Vascular cell sexual dimorphism in complex mechanical microenvironments

包括外周动脉在内的心血管疾病(CVD)的发病和进展的性别差异 疾病(PAD)，是一个令人困扰的临床观察。众所周知，心血管疾病在男性和 女性，患有这种疾病的女性比男性多。此外，在血管网络中，有 有利于心血管疾病发展的机械微环境，例如动脉粥样硬化斑块 在流动扰动和流体剪切力较低的区域形成，如血管分叉。然而，目前还没有报道 在细胞水平上将这两个不同的观察结果联系在一起，特别是 心血管疾病和局部血管力学。该项目旨在1)评估男性和女性的功能变化 人脐静脉内皮细胞对生理流体切应力和血管内皮细胞的联合作用 通过形态测量、分泌和遗传分析来评估底物硬度，2)评估 男性和女性人主动脉平滑肌细胞对联合生理周期拉伸的反应 和衬底硬度，通过形态测量、力学和遗传分析，以及3)将不同的 用统计学方法研究雄性和雌性血管细胞的功能反应。在完成这些任务后 AIMS，这是对血管细胞功能的基础性理解，因为它与性别二型性有关 将获得复杂的力学微环境。将使用实验的析因设计来 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 衬底硬度。机械微环境的影响将使用明确定义的流动和 采用RGD-共轭聚丙烯酰胺凝胶基质的拉伸生物反应器。细胞形态将通过以下方式量化 免疫荧光显微镜方法。甘露醇、血管调节剂和雌二醇的分泌产品 通过商业化验进行量化。细胞的机械性能将通过原子力显微镜进行测量。 包括与心血管疾病相关的子集在内的一大组血管细胞基因的调节将通过以下方式进行测量 定量逆转录聚合酶链式反应。将对相关基因进行统计分析 表达结果导致形态、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年美国国立卫生研究院的命令，将性作为一种生物 变量通过调查临床观察心血管疾病的性别差异在细胞 水平。这项工作的意义在于，未来量化血管力量的成像模式，如 4D核磁共振，将能够指出血管系统内易受局部细胞功能障碍影响的区域 使用相关的统计模型，并最终预测患者特定位置的疾病易感性。 这项提议是佛罗里达大学博士前培训奖学金的一部分。实习生将学会 进行提出的实验，并在全面的指导下专业成长 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的指导团队。

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.