Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm2 shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.

Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLAB's efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.

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People with diabetes suffer from accelerated, diffuse atherosclerosis. Endothelial cells are dysfunctional in high glucose and on glycated collagen, but their response to mechanical stimuli in a high glucose environment has not been examined. The aim of this study was to determine the effect of glycated collagen on aortic endothelial cell response to strain. Porcine aortic endothelial cells seeded on either native or glycated collagen coated elastic substrates were exposed to 10% cyclic strain. While cells on native collagen aligned and formed actin stress fibers perpendicular to the stretch direction after 6 h, cells on glycated collagen did not align even after 12 h of cyclic strain. Impaired actin alignment could be related to diminished focal adhesion kinase phosphorylation in cells on glycated collagen. We further show that loss of mechanotransduction affects endothelial cell barrier function. Cells on glycated collagen substrates demonstrated a threefold increase in permeability with strain, whereas cell permeability on native collagen was unchanged. Increased permeability could be related to altered cell-cell junction morphology, as evidenced by b-catenin immunofluorescence. We hypothesize that impaired endothelial cell mechanotransduction on glycated collagen is due to altered integrin interactions. These data suggest that endothelial cells exposed to diabetic hyperglycemia are unable to adapt to the mechanical environment and thereby continue to express a pro-atherosclerotic phenotype.

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen-coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm(2) in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αv β3 integrins to bind to glycated collagen instead of the typical α2 β1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm(2) higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis.