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J. Khuman, S. Roy; Downregulation of High Glucose-Induced Laminin Overexpression Reduces Integrin 3 Expression in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1355.
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To determine whether downregulation of high glucose-induced laminin (LM) overexpression with antisense LM-oligos modulates the expression of the corresponding integrin α3 (Intα3) subunit.
To determine the effect of AS-LM oligos on Intα3 expression, rat retinal endothelial (RRE) cells were grown in normal (5mM glucose) or high glucose (HG, 30mM) medium for 10 days. In parallel, cells grown in HG medium to subconfluency, were transfected with AS-LM oligos or Random (Ran) oligos. Three days after transfection, the cells were harvested and total protein isolated. Western Blot analysis was performed using total protein from the four groups of cells. The membrane was first probed with monoclonal anti-LM antibody and the signal determined using the appropriate secondary antibody. The membrane was then stripped and probed with anti-Intα3 antibody. To determine LM and Int α3 distribution and localization, parallel cell cultures grown on cover slips were subjected to immunostaining with LM and Intα3 antibodies. LM and Intα3 densitometric values from Western Blots, and LM and Intα3 immunofluorescent signals from images were analyzed using NIH image analysis software.
Western Blot analysis indicated that LM and Intα3 expression was significantly increased (220±54% of control, p=0.03, 181±54% of control p=0.03, respectively) in the RRE cells grown in HG, compared to those grown in normal medium. The RRE cells grown in HG and transfected with AS-LM oligos, showed a significant reduction in LM expression (144±38% of control, vs 220±54% of control; p=0.008). Interestingly, these cells also showed a decreased Intα3 expression (120±10% of control, vs 181±54% of control; p=0.009). Cells grown in high glucose showed intense immunostaining for LM and Intα3 compared to that of cells grown in normal medium; interestingly, the HG cells transfected with AS-LM oligos showed reduced immunostaining for both LM and Intα3.
Antisense LM oligos strategy is useful not only in reducing LM overexpression induced by high glucose but may also have an added benefit of reducing Intα3 overexpression associated with diabetic retinopathy. Thus, the antisense oligo approach may be useful in arresting vascular basement membrane (BM) thickening and inhibiting the disruption of endothelial cell attachment to the BM, thereby preventing both BM thickening and the development of acellular capillaries associated with diabetic retinopathy.
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