

Speed: 13 x g

Time: 15 minutes

Measure Concentration (2 hours in total)

• Before adding dye, make sure it is compatible to lyse (we used Bio-Rad Protein Assay-

Dye Reagent). *Always make more than you need

OUT!

• Prepare BSA dye:

o A/B Ratio of solutions is 1:50 and each well should have 200 μL of AB mix for

measurement

o Standards are prepared with 6 samples and triplicates for each

• Pipet 40 μl of BCA from Well 1 and pipet into Well 2 and mix. Move on with mixture from

Well 2, take 40 μl and pipet into Well 3, continue to Well 5 and STOP.

- 56 -

• Pipet 40 μl out of well 5 and leave Well 6 with only 40 μl of water

• In 96 well plate, pipet 10 μL of each standard and sample solution into each separate well creating triplicate sample

• Repeat for Wells 2 – 6. (This is only standard creation)

• In separate tubes, dilute total protein samples by 7 (30 water in 5 μL of total protein) and

repeat the step above with 10 μL of each sample tube in 200 μL of AB solution creating

triplicates.

• Mix the sample and reagent thoroughly then incubate for 30 min at 37 °C

• Measure absorbance using plate reader at 595 nm

• Turn on hot plate to ~100 °C

• Collect and save data to create scatter plot, find slope and R-value

• Absorbance will increase over time; samples should incubate at room temperature for NO

more than one hour

• TABLE CALC

• Once the concentration is calculated, add protein, 6X buffer, and lysis

• Heat everything combined in new labeled tubes at 98.7º C for 5 minutes. Samples now do

not have to be on ice

Gel Electrophoresis (4 hours in total)

• Grab gels from fridge and let them cool down to room temperature

• Prepare running buffer according to gels being used

• Use same buffer type in each chamber of the gel tank

• Circuit pins need to be opposite of each other

• Meanwhile prepare gels. Open from packet, rinse, and remove stickers. Insert gels, wells

facing you and clip in. Carefully remove combs without tearing gel

- 57 -

• Pour in running buffer to the fill lines marked on the bins for a total of 400 mL

• Use transfer pipet to rinse wells

• Remove all bubbles with pipet from both bins

• Collect samples from centrifuge and prepare pipet tips for loading

• Add 6mL of chlorophyll ladder into the first well of each gel.

• Load all solution of tubes using slim tip so that you do not pierce the gel as that will

interfere with your final results.

o submerge tip to bottom of well, without piercing it, and add protein

carefully until all of it is out of tip. Last push of air from micropipette

should be out of the rest of the solution already in well. Air bubble will

disturb well

• Set the electro machine at 100 volts for 1.5 hours. Process is called electrophoresis

• Life Technology

Gel of 4%-12% set voltage at 165 volts for one hour. The process is faster with a higher

voltage but the slower the better.

• BioRad

Set cables that run the current with the corresponding colors, black with black and red with

red. Run for 1.5 hours

• Transfer

1X transfer buffer recipe

for a total of 2 liters

20% methanol 400 mL

10X running buffer

water 1400 mL

- 58 -

• Create sandwich

CLEAR SIDE DOWN

sponge

filter paper

membrane (do not touch with bare hands)

gel

filter paper

sponge

• Soak sponge, push out all air, wet bottom of tray, use spatula to break open gel, cut wells

off and thick gel piece off

• Ladder should be placed with the ladder on the left and the highest molecular weight on

top

• Build sandwich submerged in water

• Roll out all bubbles using roller on each of the layers

• Place sandwich into the plastic bin

• Place ice block inside bin

• Close bin and add ice around

• Set volts to 100 for one hour, two hours for two gels

Blocking (1 hour in total)

• ALWAYS keep membrane wet/submerged

• Before taking gels out of cassette make sure to have 5% milk prepared in TBST

(for every 100 mL of TBST we need 5 grams of powdered milk)

• Before proceeding, Ponceau S. can be used to stain proteins to indicate if transfer was

successful. Wash with TBST before submerging in milk

- 59 -

• Block with milk for 1-2 hours at room temperature on a shaker

• Wash the membrane with TBST for 5 minutes

• Cut the membrane at the protein size of interest with ladder as reference

• Use 2.5% BSA/TBST for primary antibody’s dilution solution

• Place the membrane in plastic container submerged in primary antibody

Always keep the prepared antibodies on ice

• Leave in 4-degree refrigerator or cold room over night

Day 2

• Blocking continued (3+ hours)

• Recycle primary antibody up to three times before throwing away, store in -20 º C

• Remove primary antibody and rinse membrane in TBST

Wash 4Xs for 5 min each wash

• Add secondary (always keep 1:6000 ratio)

• Leave on rocker for 1 ½ hours

• Remove and recycle secondary up to three times.

• Wash in TBST for 5 min

• Make electro-chemiluminescent (ECL) substrate using 1:1 ratio of A and B solution, use

15 mL tube

• Wait 3 min

• Remove TBST and add a sufficient amount of drops onto the membrane

• Place membrane onto cassette and go to a dark room to develop results

Polymerase Chain Reaction

RNA Isolation (~three hours in total)

- 60 -

• Clean work area or bench with 10% bleach and 70% ethanol

• Materials

o Bucket of ice

o 1.5 mL tubes (one for each plate of cells)

o 100- 1000 filter pipettes

o Spill pad

o Trizol from corrosives cabinet (Quizol name brand)

o Chloroform from flammable cabinet

o Cell lifters

• Work under hood

o Add 800 mL of trizol to cell plates. Note: if using tissues or large plates of cells add

1000 mL. Cover entire surface of plate and let sit for 2 min

o Use cell lifter to wipe the plate

o Transfer trizol and cells from plate to 1.5 mL tubes

o Keep all tubes on ice and repeat for each sample

• Place 160 mL of chloroform into each 1.5 mL tube gathered. Note: if using large cells or

tissues add 200 mL; it is possible to add double the chloroform.

• Vortex all tubes

• Incubate tubes 2-3 minutes at room temperature

• Centrifuge for 15 minutes at 12 rpms or 13.8 G

• Take pipette set at 50 µL and remove the top clear layer only without disrupting any others

and place into new 1.5 mL tubes. Repeat for other tubes.

• Add 400 µL of isopropanol (found in flammable cabinet) to new tubes. Add 500 µL if

using large cells or tissues

- 61 -

• Incubate at room temperature for 10 minutes

• Centrifuge at 4°C for 10 minutes at 12 rpm

• Set heat block to 60°C

• Remove layer of isopropanol using pipette. Note: leave RNA in the tip of the tube

• Make 75% ethanol solution with 7.5 mL of ethanol and 2.5 mL of non-RNA water

• Add 1 mL of ethanol mixture to each 1.5 mL tube

• Vortex tubes

• Centrifuge at 7.5 rpm for 5 minutes at 4°C

• Take out all the ethanol and leave RNA palette with the tube cap open to let it dry for ~5-

7 minutes

• Add 10 µL of non RNA free water when tubes are dry and heat for about 10 minutes at 55-

60°C in heat block

• After heating RNA, it can be stored in -80°C

DNAse Treatment (3 hours in total)

• Thaw, vortex, and centrifuge RNA tubes if stored

• Obtain 1.5 mL or 0.25 mL centrifuge tubes (date, initial, and label accordingly)

o Add 9 µL of non-RNA water to each specific tube

o Add 1 µL of RNA to each specific tube

• Vortex and centrifuge tubes

• Measure purity and concentration of RNA on NANODrop machine

o Dispose of these tubes afterwards

• Calculate the amount of water, buffer, RNA, and DNAse to add to new PCR tubes

o 7 of H2O

o 1 RNAaa - 62 -

o 1 Buffer

o 1 DNAse

o Total should be 10 µL (Keep DNAse and Buffer consistent while adjusting

RNA and water)

o To calculate RNA divide concentration by 1000 (Ex: 1000/[x])

o Vortex and centrifuge

• Put samples in thermocycler under Alex’s protocols saved as DNAse

o Set timer to 30 minutes

o After timer goes off, open and add 1 µL of EDTA to each tube, press skip step,

enter yes to continue

o This will incubate tubes at 65°C for 10 minutes

• While waiting, take out RT buffer, DNTP, and random primers

• Make RT mixture for each tube (Use 1.5 mL tubes)

o 4.2 µL of water

o 2 µL of RT buffer

o 0.8 µL of DNTP

o 2 µL of random primers

o 1 µL of RTase

o Total of 10 µL

- 63 -

Samples 2 3 4 5 6 7 8 9 10

Water 8.4 12.6 16.8 21 25.2 29.4 33.6 37.8 42

RT buffer 4 6 8 10 12 14 16 18 20

DNTP 1.6 2.4 3.2 4 4.8 5.6 6.4 7.2 8

Random 4 6 8 10 12 14 16 18 20 Primers

RTase 2 3 4 5 6 7 8 9 10

• Put 10 µL of mixture into each NEW tube (date, initial, and label)

• Together the RT mixture and the sample are mixed for a total of 20 µL

• Place these new tubes into the thermocycler again under Alex’s protocols saved as RT

o Run for 2 hrs

o Can store in -20 when run is complete

• This is cDNAPCR Run (~1 ½ hours to make + 2 hours of downtime)

• Obtain 1.5 mL tubes, one for each primer that will be used

• For mixture 1 use the table based on how many samples will be run corresponding to each

different primer. Note: It is best to have two extra counts for each sample due to possible

pipette errors

• Mixture 1

•

- 64 -

• Obtain a 1.5 mL tube and make mix for all primer tubes. Mixture 2 will be added to each

individual PCR tube sample with a total of 9 µL.

•

• Vortex and centrifuge

• Add 1 µL of cDNA to each tube with both Mix 1 and 2

• Use PCR machine: under saved protocols, Alex, PCR, change step 5 for 34, and make sure

step 3 is at 60°C

• Store PCR products if needed

Gel

• Make 1.5g agarose gel in TBS. Add 10 µL of gel red dye

• Add 4 µL of loading dye to each sample

• Load 15 µL of sample into gel

• Load 6 µL of ladder into gel

o Ladder goes in the first well and in between genes

• Run between 110-120 V for 30 minutes

Statistical Analysis

Statistical analysis. Data are expressed as means ± SE and were analyzed for statistical significance by the unpaired Student’s t-test or one-way ANOVA for multiple groups using SigmaPlot software.

Differences were considered to be significant at P<0.05. Significant difference is expressed in the figures or figure legends as * p<0.05, ** p<0.01, and *** p<0.001.

- 65 -

RESULTS

To determine the potential differences in the expression levels of target proteins, such as

CALHM1 and CALHM2, between proliferative (synthetic) phenotype and contractile

(differentiated) phenotype of PASMCs, we used Western blot analysis to compare the protein levels in freshly isolated PA tissues with denuded endothelium and stripped-off adventitia, which contain mainly contractile PASMCs, and primary cultured PASMCs incubated in the media with

10% fetal bovine serum and various growth factors, which contain mainly proliferative PASMCs.

The contractile and proliferative PASMCs were constructed by taking PA tissue, representative of the contractile SMC phenotype, and primary cultured PASMCs, representative of the proliferative phenotype, from the same rat for project sample pairs in distinguishing PA vs. PASMC differences in expression levels of target proteins.

As shown in Figure 10, we first determined and compared a) the markers for contractile phenotype of smooth muscle cells (SMCs), myosin heavy chain 11 (MHC11), transgelin (TAGLN) and SMC α2-actin (ACTA2); b) the marker for proliferative phenotype of SMC, PDGFA (platelet- derived growth factor A) and VIM (vimentin); and c) the marker for cell proliferation, proliferating cell nuclear antigen (PCNA) in isolated PA tissues (mainly contain contractile PASMC) and primary cultured PASMC (mainly contain proliferative PASMC). Western blot analyses showed that protein expression levels of the contractile markers (e.g., MHC11, TAGLN and ACTA2) were significantly higher (p < 0.001) in freshly isolated PA than in primary cultured PASMC, while protein expression levels of the proliferative markers (e.g., PDGFA and VIM) and proliferation marker (i.e., PCNA) were significantly higher (p < 0.001) in primary cultured PASMC than in freshly isolated PA (Fig. 10). These results indicate that using freshly isolated PA and primary cultured PASMC (prepared from the same rat) is a good model to study the difference of contractile and proliferative phenotypes of PASMC.

- 66 -

Figure 10 – Contractile vs proliferative phenotype markers

A B C

kDa kDa kDa 200 MHC11 52 VIM 200 MHC11

22 TAGLN 33 PCNA 43 ACTA2

43 ACTA2 31 PDGFA 33 PCNA

42 42 Actin Actin 31 PDGFA

23 TAGLN

42 Actin PA (Contractile) D PASMC (Proliferative) 1.0 1.2 1.2 0.7 1.8 1.6 ** 1.0 1.0 0.6 ** 0.8 1.4 0.5 0.8 0.8 1.2 0.6 0.4 1.0 0.6 0.6 0.8 0.4 0.3 0.4 *** 0.4 0.6 0.2 Protein Level Protein Protein Level Protein

(Arbitrary Unit) (Arbitrary 0.4 0.2 ** 0.2 0.2 Unit) (Arbitrary *** 0.1 0.2 0.0 0.0 0.0 0.0 0.0 MHC11 TAGLN ACTA2 PDGFA PCNA

1.6 1.2 1.2 1.6 ** 25 ** 1.4 1.4 1.0 1.0 20 1.2 1.2 0.8 0.8 1.0 1.0 15 0.8 0.6 0.6 0.8 10 0.6 0.4 *** 0.4 0.6 Protein Level Protein

0.4 Level Protein 0.4 ** 0.2 0.2 5 (Normalized to PA) to (Normalized 0.2 *** PA) to (Normalized 0.2 0.0 0.0 0.0 0.0 0 MHC11 TAGLN ACTA2 PDGFA PCNA E 4.0 * 2.0 0.8 ** * PA (Contractile) 3.0 1.5 0.6 PASMC (Proliferative)

2.0 1.0 0.4

Protein Level Protein 1.0 0.5 0.2 (Arbitrary Unit) (Arbitrary

0.0 0.0 0.0 VIM PDGFA PCNA

Figure 10. Markers for contractile and proliferative smooth muscle cells (SMCs). A-C: Western blot analyses on the contractile cell markers, MHC11, TAGLN, and ACTA2, and the proliferative cell markers, VIM, PDGFA and PCNA in freshly isolated pulmonary artery (PA, containing mainly contractile PASMC) and primary cultured PASMC (containing mainly proliferative PASMC). D: Summarized data (means±SE) showing protein levels of the markers in PA and PASMC. *p<0.05, **p<0.01, ***p<0.001 vs. PA.

- 67 -

Using the same model of isolated PA and primary cultured PASMC from the same rat, we also compared protein expression level of CALHM1 and CALHM2. As shown in Figure 11, the protein expression level of CALHM1 and CALHM2 was significantly higher in primary cultured

PASMC than in freshly isolated PA. The expression level of CALHM1/2 was inversely proportional to the expression level of the contractile markers, MHC11 and TAGLN, but proportional to the expression level of the proliferation marker, PCNA (Fig. 11). These results indicate that both CALHM1 and CALHM2 are upregulated during the phenotypical transition of

PASMC from the contractile phenotype to the proliferative phenotype. The data also imply that

CALHM1/2 or Ca2+ influx through CALHM1/2 are required for or involved in stimulating and maintaining proliferation of PASMC. In addition, given the report that CALHM1/2 are also ATP channels allowing outward transportation of ATP from the cytosol to the extracellular or intercellular sites, upregulated CALHM1/2 may also be involved in enhancing ATP-mediated proliferative and migratory effects on PASMC via activation of purinergic receptors (P2Y and

P2X) in the plasma membrane. Metabolic shift and abnormalities have been implicated in the development and progression of pulmonary vascular remodeling, a major cause for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with idiopathic and associated PAH. Upregulated CALHM1/2 in proliferating PASMC may be a potential link between intracellularly accumulated ATP and extracellular ATP-mediated PASMC proliferation and migration.

- 68 -

Figure 11 – Upregulated CALHM1 and 2

A PA (contractile) B C 0.8 3.5 1.0 *** 3.0 0.8 kDa 0.6 33 PCNA 2.5 2.0 0.6 0.4 1.5 * 0.4 200 MHC11 0.2 1.0 Protein Level Protein *** 0.2 (Arbitrary Unit) (Arbitrary 0.5 22 TAGLN 0.0 0.0 0.0 MHC11 TAGLN PCNA PASMC (proliferative) 37 CALHM1 D 3.5 2.0 *** *** PA 38 CALHM2 3.0 (Contractile) 2.5 1.5 42 Actin 2.0 PASMC 1.0 1.5 (Proliferative) 1.0 0.5 Protein Level Protein (Arbitrary Unit) (Arbitrary 0.5 0.0 0.0 CALHM1 CALHM2

Figure 11. CALHM1 and CLHM2 are upregulated in proliferative PASMC in comparison to contractile PASMC. A: Images showing freshly isolated PA branches from rat (upper panel) and primary cultured PASMC from the same PA branches (lower panel). B: Western blot analyses on the contractile markers MHC11, TAGLN and the proliferating marker PCNA, as well as CALHM1 and CALHM2 in PA (containing mainly contractile PASMC) and PASMC (proliferative PASMC). C and D: Summarized data (means±SE) showing protein levels of the markers (C) and CALHM1/2 (D) in PA (contractile PASMC) and PASMC (proliferative PASMC). *p<0.05, **p<0.01, ***p<0.001 vs. PA (contractile PASMC).

