Hypoxia Down-Regulates Cyclic Guanidine Monophosphate-Dependent Protein Kinase in Fetal Pulmonary Vascular Smooth Muscle Cell Through Generation of Reactive Oxygen Species and Promotes Development of Pulmonary Hypertension

Abstract

We have previously reported that cyclic guanidine monophosphate (cGMP) mediates vasodilation primarily through protein kinase G (PKG) in fetal pulmonary vessels 1 Dhanakoti SN Gao Y Nguyen MQ et al. Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP. J Appl Physiol. 2000; 88: 1637-1642 PubMed Google Scholar and that acute hypoxia down-regulates cGMP-PKG-mediated relaxation in these vessels. 2 Gao Y Dhanakoti SN Trevino EM et al. Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L611-L618 Crossref Scopus (34) Google Scholar The mechanism by which this occurs is not known. Using a microarray, we found that in human pulmonary vascular smooth muscle cells (PVSMCs) 4 h of hypoxia altered the expression of > 900 genes fourfold or more. This was reduced to 121 genes by the reactive oxygen species (ROS) scavenger ascorbate, suggesting a role for ROS in hypoxic gene expression. Hence, first we used dichlorodihydrofluorescein (DCF) to measure ROS production in the pulmonary artery and pulmonary vein SMC during hypoxia. We determined identity of ROS by using scavengers for superoxide, hydrogen peroxide, and peroxynitrite during DCF fluorescence measurement. Ovine fetal pulmonary artery smooth muscle cells and PVSMCs were grown in Dulbecco minimum essential medium, preloaded with DCF and incubated under conditions of hypoxia (0% O2-5% CO2) or normoxia (20% O2-5% CO2). After 30 min, hypoxia ROS production increased fourfold to sixfold compared to normoxia but returned to normoxic levels after 4 h. Pretreatment of PVSMCs with ROS scavengers (Tiron for superoxide radical and Trolox for peroxynitrite radical) or with mitochondria complex III inhibitor myxothiazol significantly decreased ROS generated by hypoxia. After 4 h, hypoxia reduced PKG protein and PKG1 messenger RNA expression in PVSMCs. The scavenging of superoxide and peroxynitrite generated during hypoxia prevented the hypoxic decrease in PKG protein and PKG1 messenger RNA expression. Our findings indicate that the hypoxic generation of ROS in PVSMCs leads to the down-regulation of cGMP-mediated vasodilation by the inhibition of PKG and predisposes the patient to the development of pulmonary hypertension.