Date of Award

5-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Bioengineering

Committee Member

Dr. Agneta Simionescu, Committee Chair

Committee Member

Dr. Jeremy Mercuri

Committee Member

Dr. Jiro Nagatomi

Abstract

Diabetes mellitus is a disease that affects about 387 million people throughout the world. It is characterized by the body's inability to secrete or respond to insulin properly. As a result, diabetes mellitus is associated with hyperglycemia, the cause of many severe complications. Calcification in blood vessel walls, one of the most adverse complications of diabetes, may trigger heart failure, hypertension, plaque rupture, stroke, and coronary ischemia. In vitro and in vivo studies suggest that vascular calcification is a regulated process involving inhibitors and activators and resembling osteogenesis in bone. High glucose levels have been shown to induce endothelial cell dysfunction, expression of calcification biomarkers in vascular smooth muscle cells, and activation of fibroblasts. The purpose of this study is to investigate the interactions between co-cultured vascular endothelial cells, smooth muscle cells, and fibroblasts under diabetic/hyperglycemic and normal conditions. Human coronary artery smooth muscle cells, human aortic adventitial fibroblasts, and human aortic endothelial cells were co-cultured in pairs with alternation of one cell type on the membrane of the Transwell insert and the other on the bottom of the well. The cells were cultured under high glucose levels or normal conditions for 2 weeks. We performed immunofluorescence staining to examine the production of caspase-3 for apoptosis as well as osteocalcin and alkaline phosphatase as markers of calcification. Each surface was also stained with Alizarin Red Solution to observe the accumulation of calcium. The immunofluorescent alkaline phosphatase and Alizarin Red S stains indicated that all of the co-cultures, regardless of diabetic or nondiabetic conditions, induced expression of alkaline phosphatase and calcium. Osteocalcin was only seen on the cell surface of smooth muscle cells cultured on the membrane with fibroblasts in diabetic conditions and fibroblasts cultured on the membrane with smooth muscle cells in diabetic conditions. This study shows that intercellular communications between smooth muscle cells and fibroblasts could contribute to calcification of the vascular wall under diabetic conditions.

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