- 69 -

The PI3K/AKT/mTOR signaling cascade is a major signaling pathway involved in cell proliferation and protein synthesis in a variety of cells including cancer cells and PASMCs (26,

55, 72). Our lab and other investigators have reported that the PI3K/AKT1/mTORC1/C2 pathways are involved in the development and progression of experimental PH in mice (27, 91, 92). The next set of experiments was designed to investigate whether the transition of PASMC from contractile (isolated PA) phenotype to proliferative (primary cultured PASMC) phenotype requires upregulation or activation of the signaling proteins involved in the PI3K/AKT/mTOR signaling pathway. As shown in Figure 12, the PASMC phenotypical transition from the contractile to proliferative phenotypes was also associated with a significant upregulation of AKT and mTOR

(Fig. 12A and B) as well as a marked increase in phosphorylation of AKT (pAKT) and mTOR

(pmTOR) (Fig. 12C). The upregulated AKT/mTOR and enhanced pAKT/pmTOR in proliferative

PASMC were proportionally correlated to the proliferation maker, PCNA and were inversely proportional to the contractile markers, MHL11, ACTA2 and TAGLN (Fig. 12A and B). These data indicate that the phenotypical transition of PASMC from contractile to proliferative phenotype requires upregulation and activation (or phosphorylation) of the AKT/mTOR signaling proteins, while enhanced PI3K/AKT/mTOR signaling pathway is an important signaling cascade in PASMC proliferation and, potentially, the development and/or progression of pulmonary vascular remodeling in patients with PAH and animals with experimental PH.

- 70 -

Figure 12 – Upregulated AKT/mTOR signaling proteins

A B 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 * kDa 0.6 0.6 0.6 0.6 200 MHC11 0.4 0.4 0.4 ** 0.4 Protein Level Protein 43 ACTA2 Unit) (Arbitrary 0.2 0.2 0.2 0.2 * *** 23 TAGLN 0.0 0.0 0.0 0.0 MHC11 ACTA2 TAGLN PCNA 33 PCNA PA (Contractile) PASMC (Proliferative) C 1.0 1.2 *** 0.8 1.2 50 AKT ** ** 0.8 1.0 1.0 0.6 * 50 pAKT 0.8 0.8 0.6 0.6 0.4 0.6 250 mTOR 0.4 0.4 0.4 0.2 250 pmTOR Level Protein (Arbitrary Unit) (Arbitrary 0.2 0.2 0.2

36 GAPDH 0.0 0.0 0.0 0.0 pAKT AKT pmTOR mTOR 5.0 3.5 3.5 2.0 ** ** 3.0 *** 3.0 * 4.0 2.5 2.5 1.5 3.0 2.0 2.0 1.0 2.0 1.5 1.5 PA 1.0 1.0

Protein Level Protein 0.5 (Contractile) 1.0 0.5 0.5 (Normalized to PA) to (Normalized PASMC 0.0 0.0 0.0 0.0 D (Proliferative) pAKT AKT pmTOR mTOR 1.2 * 3.0 1.0 * 3.0 * 1.0 2.5 * 0.8 2.5 0.8 2.0 2.0 0.6 0.6 1.5 1.5 0.4 0.4 1.0 1.0 pAKT/AKT pAKT/AKT (Arbitrary Unit) (Arbitrary (Arbitrary Unit) (Arbitrary 0.2 0.5 pmTOR/mTOR 0.2 pmTOR/mTOR 0.5 (Normalized to PA) to (Normalized (Normalized to PA) to (Normalized 0.0 0.0 0.0 0.0

Figure 12. AKT/mTOR signaling pathway in PASMC phenotypical transition. A: Western blot analyses on the contractile cell markers (MHC11, ACTA2, TAGLN) and the proliferation marker (PCNA), as well as AKT, phosphorylated (p) AKT (pAKT), mTOR and pmTOR, in freshly isolated PA (containing mainly contractile PASMC) and primary cultured PASMC (containing mainly proliferative PASMC). B-D: Summarized data (means±SE) showing protein levels of the contractile and proliferative cell markers (B), pAKT, AKT, pmTOR and mTOR (C), and the ratio of pAKT/mTOR and pmTOR/mTOR (D) in PA (contractile PASMC) and PASMC (proliferative PASMC). *p<0.05, **p<0.01, ***p<0.001 vs. PA.

- 71 -

There are many membrane receptors and ion channels that are implicated in stimulating

PASMC proliferation and pulmonary vascular remodeling in patients with PAH and animals with experimental PH. Calcium-sensitive receptor (CaSR) is a G protein-coupled receptor (GPCR) that can be activated by extracellular cations (e.g., Ca2+, Mg2+), polyamines (e.g., spermine, spermidine), amino acids (e.g., L-tryptophan, glutamate), antibiotics (e.g., neomycin), amyloid-β,

3+ and inactivated by PO4 ions (22, 23, 30, 31). Our lab has previously reported that CaSR was required for acute hypoxic pulmonary vasoconstriction and upregulated CaSR was associated with the development of pulmonary hypertension in rats and mice (79, 84, 93). Interestingly, we also found that CaSR was significantly upregulated during the phenotypical transition of PASMC. As shown in Figure 13A, the protein level of CaSR in primary cultured PASMC (mainly contain proliferative PASMC) was significantly higher than in freshly isolated PA (mainly contain contractile PASMC).

Notch signaling has also been implicated in the development and progression of PAH (3,

45, 80, 84, 101, 102). Upregulated Notch ligands (e.g., Jag-1) and Notch receptors (e.g., Notch1,

Notch2 and Notch3) in PASMC are reported to contribute to the initiation and/or progression of concentric pulmonary arterial wall thickening and obliterative intimal lesions in patients with idiopathic and associated PAH and animals with experimental PH (45, 56, 65, 80). In this study, the protein expression level of Notch receptors (Notch2 and Notch3) were also significantly increased during the phenotypic transition of PASMC from the contractile phenotype to the proliferative phenotype (Fig. 13A and B). The upregulated CaSR and Notch2/3 in proliferating

PASMC were inversely proportional to protein expression levels of the contractile SMC markers

(MHC11 and ACTA2), but positively correlated to the proliferating marker (PCNA) (Fig. 13A).

Nevertheless, other signaling proteins like HIF-1 Nonetheless, not all membrane proteins or intracellular signaling proteins are upregulated during the PASMC phenotypical transition from

- 72 - the contractile to the proliferative phenotype. For example, caveolin-1 (Cav1), a membrane protein associated with endocytosis, cholesterol distribution and caveolae formation, was actually downregulated in proliferating PASMC compared with contractile PASMC (Fig. 13A and C).

Hypoxia-inducible factor 1-α (HIF-1α) was not changed during the PASMC phenotypical transition (Fig. 13AC). These results indicate that, in addition to upregulated CALHM1/2, CaSR and Notch receptors (e.g., Notch2 and Notch3), are upregulated in proliferating PASMC in comparison to contractile PASMC. The upregulated CaSR, Notch2/3 and CALHM1/2 may coordinate with each other to assure the Ca2+ signaling required for PASMC proliferation and, ultimately, pulmonary vascular remodeling in patients with PAH.

- 73 -

Figure 13 - CaSR and Notch upregulated in phenotypical transition

A B

kDa 0.6 *** 3.5 *** 100 CaSR 0.5 3.0 0.4 2.5 2.0 42 Actin 0.3 1.5 0.2 1.0 Protein Level Protein Protein Level Protein 100 Unit) (Arbitrary 0.1 0.5 Notch2 PA) to (Normalized 0.0 0.0 CaSR CaSR 36 GADPH 1.5 *** 50 *** 1.2 40 100 Notch3 0.9 30 100 HIF-1α 0.6 20 Protein Level Protein

Protein Level Protein 0.3 10 24 Cav-1 Unit) (Arbitrary 0.0 PA) to (Normalized 0 Notch2 Notch2 200 MHC11 1.2 3.5 ** 43 ACTA2 1.0 ** 3.0 0.8 2.5 2.0 33 PCNA 0.6 1.5 0.4 1.0 ProteinLevel 36 GADHP Level Protein (Arbitrary Unit) (Arbitrary 0.2 0.5 (Normalized to PA) to (Normalized 0.0 0.0 C Notch3 Notch3 1.6 1.4 1.6 1.4 1.4 1.2 1.4 1.2 1.2 1.0 1.2 1.0 1.0 1.0 0.8 0.8 0.8 0.8 * 0.6 0.6 0.6 *** *** 0.6 * 0.4 0.4 0.4 0.4 Protein Level Protein Protein Level Protein Protein Level Protein Protein Level Protein (Arbitrary Unit) (Arbitrary 0.2 0.2 Unit) (Arbitrary 0.2 0.2 (Normalized to PA) to (Normalized 0.0 PA) to (Normalized 0.0 0.0 0.0 Caveolin-1 Caveolin-1 HIF-1a HIF-1a

Figure 13. CaSR and Notch2 are upregulated in proliferative PASMC in comparison to contractile PASMC. A: Western blot analyses on CaSR, Notch2/3, HIF-1α and caveolin-1, as well as the contractile (MHC11 and ACTA2) and proliferative (PCNA) cell markers in freshly isolated PA (mainly contractile PASMC) and primary cultured PASMC (mainly proliferative PASMC). B and C: Summarized data (means±SE) showing protein expression levels of CaSR, Notch2/3, caveolin-1 and HIF-1α in contractile (PA) and proliferative (PASMC) PASMC. **p<0.01, ***p<0.001 vs. PA.

- 74 -

It is unclear whether the phenotypical switch between contractile and proliferative PASMC is pathogenically involved in the development and progression of concentric pulmonary vascular wall medial hypertrophy, arteriole muscularization and obliterative intimal lesions (14, 29, 57, 87).

