eN0S/TSP2

项目总结(见说明)： 血栓反应蛋白2(TSP2)和内皮型一氧化氮合酶(ENOS)之间的关系尚不清楚。通过血管内皮生长因子或流体切应力激活VEGFR-2，激活蛋白激酶Akt和内皮特异性Akt底物--内皮型一氧化氮合酶(ENOS)，导致动脉生成和血管生成。根据我们实验室令人兴奋的初步研究推断，TSP2是Akt-1/eNOS途径的负调控因子。此前，我们曾报道过TSP2基因敲除小鼠(TSP2-K0)在肢体缺血和促进组织愈合方面表现出增强的动脉生成和血流恢复。有趣的是，我们的初步研究表明，在eNOS-KO和Akt1-KO小鼠中，TSP2水平升高，这两种小鼠在动脉生成和血管生成方面都表现出明显的损害，而NO本身对TSP2水平具有负面调节作用。此外，TSP2/eN0S双基因敲除(DKO)小鼠在血流恢复和组织修复方面显示出显著的改善。因此，我们假设，eNOS来源的NO对TSP2表达的调节构成了先前未定义的NO的促动脉生成和促血管生成特性。为了验证这一观点，我们将：1.验证Akt1/eNOS轴部分通过抑制TSP2水平在体内介导动脉生成和血管生成反应的假设；2.确定Akt1/eNOS轴及其与ERK的串扰调节TSP2和动脉生成的机制(S)；以及3.剖析体外血管生成过程中N0/TSP2通路和其他内皮细胞功能的调节。总之，这些实验将使我们能够深入研究TSP2对动脉生成和血管生成的功能拮抗作用，并阐明NO如何调节TSP2基因的表达和肢体缺血后的血流恢复。

PROJECT SUMMARY (See instructions): The relationship between thrombospondin 2 (TSP2) and endothelial nitric oxide synthase (eNOS) are not known. Activation of VEGFR-2 via VEGF or by fluid shear stress activates the protein kinase Akt and the endothelial specific Akt substrate, endothelial nitric oxide synthase (eNOS), leading to arteriogenesis and angiogenesis. As inferred from exciting preliminary studies from our laboratories, TSP2 serves as a negative regulator of the Akt-1/eNOS pathway. Previously, we have reported that TSP2 knockout mice (TSP2-K0) demonstrate enhanced arteriogenesis and blood flow recovery in response to limb ischemia and augmented tissue healing. Interestingly, our preliminary studies show that TSP2 levels are elevated in eNOS-KO and Akt1-KO mice, which both display marked impairments in arteriogenesis and angiogenesis and NO, per se, negatively regulates TSP2 levels. Moreover, TSP2/eN0S double knockout (DKO) mice exhibit substantial improvements in blood flow recovery and tissue repair. Thus, we hypothesize that regulation of TSP2 expression by eNOS-derived NO constitutes a previously undefined pro-arteriogenic and pro-angiogenic property of NO. To test this idea, we will: 1. test the hypothesis that the Akt1/eNOS axis mediates arteriogenic and angiogenic responses in vivo, in part, by repressing TSP2 levels; 2. determine the mechanism(s) through which the Akt1/eNOS axis and crosstalk with ERK regulates TSP2 and arteriogenesis; and 3. dissect the regulation of the N0/TSP2 pathway and other endothelial cell functions during in vitro angiogenesis. Collectively, these experiments will allow us to delve deeply into the functional antagonism of arteriogenesis and angiogenesis by TSP2, and to delineate how NO regulates TSP2 gene expression and blood flow recovery after limb ischemia.