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

描述(由申请人提供)：过去十年的证据表明，脑微血管异常与AD神经病理的发生有关。更多的文献表明，EphB4/ewitinB2系统是血管系统发育和功能的重要调节因子。EphB4受体的胞外序列与其跨膜配体ewitinB2蛋白结合在血管内皮细胞表面，刺激血管生成和新血管的生长。此外，体外分析表明，用EphB4的胞外区处理表达ewitinB2的内皮细胞可刺激细胞萌发和管状形成，这些过程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，ewitinB2蛋白的胞内(细胞质)域对于ewitinB2依赖的血管生成是必要的。 我们发现EphB4刺激基质金属蛋白酶(MP)和PS/γ-分泌酶的处理，产生胞内肽，EphB4诱导内皮细胞的萌发和管状形成依赖于γ-分泌酶的活性。这些观察结果提出了内皮EphB4/ewitinB2系统通过PS/γ-分泌酶调节血管生成的可能性。为了支持这一假说，我们在体外获得了多肽ewitinB2/CTF2刺激内皮细胞萌发的数据。最近的文献表明，在血管生成因子诱导的血管生成中，Raf1/Rok-α和血管内皮细胞钙粘附素(VE-Cad-herin)形成复合体是至关重要的一步，我们做了一个新的观察(初步数据)，即用EphB4处理内皮细胞使这些血管生成复合体增加。综上所述，我们的观察表明，PS1/γ-分泌酶可能通过调节跨膜蛋白EphinB2的处理来影响血管生成，这是EphB4诱导血管生成的关键步骤。在这里，我们建议探索EphB4/EPhinB2和PS1/γ-分泌酶系统相互作用促进内皮细胞萌发和血管生成的机制，并检查这些机制中是否有任何机制在阿尔茨海默病(AD)脑中发生改变。此外，我们和其他人报道，PS1家族性AD(FAD)突变可能影响PS1/CTF2分泌酶底物的epsilon(ε)裂解，从而减少包括γB2/CTF2在内的CTF2多肽的产生(见显著意义)。因此，我们将询问PS1FAD突变体是否改变了EphB4/ewitinB2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.