pulmonary artery smooth muscle cell hif-1α regulates...

Pulmonary artery smooth muscle cell HIF-1 regulates endothelin expression 1

via microRNA-543 2

Ching-Chia Wang1,4, Lihua Ying1,2, Elizabeth A Barnes1.2, Eloa S. Adams,1,6 Francis Y. 3

Kim1,5, Karl W. Engel1, Cristina M. Alvira1,3, David N. Cornfield1,2,3 4

5

Center for Excellence in Pulmonary Biology1, Division of Pulmonary, Asthma and 6

Sleep Medicine2 and Critical Care Medicine3, Department of Pediatrics, Stanford 7

University Medical School, Stanford, CA 94305; and Department of Pediatrics, 8

National Taiwan University Children Hospital, National Taiwan University Medical 9

College, Taipei, Taiwan4, Milwaukee Children’s Hospital, Medical College of 10

Wisconsin, Milwaukee, WI5, Kaiser Oakland, Oakland, CA6. 11

12

Address for Correspondence: David N. Cornfield, M.D. 13

Center for Excellence in Pulmonary Biology 14

Stanford University School of Medicine 15

770 Welch Road, Suite 350 16

Stanford, CA 94305 17

Phone: (650)-725-8325 18

Fax: (650)-498-5560 19

Email: [email protected] 20

Running Title: HIF-1 constrains endothelin expression via microRNA 21

Author Contributions: 22

Concept and Design: ESA, CMA, DNC, LY 23

Acquisition, Analysis, and Interpretation of the Data: CW, ESA, EAB, FYK, LY, CMA, 24

DNC 25

Composing the first draft of the Manuscript: CW, LY 26

Revising the Manuscript for Important Intellectual Content: LY, CMA, DNC 27

Word count: 28

Abstract 239 words 29

Body 2945 30

31

Keywords: hypoxia-inducible factor-1, ET-1, miRNA, pulmonary arterial 32 hypertension, TWIST 33

34

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (171.066.012.093) on June 15, 2018.Copyright © 2018 American Physiological Society. All rights reserved.

mailto:[email protected]

2

Abstract 35

Pulmonary artery smooth muscle cells (PASMC) express endothelin (ET-1) which 36

modulates the pulmonary vascular response to hypoxia. Although cross-talk between 37

hypoxia-inducible factor-1HIF-1 an O2-sensitive transcription factor, and ET-1 38

is established, the cell-specific relationship between HIF-1 and ET-1 expression 39

remains incompletely understood. We tested the hypotheses that in PASMC: (i) 40

HIF-1α expression constrains ET-1 expression; and (ii) a specific microRNA (miRNA) 41

links HIF-1α and ET-1 expression. In human PASMC (hPASMC), depletion of HIF-1 42

with siRNA, increased ET-1 expression at both the mRNA and protein level (p<0.01). 43

In HIF-1-/- murine (m)PASMC, ET-1 gene and protein expression was increased 44

(p<0.0001) compared to HIF-1+/+ cells. miRNA profiles were screened in hPASMC 45

transfected with siRNA-HIF-1 and RNA hybridization performed on the Agilent 46

human miRNA microarray. With HIF-1depletion, miRNA-543 increased by 2.4 fold 47

(p<0.01). In hPASMC, miRNA-543 overexpression increased ET-1 gene (p<0.01) and 48

protein (p<0.01) expression, decreased TWIST gene expression (p<0.05) and 49

increased ET-1 gene and protein expression, compared to NTC (p<0.01). Moreover, 50

we evaluated low passage hPASMC from control and pulmonary arterial hypertension 51

(IPAH) patients. Compared to controls, protein expression of HIF-1 and TWIST1 52

was decreased (p<0.05) and miRNA-543, and ET-1 expression increased (p<0.001), 53

in hPASMC from IPAH patients. Thus, in PASMC, loss of HIF-1 increases 54

miRNA-543 which decreases Twist expression, leading to an increase in PASMC 55

ET-1 expression. This previously undescribed link between HIF-1, and ET-1 via 56

miRNA-543 mediated Twist suppression, represents another layer of molecular 57

regulation that might determine pulmonary vascular tone. 58

59

60


3

Introduction 61

At all points in mammalian life, oxygen (O2) tension plays a central role in 62

determining pulmonary vascular tone. During mammalian lung development in the 63

intrauterine environment pulmonary blood flow is constrained and O2 tension is low (1, 64

36). With the increase in O2 tension that accompanies the onset of air-breathing life, 65

pulmonary blood flow increases approximately 10 fold, as pulmonary artery pressure 66

falls to less than half of systemic levels over the first 24 hours of postnatal life(12). 67

Throughout air-breathing life, low pulmonary vascular tone is maintained even as the 68

pulmonary circulation responds to compromised ventilation with vasoconstriction to 69

prevent intrapulmonary shunting (44). If, however, pulmonary vascular tone is 70

persistently increased, as occurs in a number of disease states, the pulmonary 71

circulation remodels and right heart failure, and even death, can occur(40). 72

Though the physiology of pulmonary blood flow is relatively well described, the 73

molecular mechanisms that underlie the developmental regulation of pulmonary 74

vascular tone across fetal, neonatal and adult life remain incompletely understood. 75

Multiple lines of evidence point to a central role for the O2-sensitive transcription 76

factor, hypoxia-inducible factor-1 (HIF-1), in the regulation of pulmonary vascular 77

tone (5, 15, 22, 35). In pulmonary artery smooth muscle cells (PASMC) derived from 78

the fetal circulation, HIF-1 protein is relatively O2-insensitive, owing perhaps to 79

developmental differences in prolyl hydroxylase expression (35). However, in PASMC 80

derived from the mature pulmonary circulation, HIF-1 protein expression is highly 81

O2-sensitive (35, 50). In the context of the regulation of pulmonary vascular tone, 82

cell-specific loss of HIF-1 has been reported to either mitigate (5) or accentuate (24) 83

hypoxia-induced pulmonary hypertension. In mice wherein HIF-1 was deleted in 84

smooth muscle cells using an inducible myosin heavy chain promoter, hypoxic 85

pulmonary hypertension is attenuated (5). In contrast, data from mice with a 86

constitutive SMC-specific deletion of HIF-1 indicates that HIF-1 plays a role in 87

maintaining low pulmonary vascular resistance by constraining myosin chain 88


4

phosphorylation (24), and augmenting expression of a calcium sensitive subunit of the 89

calcium-sensitive K+ channel (2). Though data derived from human tissues generally 90

has demonstrated an overall increase in HIF-1 expression(27), including in PASMC 91

(9, 42), our lab evaluated PASMC derived from patients with pulmonary arterial 92

hypertension, demonstrated a decrease in HIF-1 expression, and an increase in 93

myosin light chain phosphorylation, kinase activity and contractility (6). Arguably, the 94

divergent findings may derive from dynamic changes in HIF-1 expression in PASMC 95

in association with increases in pulmonary vascular tone and remodeling. 96

Endothelin-1 (ET-1), a vasoactive polypeptide and known downstream target of 97

HIF-1also plays a central role in regulating pulmonary vascular tone(3). Though 98

there is wide appreciation for the role of pulmonary endothelial cell derived ET-1, the 99

physiologic significance of ET-1 produced by PASMC is less well established. Data 100

from our laboratory demonstrated that ET-1 produced by PASMC potentiates the 101

pulmonary vascular response to hypoxia (23). However, the relationship between 102

HIF-1and ET-1 in PASMC is complex with clear evidence that HIF-1 can increase 103