It is, however, evident that increased SMC proliferation and migration both contribute to the medial hypertrophy or the concentric pulmonary arterial wall thickening and arteriole muscularization. Furthermore, SMC or SMC-like cells are also found in occlusive intimal lesions and fibrotic clots in proximal and distal pulmonary arteries (36, 86). Using animal models of pulmonary hypertension, our lab and others have demonstrated that sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling both contribute to the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in rats and mice with experimental PH (e.g., hypoxia-induced PH, MCT-induced PH, Sugen/Hypoxia-induced PH), while the phenotypical transition of PASMC from a contractile phenotype to a proliferative phenotype is necessary for SMC to participate in the initiation and progression of pulmonary arterial medial hypertrophy and arteriole muscularization.

Alveolar hypoxia induces pulmonary vasoconstriction to match the perfusion with ventilation ensuring maximal oxygenation of the venous blood in pulmonary artery. Persistent hypoxia, however, causes sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling and subsequently pulmonary hypertension (54, 57, 69, 98, 106). It has been demonstrated that chronic alveolar hypoxia downregulates Kv channels and upregulates TRP channels in PASMC causing pulmonary vasoconstriction and vascular remodeling (78, 99, 103-

105). McMurtry et al. reported in 1978 (53) that lungs from chronically hypoxic rats have decreased acute hypoxia-mediated pulmonary vasoconstriction. In mice, we observed the same results that chronically exposure of mice to hypoxia for 4 weeks, recently published data from our lab in manuscript titled Revisiting the Mechanism of Hypoxic Pulmonary Vasoconstriction Using

- 75 -

Isolated Perfused/Ventilated Mouse Lung, resulted in pulmonary hypertension characterized by significant pulmonary vascular remodeling (due at least partially to increased PASMC proliferation and migration); however, the acute HPV was significantly inhibited in isolated perfused/ventilated lungs from chronically hypoxic mice (Fig. 14). These data imply that a) acute hypoxia induces pulmonary vasoconstriction by mechanisms that are shared by chronic hypoxia to induce pulmonary vasoconstriction and vascular remodeling (53, 64); b) increased pulmonary vasoconstriction occurs at the early stage of hypoxia-induced PH (98); and c) PASMC phenotypic transition from contractile to proliferative phenotype occurs at the late stage of the hypoxia- induced PH to initiate and maintain pulmonary vascular remodeling.

- 76 -

Figure 14 – Pulmonary vascular remodeling in chronic hypoxia

A B 14 Normoxic Control 6 12 5 Chronically Hypoxic 5 *** 13 4 10 4 2 min 3 12 2 8 1 3 11 0 6 6 10 5 2 4 4 ** PAP (mmHg) 3

9 Basal PAP (mmHg)

Rise in PAP (mmHg) 1

Rise in PAP (mmHg) 2 2

8 Alveolar Hypoxia-induced 1 0 0 Acute Alveolar Hypoxia-induced 0 7 Hyp Hyp Hyp Hyp Hyp 1 2 3 4 5 Normoxic Chronically Hypoxia Control Hypoxic Alveolar Hypoxia Challenge Challenge C Normoxic Control Hypoxia (4 w) D

10 500 250 ) ) 2 ) 2 2 8 400 200

6 300 150

4 *** 200 100

8x 8x ** *** 30x 30x 2 100 50 Number of Branch (/mm Number of Junction (/mm Length of Branch (mm/mm 0 0 0 Normoxic Chronically Control Hypoxic

Figure 14. Chronical hypoxia inhibits the acute hypoxia-mediated pulmonary vasoconstriction in isolated perfused/ventilated mouse lungs (A and B) and induces pulmonary vascular remodeling (C and D). A: Representative record showing changes of pulmonary arterial pressure (PAP) before, during and after acute ventilation of hypoxic gas misture (Hyp, 8% O2 in N2, for 5 min) in isolated perfused/ventilated lungs from a normoxic control mouse (blue) and a chronically hypoxic mouse (red). The averaged data (means±SE, n=8-10 mice) showing the increases in PAP induced by a series of consecutive hypoxia challenges. B: Summarized data (means±SE, n=8-10) showing the basal PAP (left) and acute hypoxia-induced increases in PAP (right) in isolated perfused/ventilated lungs from normoxic control mice (blue) and chronically hypoxic mice (red). **p<0.01, ***p<0.001 vs. normoxic control. C: Representative angiographic images showing the pulmonary vascular structure in a normoxic control mouse (left) and a chronically hypoxic mouse (right). The lower panels, 30x magnification. D: Summarized data (means±SE, n=9-10) showing the total length of pulmonary vascular branches (left), the number of branches (middle) and the number of junctions of the vascular branches (right) in normosic control mice (blue) and chronically hypoxic mice (red). **p<0.01, ***p<0.001 vs. normosic control.

- 77 -

Serum starvation causes cell growth arrest which then results in SMC differentiation and inhibits SMC proliferation. In our in vitro experiments, we found that serum starvation or decreasing serum concentration from 10% to 0.03% increased the contractile SMC marker MHC11 and downregulated CALHM2 protein expression level in human PASMC. In examining the contribution of growth and proliferation guided by SMC growth medium including 10% fetal bovine serum (FBS) and various growth factors, a gradient pattern of expression was found in contractile and proliferative cell markers. It is observed that the expression of CALHM1 and

CALHM2 also fall into this behavior of expression when treated with varying concentrations of

FBS (0.03%, 1% and 10%) over a controlled period of time. These results indicate that serum starvation-mediated growth arrest tend to induce PASMC differentiation toward to the contractile phenotype determined by an increase in MHC11 expression level.

The mTORC1 and mTORC2 play important roles in the regulation of cell proliferation and protein expression, while both are involved in the development and progression of pulmonary hypertension (26-28, 33, 34, 43). PASMC in normal PAs exhibit a differentiated, quiescent, and contractile phenotype. Imbalanced release of various growth and fibrotic factors from circulating and inflammatory cells, results in transition from contractile phenotype to synthetic or proliferative phenotype. The mTORC1 activity plays an important role in regulating phenotypical transitions of vascular SMCs (46, 61, 71). Activation of mTORC1 by PDGF-BB promotes the transition of

SMCs from a contractile phenotype to a proliferative phenotype, whereas inhibition of mTORC1 with rapamycin induces SMC differentiation and inhibits SMC proliferation (49, 71).

In proliferating PASMC, inhibition of the mTORC1 and mTORC2 using rapamycin (Rap) significantly decreased phosphorylated (p) AKT (pAKT) and mTOR (pAKT), or pAKT/AKT and pmTOR/mTOR (Fig. 15A and B). The rapamycin-associated inhibition of AKT/mTOR signaling was associated with a significant decrease in the proliferative cell marker PCNA and a marked

- 78 - downregulation of CALHM1/2 (Fig. 15A and C). These data indicate that the mTORC1/C2 activity and expression level of CALHM1/2 are required for or involved in PASMC proliferation, and likely the development and progression of pulmonary vascular remodeling in patients with

PAH and animals with experimental PH.

- 79 -

Figure 15 – Rapamycin downregulates CALHM1 and 2

A B 1.2 1.2 0.8 0.8 2.0 2.0 kDa 1.0 1.0 50 0.6 0.6 1.5 1.5 * pAKT 0.8 0.8 * 0.6 0.6 0.4 0.4 1.0 1.0 50 AKT 0.4 0.4 *** 0.2 0.2 0.5 0.5 (Arbitrary Unit) pmTOR Level Protein 250 0.2 0.2 pAKT/AKT (a.u.)

*** *** pmTOR/mTOR (a.u.) mTOR 0.0 0.0 0.0 0.0 0.0 0.0 250 pAKT AKT pmTOR mTOR

33 PCNA 1.4 1.4 1.4 1.4 2.0 2.0 38 CALHM-1 1.2 1.2 1.2 1.2 * 1.0 1.0 1.0 1.0 1.5 1.5 37 CALHM-2 0.8 0.8 0.8 0.8 1.0 1.0 * 0.6 0.6 0.6 0.6 42 Actin *** 0.4 0.4 0.4 0.4

Protein Level Protein 0.5 0.5 pAKT/AKT (a.u.) (Normalized to PA) 0.2 0.2 0.2 0.2

*** ** pmTOR/mTOR (a.u.) Control 0.0 0.0 0.0 0.0 0.0 0.0 Rapamycin pAKT AKT pmTOR mTOR

C 1.0 1.5 1.5 1.5 1.5 1.5

0.8 1.0 1.0 1.0 1.0 ** 1.0 0.6 ** ** *** *** 0.4 ** 0.5 0.5 0.5 0.5 0.5 ProteinLevel (Arbitrary Unit) Protein Level Protein ProteinLevel (Arbitrary Unit) Protein Level Protein (Arbitrary Unit) Protein Level Protein Protein Level Protein

0.2 (Normalized to PA) (Normalized to PA) (Normalized to PA)

0.0 0.0 0.0 0.0 0.0 0.0 PCNA CALHM1 CALHM2 PCNA CALHM1 CALHM2

Figure 15. Inhibition of mTORC1 and mTORC2 by rapamycin downregulates CALHM1 and CALHM2 in proliferative PASMC. A: Western blot analyses on AKT, pAKT, mTOR and pmTOR, as well as the proliferation marker PCNA in control PASMC (Control) and rapamycin-treated PASMC (Rapamycin). B: Summarized data (means±SE) showing the protein expression levels of pAKT, AKT, pmTOR and mTOR (left panels), as well as the ratio of pAKT/AKT and pmTOR/mTOR (right panels) in control and rapamycin-treated PASMC. C: Summarized data (means±SE) showing protein levels of PCNA, CALHM1 and CALHM2 in control and rapamycin-treated PASMC. *p<0.05, **p<0.01, ***p<0.001 vs. Control.

