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

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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).