ET-1 expression even as an increase in ET-1 expression can augment HIF-1 protein 104

expression by either increasing synthesis and decreasing prolyl hydroxylase 105

mediated degradation (33) or alternatively by suppressing proteasome-dependent 106

degradation(28). Whether HIF-1 might constrain ET-1 expression in PASMC 107

remains unknown. 108

HIF-1 functions as a key regulator of the response to hypoxia by regulating 109

proteins involved in essential biological processes such as erythropoiesis, cell-cycle, 110

angiogenesis, metabolism and bioenergetics (37). Recent data demonstrates that 111

miRNAs can also mediate the hypoxic response via either HIF-dependent or 112

-independent mechanisms. miRNAs, short noncoding RNA molecules of 21–24 113

nucleotides in length (7), regulate gene expression by binding the 3’UTR of mRNA 114

targets (18). For example, under hypoxic conditions miR-210, a HIF-1 target, 115


5

promotes cell cycle progression and limits apoptosis (14). Similarly, miR-191, a 116

HIF-1 target, promotes cell migration and proliferation under hypoxic conditions (31). 117

Given that endogenous miRNAs can downregulate gene expression at the 118

post-transcriptional level through mRNA degradation or promotion of targeted mRNA 119

degradation, we hypothesized that miRNAs might mechanistically link ET-1 and 120

HIF-1. We tested the hypotheses that in PASMC: (i) HIF-1α expression constrains 121

ET-1 expression; and (ii) a specific miRNA links HIF-1α and ET-1 expression. 122

123

Methods 124

Primary mouse PASMC isolation. Primary mouse PASMC (mPASMC) were isolated 125

from SM22-HIF-1-/- and littermate control mice using a modified 126

elastase/collagenase digestion protocol (23, 24). PA tissue was digested in dispersion 127

medium containing 40μmol/l CaCl2, 0.5mg/ml elastase obtained from Worthington 128

Biochemical (Lakewood, NJ), 0.5mg/ml collagenase (Worthington Biochemical), 129

0.2mg/ml soybean trypsin inhibitor (Worthington Biochemical), and 2 mg/ml albumin 130

obtained from Sigma-Aldrich (St. Louis, MO) for 20 min at 37°C. After filtration with 131

100-μm cell strainers, cells were incubated with Dynabeads purchased from Thermo 132

Fisher Scientific (Waltham, MA) coated with anti-CD31 and anti-CD102 antibodies 133

obtained from BD Biosciences (San Jose, CA) for 15 min, in order to deplete 134

endothelial cells expressing CD31 and CD102. Remaining SMC were collected 135

through centrifugation at 225g for 6 min at 4°C and cultured in DMEM obtained from 136

Thermo Fisher containing 10% fetal bovine serum (FBS) with antibiotic solution 137

obtained from Thermo Fisher Scientific (Waltham, MA). To confirm isolation of 138

PASMC, cells were stained for -smooth muscle actin (-SMA) with an antibody 139

obtained from Sigma-Aldrich (catalog number A2547, St. Louis, MO) at 1:400 dilution 140

using immunofluorescence. 141

Cell culture. In vitro studies were performed with mPASMC isolated from control and 142

transgenic C57BL/6J mice with selective deletion of HIF-1 in SMC (SM22-HIF-1-143


6

-/-) (24). hPASMC were purchased from Lonza (Basel, Switzerland) and grown 144

according to the manufacturer’s protocol. Cells from passages 4–8 were used for all 145

experiments. 146

siRNA transfection. hPASMC were transfected at 50–70% confluence using 147

Lipofectamine RNAiMAX purchased from Thermo Fischer Scientific, according to the 148

manufacturer’s protocol. Briefly, siHIF-1 purchased from Thermo Scientific 149

Dharmacon or siNTC purchased from Thermo Scientific Dharmacon was transfected 150

at a final concentration of 50nM. Twenty-four hours post-transfection, cells were 151

re-fed with fresh media. After an additional 24 h, cells were harvested and used for 152

ET-1, HIF-1α and miRNA studies. 153

HIF-1 overexpression. The HIF-1α expression plasmid, HA-HIF-1 (CA), 154

containing the double mutant P402A/P564A thereby preventing hydroxylation and 155

permitting constitutive activation(13), was a kind gift from Dr. A.J. Giaccia, Stanford 156

University. An empty vector, pcDNA3, served as a transfection control. hPASMC were 157

transfected by Lipofectamine LTX/PLUS method Thermo Fisher Scientific, per the 158

manufacturer’s instructions. In brief, cells at 50-80% confluence were transfected with 159

10μg of DNA per 100mm plate. 24h post-transfection, cells were re-fed with fresh 160

media. After an additional 24h, cells were harvested and used for ET-1, HIF-1 and 161

miRNA studies. 162

miRNA microarray assay. Total RNA from hPASMC was prepared using Trizol 163

Reagent (Invitrogen, Carlsbad, CA, USA). RNA hybridization was then performed on 164

the Agilent (Santa Clara, CA) human miRNA microarray (v3) with 15k features. 165

miRNAs with significantly different expression profiles (1.2 fold change and p<0.05) 166

were selected through statistical analysis using GeneSpring (Agilent) Software. 167

Analysis was performed using Pathway Studio and MedScan obtained from Ariadne 168

Genomics of Elsevier (Amsterdam, NL) to predict target genes. 169

miRNA overexpression and inhibition. Synthesized miRNA mimics were purchased 170

from GE-Dharmacon (Lafayette, CO) to overexpress miRNA-543 or locked nucleic 171


7

acid (LNA) inhibitors from Exiqon Qiagen (Woburn, MA) to deplete miRNA-543 and 172

each control, consisting of the parental vector without a miR target (32), were 173

purchased. hPASMC were transfected by Lipofectamine RNAiMAX method from 174

Thermo Fisher Scientific per the manufacturer’s instructions. In brief, cells at 50-80% 175

confluence were transfected with 50nM of miRNA mimics or 50nM of LNA inhibitors 176

per 100mm plate. 6 h post-transfection, cells were re-fed with fresh media. After a 177

total of 48 h, cells were harvested and used for ET-1, HIF-1 and miRNA studies. 178

Quantitative RT-PCR. To determine ET-1, HIF-1 TWIST1 and miRNA mRNA 179

expression, total RNA was isolated from cultured hPASMC and mPASMC using the 180

RNeasy Mini Kit purchased from Qiagen (Hilden, FDR). First-strand cDNA was 181

synthesized using SuperScript III Reverse Transcriptase obtained from Thermo 182

Fischer Scientific and cDNA for miRNA analysis was synthesized using specific 183

stem-loop reverse transcription primers according to the TaqMan miRNA Assay 184

protocol. Products were subsequently amplified on the C1000 Thermal Cycler CFX 185

384 Real-Time System obtained from Bio-Rad Laboratories (Hercules, CA) using 186

PCR Universal Master Mix obtained from Thermo Fischer Scientific. Quantitative 187

RT-PCR was performed using the following cycle: 95°C for 10 min, 40 cycles of 95°C 188

for 15s and 60°C for 60 s, 60°C for 5 min, followed by a dissociation curve analysis. 189

The relative expression levels of ET-1 and HIF-1 were normalized to 18S ribosomal 190

RNA and miRNAs were normalized to U6 small nuclear RNA (snRNA) expression. All 191

mRNA expression levels were analyzed using the ∆∆CT method. 192

ET-1 ELISA. Secreted ET-1 was measured using an ET-1 colorimetric immunometric 193

ELISA kit purchased from Enzo Life Sciences (Farmingdale, New York) (4, 23, 45). 194

Briefly, media from transfected hPASMC or mPASMC were plated in duplicate and 195

incubated for 1 h at room temperature. To measure intracellular ET-1 protein in 196

hPASMC and mPASMC cells were harvested and lysed. Cell lysate samples were 197

plated in duplicate, and incubated for 24 h at 4°C. Optical density was measured at 198

