MATRIX METALLOPROTEINASE EXPRESSION IN HUMAN RETINAL MICROVASCULAR CELLS

The degree of hyperglycemia correlates with the development of diabeti c retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothe lial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donor s were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, co nditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the preform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose ex posure or the disease state of the donors. CM from nondiabetic HREC cu ltures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they w ere exposed to 5 mmol/l glucose. This same activity was seen in CM fro m HREC cultures of diabetic origin in the presence of both 5 and 30 mm ol/l glucose. Western analysis confirmed the identity of the 65-kDa ba nd as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-b ased reverse transcription-polymerase chain reaction revealed that non diabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, M MP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibr onectin. After exposure to 5 or 30 mmol/l glucose, no changes were det ected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TI MP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by ex tracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.