Vascular cell sexual dimorphism in complex mechanical microenvironments

心血管疾病（CVD），包括外周动脉疾病的发病率和进展的性别差异 疾病（PAD）是一个令人不安的临床观察。众所周知，CVD在男性中表现不同， 妇女，患这种疾病的妇女多于男子。此外，在血管网络内， 有利于CVD发展的机械微环境，例如，动脉粥样硬化斑块优先 在扰动流和低流体剪切应力的区域形成，例如脉管系统分叉。然而，没有报告 在细胞水平上将这两个独立的观察结果联系起来，具体来说， CVD和局部血管力学。本研究旨在：1）评估男性和女性的功能变化 人脐静脉内皮细胞对组合的生理流体剪切应力的响应， 通过形态测量、分泌和遗传分析评估基质硬度，2）评估 男性和女性人主动脉平滑肌细胞对组合生理周期性拉伸的反应 和基板刚度的形态，机械和遗传分析，和3）相关的不同 使用统计学方法对雄性和雌性血管细胞的功能反应进行分析。在完成这些 目的是对体外血管细胞功能的基本理解，因为它涉及到性二型性， 将获得复杂的力学微环境。将采用析因实验设计， 系统地评估细胞性别、层流剪切应力或循环拉伸的影响，以及潜在的 衬底刚度机械微环境的影响将使用定义明确的流量和 用RGD-缀合聚丙烯酰胺凝胶底物拉伸生物反应器。细胞形态将通过 免疫荧光显微镜方法。糖萼，血管调节，和雌二醇分泌产物将是 通过商业测定进行定量。细胞的机械性能将通过原子力显微镜测量。 包括CVD相关亚组在内的大量血管细胞基因的调节将通过以下方法测量： 定量逆转录聚合酶链反应。将进行统计分析，以确定相关基因 表达结果的形态，分泌和机械反应，然后将它们与给定的 性和机械力的结合。这项工作遵循了2015年NIH的任务，将性视为生物学上的一种 通过研究在细胞水平上心血管疾病性别差异的临床观察， 水平这项工作的意义是，未来的成像方式，量化血管的力量，如与 4D MRI将能够指示血管内容易发生局部细胞功能障碍的区域 使用相关的统计模型，并最终预测患者对疾病的特定位置的脆弱性。 这个提议是佛罗里达大学博士前培训奖学金的一部分。学员将学习 进行建议的实验，并从全面的指导专业成长 指导团队由临床医生、细胞和分子生物学家、计算理论家和材料科学家组成。

The sex disparity in the incidence and progression of cardiovascular disease (CVD), including peripheral arterial disease (PAD), is a troubling clinical observation. It is well known that CVD manifests differently in men and women, with more women than men suffering from the disease. Further, within the vascular network, there are mechanical microenvironments that favor CVD development, for example, atherosclerotic plaques preferentially form at regions of disturbed flow and low fluid shear stresses such as, vasculature bifurcations. Yet, no reports have correlated these two separate observations at the cellular level, specifically, the sex differences in CVD and local vascular mechanics. This project aims to 1) assess the functional changes in male and female human umbilical vein endothelial cells in response to combined physiological fluid shear stresses and substrate stiffness by morphometric, secretory, and genetic analyses, 2) assess the functional changes in male and female human aortic smooth muscle cells in response to combined physiological cyclic stretching and substrate stiffness by morphometric, mechanical, and genetic analyses, and 3) correlate the disparate functional response of male and female vascular cells using statistical methods. Upon completion of these aims, a foundational understanding of vascular cell functionality in vitro as it pertains to sexual dimorphism in complex mechanical microenvironments will be gained. A factorial design of experiments will be used to systematically assess the influence of cell sex, laminar fluid shear stress or cyclic stretch, and the underlying substrate stiffness. The effects of the mechanical microenvironment will be assessed using well-defined flow and stretch bioreactors with RGD-conjugated polyacrylamide gel substrates. Cell morphology will be quantified by immunofluorescence microscopy methods. Glycocalyx, vasoregulatory, and estradiol secretory products will be quantified by commercial assays. Cellular mechanical properties will be measured by atomic force microscopy. The regulation of a large set of vascular cell genes including a subset related to CVD will be measured by quantitative reverse transcription polymerase chain reaction. Statistical analyses will be performed to relate gene expression results to morphological, secretory, and mechanical responses and then to correlate them to a given sex and mechanical force combination. This work follows the 2015 NIH mandate to consider sex as a biological variable by investigating the clinical observations of the sex disparity in cardiovascular disease at the cellular level. The significance for this work is that future imaging modalities that quantify vascular forces such as with 4D MRI, will be able to indicate areas within the vasculature that are susceptible to localized cellular dysfunction using correlative statistical models and ultimately predict a patient's location-specific vulnerability for disease. This proposal is part of a predoctoral training fellowship at the University of Florida. The trainee will learn to conduct the experiments proposed and to grow professionally from the guidance of a comprehensive mentoring team of clinicians, cell and molecular biologists, computational theorists, and material scientists.