450 nm, with the concentration of ET-1 in samples calculated from a standard curve of 199


8

recombinant ET-1. Final data were normalized to protein concentration. 200

Human PASMC from control and patients with Pulmonary Arterial Hypertension 201

(IPAH). Primary PASMC isolated from control (n=3) and IPAH (n=3) patients were 202

provided by the Pulmonary Hypertensive Breakthrough Initiative (PHBI). Funding for 203

the PHBI is provided by the Cardiovascular Medical Research and Education Fund 204

(CMREF, University of Pennsylvania, Philadelphia, USA). Isolated PASMC were 205

cultured in smooth muscle cell basic media (Lonza, Mapleton, Illinois, USA) 206

containing 5% FBS with 0.1% insulin, 0.2% hFGF-B, 0.1% hEGF, and 0.1% 207

gentamicin/amphotericin (GA-1000). PASMC between passages 2-6 were used for 208

this study. Cells were evaluated expression of -SMA, MHC11, SM-22, and calponin 209

(all markers of vascular SMC) to ensure identity as SMC (6). 210

Western immunoblotting. For Western immunoblotting, protein content was 211

quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). 212

10g of protein/sample were subjected to SDS-PAGE analysis. Immobilon-P 213

(Millipore-Sigma, St. Louis, MO) membranes were incubated with primary antibodies 214

to detect HIF-1 (10006421, Cayman Chemical, Ann Arbor, MI), -actin (A5441, 215

Sigma, St. Louis, MO), and TWIST (sc-81417, Santa Cruz Biotechnology, Inc., Dallas, 216

TX), and then incubated with horseradish peroxidase (HRP)-conjugated secondary 217

antibodies, followed by detection with ECL reagents (GE Healthcare Life Sciences, 218

Marlborough, MA). Graph represents quantification of protein expression by 219


9

densitometry with results represented relative to -actin. 220

Immunoprecipitation assay. Isolated PASMC were harvested and lysed with 0.5% 221

NP‐ 40 buffer (6). Protein content was standardized as determined by BCA assay. 222

10μg of lysates were pre‐ cleared for 1h at 4°C with 50L of protein A–Sepharose 223

beads and then incubated with 50L of TWIST antibodies (sc-81417) overnight at 224

4°C. Addition of 20L of protein A–Sepharose beads with incubation for 4h at 4°C 225

followed. Samples were washed with PBS and analyzed by SDS–PAGE, transferred 226

to immobilon membrane and detected by ECL using the TWIST antibody (sc-81417). 227

The whole cell lysate samples included 30μg of total cell lysate. Moreover, the TWIST 228

antibody used in the course of this study has been previously validated (29, 39). 229

Statistical analysis. Results are expressed as means ± SEM. Statistical significance 230

was assessed with Student’s t-test and ANOVA where appropriate. A p value of < 0.05 231

was taken as the threshold level for statistical significance. All experiments were 232

repeated a minimum of 3 times (with most being repeated ≥ 5 times). 233

234

Results 235

HIF-1 depletion or overexpression increases hPASMC ET-1 expression and 236

secretion. 237

Initially, we assessed whether deletion of HIF-1 modulates ET-1 expression levels in 238

hPASMC. siRNA directed against HIF-1 effectively depleted HIF-1 gene (Fig. 1A) 239

and protein (Fig. 1B) expression and increased ET-1 gene (Fig. 1C) expression 240


10

compared with NTC siRNA-transfected SMC. With HIF-1 depletion, ET-1 protein did 241

not change in the (Fig. 1D) cell lysates but increased significantly in the media (Fig. 242

1E) compared with NTC siRNA-transfected SMC. We then overexpressed HIF-1 by 243

transfecting hPASMC with a plasmid containing a mutant form of HIF-1 that is 244

resistant to oxygen-mediated degradation (13). Consistent with multiple prior reports 245

(11, 20) in other cell types, overexpression of HIF-1 in hPASMC (Fig. 2A) also 246

increased ET-1 gene expression (Fig. 2B), had no effect on protein expression in cell 247

lysate (Fig. 2C) but significantly increased ET-1 protein expression in the media (Fig. 248

2D), compared with SMC transfected with empty vector. These results suggest that 249

either a decrease or increase in HIF-1 expression can increase ET-1 expression in 250

PASMC. 251

Deletion of HIF-1 increases ET-1 expression in mPASMC. 252

To provide further proof-of-concept in a distinct, and complementary experimental 253

model, we measured ET-1 expression and secretion in mPASMC isolated from mice 254

with SMC specific deletion of HIF-1 (HIF-1-/-) and WT mice (HIF-1+/+) (24). As 255

shown in Fig. 3A, HIF-1 was virtually absent in HIF-1-/- mPASMC. Consistent with 256

our results in hPASMC, in HIF-1-/- mPASMC, ET-1 gene expression (Fig. 3B) and 257

protein expression (Fig. 3C) in the media were significantly increased relative to 258

HIF-1 expressing mPASMC (HIF-1+/+). 259

HIF-1 depletion in hPASMC alters expression of specific miRNA molecules. 260

To determine whether silencing of HIF-1α dysregulates miRNAs which modulate ET-1 261

expression, we performed miRNA expression analysis using the Agilent human 262

miRNAs microarray (v3) on hPAMSC transfected with NTC or HIF-1 siRNA. 263

Differentially expressed miRNA are included in Table 1. miRNA-543, with the most 264

dynamic expression profile (2.4 fold change and p<0.05), was selected through 265

statistical analysis using GeneSpring Software (Table 1). Analysis performed with 266

Pathway Studio and MedScan software suggested that miRNA-543 might link ET-1 267


11

and HIF-1 (43). 268

miRNA-543 expression is inversely proportional to HIF-1α expression. 269

We next sought to directly demonstrate that HIF-1 expression modulates PASMC 270

miRNA-543 expression. As shown in Fig. 4, knockdown of HIF-1 using siRNA in 271

hPASMC increased miRNA-543 expression compared with NTC siRNA-transfected 272

SMC. 273

Overexpression of miRNA-543 increases, and depletion of miRNA-543 274

decreases, HIF-1 and ET-1 expression 275

We next overexpressed miRNA-543 in hPASMC and measured HIF-1 and ET-1 276

expression. We confirmed that transfection with the miRNA-543 mimic increased 277

miRNA-543 expression by over 150 fold, compared to hPASMC transfected with the 278

control mimic (Fig. 5A). As depicted in Fig. 5B, miRNA-543 overexpression, 279

increased ET-1 gene expression by approximately 5-fold, and increased ET-1 protein 280

expression in the media by approximately 70% (Fig. 5C). Conversely, transfection of 281

hPASMC with differing concentrations of the miR-543 LNAs effectively depleted 282

miRNA-543 (Fig. 5D). Transfection of hPASMC with 50nM resulted in a significant 283

reduction in ET-1 (Fig. 5E) gene and protein (Fig. 5F) expression compared to 284

hPASMC treated with the control(32), a vector that did not contain any 285

oligonucleotide. 286

Overexpression of miRNA-543 decreases TWIST1 and increases ET-1 287

expression. 288

Given that miRNA molecules limit expression of target molecules through 289

post-transcriptional modification, we next sought to discover how increased 290

miRNA-543 resulted in increased ET-1 expression. Computational analysis of the 291

TWIST1 3’ UTR, identified TWIST1 as a high probability target of miR-543, and 292

miR-543 mimics repress expression in a luciferase 3’ UTR TWIST1 construct (17). 293

