Weilin Zhou

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase and myocardin

We have previously reported that in ovine fetal pulmonary venous smooth muscle cells (FPVSMC), decreased expression of cGMP-dependent protein kinase (PKG) by hypoxia could explain hypoxia-induced SMC phenotype modulation. In this study, we investigated the role of myocardin, a possible downstream effector of PKG, in SMC phenotype modulation induced by 1 and 24 h of hypoxia. Hypoxia for 1 h induced the phosphorylation of E-26-like protein 1 (Elk-1), indicating a quick activation of Elk-1 after hypoxia. Either hypoxia (1 h) or treatment with DT-3, a PKG inhibitor, increased associations of Elk-1 with myosin heavy chain (MHC) gene and serum response factor (SRF), which was paralleled by a decrease in association of myocardin with MHC gene and SRF. Exposure to hypoxia of FPVSMC for 24 h significantly decreased the promoter activity of multiple SMC marker genes, downregulated protein and mRNA expression of myocardin, and upregulated mRNA expression of Elk-1, but had no significant effects on the phosphorylation of Elk-1. Inhibition of myocardin by siRNA transfection downregulated the expression of SMC marker proteins, while overexpression of myocardin prevented the hypoxia-induced decrease in expression of SMC marker proteins. Inhibition of PKG by siRNA transfection downregulated the expression of myocardin, but upregulated that of Elk-1. Overexpression of PKG prevented hypoxia-induced effects on protein expression of myocardin and Elk-1. These data suggest that PKG induces displacement of myocardin from SRF and upregulates myocardin expression, thus activating the SMC genes transcription. The inhibitory effects of hypoxia on PKG may explain hypoxia-induced SMC phenotype modulation by decreasing the effects of PKG on myocardin.

Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia

Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po(2) approximately 100 Torr) or hypoxia (Po(2) approximately 30-40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1alpha [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1alphaR-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by alpha/beta-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by alpha(4)-, beta(1)-, and alpha(5)beta(1)-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1alpha construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.

Role of Epidermal Growth Factor Receptor in Ovine Fetal Pulmonary Vascular Remodeling Following Exposure to High Altitude Long-Term Hypoxia

The purpose of this study was to evaluate color vision during high altitude mountain climbing by applying the Mollon-Reffin Minimalist test to 14 climbers, all of whom were participating in the expedition to Ama Dablam (6,812 m) in Nepal. Before leaving for Nepal (at 300 m), all 28 eyes showed normal color vision in all 3 axes. At 1,300 m, 100% of eyes showed normal color vision in the protan and deutan axes, while 25% showed minimally reduced color discrimination in the tritan axis. At 4,000 m, 100% showed normal deutan axis, 4% minimally reduced protan axis, and 72% minimally reduced tritan axis discrimination. At 5,400 m 100% of eyes tested showed normal protan and deutan axis discrimination, while 75% showed minimally and 25% moderately reduced tritan axis discrimination. Back home at 300 m 3 days after return, 100% showed normal deutan, 4% minimally reduced protan, and 38% minimally reduced tritan axis discrimination. One year later, all eyes showed normal color vision in all three axes. Changes in tritan axis discrimination correlated well with increased heart rate (r = 0.69; p = 0.0001) and decreased oxygen saturation (r = 0.71; p = 0.001) at high altitude. This study shows that the tritan color vision axis is predominantly affected at high altitude, but that this reduced color discrimination is transient.