- 80 -

In the next set of experiments, we measured protein expression level of CALHM1 and

CALHM2 in in our disease animal model, MCT-induced PH rat model (90), and hypothesized that our proteins of interest (i.e., CALHM1/2) would align with our contractile proliferative model of

SMC and be increased to mimic the proliferative phenotype. The results were in accordance with our hypothesis indeed, CALHM1/2 are significantly upregulated in freshly isolated PA from MCT-

PH rats, in comparison to the PA from normal controls (Fig. 16A and B). The upregulated expression of CALHM1/2 in PA from MCT-PH rats was inversely proportional to the decreases in the contractile markers MHC11 and TAGLN, but positively correlated to the proliferating markers PCNA and PDGFA (Fig. 16). These results indicate that upregulation of CALHM1/2 is involved in a) the pathogenic PASMC phenotypic transition from a contractile phenotype to a proliferative phenotype and b) the initiation and progression of pulmonary arterial medial hypertrophy due to increased PASMC proliferation.

Furthermore, we also measured CALHM1 protein level in PASMC from normal subjects and patients with PAH. As shown in Figure 17, the protein expression level of CALHM1 in proliferating PASMC from patients with idiopathic PAH was significantly higher than the cells from normal healthy subjects (Fig. 17). We also measured and compared CALHM2 in normal and

PAH PASMC, but the data were not exclusive. These results indicate that upregulated CALHM1, or enhanced Ca2+ influx through CALHM1 channels and increased ATP release through CALHM1 channels, is involved in the development and progression of concentric pulmonary arterial wall thickening due to excessive PASMC proliferation (and migration).

- 81 -

Figure 16 – Upregulated CALHM1 and 2 in MCT

A PA B Nor MCT kDa Control MCT 1.0 1.2 1.6 2.0 1.2 6.0 200 MHC11 *** ** * 0.8 1.0 5.0 33 PCNA 1.2 1.5 0.9 0.8 4.0 0.6 37 CALHM1 *** 0.6 0.8 1.0 0.6 3.0 0.4 *** 0.4 2.0 38 0.4 0.5 0.3 CALHM2 Level Protein (Arbitrary Unit) (Arbitrary 0.2 Level Protein

0.2 Unit) (Arbitrary 1.0 42 Actin 0.0 0.0 0.0 0.0 0.0 0.0 MHC11 PCNA TAGLN PDGFR CALHM1 CALHM2

Figure 16. CALHM1 and CALHM2 are upregulated in pulmonary arteries from rats with MCT-induced PH. A: Western blot analyses the contractile (MHC11) and proliferating (PCNA) cell markers, as well as CALHM1 and CALHM2 in freshly isolated PA from normal control rats (Nor) and rats with MCT- induced PH (MCT). B: Summarized data (means±SE) showing protein levels of MHC11, PCNA, TAGLN and PDGFR (left panels), as well as CALHM1 and CALHM2 (right panels) in normal control (Control) and MCT-induced PH (MCT) rats. *p<0.05, **p<0.01, ***p<0.001 vs. Control.

Figure 17 – Upregulated CALHM1 in IPAH hPASMCs

A PASMC B 0.6 **

kDa 0.4 37 CALHM1 0.2 42 β-actin (Arbitrary Unit) (Arbitrary 0.0 CALHM1 Protein Level Protein CALHM1 Nor IPAH

Figure 20. CALHM1 is upregulated in PASMC patients with idiopathic PAH (IPAH). A: Western blot analysis on CALHM1 in primary cultured PASMC from normal subjects and IPAH patients. B: Summarized data (means±SE) showing protein level of CALHM1 from normal and IPAH PASMC. **p<0.01 vs. Normal (Nor).

- 82 -

CONCLUSION

In this study, we first used an ex vivo model, freshly isolated PA (containing manly contractile PASMC) and primary cultured PASMC in media containing 10% FBS and growth factors (containing mainly proliferative PASMC), to identify the membrane receptors, ion channels and intracellular signaling proteins that are associated with the PASMC phenotypic transition from a contractile phenotype to a proliferative phenotype. Our results indicate that i)

CALHM1 and CALHM2, two voltage-activated and extracellular Ca2+-inhibited Ca2+ channels that also allow ATP to go through, are significantly upregulated in proliferating PASMC in comparison to contractile PASMC; ii) CaSR, an extracellular Ca2+-activated GPCR, and Notch2/3 receptors are also significantly upregulated in proliferating PASMC compared to contractile

PASMC; iii) the upregulated CALHM1/2, CaSR and Notch2/3 are associated with significant upregulation of AKT and mTOR and increases in the ratio of pAKT/AKT and pmTOR/mTOR in proliferating PASMC compared to contractile PASMC; iv) rapamycin-mediated inhibition of mTOR, which disrupts the function of both mTORC1 and mTORC2, downregulates CALHM1/2, decreases pAKT/AKT and pmTOR/mTOR ratio and attenuates PASMC proliferation; v)

CALHM1 protein expression level in freshly isolated PA from rats with experimental PH is significantly greater than in PA from normal control rats; and vi) CALHM1 in primary cultured

PASMC from patients with PAH is greater than in PASMC from normal subjects. These observations indicate that a) upregulation of CALHM1 (and CALHM2), along with CaSR and

Notch2/3, is involved in PASMC phenotypical switch from the contractile phenotype to the synthetic or proliferative phenotype; and b) upregulated CALHM1 (and CALHM2) is potentially involved in augmenting Ca2+ influx and enhancing ATP release to stimulate PASMC proliferation and develop pulmonary vascular remodeling due to PASMC proliferation and migration in patients with PAH and animals with experimental PH.

- 83 -

The evidence summarized above demonstrates that SMC differentiation can produce many types of intermediates within the gradient of a contractile to a proliferative phenotype within a progressing pathological state. It is understood that the SMCs in the normal vessel wall are absolutely necessary for their function towards maintenance of vascular tone and also contribute to the physiological vascular remodeling and proliferative process as a protective agent from the increased pulmonary arterial pressure (PAP) powered as a consequence by the heart. Both contractile and proliferative cells can thus be considered differentiated cells, although we present here that under a changed programmed environment, regardless of cells' initial known characterization with contractile markers to as defines, we can induce a change that shifts with the influences of its local environment and adapt towards a synthetic and proliferative phenotype that can be characterized by its known proliferative markers.

2+ 2+ The increased cytosolic Ca concentration ([Ca ]cyt) in PASMCs generate a local environment in which the cell contractile or proliferative phenotype is influenced by. This determines modulation within the initial epigenetic program contributing as a force where upregulation of CALHM1 and CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype.

Alternatively, upregulated CALHM1 and/or CALHM2 are required for or involved in initiating and maintaining excessive PASMC proliferation occurring in the pathophysiological and pathological conditions, such as in patients with PAH or animals with experimental PH. We can determine that Ca2+ influx through voltage- and extracellular Ca2+-regulated CALHM1/2 channels in the plasma membrane of PASMCs is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.

Additionally, due to PASMCs and PAECs difference physically, location among the lining of the blood vessel may be an indication that there is efficient communication happening

- 84 - intracellularly that allows for the proper flow of Ca2+ into and between the cells undergoing IPAH.

Mechanistically, there is an affinity and a localization of CALHM1 and CALHM2 being expressed in SMCs therefore sensitive to the presence of Ca2+ within the cell. Cl- and K+ channels enable the exiting of Ca2+ into the cytosol which may be the misalignment in the IPAH process if the receptors for CALHM are consistent between the two but also nonselective to the ions (Cl-, and

K+ ) or ATP, therefore leading to investigate if there is a upregulation of Ca2+ transporter (e.g.,

Ca2+/Mg2+ ATPase, Na+/Ca2+ exchanger) attempting to compensate for outward Ca2+ transportation that enable the build-up of Ca2+ in the cytosol of the cell. There may be a connection between the sustained CALHM protein level between normal and IPAH PASMCs due to an increase of gap junctions and pannexins’ role in intercellular communication, this is also further investigation for the future of this project. This coordinated communication in blood vessels is similar to that of systemic vascular smooth muscle cells or PASMCs which allow a direct transfer of electric signals and signaling molecules from cell to cell.

As a result, blood vessels within a pathological state or healthy state contain different compositions of SMCs' plasticity that enable them to perform and satisfy physiological needs. The

2+ diversity found within PASMCs considers the overall environment (increased [Ca ]cyt in PAH for example) and is dependent upon local changes and conditions that although affect the resulting normal function, do not allow it to cease or weaken given a combination of increased stimulus and pressure. This in turn is what pulmonary vascular biologists must further investigate carefully when an overflow of proliferation, migration, and change occurs against what natural physiologically responds to effect and is in fact creating layers to protect, but rather is actually leading to progressing disease.