Moreover, in the context of osteosarcoma, TWIST inversely correlates with ET-1 294

expression (53). In keeping with these data, overexpression of miRNA-543 in 295


12

hPASMC significantly decreased TWIST1 expression (Fig. 6A). Moreover, with 296

effective silencing of TWIST1 with siRNA (Fig. 6B), ET-1 gene expression increased 297

by approximately 3-fold (Fig. 6C). Although ET-1 protein expression did not change in 298

the cell lysate (Fig. 6D), it increased 2-fold in the conditioned media (Fig. 6E). 299

in hPASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) 300

compared to control patients, HIF-1 and TWIST1 are decreased and 301

miRNA-543 and ET-1 are increased. 302

To determine whether the observations in cell culture and murine models have fidelity 303

with human pathobiology, we measured expression HIF-1, TWIST1 and ET-1 in 304

hPASMC derived from controls (n=3) and patients with idiopathic pulmonary arterial 305

hypertension (IPAH) (n=3; Table 2). HIF-1306

expression were decreased in PASMC from patients with IPAH, compared to control 307

patients. Consistent with our in vitro and murine studies, TWIST1 gene (Fig. 7C) and 308

protein (Fig. 7D) expression were significantly decreased. Moreover, miRNA-543 309

expression was increased in hPASMC from patients with IPAH compared to controls 310

(Fig. 7E). Finally, as further proof of principle, we demonstrated that ET-1 expression 311

was significantly increased in the cell media of hPASMC from patients with IPAH, as 312

compared to control patients (Fig. 7F). 313

314

Discussion 315

The present report is the first to demonstrate that deletion of HIF-1in PASMC 316

increases ET-1 gene and protein expression. Specifically, these results demonstrate 317

that in PASMC the transcription factor HIF-1 modulates expression of a powerful 318

pulmonary vasoconstrictor, ET-1, via a specific miRNA molecule, miRNA-543. The 319

strength of the evidence is buttressed by observations in both human and murine 320

systems wherein with HIF-1 depletion, ET-1 expression is increased. In hPASMC 321

HIF-1 depletion increases miRNA-543 expression as well. Our data demonstrate 322


13

that in hPASMC an increase in miRNA-543 decreased TWIST1 gene expression, and 323

increased both ET-1 gene and protein expression. Consistent with this construct, 324

depletion of miRNA-543 in hPASMC increased TWIST1 gene expression, and 325

decreased ET-1 gene and protein expression. Further evidence for the biologic 326

relevance of the findings derive from experiments undertaken in low passage 327

hPASMC from control patients and from patients with IPAH. The findings were entirely 328

consistent with those from the in vitro and murine experiments. Specifically, HIF-1 329

and TWIST1 were decreased while miRNA-543 and ET-1 were increased. Taken 330

together, these results provide evidence of a previously undescribed mechanistic link 331

wherein a decrease in HIF-1 expression can result in an increase in ET-1, molecules 332

that play a central role in the regulation of both physiologic and pathophysiologic 333

pulmonary vascular tone. 334

Although HIF-1 is a molecule that determines the cellular response to hypoxia in 335

multiple ways, including cell proliferation, migration, metabolism and hypertrophy (26, 336

30, 35, 38, 51), increasing evidence suggests a cell-specific role in the pulmonary 337

vasculature. For example, mice globally haploinsufficient for HIF-1 demonstrated an 338

attenuated response to both acute and chronic hypoxia(51). Similarly, in mice with 339

conditional, cell specific deletion of HIF-1 in myosin heavy chain expressing cells, 340

hypoxia-induced pulmonary hypertension was mitigated (5). Conversely, in another 341

report wherein HIF-1 was deleted in SM-22 expressing cells, pulmonary vascular 342

tone was increased in both normoxic and hypoxic conditions, and myosin light chain 343

phosphorylation in PASMC was increased, even in the absence of vascular 344

remodeling (24). Even more recent reports indicate that overexpression of HIF-1 in 345

pulmonary artery endothelial cells leads to dramatic elevations in pulmonary artery 346

pressures and marked vascular remodeling (10, 15, 40). Thus, the present report 347

adds to the accumulating evidence for a cell-specific role for HIF-1 in the pulmonary 348

circulation. 349


14

There is a large body of evidence, predominately in endothelial cells, indicating 350

that ET-1 is a downstream target of HIF-1, which is upregulated during hypoxia (11, 351

20, 46). Moreover, there is a significant body of evidence supporting the notion that in 352

PASMC, an increase in ET-1 leads to an increase in HIF-1 expression through either 353

an increase in synthesis, a decrease in hydroxylation (33) or a decrease in 354

proteosomal degradation(28). However, the present data focus on a mechanism 355

whereby a decrease in HIF-1 expression can drive ET-1 expression in PASMC or 356

stated more directly, HIF-1 expression might normally act to constrain ET-1 357

expression. Previously, we demonstrated that PASMC ET-1 expression modulates the 358

pulmonary vascular response, including tone, structural remodeling, cell proliferation 359

and migration, to chronic hypoxia (23). In this report, we outline an interaction 360

between molecules with physiologically significant roles in the pulmonary circulation 361

via miRNA-543. The observation that in both human and murine PASMC either 362

HIF-1 overexpression or depletion can increase ET-1 expression, via distinct 363

pathways, argues for the importance of closely regulated HIF-1 expression. The 364

physiologic significance of the observation is amplified by the marked increase in ET-1 365

secretion by HIF-1-/- cells. That ET-1 expression is increased in the media but not the 366

cell lysate of SMC is consistent with ET-1 expression in endothelial cells wherein ET-1 367

is synthesized and continuously released. In endothelial cells ET-1 is predominately 368

regulated at the transcriptional level(41). 369

Previous studies have demonstrated a role for miRNA-543, located on human 370

chromosome 14, in tumorigenesis and metastasis (21). Recent studies have showed 371

that miRNA-543 can suppress tumorigenesis and metastasis by inhibiting focal 372

adhesion kinase and TWIST in endometrial cancer cell lines (8). However, in 373

osteosarcoma cells TWIST expression is inversely correlated with ET-1 expression 374

(53). Moreover, miRNA-543 decreases Twist expression by directly targeting the 3’–375

UTR of TWIST (8). This represents the first report of a role for miRNA-543 mediated 376


15

regulation of ET-1 expression in PASMC. Given the capacity of ET-1 to modulate 377

vascular development (46, 47), the data in the present report suggest the potential of 378

a pro-angiogenic role for miRNA-543 via augmented ET-1 expression (52). 379

The results have implications for the regulation of pulmonary vascular tone under 380

both physiologic and pathophysiologic conditions. Data from our laboratory indicate 381

that in PASMC, HIF-1 is present under both hypoxic and normoxic conditions (35). In 382

a murine model of SM-22-specific deletion of HIF-1, compared to controls, 383

pulmonary vascular tone is increased under normoxic conditions and hypoxia-induced 384

pulmonary hypertension is exaggerated relative to controls(24). As PASMC do 385

express ET-1(23), the loss of HIF-1 may increase pulmonary vascular tone through 386

direct effects that entail an increase in myosin light chain phosphorylation(6, 24), as 387

well as via indirect effects wherein ET-1 production is increased via miR-543 mediated 388

suppression of the transcription factor TWIST. Thus, the result of decreased HIF-1 in 389

PASMC, such as in the present report and a prior report from our lab in PASMC from 390

patients with pulmonary hypertension(6), is an increase pulmonary vascular tone. 391

Conversely, with well-preserved HIF-1 expression in PASMC, myosin light chain 392

phosphorylation and ET-1 production is constrained, thereby maintaining the low tone 393

of the normal pulmonary circulation. 394

To our knowledge this represents the initial report of the association between 395

miRNA-543, Twist expression and ET-1 in the pulmonary vasculature (Fig. 7). These 396

data detail a relationship between HIF-1 and miRNA-543 expression in PASMC, 397

where in the absence of HIF-1, miRNA-543 and ET-1 expression both increase, with 398

