Biomechanical Signaling in Vascular Smooth Muscle Cell Proliferative Disease Following Functional Loss of Elastin

弹性蛋白功能丧失后血管平滑肌细胞增殖性疾病的生物力学信号传导

• 批准号: 10058762
• 负责人: Matthew W Ellis
• 金额: $ 4.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058762
• 关键词: 3-Dimensional; ADAMTS1 gene; Address; Affect; Aging; Arterial Medias; Biomechanics; Blood Vessels; CRISPR/Cas technology; Cardiovascular Diseases; Cause of Death; Cell Culture Techniques; Cell model; Cells; Chondroitin Sulfates; Collaborations; Complement; Complementary DNA; Data; Defect; Deposition; Development; Disease; Disease Progression; Dissection; Down-Regulation; Doxycycline; Elastin; Elastin Fiber; Environment; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Focal Adhesion Kinase 1; Focal Adhesions; Foundations; Genes; Genetic; Glucose; Glycosaminoglycans; Heart failure; Human; ITGB3 gene; Immunofluorescence Immunologic; Impairment; In Vitro; Investigation; Letters; Link; Mechanics; Messenger RNA; Metalloproteases; Methods; Modeling; Mus; Mutation; Neural Crest; Pathogenesis; Patients; Phenocopy; Phenotype; Phosphorylation; Plasmids; Play; Population; Production; Proteins; Proteoglycan; Role; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Somatic Cell; Stem Cell Factor; Stretching; Supravalvular aortic stenosis; Talin; Testing; Tissues; United States; Up-Regulation; Vascular Proliferation; Vascular Smooth Muscle; Western Blotting; ascending aorta; chondroitin sulfate glycosaminoglycan; effective therapy; experience; experimental study; functional loss; growth factor receptor-bound protein 2; in utero; in vivo; induced pluripotent stem cell; inhibitor/antagonist; loss of function; mechanotransduction; member; mouse model; neonatal period; novel; overexpression; restenosis; small hairpin RNA; small molecule; stem cell model; therapeutic development; three-dimensional modeling; vascular smooth muscle cell proliferation; versican

PROJECT SUMMARY Loss of the extracellular matrix (ECM) protein elastin leads to the uncontrolled proliferation of vascular smooth muscle cells (VSMCs). This proliferation is accompanied by a loss in contractility of VSMCs and in humans manifests as the condition supravalvular aortic stenosis (SVAS). SVAS patients experience progressive occlusion of their arterial lumen, ultimately leading to vessel stiffening and heart failure, for which the mechanisms remain largely unknown. Preliminary data has shown an upregulation of both chondroitin sulfate and the associated proteoglycan versican in the ascending aortas of elastin haploinsufficient mouse models, and both have been linked to impaired elastogenesis and hyperproliferation. Further, as signaling by integrin β3 and FAK is upregulated in SVAS, while expression of smooth muscle contractile markers is reduced, I hypothesize that abnormal ECM composition under elastin haploinsufficiency is recognized and transduced by the internal VSMC environment, leading to a shift from a differentiated to a proliferative phenotype. I will assess this hypothesis using induced pluripotent stem cells (iPSCs) as a model platform, as iPSCs present a limitless supply of patient-specific cells unattainable through other conventional methods and complement my findings with an in vivo mouse model of SVAS. In Aim 1, I will phenotypically validate elastin haploinsufficient iPSC-derived VSMCs specific to the neural crest lineage to phenocopy SVAS and try to rescue the phenotype through interfering with versican assembly in vivo and in vitro. In Aim 2, I will study the biomechanical signal transduction involved in the phenotypic switching of elastin haploinsufficient VSMCs from a differentiated, contractile state to a dedifferentiated, proliferative state in both iPSC and mouse models of SVAS. These aims involve in vivo studies of murine models, immunofluorescence, western blotting, qPCR, 2D- and 3D-modeling using iPSCs. The objectives of these aims are to identify key factors in the uncontrolled proliferation observed by VSMCs under elastin haploinsufficiency, and methods of rescuing this defect. This proposal addresses important, understudied mechanisms of vascular proliferation, and has the potential to facilitate the development of therapeutics for these currently incurable diseases.

项目概要 细胞外基质（ECM）蛋白弹性蛋白的损失导致血管平滑肌不受控制的增殖 这种增殖伴随着 VSMC 和人类的收缩性丧失。 表现为瓣膜上主动脉瓣狭窄 (SVAS) 患者病情进展。 动脉腔闭塞，最终导致血管僵硬和心力衰竭，为此 初步数据显示硫酸软骨素的上调机制仍不清楚。 以及弹性蛋白单倍体不足小鼠模型升主动脉中相关的蛋白聚糖多功能蛋白聚糖，以及 此外，整合素 β3 和 β3 的信号传导均与弹性生成受损和过度增殖有关。 FAK 在 SVAS 中上调，而平滑肌收缩标志物的表达降低，I 弹性蛋白单倍体不足下的异常 ECM 成分被内部识别并转导 VSMC 环境导致从分化表型转变为增殖表型，我将对此进行评估。 使用诱导多能干细胞 (iPSC) 作为模型平台的假设，因为 iPSC 的供应量是无限的 通过其他传统方法无法获得的患者特异性细胞，并用 SVAS 的体内小鼠模型 在目标 1 中，我将通过表型验证 iPSC 衍生的弹性蛋白单倍体不足。 特定于神经嵴谱系的 VSMC 对 SVAS 进行表型复制，并尝试通过以下方式挽救表型 在目标 2 中，我将研究生物力学信号转导。 参与弹性蛋白单倍体不足的 VSMC 从分化的收缩状态到 iPSC 和 SVAS 小鼠模型中的去分化、增殖状态这些目标涉及体内研究。 小鼠模型、免疫荧光、蛋白质印迹、qPCR、使用 iPSC 进行 2D 和 3D 建模。 这些目的的目的是确定 VSMC 在条件下观察到的不受控制的增殖的关键因素。 弹性蛋白单倍体不足，以及挽救这种缺陷的方法该提案解决了重要的、尚未充分研究的问题。 血管增殖机制，并有可能促进这些治疗方法的开发 目前的疑难杂症。