3-D PEG-BASED SYNTHETIC ECMS TO REGULATE SMOOTH MUSCLE CELL DIFFERENTIATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 心血管疾病目前是美国的头号杀手，仅今年一年就将导致超过150万人死亡。 在各种心血管病理学中，例如肥大、高血压和动脉粥样硬化，血管和小动脉的机械性质可能会显著改变。这些机械变化可能会引发这些病变组织中平滑肌细胞（SMC）基因表达模式的显着变化，但发生这种情况的机制仍然知之甚少。由于胆固醇沉积、流体剪切力变化或血压变化而导致的动脉壁硬化可以通过三种方式影响局部细胞行为：1.）通过诱导平滑肌细胞从动脉的中间层迁移到内膜层; 2.）通过触发内膜层中细胞的过度增殖;和3.）通过增加内膜层中细胞的细胞外基质（ECM）沉积。 拟议研究的总体目标是利用体外工程平滑肌组织，进一步研究通过ECM在3-D中传递到SMC的机械应力的影响。具体而言，本研究旨在研究ECM的静态，内在的机械性能如何影响SMC表型在3-D PEG-纤维蛋白原系统。 为此，我们将使用共聚焦显微镜检查SMC特异性分化标志物（例如α-肌动蛋白、钙调蛋白、钙调蛋白等）的表达。通过FITC和TRITC结合的抗体。 结合这些分化研究，我们还将研究这些PEG-纤维蛋白原构建体的刚度如何通过观察FITC结合的鬼笔状突起（f-肌动蛋白原纤维的标记物）和TRITC染色的黏着斑蛋白（粘着斑大小和成熟度的标记物）来调节SMC的细胞骨架组装。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cardiovascular disease is currently the number one killer in the United States and will account for over 1.5 million deaths in the U.S. this year alone. In a variety of cardiovascular pathologies, such as hypertrophy, hypertension, and atherosclerosis, the mechanical properties of blood vessels and small arteries may be dramatically altered. These mechanical changes may trigger significant changes in gene expression patterns in the smooth muscle cells (SMCs) residing in these diseased tissues, but the mechanisms by which this occurs remain poorly understood. The hardening of the arterial wall due to cholesterol deposition, changes in fluid shear, or changes in blood pressure can affect local cell behavior in three ways: 1.) by inducing the migration of smooth muscle cells from the medial to intimal layers of the artery; 2.) by triggering the hyperproliferation of cells in the intimal layer; and 3.) by increasing the deposition of extracellular matrix (ECM) by cells in the intimal layer. The overall goal of the proposed research is to utilize engineered smooth muscle tissues in vitro to further investigate the effects of mechanical stresses transmitted to SMCs via the ECM in 3-D. Specifically, this research intends to examine how the static, intrinsic mechanical properties of the ECM affect SMC phenotype in a 3-D PEG-Fibrinogen system. To do this, we will be using confocal microscopy to examine the expression of SMC specific differentiation markers (e.g. alpha-actin, calponin, caldesmon, etc.) by FITC and TRITC-conjugated antibodies. In conjunction with these differentiation studies, we will also be examining how the stiffness of these PEG-Fibrinogen constructs regulates the cytoskeletal assembly of SMCs by looking at FITC-conjugated phalloiden (a marker for f-actin fibrils) and TRITC-stained vinculin (a marker of focal adhesion size and maturity).