Zhou Weilin, Dasgupta Chiranjib, Negash Sewite, Raj J Usha
Division of Neonatology, Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, 1124 West Carson St., Torrance, CA 90502, USA.
Am J Physiol Lung Cell Mol Physiol. 2007 Jun;292(6):L1459-66. doi: 10.1152/ajplung.00143.2006. Epub 2007 Feb 23.
Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, alpha-smooth muscle actin (alphaSMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on alphaSMA and TSP. Treatment with 30 microM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, alphaSMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.
慢性缺氧会引发肺血管重塑，这与血管平滑肌细胞（SMC）表型从收缩性、分化状态转变为合成性、去分化状态有关。我们之前报道过，急性缺氧会抑制绵羊胎儿肺静脉平滑肌细胞（FPVSMCs）中依赖环磷酸鸟苷（cGMP）的蛋白激酶（PKG）的表达。因此，我们测试了PKG表达的改变是否能解释缺氧暴露后SMC表型的调节。缺氧诱导的PKG蛋白表达降低与SMC表型标志物肌球蛋白重链（MHC）、钙调蛋白、波形蛋白、α-平滑肌肌动蛋白（αSMA）和血小板反应蛋白（TSP）的表达受抑制密切相关，表明SMC的缺氧暴露诱导了表型调节至去分化状态，且PKG可能参与了SMC表型调节。在FPVSMCs中进行PKG特异性小干扰RNA（siRNA）转染可显著减弱钙调蛋白、波形蛋白和MHC的表达，而对αSMA和TSP没有影响。用30微摩尔的果蝇触角足蛋白（DT-3）（一种PKG的膜通透性肽抑制剂）处理可减弱TSP、MHC、αSMA、波形蛋白和钙调蛋白的表达。PKG siRNA和DT-3研究的结果表明，PKG抑制对缺氧诱导的蛋白表达降低也有类似影响。通过用绿色荧光融合蛋白标记的全长PKG构建体（PKG-GFP）转染在FPVSMCs中过表达PKG，可逆转缺氧对SMC表型标志物蛋白表达的影响。这些结果表明，PKG可能是SMC表型标志物蛋白表达的决定因素之一，并且可能参与缺氧状态下肺血管SMC分化表型的维持。