Twist linking the two molecules. These results underscore the importance of PASMC 399

HIF-1 in the regulation of pulmonary vascular tone under not only hypoxic, but also 400

normoxic conditions. An important limitation of the present series is that the 401

experiments were conducted in normoxia and a hypoxic environment may well alter 402

the findings. Notwithstanding that caveat, the biologic fidelity between the findings 403


16

from in vitro studies, murine models and the hPASMC derived from patients increases 404

the likelihood that the present findings possess clinically relevant implications for both 405

pulmonary vascular development and the regulation of pulmonary vascular tone. 406

Modulation of either miRNA-543 or Twist expression might represent a viable 407

therapeutic strategy to address pathophysiologic elevations of pulmonary vascular 408

tone. 409

TWIST is a direct transcriptional target of HIF-1 (49). In the context of cancer, an 410

increase in HIF-1 expression can promote epithelial-mesenchymal transition (EMT) 411

and metastatic disease(48, 49). Recent data indicate that HIF-1 regulates TWIST 412

expression by directly binding to the hypoxia response element in the proximal region 413

of the TWIST promoter. In the presence of both HIF-1 and TWIST expression, 414

squamous cell cancers of the head and neck are more likely to metastasize providing 415

evidence that HIF-1 signals, in part, via TWIST (48, 49). Conversely, data in the 416

present report indicate that loss of HIF-1 can decrease TWIST expression, albeit 417

indirectly. With loss of HIF-1 expression, miR-543 expression increases, which in 418

turn, represses TWIST expression. The relatively recent reports demonstrating a role 419

for TWIST in EMT (34), myogenesis(25), inflammation (16) and BMP signaling (19), 420

processes all involved in the pathogenesis of pulmonary hypertension, suggest that 421

manipulating miR-543, a molecule that links HIF-1 to TWIST, might represent a 422

worthwhile and novel therapeutic strategy. 423

424

425

Acknowledgments 426

Funding: The work has been supported by the National Institutes of Health (HL060784 427

(DNC), HL0706280 (DNC), and HL122918 (CMA). Contributing investigators have also 428

been supported by Burroughs Welcome Fund Preterm Birth Initiative (DNC) and the 429

Stanford Child Health Research Institute Tashia and John Morgridge Faculty Scholar 430


17

Award (CMA). 431

432


18

References 433

1. Accurso F, Albert B, Wilkening R, Peterson R, and Meschia G. 434

Time-dependent response of fetal pulmonary blood flow to an increase in fetal oxygen 435

tension. Respir Physiol 63: 43-52, 1986. 436

2. Ahn YT, Kim YM, Adams E, Lyu SC, Alvira CM, and Cornfield DN. 437

Hypoxia-inducible factor-1alpha regulates KCNMB1 expression in human pulmonary 438

artery smooth muscle cells. American journal of physiology Lung cellular and 439

molecular physiology 302: L352-359, 2012. 440

3. Allen SW, Chatfield BA, Koppenhafer SA, Schaffer MS, Wolfe RR, and 441

Abman SH. Circulating immunoreactive endothelin-1 in children with pulmonary 442

hypertension. Association with acute hypoxic pulmonary vasoreactivity. The American 443

review of respiratory disease 148: 519-522, 1993. 444

4. Alvarez D, Briassouli P, Clancy RM, Zavadil J, Reed JH, Abellar RG, 445

Halushka M, Fox-Talbot K, Barrat FJ, and Buyon JP. A novel role of endothelin-1 in 446

linking Toll-like receptor 7-mediated inflammation to fibrosis in congenital heart block. 447

The Journal of biological chemistry 286: 30444-30454, 2011. 448

5. Ball MK, Waypa GB, Mungai PT, Nielsen JM, Czech L, Dudley VJ, Beussink 449

L, Dettman RW, Berkelhamer SK, Steinhorn RH, Shah SJ, and Schumacker PT. 450

Regulation of hypoxia-induced pulmonary hypertension by vascular smooth muscle 451

hypoxia-inducible factor-1alpha. Am J Respir Crit Care Med 189: 314-324, 2014. 452

6. Barnes EA, Chen CH, Sedan O, and Cornfield DN. Loss of smooth muscle cell 453

hypoxia inducible factor-1alpha underlies increased vascular contractility in 454

pulmonary hypertension. FASEB journal : official publication of the Federation of 455

American Societies for Experimental Biology 31: 650-662, 2017. 456

7. Bienertova-Vasku J, Novak J, and Vasku A. MicroRNAs in pulmonary arterial 457

hypertension: pathogenesis, diagnosis and treatment. Journal of the American 458

Society of Hypertension : JASH 9: 221-234, 2015. 459

8. Bing L, Hong C, Li-Xin S, and Wei G. MicroRNA-543 suppresses endometrial 460

cancer oncogenicity via targeting FAK and TWIST1 expression. Archives of 461

gynecology and obstetrics 290: 533-541, 2014. 462

9. Bonnet S, Michelakis ED, Porter CJ, Andrade-Navarro MA, Thebaud B, 463

Haromy A, Harry G, Moudgil R, McMurtry MS, Weir EK, and Archer SL. An 464

abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts 465

oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: 466

similarities to human pulmonary arterial hypertension. Circulation 113: 2630-2641, 467

2006. 468

10. Bryant AJ, Carrick RP, McConaha ME, Jones BR, Shay SD, Moore CS, 469

Blackwell TR, Gladson S, Penner NL, Burman A, Tanjore H, Hemnes AR, 470


19

Karwandyar AK, Polosukhin VV, Talati MA, Dong HJ, Gleaves LA, Carrier EJ, 471

Gaskill C, Scott EW, Majka SM, Fessel JP, Haase VH, West JD, Blackwell TS, and 472

Lawson WE. Endothelial HIF signaling regulates pulmonary fibrosis-associated 473

pulmonary hypertension. American journal of physiology Lung cellular and molecular 474

physiology 310: L249-262, 2016. 475

11. Camenisch G, Stroka DM, Gassmann M, and Wenger RH. Attenuation of 476

HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression. Pflugers 477

Archiv : European journal of physiology 443: 240-249, 2001. 478

12. Cassin S, Dawes GS, Mott JC, Ross BB, and Strang LB. The vascular 479

resistance of the fetal and newly ventilated lung of the lamb. J Physiol 171: 61-79, 480

1964. 481

13. Chan DA, Sutphin PD, Yen SE, and Giaccia AJ. Coordinate regulation of the 482

oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha. 483

Molecular and cellular biology 25: 6415-6426, 2005. 484

14. Chan YC, Banerjee J, Choi SY, and Sen CK. miR-210: the master hypoxamir. 485

Microcirculation 19: 215-223, 2012. 486

15. Dai Z, Li M, Wharton J, Zhu MM, and Zhao YY. Prolyl-4 Hydroxylase 2 (PHD2) 487

Deficiency in Endothelial Cells and Hematopoietic Cells Induces Obliterative Vascular 488

Remodeling and Severe Pulmonary Arterial Hypertension in Mice and Humans 489

Through Hypoxia-Inducible Factor-2alpha. Circulation 133: 2447-2458, 2016. 490

16. Dobrian AD. A tale with a Twist: a developmental gene with potential relevance 491

for metabolic dysfunction and inflammation in adipose tissue. Frontiers in 492

endocrinology 3: 108, 2012. 493

17. Haga CL, and Phinney DG. MicroRNAs in the imprinted DLK1-DIO3 region 494

repress the epithelial-to-mesenchymal transition by targeting the TWIST1 protein 495

signaling network. The Journal of biological chemistry 287: 42695-42707, 2012. 496

