Annexin II in angiogenesis

膜联蛋白II是一种表达于内皮细胞表面的磷脂结合钙调节蛋白。它具有结合内皮细胞分泌产物组织型纤溶酶原激活剂(t-PA)和纤溶酶(一种具有广泛底物特异性的纤溶丝氨酸蛋白酶)的能力60倍。我们最近创造了一种膜联蛋白II零缺失小鼠，它显示出毛细血管密度降低的心肌病。更多的初步数据表明，在选定的体内环境中，如Matrigel植入物的微血管侵入和氧诱导的视网膜新生血管的形成，Annexin II是出生后血管生成所必需的。在这项提案中，我们计划检查膜联蛋白II缺乏症的血管生成缺陷的范围，并确定其分子机制。我们将采用各种遗传和分子方法来解决这个问题。在特定目标I下，我们将在体内研究Annexin II在Matrigel侵袭试验、氧诱导的视网膜病变、角膜袋试验、颈动脉结扎模型以及使用Annexin II“敲除”小鼠的耳廓翻译试验中的作用。此外，我们将使用AnnII-LacZ转基因小鼠来确定Annin II启动子在这些新生血管环境中是否上调。最后，我们将在这些检测中检查纤溶系统其他成分(纤溶酶原、t-PA、尿激酶)缺乏的小鼠的行为，以确定膜联蛋白II在血管生成中的作用是否依赖于纤溶酶的生成。在AIM II中，我们将测定从Annexin II缺陷动物分离的微血管内皮细胞的增殖能力、存活情况、蛋白分解活性和迁移能力，并研究它们对已知血管生成调节剂的反应。在Aim III中，我们将在体内检测同型半胱氨酸(HC)对膜联蛋白II功能的影响。HC是一种含有硫醇的氨基酸，能特异性地阻断Annexin II“尾部”区域内的t-PA结合域。我们将确定HC诱导的Annexin II阻断对因半胱氨酸β-合成酶缺乏而导致高同型半胱氨酸血症的小鼠血管生成的影响，以及这种缺陷是否反映了纤溶酶的生成受损。综上所述，这些实验将提供对膜联蛋白II在出生后新生血管发育中的作用的详细、机械性的理解，并可能为未来血管生成的治疗干预提供新的策略。

Annexin II is a phospholipid-binding calcium-regulated protein expressed on the surface of endothelial cells. It possesses the to bind tissue plasminogen activator (t-PA), an endothelial cell secretory product, as well as plasmin, a fibrinolytic serine protease of broad substrate specificity, by 60-fold. We recently created an annexin II null deletion mouse which displays cardiomyopathy with reduced capillary density. Additional preliminary data suggest that annexin II is required for post- natal angiogenesis in selected in vivo settings such as microvascular invasion of Matrigel implants and development of oxygen-induced retinal neovascularization. In this proposal, we plan to examine the scope of the angiogenic defect in annexin II deficiency, and to determine its molecular mechanism. We will employ a variety of genetic and molecular approaches to address this question. Under Specific Aim I, we will study the role of annexin II in vivo in the Matrigel invasion assay, in oxygen- induced retinopathy, in the corneal pocket assay, in a carotid artery ligation model, and in an auricular translation assay using annexin II "knockout" mice. In addition, we will determine whether the annexin II promoter is up-regulated in these settings of neovascularization using AnnII-LacZ transgenic mice. Finally, we will examine the behavior of mice deficient in other elements of the fibrinolytic system (plasminogen, t-PA, urokinase) in these assays to determine whether the role of annexin II in angiogenesis is dependent upon plasmin generation. Under Aim II, we will determine the proliferative capacity, survival profile, proteolytic activity, and migratory potential of microvascular endothelial cells isolated from annexin II-deficient animals, and study their response to known angiogenic modulators. Under Aim III, we will examine the influence of homocysteine (HC) on annexin II function in vivo. HC is a thiol-containing amino acid that specifically blocks the t-PA binding domain within the "tail" region of annexin II. We will determine the effect of HC-induced annexin II blockade on angiogenesis in mice that are hyperhomocysteinemic due to cystathionine beta-synthase deficiency, and whether this defect reflects impaired plasmin generation. Taken together, these experiments should provide a detailed, mechanistic understanding of the role of annexin II in post-natal neovascular development, and will likely provide new strategies for therapeutic interventions in angiogenesis in the future.