- 85 -

REFERENCES

1. Alexander MR and Owens GK. Epigenetic control of smooth muscle cell differentiation

and phenotypic switching in vascular development and disease. Annu Rev Physiol 74: 13-

40, 2012.

2. Awad KS, West JD, de Jesus Perez V, and MacLean M. Novel signaling pathways in

pulmonary arterial hypertension (2015 Grover Conference Series). Pulm Circ 6: 285-294,

2016.

3. Babicheva A and Yuan JX. Endothelial Notch1 in pulmonary hypertension. Circ Res 124:

176-179, 2019.

4. Batton KA, Austin CO, Bruno KA, Burger CD, Shapiro BP, and Fairweather D. Sex

differences in pulmonary arterial hypertension: role of infection and autoimmunity in the

pathogenesis of disease. Biol Sex Differ 9: 15, 2018.

5. Beamish JA, Fu AY, Choi AJ, Haq NA, Kottke-Marchant K, and Marchant RE. The

influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth

muscle cell phenotype. Biomaterials 30: 4127-4135, 2009.

6. Beamish JA, He P, Kottke-Marchant K, and Marchant RE. Molecular regulation of

contractile smooth muscle cell phenotype: implications for vascular tissue engineering.

Tissue Eng Part B Rev 16: 467-491, 2010.

7. Bennett MR, Sinha S, and Owens GK. Vascular Smooth Muscle Cells in Atherosclerosis.

Circ Res 118: 692-702, 2016.

8. Braganza M, Shaw J, Solverson K, Vis D, Janovcik J, Varughese RA, Thakrar MV,

Hirani N, Helmersen D, and Weatherald J. A Prospective Evaluation of the Diagnostic

Accuracy of the Physical Examination for Pulmonary Hypertension. Chest 155: 982-990,

2019.

- 86 -

9. Brown LM, Chen H, Halpern S, Taichman D, McGoon MD, Farber HW, Frost AE,

Liou TG, Turner M, Feldkircher K, Miller DP, and Elliott CG. Delay in recognition

of pulmonary arterial hypertension: factors identified from the REVEAL Registry. Chest

140: 19-26, 2011.

10. Chakraborty R, Saddouk FZ, Carrao AC, Krause DS, Greif DM, and Martin KA.

Promoters to Study Vascular Smooth Muscle. Arterioscler Thromb Vasc Biol 39: 603-612,

2019.

11. Cisneros-Mejorado A, Gottlieb M, Ruiz A, Chara JC, Perez-Samartin A,

Marambaud P, and Matute C. Blockade and knock-out of CALHM1 channels attenuate

ischemic brain damage. J Cereb Blood Flow Metab 38: 1060-1069, 2018.

12. Dreses-Werringloer U, Lambert JC, Vingtdeux V, Zhao H, Vais H, Siebert A, Jain A,

Koppel J, Rovelet-Lecrux A, Hannequin D, Pasquier F, Galimberti D, Scarpini E,

Mann D, Lendon C, Campion D, Amouyel P, Davies P, Foskett JK, Campagne F, and

Marambaud P. A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels,

and Alzheimer's disease risk. Cell 133: 1149-1161, 2008.

13. Dreses-Werringloer U, Vingtdeux V, Zhao H, Chandakkar P, Davies P, and

Marambaud P. CALHM1 controls the Ca(2)(+)-dependent MEK, ERK, RSK and MSK

signaling cascade in neurons. J Cell Sci 126: 1199-1206, 2013.

14. Durmowicz AG and Stenmark KR. Mechanisms of structural remodeling in chronic

pulmonary hypertension. Pediatr Rev 20: e91-e102, 1999.

15. Farber HW, Miller DP, Poms AD, Badesch DB, Frost AE, Muros-Le Rouzic E,

Romero AJ, Benton WW, Elliott CG, McGoon MD, and Benza RL. Five-Year

outcomes of patients enrolled in the REVEAL Registry. Chest 148: 1043-1054, 2015.

- 87 -

16. Fernandez RA, Wan J, Song S, Smith KA, Gu Y, Tauseef M, Tang H, Makino A,

Mehta D, and Yuan JX. Upregulated expression of STIM2, TRPC6, and Orai2

contributes to the transition of pulmonary arterial smooth muscle cells from a contractile

to proliferative phenotype. Am J Physiol Cell Physiol 308: C581-593, 2015.

17. Firth AL, Won JY, and Park WS. Regulation of ca(2+) signaling in pulmonary

hypertension. Korean J Physiol Pharmacol 17: 1-8, 2013.

18. Frost A, Badesch D, Gibbs JSR, Gopalan D, Khanna D, Manes A, Oudiz R, Satoh T,

Torres F, and Torbicki A. Diagnosis of pulmonary hypertension. Eur Respir J 53, 2019.

19. Gallant C, You JY, Sasaki Y, Grabarek Z, and Morgan KG. MARCKS is a major

PKC-dependent regulator of calmodulin targeting in smooth muscle. J Cell Sci 118: 3595-

3605, 2005.

20. Gallego-Sandin S, Alonso MT, and Garcia-Sancho J. Calcium homoeostasis modulator

1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers

ER stress. Biochem J 437: 469-475, 2011.

21. Gees M, Colsoul B, and Nilius B. The role of transient receptor potential cation channels

in Ca2+ signaling. Cold Spring Harb Perspect Biol 2: a003962, 2010.

22. Geng Y, Bush M, Mosyak L, Wang F, and Fan QR. Structural mechanism of ligand

activation in human GABA(B) receptor. Nature 504: 254-259, 2013.

23. Geng Y, Mosyak L, Kurinov I, Zuo H, Sturchler E, Cheng TC, Subramanyam P,

Brown AP, Brennan SC, Mun HC, Bush M, Chen Y, Nguyen TX, Cao B, Chang DD,

Quick M, Conigrave AD, Colecraft HM, McDonald P, and Fan QR. Structural

mechanism of ligand activation in human calcium-sensing receptor. Elife 5, 2016.

24. Giannotti KC, Weinert S, Viana MN, Leiguez E, Araujo TLS, Laurindo FRM,

Lomonte B, Braun-Dullaeus R, and Teixeira C. A Secreted Phospholipase A2 Induces

- 88 -

Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like

State. Molecules 24, 2019.

25. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ,

and Yuan JX. Upregulated TRP and enhanced capacitative Ca(2+) entry in human

pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280:

H746-755, 2001.

26. Goncharov DA, Kudryashova TV, Ziai H, Ihida-Stansbury K, DeLisser H,

Krymskaya VP, Tuder RM, Kawut SM, and Goncharova EA. Mammalian target of

rapamycin complex 2 (mTORC2) coordinates pulmonary artery smooth muscle cell

metabolism, proliferation, and survival in pulmonary arterial hypertension. Circulation 129:

864-874, 2014.

27. Goncharova EA, Simon MA, and Yuan JX. mTORC1 in pulmonary arterial

hypertension. At the crossroads between vasoconstriction and vascular remodeling? Am J

Respir Crit Care Med 201: 1177-1179, 2020.

28. Guo X, Fereydooni A, Isaji T, Gorecka J, Liu S, Hu H, Ono S, Alozie M, Lee SR,

Taniguchi R, Yatsula B, Nassiri N, Zhang L, and Dardik A. Inhibition of the Akt1-

mTORC1 axis alters venous remodeling to improve arteriovenous fistula patency. Sci Rep

9: 11046, 2019.

29. Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones

PL, Maitland ML, Michelakis ED, Morrell NW, Newman JH, Rabinovitch M,

Schermuly R, Stenmark KR, Voelkel NF, Yuan JX, and Humbert M. Inflammation,

growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 54: S10-19, 2009.

30. Hendy GN and Canaff L. Calcium-sensing receptor, proinflammatory cytokines and

calcium homeostasis. Semin Cell Dev Biol 49: 37-43, 2016.

- 89 -

31. Hendy GN, Guarnieri V, and Canaff L. Calcium-sensing receptor and associated

diseases. Prog Mol Biol Transl Sci 89: 31-95, 2009.

32. Hill-Eubanks DC, Werner ME, Heppner TJ, and Nelson MT. Calcium signaling in

smooth muscle. Cold Spring Harb Perspect Biol 3: a004549, 2011.

33. Houssaini A, Abid S, Derumeaux G, Wan F, Parpaleix A, Rideau D, Marcos E, Kebe

K, Czibik G, Sawaki D, Treins C, Dubois-Rande JL, Li Z, Amsellem V, Lipskaia L,

Pende M, and Adnot S. Selective tuberous sclerosis complex 1 gene deletion in smooth

muscle activates mammalian target of rapamycin signaling and induces pulmonary

hypertension. Am J Respir Cell Mol Biol 55: 352-367, 2016.

34. Houssaini A, Abid S, Mouraret N, Wan F, Rideau D, Saker M, Marcos E, Tissot CM,

Dubois-Rande JL, Amsellem V, and Adnot S. Rapamycin reverses pulmonary artery

smooth muscle cell proliferation in pulmonary hypertension. Am J Respir Cell Mol Biol 48:

568-577, 2013.

