miR-214-Mediated Attenuation of hPAEC p53; Role in PAH?&@ Max Alvarez, *# Sanghamitra Sahoo, *# Eugenia Cifuentes-Pagano, and *# Patrick J. Pagano.

*Department of Pharmacology and Chemical Biology and #Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh. &PURDiP. @Palm Beach Atlantic University.

HYPOTHESIS

INTRODUCTION

Hypoxia-induced miR-214 attenuates p53 expression in human pulmonary artery endothelial

cells (hPAECs) leading to EC proliferation.

METHODS RESULTS

CONCLUSIONS

RESULTShPAEC: Scrambled & miR-214 Mimic

β-actin

Scrm Mimic

Notch2

p53

Prot

ein

expr

essi

on(fo

ld c

hang

e vs

. Nor

m)

p53 Notch20.0

0.5

1.0

1.5

2.0

2.5 ScrmMimic

*

*

Figure 1. miR-214 expression by qRT-PCR in hPAECs exposed (24hrs) to hypoxia (1% O2) or normoxia (21% O2) (n=6-7).

hPAEC 24h Hypoxia

Prot

ein

expr

essi

on(fo

ld c

hang

e vs

. Nor

m)

p53 Bax0.0

0.5

1.0

1.5NormoxiaHypoxia

**

β-actin

HypoxiaNormoxiap53

Bax

Figure 3. In response to 24h hypoxia, no change in p53 was found. Hypoxia decreases expression of Bax (n=3).

hPAEC 48h Hypoxia

Prot

ein

expr

essio

n(fo

ld c

hang

e vs

. Nor

m)

p53 Bax0.0

0.5

1.0

1.5

2.0NormoxiaHypoxia

ns

p53HypoxiaNormoxia

β-actinBax

Figure 4. In response to 48h hypoxia, no change in p53, and Bax expression (n=3) was observed.

hPASMC: Scrambled & miR-214 Mimic

p53β-actin

Scrm Mimic

Pro

tein

exp

ress

ion

(fo

ld c

ha

ng

e v

s. N

orm

)

p530.0

0.5

1.0

1.5

2.0ScrmMimic

***

hPASMC 24h Hypoxia

HypoxiaNormoxiap53β-actinBax

hPASMC 72h Hypoxia

Normoxia Hypoxiap53β-actinBax Pr

otein

exp

ress

ion(fo

ld ch

ange

vs. N

orm)

p53 Bax0.0

0.5

1.0

1.5 NormoxiaHypoxia

** *

Effects of Pulmonary Arterial Hypertension Pulmonary arterial hypertension (PAH) ensues when the

pulmonary arterioles become partially or totally occluded due toproliferation and aberrant migration of pulmonary vascularendothelial and smooth muscle cells.

Occlusion in vessels lead to increase in pulmonary arterialpressure and eventually to right ventricular heart failure anddeath.

MicroRNA-214 miR-214 is implicated in cancer, ischemia injury, and heart failure.

Tumor Suppressor protein 53 Inhibition of p53 has been linked to PAH. p53 regulates a variety of cellular response , such as apoptosis and

proliferation. Bax is downstream of p53 and promotes apoptosis

Lai 2014 Circ Res. 115:115-130.

Cell Culture• hPAECs and hPASMCs (Lonza, MD) were cultured in EBM-2 and SMBM

(Lonza), respectively. Cells were grown to 80-90% confluence, and serum-deprived a priori hypoxic (1% O2) or Normoxic (21% O2) exposure.

Oligonucleotide Transfections• Transfections with miR-214 mimic and scrambled control (scrm)

(Invitrogen Inc.) were performed at a final concentration of 25pmoles, using Lipofectamine 3000 according to the manufacturer’s instructions.

Western Blotting• Immunoblotting of the cell lysates were performed using the following

antibodies: p53, Notch2, Bax, and β-actin.

• The data suggest a significant decrease in hPASMC expression of p53 after 72 h exposure to hypoxia.

• The data are also consistent with miR214 not being causally involved in the attenuation in p53 expression (in both hPAECs and hPASMCs); in fact, p53 was increased in both cell types.

• Though we know that under hypoxia conditions there is an increase in expression of miR-214, it appears to not be responsible for the attenuation of p53.

0.0

0.5

1.0

1.5

2.0*

Normoxia Hypoxia

miR

-214

exp

ress

ion

Fold

Cha

nge

(Nor

mal

ized

to R

NU43

)

Decrease in p53 Expression

Hypoxic Environment

Occluded VesselNormal Vessel

miR-214 is Upregulated in hPAECs in Hypoxia

Figure 2. miR-214 mimic transfected cells show an increase in p53 and a decrease in Notch2 expression (n=3).

Figure 5. In response to 24h hypoxia, no change in p53 and Bax expression (n=3) was observed.

Prot

ein

expr

essi

on(fo

ld c

hang

e vs

. Nor

m)

p53 Bax0.0

0.5

1.0

1.5 NormoxiaHypoxia

Figure 6. Following 72h hypoxia, levels of p53 and Bax (n=3) are reduced.

Figure 7. miR-214 mimic transfected cells display an increase in p53 (n=6).

ACKNOWLEDGEMENTSThe Center for Biologic Imaging at the University of Pittsburgh is gratefullyacknowledged as are the generous support of the VMI, the Hemophilia Center ofWestern PA, ITxM & NIH-NHLBI Grants (specified below).

NIH R01 HL112914, P01HL103455, R42-AA024003 & R01-CA162306, R01HL113178

Disclosures: None