eN0S/TSP2

项目总结（见说明）： 血小板反应蛋白2（TSP 2）与内皮型一氧化氮合酶（eNOS）之间的关系尚不清楚。通过VEGF或通过流体剪切应力激活VEGFR-2激活蛋白激酶Akt和内皮特异性Akt底物内皮一氧化氮合酶（eNOS），导致动脉生成和血管生成。从我们实验室令人兴奋的初步研究中推断，TSP 2是Akt-1/eNOS通路的负调节因子。先前，我们已经报道了TSP 2敲除小鼠（TSP 2-K 0）表现出增强的动脉生成和血流恢复，以响应肢体缺血和增强的组织愈合。有趣的是，我们的初步研究表明，TSP 2水平在eNOS-KO和Akt 1-KO小鼠中升高，这两种小鼠都显示出动脉生成和血管生成的显著损伤，而NO本身负调节TSP 2水平。此外，TSP 2/eNOS双敲除（DKO）小鼠表现出血流恢复和组织修复的显著改善。因此，我们假设eNOS衍生的NO对TSP 2表达的调节构成了NO先前未定义的促动脉生成和促血管生成特性。检验Akt 1/eNOS轴部分通过抑制TSP 2水平介导体内动脉生成和血管生成反应的假设; 2.确定Akt 1/eNOS轴和与ERK的串扰调节TSP 2和动脉生成的机制;和3.剖析了在体外血管生成过程中N 0/TSP 2通路和其他内皮细胞功能的调节。总的来说，这些实验将使我们能够深入研究TSP 2对动脉生成和血管生成的功能性拮抗作用，并描述NO如何调节TSP 2基因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.