35. Humbert M and Ghofrani HA. The molecular targets of approved treatments for

pulmonary arterial hypertension. Thorax 71: 73-83, 2016.

36. Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM,

Christman BW, Weir EK, Eickelberg O, Voelkel NF, and Rabinovitch M. Cellular and

molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 43: 13S-

24S, 2004.

37. Ingber DE. Tensegrity-based mechanosensing from macro to micro. Prog Biophys Mol

Biol 97: 163-179, 2008.

38. Jain M, Dhanesha N, Doddapattar P, Chorawala MR, Nayak MK, Cornelissen A,

Guo L, Finn AV, Lentz SR, and Chauhan AK. Smooth muscle cell-specific fibronectin-

- 90 -

EDA mediates phenotypic switching and neointimal hyperplasia. J Clin Invest 130: 295-

314, 2020.

39. Koledova VV and Khalil RA. Ca2+, calmodulin, and cyclins in vascular smooth muscle

cell cycle. Circ Res 98: 1240-1243, 2006.

40. Kreusser MM and Backs J. Integrated mechanisms of CaMKII-dependent ventricular

remodeling. Front Pharmacol 5: 36, 2014.

41. Kumar A and Neema PK. Severe pulmonary hypertension and right ventricular failure.

Indian J Anaesth 61: 753-759, 2017.

42. Lacolley P, Regnault V, Nicoletti A, Li Z, and Michel JB. The vascular smooth muscle

cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res 95: 194-

204, 2012.

43. Lee M, San Martin A, Valdivia A, Martin-Garrido A, and Griendling KK. Redox-

sensitive regulation of myocardin-related transcription factor (MRTF-A) phosphorylation

via palladin in vascular smooth muscle cell differentiation marker gene expression. PLoS

One 11: e0153199, 2016.

44. Leggett K, Maylor J, Undem C, Lai N, Lu W, Schweitzer K, King LS, Myers AC,

Sylvester JT, Sidhaye V, and Shimoda LA. Hypoxia-induced migration in pulmonary

arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1. Am

J Physiol Lung Cell Mol Physiol 303: L343-353, 2012.

45. Li X, Zhang X, Leathers R, Makino A, Huang C, Parsa P, Macias J, Yuan JX,

Jamieson SW, and Thistlethwaite PA. Notch3 signaling promotes the development of

pulmonary arterial hypertension. Nat Med 15: 1289-1297, 2009.

46. Liu M and Gomez D. Smooth muscle cell phenotypic diversity. Arterioscler Thromb Vasc

Biol 39: 1715-1723, 2019.

- 91 -

47. Ma Z, Siebert AP, Cheung KH, Lee RJ, Johnson B, Cohen AS, Vingtdeux V,

Marambaud P, and Foskett JK. Calcium homeostasis modulator 1 (CALHM1) is the

pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of

neuronal excitability. Proc Natl Acad Sci U S A 109: E1963-1971, 2012.

48. Ma Z, Tanis JE, Taruno A, and Foskett JK. Calcium homeostasis modulator (CALHM)

ion channels. Pflugers Arch 468: 395-403, 2016.

49. Majed BH and Khalil RA. Molecular mechanisms regulating the vascular prostacyclin

pathways and their adaptation during pregnancy and in the newborn. Pharmacol Rev 64:

540-582, 2012.

50. Makino A, Firth AL, and Yuan JX. Endothelial and smooth muscle cell ion channels in

pulmonary vasoconstriction and vascular remodeling. Compr Physiol 1: 1555-1602, 2011.

51. Malenfant S, Margaillan G, Loehr JE, Bonnet S, and Provencher S. The emergence of

new therapeutic targets in pulmonary arterial hypertension: from now to the near future.

Expert Rev Respir Med 7: 43-55, 2013.

52. Mandegar M, Fung YC, Huang W, Remillard CV, Rubin LJ, and Yuan JX. Cellular

and molecular mechanisms of pulmonary vascular remodeling: role in the development of

pulmonary hypertension. Microvasc Res 68: 75-103, 2004.

53. McMurtry IF, Petrun MD, and Reeves JT. Lungs from chronically hypoxic rats have

decreased pressor response to acute hypoxia. Am J Physiol 235: H104-109, 1978.

54. McMurtry IF, Rounds S, and Stanbrook HS. Studies of the mechanism of hypoxic

pulmonary vasoconstriction. Adv Shock Res 8: 21-33, 1982.

55. Meng Q, Lai YC, Kelly NJ, Bueno M, Baust JJ, Bachman TN, Goncharov D,

Vanderpool RR, Radder JE, Hu J, Goncharova E, Morris AM, Mora AL, Shapiro

SD, and Gladwin MT. Development of a mouse model of metabolic syndrome, pulmonary

- 92 -

hypertension, and heart failure with preserved ejection fraction. Am J Respir Cell Mol Biol

56: 497-505, 2017.

56. Miyagawa K, Shi M, Chen PI, Hennigs JK, Zhao Z, Wang M, Li CG, Saito T, Taylor

S, Sa S, Cao A, Wang L, Snyder MP, and Rabinovitch M. Smooth muscle contact drives

endothelial regeneration by BMPR2-Notch1-mediated metabolic and epigenetic changes.

Circ Res 124: 211-224, 2019.

57. Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, McMurtry IF,

Stenmark KR, Thistlethwaite PA, Weissmann N, Yuan JX, and Weir EK. Cellular and

molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 54: S20-31, 2009.

58. Ogata T and Iijima T. Structure and pathogenesis of plexiform lesion in pulmonary

hypertension. Chin Med J (Engl) 106: 45-48, 1993.

59. Oka Y. Opening a "Wide" Window onto Taste Signal Transmission. Neuron 98: 456-458,

2018.

60. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75:

487-517, 1995.

61. Owens GK, Kumar MS, and Wamhoff BR. Molecular regulation of vascular smooth

muscle cell differentiation in development and disease. Physiol Rev 84: 767-801, 2004.

62. Perros F, Sentenac P, Boulate D, Manaud G, Kotsimbos T, Lecerf F, Lamrani L,

Fadel E, Mercier O, Londono-Vallejo A, Humbert M, and Eddahibi S. Smooth Muscle

Phenotype in Idiopathic Pulmonary Hypertension: Hyper-Proliferative but not Cancerous.

Int J Mol Sci 20, 2019.

63. Pfitzner J. Poiseuille and his law. Anaesthesia 31: 273-275, 1976.

64. Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto

K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, and Archer SL. In vivo gene

- 93 -

transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and

restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107:

2037-2044, 2003.

65. Proweller A, Pear WS, and Parmacek MS. Notch signaling represses myocardin-

induced smooth muscle cell differentiation. J Biol Chem 280: 8994-9004, 2005.

66. Raines EW and Ferri N. Thematic review series: The immune system and atherogenesis.

Cytokines affecting endothelial and smooth muscle cells in vascular disease. J Lipid Res

46: 1081-1092, 2005.

67. Rasmussen G and Rasmussen C. Calmodulin-dependent protein kinase II is required for

G1/S progression in HeLa cells. Biochem Cell Biol 73: 201-207, 1995.

68. Rensen SS, Doevendans PA, and van Eys GJ. Regulation and characteristics of vascular

smooth muscle cell phenotypic diversity. Neth Heart J 15: 100-108, 2007.

69. Rodman DM, Yamaguchi T, O'Brien RF, and McMurtry IF. Hypoxic contraction of

isolated rat pulmonary artery. J Pharmacol Exp Ther 248: 952-959, 1989.

70. Runo JR and Loyd JE. Primary pulmonary hypertension. Lancet 361: 1533-1544, 2003.

71. Rzucidlo EM, Martin KA, and Powell RJ. Regulation of vascular smooth muscle cell

differentiation. J Vasc Surg 45 Suppl A: A25-32, 2007.

72. Sahoo S, Meijles DN, Al Ghouleh I, Tandon M, Cifuentes-Pagano E, Sembrat J, Rojas

M, Goncharova E, and Pagano PJ. MEF2C-MYOCD and leiomodin1 suppression by

miRNA-214 promotes smooth muscle cell phenotype switching in pulmonary arterial

hypertension. PLoS One 11: e0153780, 2016.

73. Sakao S and Tatsumi K. Vascular remodeling in pulmonary arterial hypertension:

multiple cancer-like pathways and possible treatment modalities. Int J Cardiol 147: 4-12,

2011.

- 94 -

74. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A,

Lai YJ, Weissmann N, Seeger W, and Grimminger F. Reversal of experimental

pulmonary hypertension by PDGF inhibition. J Clin Invest 115: 2811-2821, 2005.

75. Sclafani A and Ackroff K. Greater reductions in fat preferences in CALHM1 than CD36

knockout mice. Am J Physiol Regul Integr Comp Physiol 315: R576-R585, 2018.

76. Siebert AP, Ma Z, Grevet JD, Demuro A, Parker I, and Foskett JK. Structural and

functional similarities of calcium homeostasis modulator 1 (CALHM1) ion channel with

connexins, pannexins, and innexins. J Biol Chem 288: 6140-6153, 2013.

