CD45-mediated endothelial-to-mesenchymal transition in cardiovascular disease

项目摘要/摘要 动脉粥样硬化是导致冠心病、缺血性中风和外周动脉疾病的主要原因 疾病。这种进行性血管疾病仍然是美国死亡的主要原因，尽管 广泛使用有效的降脂疗法和预防方案。当内皮细胞激活时 而新生内膜增生是动脉粥样硬化发生和发展的标志，鉴定 内皮功能障碍的分子标志物既是一个生物学挑战，也是一个机会 开发新的治疗靶点。在这一应用中，我们假设探索内皮表达的功能 CD45在动脉粥样硬化中的作用CD45是最广为人知的造血细胞标志物。我们最近的数据显示， CD45在推动心肌梗死后内皮间充质转化(EndMT)中的作用 脑梗塞。EndMT被认为代表了一种新的内皮扰动和一种关键的调节因子 由于EndMT通过内皮细胞分层破坏内皮的正常功能，导致动脉粥样硬化的发生 并产生新的内膜间充质细胞，它分泌促炎分子和细胞外 促进动脉粥样硬化进展的基质蛋白。血管内皮细胞CD45是否参与血管病变的进展 促进EndMT导致的动脉粥样硬化是一个完全未知但意义重大的问题。在我们最新的 在研究中，我们给CD45缺失的小鼠注射了动脉粥样硬化加速器PCSK9 AAV8，并喂食了 西式饮食在动脉粥样硬化形成方面有明显的减少。相反，我们观察到CD45显著增加 动脉粥样硬化病变中经历EndMT的阳性内皮细胞(ECs)。我们还发现， CRISPR/Cas9介导的人内皮细胞CD45活化诱导EndMT。有趣的是，CD45的表达 在HDAC2基因敲除的血液内皮细胞中显著增加，提示HDAC2介导的缺失 EndMT过程中CD45的抑制。此外，在miR-155缺陷小鼠中，CD45的表达发生了变化，表明 在EndMT过程中，microRNAs可能调节CD45的表达和功能。我们的试点核糖核酸序列研究显示 上调内毒素和下调KLF对CD45过表达的影响。鉴于Espins促进和 KLFS抑制动脉粥样硬化，CD45是否诱导EndMT并通过调节促进动脉粥样硬化 Eepsin和KLF函数是一个全新的问题。为了测试这一点，我们将确定内皮细胞的作用 CD45在体内调节动脉粥样硬化中的作用及分子机制探讨1)血管内皮细胞 CD45通过控制Eepsin和KLF的表达来调节动脉粥样硬化中的EndMT和2)潜在的方式 内皮细胞在EndMT过程中CD45的表达受表观遗传调控以及是否靶向内皮CD45 促进动脉粥样硬化的分辨率。我们的初步数据提供了强有力的证据，这是一个强有力的科学证据 这是我们提出研究的前提。如果成功，我们的研究不仅将提供广泛的机械证据 CD45介导的信号在调节EndMT中的核心作用，但将提供一种创新的方法 用于治疗动脉粥样硬化，并启动研究范式的转变，以对抗心血管疾病。

PROJECT SUMMARY/ABSTRACT Atherosclerosis is the primary cause of coronary heart disease, ischemic stroke, and peripheral arterial disease. This progressive vascular disease remains a leading cause of mortality in the United States despite wide-spread use of effective lipid-lowering therapies and prevention programs. While endothelial cell activation and neointimal hyperplasia is a hallmark of the initiation and progression of atherosclerosis, identification of molecular markers of dysfunctional endothelium represents a biological challenge as well as an opportunity to develop new therapeutic targets. In this application, we posit to explore the function of endothelial expressed CD45 in atherosclerosis. CD45 is best known as marker of hematopoietic cells. Our recent data shows that CD45 is indispensable in driving the endothelial-to-mesenchymal transition (EndMT) following myocardial infarction. It is thought that EndMT represents a novel endothelial perturbation and a critical regulator of atherogenesis given that EndMT disrupts the normal function of endothelium by delaminating endothelial cells and giving rise to neointimal mesenchymal cells, which secrete proinflammatory molecules and extracellular matrix proteins that fuel atherosclerosis progression. Whether endothelial CD45 contributes to progression of atherosclerosis by facilitating the EndMT is a completely unknown but highly significant question. In our latest studies, we showed CD45 null mice injected with the atherosclerosis accelerator PCSK9 AAv8 and fed a western diet had a marked reduction in atherogenesis. Conversely, we observed a significant increase in CD45 positive endothelial cells (ECs) that undergo the EndMT in atherosclerotic lesions. We also found that CRISPR/Cas9-mediated activation of CD45 in human ECs induces the EndMT. Intriguingly, CD45 expression is dramatically increased in HDAC2 knockout hemogenic endothelium, suggesting loss of HDAC2-mediated repression of CD45 during EndMT. Further, CD45 expression is altered in miR-155 deficient mice, indicating microRNAs may regulate the expression and function of CD45 during EndMT. Our pilot RNA seq study shows upregulation of epsins and downregulation of KLFs upon CD45 overexpression. Given that epsins promote and KLFs impede atherosclerosis, whether CD45 induces EndMT and promotes atherosclerosis by modulating epsin and KLF function is an entirely novel question. To test this, we will determine the role of endothelial CD45 in regulating atherosclerosis in vivo and interrogate molecular mechanisms 1) by which endothelial CD45 regulates EndMT in atherosclerosis by controlling epsin and KLF expression and 2) underlying how CD45 expression is epigenetically regulated in ECs during EndMT and whether targeting endothelial CD45 promotes atheroma resolution. Our preliminary data presents robust evidence that serves as a strong scientific premise for our proposed study. If successful, our study will not only provide extensive mechanistic evidence for a central role of CD45-mediated signaling in regulating the EndMT, but will provide an innovative approach for the treatment of atherosclerosis and launch a paradigm shift in research to combat cardiovascular disease.