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Matrix metalloproteinase expression in human retinal microvascular cells
M B Grant1, S Caballero, R W Tarnuzzer
1Department of Medicine, University of Florida, Gainesville 32610-0226, USA. grantmb@medicine.ufl.edu
Abstract:
The degree of hyperglycemia correlates with the development of diabetic 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 endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.
Insights
High glucose levels alter matrix metalloproteinase (MMP) activity in retinal cells, potentially contributing to diabetic retinopathy. This study observed changes in MMP-2 activity, not its mRNA, in response to glucose in HRECs.
Area of Science:
- Ophthalmology
- Endocrinology
- Molecular Biology
Background:
- Diabetic retinopathy (DR) is a microvascular complication of diabetes.
- Hyperglycemia is a key factor in DR development.
- Matrix metalloproteinases (MMPs) are implicated in extracellular matrix remodeling and disease pathogenesis.
Purpose of the Study:
- To investigate the effect of glucose on matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity in human retinal microvascular endothelial cells (HRECs).
- To explore the role of glucose in the context of diabetic retinopathy development.
Main Methods:
- Cultured HRECs from nondiabetic and diabetic donors were exposed to varying glucose concentrations (5 and 30 mmol/l).
- Gelatin zymography and Western analysis were used to assess MMP activity and identify specific proteins.
- Reverse transcription-polymerase chain reaction (RT-PCR) was employed to evaluate mRNA levels of MMPs, their inhibitors (TIMPs), and fibronectin.
Main Results:
- Glucose exposure did not alter the expression of MMP-2 or MMP-9 mRNA in HRECs from either diabetic or nondiabetic donors.
- An increase in proteolytic activity at 90 kDa, identified as MMP-2 associated with a fibronectin fragment, was observed in HRECs exposed to high glucose (30 mmol/l) and in diabetic HRECs at both glucose levels.
- Constitutive mRNA expression for MMP-2, MMP-9, TIMP-1, TIMP-2, and fibronectin was present in all HREC cultures, with no significant changes upon glucose exposure.
Conclusions:
- Glucose modulates MMP-2 activity, potentially through association with extracellular matrix components like fibronectin, rather than altering its mRNA levels.
- These glucose-induced changes in MMP activity may play a role in the pathogenesis of diabetic retinopathy.
- Further research is needed to elucidate the precise mechanisms of MMP modulation in diabetic microvascular complications.