18. Hausser J, and Zavolan M. Identification and consequences of miRNA-target 497

interactions--beyond repression of gene expression. Nature reviews Genetics 15: 498

599-612, 2014. 499

19. Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, Kato H, Tamai K, 500

and Kaneda Y. Comparative roles of Twist-1 and Id1 in transcriptional regulation by 501

BMP signaling. Journal of cell science 120: 1350-1357, 2007. 502

20. Hu J, Discher DJ, Bishopric NH, and Webster KA. Hypoxia regulates 503

expression of the endothelin-1 gene through a proximal hypoxia-inducible factor-1 504

binding site on the antisense strand. Biochemical and biophysical research 505

communications 245: 894-899, 1998. 506

21. Hu X, Chi L, Zhang W, Bai T, Zhao W, Feng Z, and Tian H. Down-regulation of 507

the miR-543 alleviates insulin resistance through targeting the SIRT1. Biochemical 508

and biophysical research communications 468: 781-787, 2015. 509


20

22. Kapitsinou PP, Rajendran G, Astleford L, Michael M, Schonfeld MP, Fields T, 510

Shay S, French JL, West J, and Haase VH. The Endothelial Prolyl-4-Hydroxylase 511

Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in 512

Mice. Molecular and cellular biology 36: 1584-1594, 2016. 513

23. Kim FY, Barnes EA, Ying L, Chen C, Lee L, Alvira CM, and Cornfield DN. 514

Pulmonary artery smooth muscle cell endothelin-1 expression modulates the 515

pulmonary vascular response to chronic hypoxia. American journal of physiology 516

Lung cellular and molecular physiology 308: L368-377, 2015. 517

24. Kim YM, Barnes EA, Alvira CM, Ying L, Reddy S, and Cornfield DN. 518

Hypoxia-inducible factor-1alpha in pulmonary artery smooth muscle cells lowers 519

vascular tone by decreasing Myosin light chain phosphorylation. Circulation research 520

112: 1230-1233, 2013. 521

25. Koutalianos D, Koutsoulidou A, Mastroyiannopoulos NP, Furling D, and 522

Phylactou LA. MyoD transcription factor induces myogenesis by inhibiting Twist-1 523

through miR-206. Journal of cell science 128: 3631-3645, 2015. 524

26. Krick S, Hanze J, Eul B, Savai R, Seay U, Grimminger F, Lohmeyer J, 525

Klepetko W, Seeger W, and Rose F. Hypoxia-driven proliferation of human 526

pulmonary artery fibroblasts: cross-talk between HIF-1alpha and an autocrine 527

angiotensin system. FASEB J 19: 857-859, 2005. 528

27. Lei W, He Y, Shui X, Li G, Yan G, Zhang Y, Huang S, Chen C, and Ding Y. 529

Expression and analyses of the HIF-1 pathway in the lungs of humans with pulmonary 530

arterial hypertension. Molecular medicine reports 14: 4383-4390, 2016. 531

28. Li M, Liu Y, Jin F, Sun X, Li Z, Liu Y, Fang P, Shi H, and Jiang X. Endothelin-1 532

induces hypoxia inducible factor 1alpha expression in pulmonary artery smooth 533

muscle cells. FEBS letters 586: 3888-3893, 2012. 534

29. Matsuo N, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Tanaka S, Nishina S, 535

Nakanishi Y, Uemura M, Takaki A, Nakamura S, Kobayashi Y, Nouso K, Yagi T, 536

and Yamamoto K. Twist expression promotes migration and invasion in 537

hepatocellular carcinoma. BMC cancer 9: 240, 2009. 538

30. Mazure NM, Chen EY, Laderoute KR, and Giaccia AJ. Induction of vascular 539

endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 540

3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia 541

inducible factor-1 transcriptional element. Blood 90: 332-331, 1997. 542

31. Nagpal N, Ahmad HM, Chameettachal S, Sundar D, Ghosh S, and 543

Kulshreshtha R. HIF-inducible miR-191 promotes migration in breast cancer through 544

complex regulation of TGFbeta-signaling in hypoxic microenvironment. Scientific 545

reports 5: 9650, 2015. 546

32. Orom UA, Kauppinen S, and Lund AH. LNA-modified oligonucleotides mediate 547

specific inhibition of microRNA function. Gene 372: 137-141, 2006. 548


21

33. Pisarcik S, Maylor J, Lu W, Yun X, Undem C, Sylvester JT, Semenza GL, and 549

Shimoda LA. Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth 550

muscle cells by endothelin-1. American journal of physiology Lung cellular and 551

molecular physiology 304: L549-561, 2013. 552

34. Ranchoux B, Antigny F, Rucker-Martin C, Hautefort A, Pechoux C, Bogaard 553

HJ, Dorfmuller P, Remy S, Lecerf F, Plante S, Chat S, Fadel E, Houssaini A, 554

Anegon I, Adnot S, Simonneau G, Humbert M, Cohen-Kaminsky S, and Perros F. 555

Endothelial-to-mesenchymal transition in pulmonary hypertension. Circulation 131: 556

1006-1018, 2015. 557

35. Resnik ER, Herron JM, Lyu SC, and Cornfield DN. Developmental regulation 558

of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth 559

muscle cells. Proceedings of the National Academy of Sciences of the United States 560

of America 104: 18789-18794, 2007. 561

36. Rudolph A. Distribution and regulation of blood flow in the fetal and neonatal 562

lamb. Circ Res 57: 811-821, 1985. 563

37. Semenza G. Perspectives on oxygen sensing. Cell 98: 281–284, 1999. 564

38. Semenza GL. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms 565

of blood vessel formation and remodeling. J Cell Biochem 102: 840-847, 2008. 566

39. Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E, Kidani A, 567

Yokomizo A, Naito S, and Kohno K. Twist promotes tumor cell growth through YB-1 568

expression. Cancer research 68: 98-105, 2008. 569

40. Stenmark KR, Fagan KA, and Frid MG. Hypoxia-induced pulmonary vascular 570

remodeling: cellular and molecular mechanisms. Circulation research 99: 675-691, 571

2006. 572

41. Stow LR, Jacobs ME, Wingo CS, and Cain BD. Endothelin-1 gene regulation. 573

FASEB journal : official publication of the Federation of American Societies for 574

Experimental Biology 25: 16-28, 2011. 575

42. Tang H, Babicheva A, McDermott KM, Gu Y, Ayon RJ, Song S, Wang Z, 576

Gupta A, Zhou T, Sun X, Dash S, Wang Z, Balistrieri A, Zheng Q, Cordery AG, 577

Desai AA, Rischard F, Khalpey Z, Wang J, Black SM, Garcia JGN, Makino A, and 578

Yuan JX. Endothelial HIF-2alpha contributes to severe pulmonary hypertension due 579

to endothelial-to-mesenchymal transition. American journal of physiology Lung 580

cellular and molecular physiology 314: L256-L275, 2018. 581

43. Thomas S, and Bonchev D. A survey of current software for network analysis in 582

molecular biology. Human genomics 4: 353-360, 2010. 583

44. Weir EK, and Archer SL. The mechanism of hypoxic pulmonary 584

vasoconstriction: The tale of two channels. FASEB journal : official publication of the 585

Federation of American Societies for Experimental Biology 9: 183-189, 1995. 586

45. Whitman EM, Pisarcik S, Luke T, Fallon M, Wang J, Sylvester JT, Semenza 587


22

GL, and Shimoda LA. Endothelin-1 mediates hypoxia-induced inhibition of 588

voltage-gated K+ channel expression in pulmonary arterial myocytes. American 589

journal of physiology Lung cellular and molecular physiology 294: L309-318, 2008. 590

