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

补编的项目摘要 母项目是为了研究SRF的激活在主动脉硬化中的作用， 然而，高血压VSMC中SRF增加的机制仍不清楚。这项工作 在父母赠款上进行的操作直接导致了白细胞介素36受体(IL-36R)的鉴定，以及 炎症调节因子，在高血压的主动脉组织和VSMCs中显著增加，并且 建立了IL-36R与高血压VSMC中SRF激活之间的潜在联系。这些新的发现建立了 这是本补充申请的基础。本附录的目标之一是扩展 父母资助探讨IL-36R在高血压大鼠主动脉硬化发病中的作用 高血压及其与SRF介导的信号转导的关系。本附录的另一个目标是支持 候选人在这一新研究领域的调查是从父母的资助延伸出来的，并帮助她成为 在1-2年内成为R01资助的首席研究员。根据我们的初步数据，我们假设 IL-36R上调通过增加VSMC僵硬度参与高血压患者的主动脉僵硬 SRF介导的信号转导。我们计划通过追求以下具体目标来检验这一假设：目标1： 体外测定IL-36R在VSMC僵硬中的作用及其对SRF介导的信号转导的调节。 我们将使用功能增减策略在细胞水平识别IL-36R依赖的效应。我们会 检测WKY VSMC过度表达IL-36R是否增加VSMC的刚性，以及敲击是否增加VSMC的刚性。 下调IL-36R可逆转自发性高血压大鼠血管平滑肌细胞的刚性增强。此外，我们将使用IL-36R 激动剂和拮抗剂，以确定IL-36R的作用是否需要由IL-36细胞因子激活。 VSMCs的硬度将通过原子力显微镜和3D构建的组织模型来测量，这是我们在父代中提出的 格兰特。我们还将确定在VSMC中过度表达或敲除IL-36R是否会导致平行的 通过检测亲本中的关键基因和蛋白改变SRF介导的信号转导 格兰特。具体目标2：确定IL-36R在主动脉硬化和高血压发病中的作用 活体高血压。要做到这一点，我们将确定IL-36R是否需要调节 使用VSMC特异性IL-36R KO小鼠模型。我们将测试IL-36R的缺失是否 对血管紧张素II诱导的小鼠主动脉硬化和血压有影响。主动脉僵硬会 在体内和体外进行测量，正如我们在父提案中所描述的那样。VSMC的刚度和 根据Aim1中的建议，将在这些小鼠分离的TA VSMCs中确定涉及的信号转导。我们 期待这一补充将发现可能将IL-36R与SRF中介联系起来的新机制 细胞内信号转导将有助于更好地理解主动脉僵直的发病机制 并帮助应聘者S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.