Endothelial cell structure changes on native compared to glycated collagen in response to substrates of different stiffness

Many diseases alter both the stiffness and composition of the extracellular matrix. Of particular interest to us is that diabetic hyperglycemia leads to collagen glycation, which both stiffens the extracellular matrix and alters cell-matrix interactions. Endothelial cells attach to matrix proteins via integrins clustered at focal adhesions. Cells transmit forces to integrins and extracellular matrix through the cytoskeleton, which deform substrates that are within a physiological stiffness range but not rigid substrates. As a consequence, cell morphology and function depend on substrate stiffness.We hypothesized that collagen glycation affects endothelial response to substrates of different stiffness by altering cell-matrix interactions. To investigate this hypothesis, we seeded porcine aortic endothelial cells for 24 hours on native and glycated collagencoated polyacrylamide gels with varying shear moduli (0.3 kPa – 30 kPa), as well as coated glass cover slips as a rigid control. Cells were labeled with rhodamine phalloidin and a vinculin antibody to visualize the actin cytoskeleton and focal adhesions, respectively. Cell morphology, including area, aspect ratio, and focal adhesion size and number were measured.Cell area and focal adhesion number and size increased with substrate stiffness on both native and glycated collagen. However, cell area on native collagen increased 625% as gel stiffness increased from 0.3 kPa to 30 kPa, while cell area on glycated collagen coated gels increased only 139%. In contrast, cells on gels coated with glycated collagen increased focal adhesion number by 32% and size by nearly 439%, whereas cells on native collagen increased focal adhesion number by over 100% and size by 1140% as gel stiffness increased from 5 kPa to 30 kPa. No focal adhesion sites were observed on cells seeded on 0.3 kPa gels coated with either native or glycated collagen. Cells on glass had the largest cell area, with no difference in area or focal adhesion size and number between native and glycated collagen coatings. Preliminary results from cell traction force studies revealed that on a 5 kPa gel, the average cell traction stress was 78% higher on native compared to glycated collagen and on the 10 kPa gel, the average cell traction stress was 33% higher on native compared to glycated collagen.These data suggest that endothelial cell response to substrate stiffness is altered in disease conditions as cells are unable to properly spread, form focal adhesions and exert traction forces on glycated compared to native collagen coated substrates. These findings shed new light on how endothelial cell mechanotransduction can be impaired by disease conditions.M.S., Mechanical Engineering -- Drexel University, 201