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

HEG1-KRIT1 蛋白复合物的遗传和药理学操作调节内皮血管保护功能

• 批准号: 10229372
• 负责人: Miguel Alejandro Lopez-Ramirez
• 金额: $ 37.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-05 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10229372
• 关键词: Actomyosin; Adhesions; Adult; Affect; Animal Model; Anti-Inflammatory Agents; Anticoagulants; Aorta; Atherosclerosis; Binding; Blood Vessels; Blood flow; CCM1 gene; Cardiovascular Diseases; Cell physiology; Cells; Collaborations; Complex; Cultured Cells; Data; Endothelial Cells; Endothelium; Exhibits; GKLF protein; Gene Expression; Generations; Genes; Genetic; Health; Heart; Hemostatic function; Homeostasis; Inflammation; Intercellular Junctions; Knock-out; Knowledge; Leukocyte Adhesion Molecules; Leukocytes; Mediating; Modeling; Mus; NOS3 gene; Outcome; Perinatal; Pharmacology; Physiology; Platelet Inhibitors; Property; Protein C; Proteins; Regulation; Structure; Study models; Testing; Therapeutic; Thrombomodulin; Thrombosis; Up-Regulation; Vascular Endothelial Cell; activated Protein C; activated protein C receptor; base; cerebral cavernous malformations; cofactor; cytokine; endothelial dysfunction; hemodynamics; in vivo; insight; novel; platelet function; prevent; protein complex; protein expression; response; small molecule; small molecule inhibitor; transcription factor; vascular bed; vascular inflammation

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.

在围产期小鼠中，Krit 1（Krev 1相互作用捕获基因1）的内皮特异性失活导致大脑 海绵状血管畸形（CCM），而Krit 1（Krit 1 ECKO）或其结合伴侣HEG 1在成人中失活 并没有。Krit 1 ECKO或Heg 1 ECKO导致转录因子KLF 2和KLF 4的表达增加（Krüppel- 如因子2和4）;这些转录因子在层流调节血流的能力中是重要的 血管活性、抗凝和抗炎因子的内皮表达导致血管保护 炎症和血栓形成在初步研究中，我们发现成人Krit 1 ECKO内皮细胞 (EC)增加由KLF 2和KLF 4调节的血管保护基因的表达，包括eNOS和蛋白质 C激活辅因子，血栓调节蛋白（TM）。成年Krit 1 ECKO小鼠显示KLF 4表达显著升高， 动脉粥样硬化提示可能的血管保护作用。此外，内皮TM和 KRIT 1缺失后的EPCR伴随着EC产生活化蛋白的能力增加 C（APC），一种天然的抗血栓和抗炎蛋白。这一提议的科学前提是， 通过上调KLF 2和KLF 4，Krit 1的基因失活可能模拟层流的血管保护作用。 在内皮上流动，从而减少血栓形成、动脉粥样硬化和血管炎症。而且我们 表明这种流动效应也可以通过破坏KRIT 1-HEG 1相互作用的小分子来模拟。 为了检验这些想法：具体目标1将测试Krit 1的遗传失活将 保护小鼠免于血栓形成、动脉粥样硬化和血管炎症。在核心A（Bergmeier）中，我们将 研究Krit 1 ECKO或Heg 1 ECKO在体内止血和血栓形成方面的作用。由于eNOS的上调 表达导致NO水平增加，NO是已知的血小板功能和白细胞粘附的抑制剂， 与项目2和核心A合作，我们将研究炎症诱导的血管激活模型， Krit 1 ECKO小鼠的动脉粥样硬化。我们将主要关注内皮Krit 1的基因失活，但也将 必要时使用内皮Heg 1基因失活。具体目标2将检验以下假设： EC中HEG 1-KRIT 1蛋白复合物的药理学破坏可促进血管保护。我们 将检测小分子HKi 002对血管保护基因eNOS和TM表达的影响， 在EC中。将使用来自不同血管内皮细胞的EC研究HKi 002对NO和APC产生的影响。 床在这种情况下，我们将确定HKi 002对马槟榔诱导的炎症和内皮细胞的影响。 通过评估粘附分子/白细胞粘附的表达和通过分析 细胞间连接完整性/肌动球蛋白收缩性。总的来说，通过结合生物体和细胞 方法，我们将提供一个分析的影响，破坏KRIT 1-HEG 1蛋白复合物在成人 血管和血管保护，并为测试药理学破坏血管的想法奠定了基础。 复合物可抑制炎症和血栓形成。