Abstract 451: Oxygen Regulation of Human Coronary Artery Smooth Muscle Cell Proliferation and Migration

Abstract

Introduction: Intimal thickening of blood vessels, a hallmark of several vascular diseases including atherosclerosis and a potential point of therapeutic intervention, is caused by vascular smooth muscle cell proliferation and migration. It has been suggested that oxygen availability in vessels not only regulates behavior of smooth muscle cells but also serves as a trigger that may lead to pathological responses. In this study we determined whether hypoxia elicits proliferative and migratory responses in Human Coronary Artery SMCs (HCASMCs). Methods: Proliferation of HCASMCs was assessed using a 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. SMCs were plated in 96-well plates (n=5), serum starved, and then placed under hypoxic or normoxic conditions for 2, 4 and 6 days (2D/4D/6D) before MTT was added to each well. Absorbance at the wavelength 570 nm was read on an ELISA plate reader, and percent change in cell viability was determined and normalized to control (cell viability under normoxia). Cell migration was characterized by scratch-wound assay. SMCs were seeded in 6 well plates overnight (n=3), then a ‘scratch’ on the cell monolayer was created for each well before putting into different oxygen levels for 4 hours, 12 hours and 24 hours. Images were captured at the beginning and at intervals during cell migration to close the scratch, and the degree of migration was determined by comparing the images. Results: Compared to normoxic condition, cell number changed to 118.1%±1.3% in 5% O 2 (p<0.05) and 98.2%%±1.9% in 1% O 2 after 2D; to 151.9% ±8.5% in 5% O 2 (p<0.001) and 119.4%±5.0% in 1% O 2 (p<0.05) after 4D; and to 163.0%±4.3% in 5% O 2 (p<0.001) and 120.3%±2.2% in 1% O 2 (p<0.05) after 6D. In the cell migration assay, the difference in migration rate between different groups after 4 hours was not obvious, but there was a significant difference after 12 hours (29.3%±1.3% closure in normoxia vs 39.8%±1.9% in 5% O 2 vs 40.9%±3.5% in 1% O 2 , p<0.05) and 24 hours (71.5%±4.4% in normoxia vs 87.2%±2.2% in 5% O 2 vs 87.5%±3.1% in 1% O 2 , p<0.05). Conclusion: Our studies reveal that hypoxia induces both proliferation and migration of HCASMCs.