Genetic and pharmacological manipulation of HEG1-KRIT1 protein complex modulates endothelial vasoprotective functions

在围产期小鼠中，Krit1(Krev1相互作用捕获基因1)的内皮特异性失活导致脑组织 海绵状畸形(CCM)，而Krit1(Krit1ECKO)或其结合伙伴HEG1在成人中失活 不会的。Krit1ECKO或Heg1ECKO导致转录因子KLF2和KLF4表达增加(Krüppel- 就像因子2和因子4一样；这些转录因子对层流血流的调节能力很重要 血管活性、抗凝剂和抗炎因子的内皮表达导致血管保护作用 来自炎症和血栓形成。在初步研究中，我们发现成年Krit1ECKO内皮细胞 (EC)增加受KLF2和KLF4调控的血管保护基因的表达，包括eNOS和蛋白 C激活辅因子、血栓调节蛋白(TM)。成年Krit1ECKO小鼠KLF4表达显著上调 大动脉提示可能对动脉粥样硬化有血管保护作用。此外，内皮细胞TM和Tm的增加 KRIT1缺失后的EPCR伴随着EC产生活化蛋白的能力的增加 C(APC)，一种天然的抗血栓和抗炎蛋白。这项提议的科学前提是 Krit1基因失活，通过上调KLF2和KLF4，可能模仿板层的血管保护作用 在血管内皮细胞上流动，从而减少血栓形成、动脉粥样硬化和血管炎症。此外，我们 这表明，这种流动的影响也可以被破坏KRIT1-HEG1相互作用的小分子模仿。 为了检验这些想法：特定目标1将测试Krit1基因失活将 保护小鼠免受血栓、动脉粥样硬化和血管炎症的影响。在核心A(Bergmeier)中，我们将 观察Krit1ECKO和Heg1ECKO对体内止血和血栓形成的影响。自eNOS上调以来 表达导致NO水平升高，NO是一种已知的血小板功能和白细胞黏附的抑制物，在 与项目2和CORE A合作，我们将研究炎症诱导的血管激活和 Krit1ECKO小鼠的动脉粥样硬化。我们将主要关注内皮细胞Krit1的遗传失活，但也将 必要时使用内皮细胞Heg1基因失活。《特定目标2》将检验这一假设 在EC中药物破坏HEG1-KRIT1蛋白复合体可以促进血管保护。我们 将研究小分子HKi002对血管保护基因eNOS和TM表达的影响， 在欧洲议会。用不同来源的血管内皮细胞研究HKi002对NO和APC生成的影响 床。在此背景下，我们将确定HKi002在细胞因子诱导的炎症和内皮细胞中的作用 通过评估黏附分子/白细胞黏附的表达和分析 细胞间连接完整性/肌动球蛋白收缩能力。通过将有机体和细胞相结合 方法，我们将提供破坏KRIT1-HEG1蛋白复合体对成人的影响的分析 血管系统和血管保护，并为测试药物对血管的破坏 复合体可以抑制炎症和血栓形成。

In perinatal mice, endothelial-specific inactivation of Krit1 (Krev1 interaction trapped gene1) leads to cerebral cavernous malformations (CCM), whereas inactivation of Krit1 (Krit1ECKO) or its binding partner HEG1 in adults does not. Krit1ECKO or Heg1ECKO results in increased expression of transcription factors KLF2 and KLF4 (Krüppel- like factors 2 and 4); these transcription factors are important in the capacity of laminar blood flow to modulate endothelial expression of vasoactive, anticoagulant, and anti-inflammatory factors resulting in vasoprotection from inflammation and thrombosis. In preliminary studies, we have found that adult Krit1ECKO endothelial cells (EC) increase expression of vasoprotective genes regulated by KLF2 and KLF4, including eNOS and the protein C activation cofactor, thrombomodulin (TM). Adult Krit1ECKO mice show marked elevation of KLF4 expression in aortas suggesting possible vasoprotection from atherosclerosis. In addition, the increase in endothelial TM and EPCR following loss of KRIT1 was accompanied by an increased capacity of EC to generate activated protein C (APC), a natural anti-thrombotic and anti-inflammatory protein. The scientific premise of this proposal is that genetic inactivation of Krit1, by upregulating KLF2 and KLF4, may mimic the vasoprotective effects of laminar flow on endothelium, thereby reducing thrombosis, atherosclerosis, and vascular inflammation. Moreover, we suggest that this effect of flow can also be mimicked by small molecules that disrupt the KRIT1-HEG1 interaction. To examine these ideas: Specific Aim 1 will test the hypothesis that genetic inactivation of Krit1 will protect mice from thrombosis, atherosclerosis, and vascular inflammation. In core A (Bergmeier), we will investigate the effect of Krit1ECKO or Heg1ECKO in hemostasis and thrombosis in vivo. Since upregulation in eNOS expression leads to increased levels of NO, a known inhibitor of platelet function and leukocyte adhesion, in collaboration with project 2 and core A, we will study models of inflammation-induced vascular activation and atherosclerosis in Krit1ECKO mice. We will primarily focus on genetic inactivation of endothelial Krit1 but will also use endothelial Heg1 genetic inactivation when warranted. Specific Aim 2 will test the hypothesis that pharmacologic disruption of the HEG1-KRIT1 protein complex in EC can promote vasoprotection. We will examine the effect of a small-molecule, HKi002, on the expression of vasoprotective genes, eNOS and TM, in ECs. The effect of HKi002 on NO and APC generation will be investigated using ECs from different vascular beds. In this context, we will determine the effect of HKi002 on cytokine-induced inflammation and endothelial barrier properties by assessing expression of adhesion molecules/leukocyte adhesion and by analyzing intercellular junction integrity/actomyosin contractility. Altogether by combining organismal and cell-based approaches, we will provide an analysis of the effects of disrupting KRIT1-HEG1 protein complex in adult vasculature and vasoprotection and set the stage for testing the idea that pharmacological disruption of the complex can suppress inflammation and thrombosis.