Abstract 46: The Effects of Membrane Cholesterol on Vascular Smooth Muscle Cell Stiffness and Adhesion to Fibronectin

Atherosclerosis remains a major cause of cardiovascular disease (CVD). Cholesterol has been identified as a major contributor to the cause of atherosclerosis. It is well known that the cholesterol accumulation in macrophage-derived foam cells is the major component of atherosclerotic plaque. However, growing evidences suggests that cholesterol loading into vascular smooth muscle cells (VSMC) in atherosclerosis is much larger than previously known, and about 40% of total foam cells in the atherosclerotic plaque are VSMC-derived. Cholesterol may not only contribute as the fatty deposition in the atherosclerotic lesion, but also play a critical role in the VSMC migration toward the intima of the blood vessel wall. In addition, the arterial wall becomes stiffer during atherosclerosis altering the micromechanical environment experienced by the VSMCs leading to changes in VSMC stiffens, adhesion, and phenotype. Migration of VSMCs is a complex process including proliferation and phenotypic switching of VSMCs, thus contributing too many changes in cell membrane adhesion molecules. We tested the hypothesis that membrane cholesterol in VSMCs may play an important role in α 5 β 1 -integrin mediated adhesion, and alter the sensory function of VSMCs to ECM mechanical properties. In this study cholesterol manipulation was achieved using methyl-β-cyclodextrin, and gel substrates with varying stiffness were used to mimic the changing environment in atherosclerosis. Atomic force microscopy (AFM) was used to determine integrin-fibronectin adhesion force and cell stiffness. A custom-written MATLAB program was used to interpret the elasticity of the VSMC cytoskeleton and adhesion force. Cellular adhesion was measured for 50%-70% confluent cells with a sample size of 50 cells on a fibronectin coated AFM stylus probe. Our results show that there is a significant decrease in α5β1-integrin adhesion of VSMCs on substrates above 9 kPa upon membrane cholesterol depletion. Additionally, mechanotransduction of VSMCs upon cholesterol depletion is less efficient. In conclusion, cell membrane cholesterol and extracellular mechanical signals may synergistically regulate cellular mechanical functions of VSMCs and their migration in the progression of atherosclerosis.