77. Simonneau G, Montani D, Celermajer DS, Denton CP, Gatzoulis MA, Krowka M,

Williams PG, and Souza R. Haemodynamic definitions and updated clinical classification

of pulmonary hypertension. Eur Respir J 53, 2019.

78. Smirnov SV, Robertson TP, Ward JP, and Aaronson PI. Chronic hypoxia is associated

with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am J Physiol

266: H365-370, 1994.

79. Smith KA, Ayon RJ, Tang H, Makino A, and Yuan JX. Calcium-Sensing Receptor

Regulates Cytosolic [Ca (2+) ] and Plays a Major Role in the Development of Pulmonary

Hypertension. Front Physiol 7: 517, 2016.

80. Smith KA, Voiriot G, Tang H, Fraidenburg DR, Song S, Yamamura H, Yamamura

A, Guo Q, Wan J, Pohl NM, Tauseef M, Bodmer R, Ocorr K, Thistlethwaite PA,

Haddad GG, Powell FL, Makino A, Mehta D, and Yuan JX. Notch activation of Ca2+

signaling in the development of hypoxic pulmonary vasoconstriction and pulmonary

hypertension. Am J Respir Cell Mol Biol 53: 355-367, 2015.

- 95 -

81. Solik P, Lesny P, Luknar M, Varga I, and Goncalvesova E. The long-term response to

treatment with calcium channel blockers in a patient with idiopathic pulmonary arterial

hypertension. Bratisl Lek Listy 114: 283-286, 2013.

82. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein

phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522 Pt 2: 177-185,

2000.

83. Song MY, Makino A, and Yuan JX. STIM2 Contributes to Enhanced Store-operated Ca

Entry in Pulmonary Artery Smooth Muscle Cells from Patients with Idiopathic Pulmonary

Arterial Hypertension. Pulm Circ 1: 84-94, 2011.

84. Song S, Babicheva A, Zhao T, Ayon RJ, Rodriguez M, Rahimi S, Balistrieri F,

Harrington A, Shyy JY, Thistlethwaite PA, Makino A, and Yuan JX. Notch enhances

Ca2+ entry by activating calcium-sensing receptors and inhibiting voltage-gated K+

channels. Am J Physiol Cell Physiol 318: C954-C968, 2020.

85. Song S, Carr SG, McDermott KM, Rodriguez M, Babicheva A, Balistrieri A, Ayon

RJ, Wang J, Makino A, and Yuan JX. STIM2 (Stromal Interaction Molecule 2)-

Mediated Increase in Resting Cytosolic Free Ca(2+) Concentration Stimulates PASMC

Proliferation in Pulmonary Arterial Hypertension. Hypertension 71: 518-529, 2018.

86. Stenmark KR, Fagan KA, and Frid MG. Hypoxia-induced pulmonary vascular

remodeling: cellular and molecular mechanisms. Circ Res 99: 675-691, 2006.

87. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary

vascular remodeling. Annu Rev Physiol 59: 89-144, 1997.

88. Strasser C, Grote P, Schauble K, Ganz M, and Ferrando-May E. Regulation of nuclear

envelope permeability in cell death and survival. Nucleus 3: 540-551, 2012.

- 96 -

89. Sweeney M, McDaniel SS, Platoshyn O, Zhang S, Yu Y, Lapp BR, Zhao Y,

Thistlethwaite PA, and Yuan JX. Role of capacitative Ca2+ entry in bronchial contraction

and remodeling. J Appl Physiol (1985) 92: 1594-1602, 2002.

90. Tang H, Babicheva A, McDermott KM, Gu Y, Ayon RJ, Song S, Wang Z, Gupta A,

Zhou T, Sun X, Dash S, Wang Z, Balistrieri A, Zheng Q, Cordery AG, Desai AA,

Rischard F, Khalpey Z, Wang J, Black SM, Garcia JGN, Makino A, and Yuan JX.

Endothelial HIF-2alpha contributes to severe pulmonary hypertension due to endothelial-

to-mesenchymal transition. Am J Physiol Lung Cell Mol Physiol 314: L256-L275, 2018.

91. Tang H, Chen J, Fraidenburg DR, Song S, Sysol JR, Drennan AR, Offermanns S, Ye

RD, Bonini MG, Minshall RD, Garcia JG, Machado RF, Makino A, and Yuan JX.

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension.

Am J Physiol Lung Cell Mol Physiol 308: L208-220, 2015.

92. Tang H, Wu K, Wang J, Vinjamuri S, Gu Y, Song S, Wang Z, Zhang Q, Balistrieri

A, Ayon RJ, Rischard F, Vanderpool R, Chen J, Zhou G, Desai AA, Black SM, Garcia

JGN, Yuan JX, and Makino A. Pathogenic Role of mTORC1 and mTORC2 in

Pulmonary Hypertension. JACC Basic Transl Sci 3: 744-762, 2018.

93. Tang H, Yamamura A, Yamamura H, Song S, Fraidenburg DR, Chen J, Gu Y, Pohl

NM, Zhou T, Jimenez-Perez L, Ayon RJ, Desai AA, Goltzman D, Rischard F,

Khalpey Z, Black SM, Garcia JG, Makino A, and Yuan JX. Pathogenic role of calcium-

sensing receptors in the development and progression of pulmonary hypertension. Am J

Physiol Lung Cell Mol Physiol 310: L846-859, 2016.

94. Tansey MG, Luby-Phelps K, Kamm KE, and Stull JT. Ca(2+)-dependent

phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain

phosphorylation within smooth muscle cells. J Biol Chem 269: 9912-9920, 1994.

- 97 -

95. Taruno A. ATP Release Channels. Int J Mol Sci 19, 2018.

96. Thenappan T, Ormiston ML, Ryan JJ, and Archer SL. Pulmonary arterial hypertension:

pathogenesis and clinical management. BMJ 360: j5492, 2018.

97. Tuder RM. Pulmonary vascular remodeling in pulmonary hypertension. Cell Tissue Res

367: 643-649, 2017.

98. Wan J, Yamamura A, Zimnicka AM, Voiriot G, Smith KA, Tang H, Ayon RJ,

Choudhury MS, Ko EA, Wang J, Wang C, Makino A, and Yuan JX. Chronic hypoxia

selectively enhances L- and T-type voltage-dependent Ca2+ channel activity in pulmonary

artery by upregulating Cav1.2 and Cav3.2. Am J Physiol Lung Cell Mol Physiol 305: L154-

164, 2013.

99. Wang J, Juhaszova M, Rubin LJ, and Yuan XJ. Hypoxia inhibits gene expression of

voltage-gated K+ channel alpha subunits in pulmonary artery smooth muscle cells. J Clin

Invest 100: 2347-2353, 1997.

100. Wang Y, Mishra R, and Simonson MS. Ca2+/calmodulin-dependent protein kinase II

stimulates c-fos transcription and DNA synthesis by a Src-based mechanism in glomerular

mesangial cells. J Am Soc Nephrol 14: 28-36, 2003.

101. Yamamura H, Yamamura A, Ko EA, Pohl NM, Smith KA, Zeifman A, Powell FL,

Thistlethwaite PA, and Yuan JX. Activation of Notch signaling by short-term treatment

with Jagged-1 enhances store-operated Ca2+ entry in human pulmonary arterial smooth

muscle cells. Am J Physiol Cell Physiol 306: C871-878, 2014.

102. Yamamura K, Ohishi K, Katayama N, Kato K, Shibasaki T, Sugimoto Y, Miyata E,

Shiku H, Masuya M, Nishioka J, Nobori T, Nishikawa M, Inagaki Y, Hiramatsu H,

and Nakahata T. Notch ligand Delta-1 differentially modulates the effects of gp130

- 98 -

activation on interleukin-6 receptor a-positive and -negative human hematopoietic

progenitors. Cancer Sci 98: 1597-1603, 2007.

103. Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, Tigno

DD, Thistlethwaite PA, Rubin LJ, and Yuan JX. Enhanced expression of transient

receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad

Sci U S A 101: 13861-13866, 2004.

104. Yu Y, Keller SH, Remillard CV, Safrina O, Nicholson A, Zhang SL, Jiang W, Vangala

N, Landsberg JW, Wang JY, Thistlethwaite PA, Channick RN, Robbins IM, Loyd JE,

Ghofrani HA, Grimminger F, Schermuly RT, Cahalan MD, Rubin LJ, and Yuan JX.

A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with

idiopathic pulmonary arterial hypertension. Circulation 119: 2313-2322, 2009.

105. Yuan XJ, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Hypoxia reduces

potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J

Physiol 264: L116-123, 1993.

106. Yuan XJ, Tod ML, Rubin LJ, and Blaustein MP. Contrasting effects of hypoxia on

tension in rat pulmonary and mesenteric arteries. Am J Physiol 259: H281-289, 1990.

107. Zhang M, Li F, Wang X, Gong J, Xian Y, Wang G, Zheng Z, Shang C, Wang B, He

Y, Wang W, and Lin R. MiR-145 alleviates Hcy-induced VSMC proliferation, migration,

and phenotypic switch through repression of the PI3K/Akt/mTOR pathway. Histochem

Cell Biol 153: 357-366, 2020.

- 99 -