46. Yamashita K, Discher DJ, Hu J, Bishopric NH, and Webster KA. Molecular 591

Regulation of the Endothelin-1 Gene by Hypoxia 592

Ccontributions of Hypoxia-Inducible factor-1, Activator protein-1, GATA-2 and 593

p300/CBP. J Biol CHem 276: 12645-12653., 2001. 594

47. Yanagisawa H, Hammer RE, Richardson JA, Emoto N, Williams SC, Takeda 595

S, Clouthier DE, and Yanagisawa M. Disruption of ECE-1 and ECE-2 reveals a role 596

for endothelin-converting enzyme-2 in murine cardiac development. J Clin Invest 105: 597

1373-1382., 2000. 598

48. Yang MH, and Wu KJ. TWIST activation by hypoxia inducible factor-1 (HIF-1): 599

implications in metastasis and development. Cell cycle 7: 2090-2096, 2008. 600

49. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, Teng SC, and Wu 601

KJ. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nature cell 602

biology 10: 295-305, 2008. 603

50. Yu AY, Frid MG, Shimoda LA, Wiener CM, Stenmark K, and Semenza GL. 604

Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in 605

the lung. Am J Physiol 275: L818-826, 1998. 606

51. Yu AY, Shimoda LA, Iyer NV, Huso DL, Sun X, McWilliams R, Beaty T, Sham 607

JS, Wiener CM, Sylvester JT, and Semenza GL. Impaired physiological responses 608

to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha. J Clin 609

Invest 103: 691-696, 1999. 610

52. Zhang J, Yang W, Hu B, Wu W, and Fallon MB. Endothelin-1 activation of the 611

endothelin B receptor modulates pulmonary endothelial CX3CL1 and contributes to 612

pulmonary angiogenesis in experimental hepatopulmonary syndrome. The American 613

journal of pathology 184: 1706-1714, 2014. 614

53. Zhou Y, Zang X, Huang Z, and Zhang C. TWIST interacts with 615

endothelin-1/endothelin A receptor signaling in osteosarcoma cell survival against 616

cisplatin. Oncology letters 5: 857-861, 2013. 617

618

619


23

Figure legends 620

Fig. 1. HIF-1 depletion increases human pulmonary artery smooth muscle cell 621

(hPASMC) ET-1 expression and secretion. hPASMC were transfected with 622

nontargeted control small interfering RNA (siNTC) (n=6) or siRNA directed against 623

HIF-1. In hPASMC transfected with siHIF-1(n=6) (A) HIF-1mRNA was 624

decreased; (B) Expression of HIF-1 in hPASMC by Western immunoblot after 625

transfection with NTC siRNA or HIF-1 siRNA. -actin serves as a loading control 626

(n=3); (C) HIF-1depletion markedly increased ET-1 mRNA (n=6) compared to cells 627

transfected with siNTC; ET-1 protein expression did not change in (D) cell lysates of 628

hPASMC transfected with siHIF-1(n=9) but did increase significantly in the (E) 629

media (n=9). Results are presented as means ± SEM. **p<0.01, ***p<0.001, 630

****p<0.0001 by unpaired Student’s t-test, NTC siRNA vector versus HIF-1 siRNA 631

632

Fig. 2. HIF-1 overexpression increases human pulmonary artery smooth muscle cell 633

(hPASMC) ET-1 expression and secretion. Compared to hPASMC transfected with 634

empty vector (pcDNA3) (n=6), hPASMC, transfected with constitutively active (CA) 635

HIF-1demonstrated: an increase in (A) HIF-1 (n=4) and (B) ET-1 mRNA (n=6) and 636

ET-1 protein expression did not change in the (C) cell lysates (n=3) but increased in 637

the (D) media (n=3). Results are shown as means ± SEM. ***p<0.001, ***p<0.0001, 638

by unpaired Student’s t-test, control vector versus HIF-1 or CA-HIF-1. 639

640

Fig. 3. In murine PASMC (mPASMC)., HIF-1 deletion increases ET-1 expression. 641

(A) HIF-1 mRNA expression in mPASMC isolated from SM22-HIF-1α-/- (n=3) and 642

WT (n=3) mice. (B) Expression of ET-1 mRNA (n=3) in isolated mPASMC. (C) 643

Expression of ET-1 protein (n=3) in isolated mPASMC. Results are presented as 644

means ± SEM. HIF-1+/+ vs. HIF-1-/-. **p<0.01, by unpaired Student’s t-test, 645

HIF-1+/+ vs. HIF-1-/- 646


24

647

Fig. 4. In human PASMC (hPASMC), HIF-1 depletion increases miRNA-543 648

expression. In hPASMC with HIF-1 depletion (n=3) compared to siNTC (n=3). 649

Results are presented as means ± SEM. *p<0.05, by non-parametric Mann-Whitney U 650

test, siHIF-1 vs. siNTC). 651

652

Fig. 5. Overexpression of miRNA-543 increases, and depletion decreases, ET-1 653

expression in hPASMC. With miRNA-543 overexpression in hPASMC using a 654

constitutively active miRNA-543 mimetic (n=6), (A) miRNA-543 expression was 655

dramatically increased compared to miR-543 mRNA expression in hPASMC 656

transfected with a negative control (n=6). In the presence of miRNA-543 657

overexpression in hPASMC, (B) ET-1 mRNA (n=4) and (C) protein expression (n=3) 658

were all increased compared to control (*p<0.05, by non-parametric Mann-Whitney U 659

test, miRNA-543 mimic (n=3) vs. negative control (n=3)). (D) hPASMC were 660

transfected with negative control (n=6) or locked nucleic acid miRNA-543 inhibitor 661

(12.5, 25, 50, and 100nM) (n=6). Inhibition decreased miRNA-543 expression. 662

Inhibition of miRNA-543 expression by miRNA-543 inhibitor (50nm) decreased (E) 663

ET-1 mRNA expression (n=18), and (F) ET-1 protein expression (n=3) in the 664

conditioned media. Results are presented as means ± SEM. *p<0.05, **p<0.01, 665

****p<0.0001, by non-parametric Mann-Whitney U test, miRNA-543 inhibitor vs. 666

control 667

668

Fig. 6. TWIST1 depletion increases human pulmonary artery smooth muscle cell 669

(hPASMC) ET-1 expression and secretion. With miRNA-543 overexpression in 670

hPASMC using a constitutively active miRNA-543 mimetic, (A) TWIST mRNA 671

expression (n=8) decreased. (B) siRNA directed against TWIST effectively decreased 672

mRNA (n=5); (C) ET-1 mRNA (n=5) was increased while protein expression (n=5) 673

was unchanged in the (E) cell lysate but increased significantly, compared to control in 674


25

the (E) conditioned media (n=5). Results are presented as means ± SEM. **p<0.01, 675

***<0.001, ****p<0.0001, by non-parametric Mann-Whitney U test, NTC vector versus 676

TWIST1 mimic or siRNA. 677

678

Fig. 7. In PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) 679

(n=3) compared to control (n=3) patients, HIF-1 and TWIST1 are decreased and 680

miRNA-543 and ET-1 are increased. (A) HIF-1 mRNA expression in PASMC isolated 681

from control and patients with IPAH; (B) Western immunoblot of HIF-1 protein 682

expression in PASMC from control and patients with IPAH with -actin loading control. 683

Graph represents means ± SEM, *** p < 0.001, IPAH vs. Control; (C) TWIST1 684

mRNA expression in mPASMC from control and patients with IPAH, Graph represents 685

means ± SEM, *p < 0.05, IPAH vs. Control; (D) Western immunoblot of TWIST1 686

protein expression in PASMC from control and IPAH patients with -actin loading 687

control, Graph represents means ± SEM (n=3). ***p < 0.001, IPAH vs. Control. 688

Expression of TWIST1 in whole cell lysates (WCLs) in isolated PASMC and 689

immunoprecipitated TWIST in control PASMC by Western immunoblot; (E) 690

miRNA-543 expression in mPASMC isolated from controls and patients with IPAH. *p 691

