Department of Molecular Biology, Section of Pharmacology, University of Siena, Siena, Italy.
Am J Physiol Regul Integr Comp Physiol. 2010 Mar;298(3):R824-32. doi: 10.1152/ajpregu.00222.2009. Epub 2009 Dec 23.
Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.
一氧化氮 (NO) 已知可促进血管内皮生长因子 (VEGF) 刺激的通透性和血管生成。然而，该途径中 NO 下游的效应分子仍未得到很好的描述。在此，我们确定了可溶性鸟苷酸环化酶 (sGC) 抑制对 VEGF 体外和体内反应的影响。VEGF 处理内皮细胞 (EC) 可刺激 eNOS 磷酸化和 cGMP 积累；用 sGC 抑制剂 4H-8-溴-1,2,4-噁二唑并[3,4-d]苯并b恶嗪-1-酮 (NS-2028) 预处理会削弱 cGMP 水平，而不影响 VEGF 受体磷酸化。细胞孵育 NS-2028 可阻断 VEGF 的有丝分裂作用。此外，抑制 sGC 的细胞对 VEGF 没有迁移和发芽反应。为了研究 NS-2028 抑制 EC 迁移的机制，我们确定了 cGMP 水平变化对 p38 MAPK 的影响。最初，我们观察到抑制 sGC 可减弱 VEGF 刺激的 p38 激活。相比之下，向 EC 添加 8-Br-cGMP 可刺激 p38 磷酸化。添加 cGMP 升高剂（BAY 41-2272、DETA-NO 和 YC-1）可增强 EC 迁移。为了测试 sGC 是否也介导 VEGF 在体内的血管生成作用，我们使用了兔角膜试验。口服接受 NS-2028 的动物对 VEGF 的血管生成反应降低。由于新血管形成之前发生血管通透性增加，因此我们确定了 NS-2028 在血管通透性中的作用。使用改良的 Miles 试验，我们观察到 NS-2028 可减弱 VEGF 诱导的通透性。总的来说，我们提供的证据表明 sGC 介导了 VEGF 的血管生成和促通透性活性，表明 sGC 作为 VEGF 触发反应的下游效应物的重要性。
