| People with diabetes suffer from accelerated atherosclerosis, which is linked to endothelial cell dysfunction in high glucose. Glucose affects endothelial cells directly and glycates basement membrane collagen. Endothelial cells are exposed to cyclic stretch in vivo and align their actin cytoskeleton and remodel the basement membrane in response to this stimulus. However, the effect of collagen glycation on stretch response is not known. The central hypothesis of this thesis is that glycated collagen alters endothelial cell mechanotransduction by disrupting focal adhesions, which prevents both the cell and basement membrane response to cyclic stretch.;Cyclic stretch was applied to porcine aortic endothelial cells seeded on flexible membranes coated with native or glycated collagen. While cells on native collagen aligned and formed actin stress fibers perpendicular to the stretch direction, cells on glycated collagen did not align. FAK phosphorylation was diminished in cells on glycated collagen in response to cyclic stretch, which could contribute to loss of alignment. Inhibited mechanotransduction also affected endothelial cell barrier function. Cells on glycated collagen demonstrated threefold higher permeability with stretch compared to cells on native collagen. Increased permeability could relate to altered cell-cell junction morphology, as evidenced by invaginated cell-cell junctions and increased β-catenin translocation to the nucleus in cells on glycated collagen.;Similar to actin, cells on glycated collagen substrates did not align basement membrane fibers in response to stretch. While basement membrane protein content did not change in cells on native or glycated collagen in response to cyclic stretch, there was decreased degradation activity in cells on glycated collagen. MMP-2 and MT1-MMP protein levels decreased, and therefore MMP-2 activity also decreased. We have shown that these MMP changes could relate to the inhibition of Jnk phosphorylation with cyclic stretch, which has previously been linked to FAK.;These data suggest that collagen glycation elicits structural and biochemical changes that disrupt the endothelial cell-basement membrane co-regulatory unit. Endothelial cells that cannot adapt to the mechanical environment may express a pro-atherosclerotic phenotype, and thereby contribute to accelerated atherosclerosis in people with diabetes. Understanding the role of collagen glycation in endothelial dysfunction could lead to target-specific drug therapies and help to reduce the high associated risk of vascular morbidity and mortality seen in diabetic patients. |