< 0.05, IPAH vs. Control; (F) ET-1 protein expression did not change in the cell media 692

(n=3) of hPASMC from control and IPAH patients. Results are shown as means ± 693

SEM. *p<0.05, by unpaired Student’s t-test. 694

695


26

Fig. 8. Schematic representation of the proposed HIF-1 and ET-1 interaction. In the 696

pulmonary artery smooth muscle cell, HIF-1 is constitutively active leading to a low 697

level of endothelin (EDNI) gene expression. With a decrease in HIF-1 expression, 698

miRNA-543 expression increases which serves to decrease TWIST1 expression and 699

nuclear accumulation to thereby derepress endothelin gene expression and augment 700

secretion of the peptide endothelin (ET-1), a powerful vasoconstrictor molecule. 701

Under hypoxic conditions, HIF-1 protein is stable and translocates into the nucleus, 702

and increases EDNI transcription and ET-1 secretion. 703

704

705


27

Table 1. 706

hPASMC HIF-1 depletion alters expression of specific microRNA (miRNA) 707

molecules. RNA hybridization was performed on the Agilent human miRNAs 708

microarray (v3) with 15k features (Agilent). miRNA-543 demonstrated a divergent 709

expression profile after HIF-1 depletion (2.4 fold change and p<0.05) and was 710

selected through statistical analysis using GeneSpring Software. 711

712

713


28

714

Table 1. 715

716

717

718


29

719

720

721

722

Table 2.

Demographic characteristics of patients. Isolated PASMC were derived from

the above listed control and IPAH patients (initial passage number 1-2).

Subsequent studies were performed on cells between passage numbers 2-6.

HT, head trauma; IH, intracranial hemorrhage; IPAH, idiopathic pulmonary

arterial hypertension.


Figure 1

NTC siRNA

HIF-1α siRNA

HIF

-1α

RN

A E

xpre

ssio

n (n

orm

aliz

ed to

18s

)

****

1.0

0.5

0.0

****

ET-

1 R

NA

Exp

ress

ion

(nor

mal

ized

to 1

8s)

0NTC

siRNA HIF-1α siRNA

cell media

ET-

1 P

rote

in E

xpre

ssio

n

2

3

0

4

NTC siRNA

HIF-1α siRNA

cell lysate

NTC siRNA

HIF-1α siRNA

ET-

1 P

rote

in E

xpre

ssio

n

1.5

1.0

0.5

0.0

2.0

A

1

C D

1.5

**

2

3

5

4

1

HIF-1α

β-actin

NTC siRNA

HIF-1α siRNA

***

HIF

-1α

Pro

tein

Exp

ress

ion

(nor

mal

ized

to β

-act

in)

2.0

1.0

0.5

0

1.5

NTC siRNA

HIF-1α siRNA

B

E


Figure 2

HIF

-1α

RN

A E

xpre

ssio

n (n

orm

aliz

ed to

18s

)

2

3

0

4

1

Vector HA-HIF-1α

cell lysate 1.5

1.0

0.5

0.0 Vector HA-

HIF-1α

ET-

1 P

rote

in E

xpre

ssio

n

20

15

10

5

0Vector HA-

HIF-1α

ET-

1 R

NA

Exp

ress

ion

(nor

mal

ized

to 1

8s)

**** ***

cell media

3

1

2

0

4

Vector HA-HIF-1α

ET-

1 P

rote

in E

xpre

ssio

n

***

A B

C D


Figure 3

**

1.5

1.0

0.5

0.0

HIF

-1α

RN

A E

xpre

ssio

n (n

orm

aliz

ed to

18s

)

10

15

0

20

5

25 **

ET-

1 R

NA

Exp

ress

ion

(nor

mal

ized

to 1

8s)

A B

ET-

1 P

rote

in E

xpre

ssio

n

1.0

1.5

0

2.0

0.5

2.5 *

cell media C

HIF-1α+/+ HIF-1α-/- HIF-1α+/+ HIF-1α-/- HIF-1α+/+ HIF-1α-/-


Figure 4

miR

-543

Exp

ress

ion

(nor

mal

ized

to U

6) *

2

4

0

6

NTC siRNA

HIF-1α siRNA


Figure 5 m

iR-5

43 E

xpre

ssio

n (n

orm

aliz

ed to

U6)

100

250

0

300

Control miR-543 mimic

****

ET-

1 E

xpre

ssio

n (n

orm

aliz

ed to

18s

)

4

6

0

8

2

10 *

A

ET-

1 P

rote

in E

xpre

ssio

n

*

10

15

0

20

5


1.0

1.5

0

0.5

Control

miR-543 inhibitor

12.5nm 25nm

50nm

miR

-543

Exp

ress

ion

(nor

mal

ized

to U

6)

ET-

1 R

NA

Exp

ress

ion

(nor

mal

ized

to 1

8s)

1.0

2.0

0

0.5

Control miR-543 inhibitor

* 4

6

0

2

Control miR-543 inhibitor

ET-

1 P

rote

in E

xpre

ssio

n

E

cell media

cell media

100nm

**

****

1.5

B C

D F


+ +

+ +

+


NTC siRNA

TWIST1 siRNA

***

TWIS

T1 R

NA

Exp

ress

ion

(nor

mal

ized

to β

-act

in)

ET-

1 R

NA

Exp

ress

ion

(nor

mal

ized

to β

-act

in)

**

2

3

0

4

1

NTC siRNA

TWIST1 siRNA

cell lysate

ET-

1 P

rote

in E

xpre

ssio

n

1.5

1.0

0.5

0.0

2

3

0

1

NTC siRNA

TWIST1 siRNA

ET-

1 P

rote

in E

xpre

ssio

n cell media

***


TWIS

T1 R

NA

Exp

ress

ion

(nor

mal

ized

to β

-act

in)

**

2.0

1.0

0.5

0.0

1.5

A

E

B

C D

1.5

1.0

0.5

0.0

NTC siRNA

TWIST1 siRNA

Figure 6


control

TWIS

T1 e

xpre

ssio

n (n

orm

aliz

ed to

18s

)

* 1.0

1.5

0

0.5

IPAH

Figure 7 H

IF-1α

Exp

ress

ion

(nor

mal

ized

to 1

8s)

1.0

1.5

0

0.5

IPAH Control

A!

100kDa HIF-1α

IPAH 3 4 1 2 6 5

Control

50kDa β-actin

37kDa

B!

Control IPAH

1.0

1.5

0

0.5

***

HIF

-1α

Pro

tein

Exp

ress

ion

(nor

mal

ized

to β

-act

in)

miR

-543

Exp

ress

ion

(nor

mal

ized

to U

6)

Control

2.0

1.0

0.5

0

1.5

*

IPAH

1.0

1.5

0

0.5

Control IPAH TWIS

T1 P

rote

in E

xpre

ssio

n (n

orm

aliz

ed to

β-a

ctin

)

***

50kDa

37kDa

TWIST1 37kDa

β-actin

25kDa

IPAH 3 4 1 2 6 5

Control D!

25kDa TWIST1

TWIST IP Control IPAH

WCLs

37kDa

E!

ET-

1 P

rote

in E

xpre

ssio

n cell media

2

0

4

6 *

Control IPAH

F!

C!


Figure 8

TWIST1

HIF-1αMir-543

PulmonaryArterySmoothCellMembrane

ET-1

HIF-1α

HIF1α HIF1β

HRE

Nucleus

Hypoxia

EDN1VEGF

ET-1

O2

O2


pulmonary artery smooth muscle cell hif-1α regulates...

Documents