Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function
Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function
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DOI：
10.1152/ajplung.00417.2007
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发表时间：
2008-04-01
影响因子：
4.9
通讯作者：
Pearse, David B.
中科院分区：
文献类型：
--
作者：
Rentsendorj, Otgonchimeg;Mirzapoiazova, Tamara;Pearse, David B.
增加肺内皮 cGMP 可以通过激活蛋白激酶 G (PKG(I)) 来预防内皮屏障功能障碍。血管舒张刺激磷蛋白 (VASP) 被假设可介导 PKGI 屏障保护，因为 VASP 是细胞-细胞连接中表达的 PKGI 的细胞骨架磷酸化靶标。未磷酸化的 VASP 被认为通过肌动蛋白聚合和应力纤维成束来增加细胞旁通透性，这一过程受到 PKG(I) 介导的 Ser(157) 和 Ser(239) 磷酸化的抑制。为了检验这一假设，我们检查了 VASP 在人肺动脉内皮细胞 (HPAEC) 单层中由 H2O2 引起的短暂性屏障功能障碍中的作用，这些细胞在没有和有腺病毒感染 (Ad.PKG) 引入 PKGI 表达的情况下进行研究。在缺乏 PKGI 表达的情况下，H2O2 (100-250 μM) 会导致通透性短暂增加和 pSer(157)-VASP 形成，而这两种现象均会因蛋白激酶 C 抑制而减弱。通过使用环孢菌素抑制磷酸酶 2B 或使用毛喉素激活蛋白激酶 A 来增强 VASP Ser(157) 磷酸化，延长而不是抑制 H2O2 引起的通透性增加。 Ad.PKG 感染后，用小干扰 RNA 抑制 VASP 表达会加剧 H2O2 诱导的屏障功能障碍，但对 cGMP 介导的屏障保护没有影响。此外，Ser-双磷酸模拟突变体VASP的表达未能再现激活的PKGI的保护作用。最后，抗 Ser-双磷酸化的 VASP 的表达未能干扰 cGMP/PKG(I) 减弱 H2O2 诱导的 VE-钙粘蛋白同型结合破坏的能力。我们的结果表明，VASP 磷酸化并不能解释 cGMP/PKG(I) 对 H2O2 诱导的 HPAEC 内皮屏障功能障碍的保护作用。
Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKGI barrier protection because VASP is a cytoskeletal phosphorylation target of PKGI expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H2O2 in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKGI expression introduced by adenoviral infection (Ad.PKG). In the absence of PKGI expression, H2O2 (100-250 mu M) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H2O2. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H2O2-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKGI. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG(I) to attenuate H2O2-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H2O2-induced endothelial barrier dysfunction in HPAEC.