Studying the effect of shear stress on human corneal epithelial cells

Transcription

1 Studying the effect of shear stress on human corneal epithelial cells Wulff, David J (University of Waterloo) Molladavoodi, Sara (University of Waterloo) Gorbet, Maud (University of Waterloo) Introduction The cornea is the most important refractive component of the ocular system (1). Corneal epithelial cells are exposed to shear stresses that are caused by the eyelids during blinking (2). These shear stresses can vary with specific ocular conditions, such as in keratoconus (KC), a progressive bilateral distension of the cornea accompanied by corneal thinning (3) and scarring (4). Excessive eye rubbing is also a common phenomenon in individuals suffering from keratoconus. While the effects of shear stress are well documented in blood cells and endothelial cells (5,6), limited knowledge currently exists on corneal epithelial cells. To develop better therapeutic strategies to preserve vision, there is a need to gain an understanding on how corneal epithelial cells respond to mechanical stimuli, such as exposure to shear. In this study, the effect of shear stress

2 (magnitude and exposure times) on human corneal epithelial cells (HCECs) was investigated in vitro. Materials and Methods HPV16 E6/E7 immortalized Human Corneal Epithelial Cells (HCECs) (5 105) were cultured in serum-free medium (KM, ScienCell) as a confluent monolayer on collagen coated coverslips (35mm; Glycotech, MA) for hours prior to the experiments. To expose cells to shear stress, cell-seeded coverslips were placed in a parallel plate flow chamber (Glycotech, MA) (Figure 1). Bi-directional flow (using KM) was applied to the cells using a syringe pump (Cole-Parmer, Montreal). Cells were exposed to shear of 4 dyne/cm2 (low shear stress) and 8 dyne/cm2 (high shear stress) for 6, 14, and 24 hours. Cells cultured on a collagen-coated coverslip, not exposed to shear stress, were used as controls. All experiments were performed at 37 C (5% CO2). Following shear stress exposure, cells were detached from the coverslips, washed and incubated with fluorescently labeled antibodies against integrin-β1 (CD29), integrin-α3 (CD49c) and ICAM-1 (CD54) (BD Biosciences, CA) for 30 minutes

3 at room temperature in the dark. Control samples were treated the same way. Cells were fixed with paraformaldehyde and levels of expression were then measured using flow cytometry (BD Biosciences, CA) within 5 days. To determine whether exposure to shear stress induced apoptosis or necrosis in HCECs, the FITC-pan caspase kit (Immunochemistry Technologies, MN) was used: FITC-VAD-FMK binds to activated caspases labeling cells undergoing apoptosis while propidium iodine is used to assess necrosis. Results To assess the effect of shear stress on cellsubstrate adhesion, the level of expression of integrin-α3 and integrin-β1 were determined. Integrins are heterodimer molecules that connect cell cytoplasm to the extracellular matrix. In corneal epithelial cells, integrin-α3β1 is highly involved in spreading and adhesion of these cells (7). As shown in Figure 2, our results suggest that integrin-β1 is upregulated by exposure to shear stress and that exposure time also has an effect on the level of expression. On the other hand, minimal changes

4 were observed with expression of integrin-α3 (Figure 2). Both 4 (low shear) and 8 (high shear) dynes/cm2 led to similar results. Intercellular adhesion molecule-1 (ICAM-1 or CD54) is a transmembrane molecule that is related to both cytoskeleton components (8) and the inflammatory response of corneal epithelial cells (9). While some fluctuations were observed, the levels of expression in the presence of shear were similar to the levels observed in control cells (cells not exposed to shear). Our preliminary results indicate that exposure to low shear stress can induce necrosis in HCECs. Further experiments are needed to confirm these results. Discussion and Conclusion Based on our knowledge, this is the first study that aimed to characterize how HCECs respond to various levels of shear stress. Our results indicate that corneal epithelial cells are sensitive to shear stress and that exposure time also plays a significant role in cell response. Further studies will aim at confirming how certain levels of shear stress may induce necrosis in cells but also how different

5 combination of shear stress levels and exposure times may affect corneal epithelial cells. Figure 1. Experimental setup Figure 2. Relative integrin expression of HCECs. Relative integrin expression is normalized to the control cells, results are representative of 3 experiments.

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