Hypoxia stimulates pulmonary artery smooth muscle cell (PASMC) proliferation. Recent studies have implicated an important role for microRNAs (miRNAs) in hypoxia-mediated responses in various cellular processes, including cell proliferation. In this study, we investigated the role of microRNA-21 (miR-21) in hypoxia-induced PASMC proliferation and migration. We first demonstrated that miR-21 expression increased by ∼3-fold in human PASMC after 6 h of hypoxia (3% O₂) and remained high (∼2-fold) after 24 h of hypoxia. Knockdown of miR-21 with anti-miR-21 inhibitors significantly reduced hypoxia-induced cell proliferation, whereas miR-21 overexpression in normoxia enhanced cell proliferation. We also found that miR-21 is essential for hypoxia-induced cell migration. Protein expression of miR-21 target genes, specifically programmed cell death protein 4 (PDCD4), Sprouty 2 (SPRY2), and peroxisome proliferator-activated receptor-α (PPARα), was decreased in hypoxia and in PASMC overexpressing miR-21 in normoxia and increased in hypoxic cells in which miR-21 was knocked down. In addition, PPARα 3'-untranslated region (UTR) luciferase-based reporter gene assays demonstrated that PPARα is a direct target of miR-21. Taken together, our findings indicate that miR-21 plays a significant role in hypoxia-induced pulmonary vascular smooth muscle cell proliferation and migration by regulating multiple gene targets.

Chronic hypoxia is an important contributing factor to the development of pulmonary hypertension. Hypoxia triggers pulmonary vascular smooth muscle cell (PVSMC) remodeling from a differentiated state to a proliferative, dedifferentiated state. cGMP‐dependent protein kinase type 1 (Prkg1) is involved in the regulation of vasomotor tone and remodelling. MicroRNAs play important roles in various biologic processes in cells, where they regulate apoptosis, proliferation, differentiation, development & metabolism. However, the role of microRNAs in hypoxia‐mediated Prkg1 down‐regulation in smooth muscle cells and SMC proliferation is unknown. The objective of the study was to characterize miRNA regulation in PVSMC response to hypoxia, affecting cell proliferation. This is important in understanding the molecular mechanisms triggered by cell exposure to low oxygen tension in Persistent Pulmonary Hypertension (PPH) in neonates. We used human PVSMCs as an in vitro model system and studied the effects of hypoxic (3% oxygen) exposure. Cells were processed for mRNA, protein and microRNA studies. We found reduced mRNA and protein expression of SMC markers such as α‐Smooth muscle actin and calponin as well as Prkg1. Quantitative PCR analyses showed that 24h hypoxia treatment induced miR‐21 upregulation. Our studies suggest that miR‐21 plays a role in hypoxia‐induced smooth muscle cell proliferation. This work was supported by R01 HL075187‐05A1

We have shown in the fetus and newborn, that in pulmonary vascular relaxation induced by cGMP‐elevating agents, cGMP‐dependent protein kinase I(Prkg1) plays a primary role. Hypoxia attenuates cGMP‐dependent protein kinase(Prkg1)‐mediated relaxation in pulmonary vessels. We have shown that peroxynitrite, an important effector of oxidative stress, produced in pulmonary vascular smooth muscle cells (PVSMC) during hypoxia, decreases Prkg1 protein levels as well as Prkg1 activity. In this study we test the hypothesis that hypoxia alters Prkg1 protein by ROS/RNS‐dependent covalent modification of critical amino acids which decreases its kinase activity. Ovine PVSMCs were exposed to hypoxia (3 % O2) or normoxia (20% O2) and the purified Prkg1 protein was analyzed by FTMS. Two peptides containing cysteine were among those identified for Prkg1 and were found to exhibit definitive transformation under hypoxic conditions. These results suggest that hypoxia‐induced reactive oxygen/nitrogen species can modulate the activity of Prkg1 by modifying the cysteine residues which may be relevant to the pathophysiological conditions associated with increased hypoxia‐induced oxidative stress. Support NIH R01 HL075187‐05A1