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

点击翻译按钮获取中文摘要

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.