Regulation of ephrinB2-dependent angiogenesis by PS1 in normal and AD

描述（由申请方提供）：过去十年的证据表明，脑微血管异常与AD神经病理学的发生有关。另外的文献显示EphB 4/ephrinB 2系统是血管系统发育和功能的重要调节剂。EphB 4受体的细胞外序列与血管内皮细胞表面上的其跨膜配体ephrinB 2蛋白的结合刺激血管生成和来自现有脉管系统的新血管的生长。此外，体外试验表明，用EphB 4的细胞外结构域处理表达ephrinB 2的内皮细胞刺激细胞发芽和管形成，这被认为是血管生成的关键初始步骤，而转基因小鼠实验表明ephrinB 2蛋白的细胞内（细胞质）结构域对于ephrinB 2依赖性血管生成是必需的。 我们发现EphB 4刺激ephrinB 2的金属蛋白酶（MP）和PS/γ-分泌酶加工产生胞质肽ephrinB 2/CTF 2，并且EphB 4诱导的内皮细胞出芽和管形成依赖于γ-分泌酶活性。这些观察结果提高了内皮EphB 4/ephrinB 2系统通过PS/γ-分泌酶调节血管生成的可能性。为了支持这一假设，我们获得的数据表明肽ephrinB 2/CTF 2在体外刺激内皮细胞发芽。最近的文献表明，血管生成因子诱导的血管生成中的关键步骤是Raf 1/ Rok-α和血管内皮钙粘蛋白（VE-钙粘蛋白）之间的复合物的形成，并且我们进行了新的观察（初步数据），即用EphB 4处理内皮细胞培养物增加了这些血管生成复合物。总之，我们的观察结果表明，PS1/γ-分泌酶可能通过调节跨膜蛋白ephrinB 2的加工来影响血管生成，ephrinB 2是EphB 4诱导血管生成的关键步骤。在这里，我们打算探索EphB 4/ephrinB 2和PS1/γ-分泌酶系统相互作用促进内皮细胞发芽和血管生成的机制，并检查这些机制是否在阿尔茨海默病（AD）脑中发生改变。此外，我们和其他人报道了PS1家族性AD（FAD）突变可能影响PS1/γ-分泌酶底物的ε（ε）切割，从而减少CTF 2肽（包括ephrinB 2/CTF 2）的产生（见显著性）。因此，我们将询问PS1 FAD突变体是否改变EphB 4/ephrinB 2依赖的血管生成。

DESCRIPTION (provided by applicant): Evidence in the last decade implicates cerebral microvasculature abnormalities in the genesis of AD neuropathology. Additional literature shows that the EphB4/ephrinB2 system is an important regulator of the development and function of the vascular system. Binding of the extracellular sequence of EphB4 receptor to its transmembrane ligand ephrinB2 protein on the surface of endothelial cells of blood vessels, stimulates angiogenesis and growth of new vessels from existing vasculature. Furthermore, in vitro assays show that treatment of ephrinB2expressing endothelial cells with the extracellular domain of EphB4 stimulates cell sprouting and tube formation, processes considered crucial initial steps in angiogenesis, while transgenic mouse experiments indicate that the intracellular (cytoplasmic) domain of ephrinB2 protein is necessary for ephrinB2-dependent angiogenesis. We found that EphB4 stimulates the metalloproteinase (MP) and PS/γ-secretase processing of ephrinB2 producing cytosolic peptide ephrinB2/CTF2 and that the EphB4-induced sprouting and tube formation of endothelial cells depends on γ-secretase activity. These observations raise the possibility that the endothelial EphB4/ephrinB2 system regulates angiogenesis through PS/γ-secretase. In support of this hypothesis, we obtained data that peptide ephrinB2/CTF2 stimulates sprouting of endothelial cells in vitro. Recent literature shows that a crucial step in angiogenic factor-induced angiogenesis is formation of complexes between Raf1/ Rok-α and Vascular Endothelial cadherin (VE-cadherin) and we made the novel observation (preliminary data) that treatment of endothelial cell cultures with EphB4 increases these angiogenic complexes. Together, our observations suggest that PS1/γ-secretase may affect angiogenesis by regulating processing of transmembrane protein ephrinB2, a critical step in EphB4-induced angiogenesis. Here we propose to explore the mechanisms via which the EphB4/ephrinB2 and PS1/γ-secretase systems interact to promote endothelial cell sprouting and angiogenesis and to examine whether any of these mechanisms are altered in Alzheimer disease (AD) brains. Furthermore, we and others reported that PS1 familial AD (FAD) mutations may affect the epsilon (ε) cleavage of PS1/γ-secretase substrates thus decreasing production of CTF2 peptides including ephrinB2/CTF2 (see Significance). Thus, we will ask whether PS1 FAD mutants alter the EphB4/ephrinB2-dependent angiogenesis.