Intrinsic Stiffness of Aortic Vascular Smooth Muscle Cell in the Development of Hypertension

补编项目摘要 母项目旨在研究SRF激活在主动脉硬化中的作用， 然而，高血压VSMCs中SRF增加的潜在机制尚不清楚。工作 在父母资助下进行的研究直接导致了白细胞介素36受体（IL-36 R）的鉴定， 炎症调节因子，在高血压主动脉组织和VSMC中显著增加， 建立了IL-36 R与高血压VSMCs中SRF激活的潜在联系。这些新发现建立了 本补充申请的基础。这一补充的目标之一是扩大 父母资助的研究，以探索IL-36 R在主动脉硬化发展中的潜在作用， 高血压及其与SRF介导的信号传导的关联。该补充的另一个目标是支持 候选人在这个新的研究领域的调查从父母的资助延伸，并帮助她成为 R 01资助的首席研究员在1-2年。根据我们的初步数据，我们假设 IL-36 R的上调通过以下途径增加VSMC的僵硬度，从而导致高血压患者的主动脉僵硬度 SRF介导的信号传导。我们计划通过追求以下具体目标来测试这一假设：目标1： 确定IL-36 R在VSMC僵硬中的作用及其对体外SRF介导的信号传导的调节。 我们将使用功能获得和功能丧失策略来确定细胞水平上的IL-36 R依赖性效应。我们将 测试WKY VSMC中过表达IL-36 R是否增加VSMC的刚度，以及敲- 下调IL-36 R可逆转SHR VSMCs的刚度增强。此外，我们将使用IL-36 R 激动剂和拮抗剂，以确定IL-36 R的作用是否需要IL-36细胞因子的激活。 将通过AFM和3D构建的组织模型测量VSMCs的刚度，如我们在母体中提出的 格兰特.我们还将确定VSMCs中IL-36 R的过表达或敲低是否诱导了平行的 通过测试亲本中证明的关键基因和蛋白质，改变SRF介导的信号传导 格兰特.具体目标2：确定IL-36 R在主动脉硬化发展中的作用， 体内高血压。为了实现这一点，我们将确定IL-36 R是否是调节 通过使用VSMC特异性IL-36 R KO小鼠模型测量主动脉僵硬度。我们将测试IL-36 R的缺失是否 影响这些小鼠中血管紧张素II诱导的主动脉硬化和血压。主动脉僵硬 如我们在母提案中所述，可以在体内和离体测量。VSMC刚度和 如Aim 1中所提出的，将在来自这些小鼠的分离的TA VSMC中确定所涉及的信号传导。我们 我希望这种补充剂能发现新的机制，将IL-36 R与SRF介导的 细胞内信号传导，这将导致更好地了解主动脉僵硬的发病机制， 高血压和帮助候选人的职业发展。

PROJECT SUMMARY of the Supplement The parent project was proposed to investigate the role of the activation of SRF in aortic stiffening, however, the mechanisms underlying the increase of SRF in hypertensive VSMCs remains unknown. The work performed on the parent grant directly led to the identification of interleukin 36 receptor (IL-36R), an inflammatory regulator, which was dramatically increased in the hypertensive aortic tissue and VSMCs, and established a potential link of the IL-36R with SRF activation in hypertensive VSMCs. These new findings built the foundation of the present supplemental application. One of the goals of this supplement is to expand the research of the parent grant to explore the potential role of IL-36R in the development of aortic stiffening in hypertension and its association with SRF-mediated signaling. Another goal of this supplement is to support the candidate’s investigation in this new research area extended from the parent grant, and help her to become an R01-funded principal investigator in 1-2 years. Based on our preliminary data, we hypothesize that the upregulation of IL-36R contributes to aortic stiffness in hypertension by increasing VSMC stiffness through SRF-mediated signaling. We plan to test this hypothesis by pursuing the following specific aims: Aim 1: Determine the role of IL-36R in VSMC stiffness and its regulation on SRF–mediated signaling in vitro. We will use a gain- and loss-of-function strategy to identify IL-36R-dependent effects at the cell level. We will test whether overexpressing IL-36R in WKY VSMCs increases the VSMC stiffness, and whether knocking- down IL-36R results in a reversal of the enhanced stiffness of SHR VSMCs. In addition, we will use IL-36R agonists and antagonists to determine whether the effect of IL-36R requires activation by an IL-36 cytokine. The VSMCs stiffness will be measured by AFM and 3D constituted tissue models as we proposed in the parent grant. We will also determine whether overexpressing or knocking down IL-36R in VSMCs induces a parallel alteration on the SRF-mediated signaling by testing the key genes and proteins as demonstrated in the parent grant. Specific Aim 2: Determine the role of IL-36R in the development of aortic stiffening and hypertension in vivo. To accomplish this, we will determine whether IL-36R is necessary for the regulation of aortic stiffness by using a VSMC-specific IL-36R KO mouse model. We will test whether the deletion of IL-36R affects the aortic stiffening and blood pressure induced by Angiotensin II in these mice. The aortic stiffness will be measured both in vivo and ex vivo as we described in the parent proposal. The VSMC stiffness and involved signaling will be determined in the isolated TA VSMCs from these mice as proposed in the Aim1. We expect that this supplement will discover novel mechanisms that may link the IL-36R with SRF-mediated intracellular signaling which will lead to a better understanding in the pathogenesis of aortic stiffness in hypertension and to help the candidate ‘s career development.