Role of matrix metalloproteinase-9 in the development of diabetic retinopathy and its regulation by H-Ras

Abstract

Purpose: Diabetes activates a small molecular weight G-protein, H-Ras, in the retina and its capillary cells, and H-Ras activation is implicated in the apoptosis of retinal capillary cells. Matrix metalloproteinase (MMP)-9 is regulated by H-Ras, and in diabetes its activation is associated with increased vascular permeability. The goal of this study was to investigate the role of sustained activation of MMP-9 in the pathogenesis of diabetic retinopathy and to illustrate the mechanism through which it is upregulated in diabetes.

Methods: Retinal MMP-9 activation and its tissue inhibitor, TIMP-1, were quantified in streptozotocin-induced diabetic rats. Inhibition of H-Ras by simvastatin on diabetes-induced activation of H-Ras was evaluated. The mechanism by which diabetes regulates retinal MMP-9 was confirmed by determining the effect of genetic or pharmacologic regulation of H-Ras on its activation in retinal endothelial cells.

Results: In rats, MMP-9 was activated and expression of TIMP-1 was decreased in the retina and its microvasculature at both 2 months and 12 months of diabetes. In retinal endothelial cells, high glucose activated MMP-9, and inhibition of its activation (by pharmacologic inhibitor or siRNA) ameliorated accelerated apoptosis. Inhibition of H-Ras, both in diabetic rats (simvastatin) and in isolated endothelial cells (H-Ras siRNA), abrogated the activation of MMP-9 and prevented the reduction of TIMP-1.

Conclusions: Hyperglycemia-induced activation of MMP-9 accelerates apoptosis of retinal capillary cells, a phenomenon that predicts the development of diabetic retinopathy, and the activation of MMP-9 is downstream of H-Ras. Characterizing the role of MMP-9 in the development of diabetic retinopathy will help explore novel molecular targets for future pharmacological interventions.

Figure 1.

MMP-9 is upregulated in the retina in diabetes. (a) MMP-9 gelatinase activity and gene expression (by real-time PCR) were measured in retinas obtained from rats that had diabetes for 2 months (Diab-2) or 12 months (Diab-12). Histogram represents gene expression of MMP-9 adjusted to that of 18sRNA. (b) TIMP-1 gene expression was measured in the same retina samples by real-time PCR using assays for the bovine species. (c) Gelatinase activity and gene expression of MMP-9 were measured in the retinal microvessels prepared from rats that had been diabetic for 2 months. Values obtained from normal rat retina/microvessels are considered as 100%. Each measurement was made in duplicate, and the values are represented as mean ± SD obtained from six or more rats in each group. *P < 0.05 compared with normal.

Figure 2.

Simvastatin inhibits diabetes-induced activation of retinal H-Ras and MMP-9. Retinas harvested from diabetic rats receiving diet supplemented with or without simvastatin were used to measure the activation of H-Ras by Raf-1 binding assay and that of MMP-9 by measuring its gelatinase activity. Each sample was measured in duplicate using retinas from five to seven rats in each group, and the graph represents mean ± SD. *P < 0.05 compared with normal. #P < 0.05 compared with diabetes.

Figure 3.

MMP-9 is activated in retinal endothelial cells in high-glucose conditions. Bovine retina endothelial cells from the third to the sixth passages were incubated in 5 mM glucose or 20 mM glucose medium for 4 days in the presence or absence of 4 nM MMP-I. For siRNA experiments, the cells were transfected with MMP-9-siRNA (MMP-siR) or scrambled siRNA (Scramb), followed by incubation in 5 mM or 20 mM glucose for 4 days. Cells treated with the transfection reagents alone are identified as Mock. At the end of the incubation, the medium was collected to quantify the gelatinase activity of MMP-9. Each measurement was made in duplicate in at least three different cell preparations. Values obtained from the untransfected cells incubated in 5 mM glucose are considered as 100% (control). 5G, 5 mM glucose; 20 G, 20 mM glucose; 20 mannitol, 20 mM mannitol. *P < 0.05 compared with the values obtained from the cells incubated in 5 mM glucose.

Figure 4.

High glucose attenuates TIMP-1 in retinal endothelial cells. Gene expression of TIMP-1 was quantified in the retinal endothelial cells incubated in 5 mM or 20 mM glucose for 4 days in the presence or absence of 25 μM FTI-277 or 10 μM manumycin (by real-time PCR using assays for bovine species). The level of TIMP-1 gene expression was adjusted to that of 18sRNA in each sample. Each measurement was performed in three to four different cell preparations. Values obtained from the cells incubated in 5 mM glucose are considered as 100% (control). *P < 0.05 compared with the values obtained from the cells incubated in 5 mM glucose.

Figure 5.

Inhibition of glucose-induced activation of MMP-9 prevents apoptosis of retinal endothelial cells. (a) Apoptosis was measured with cell death ELISA by measuring cytoplasmic histone-associated DNA fragments using a commercial assay kit. (b) Caspase-3 activity was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different cell preparations and measurements performed in duplicate. Values obtained from the cells incubated in 5 mM glucose are considered as 100% (control). *P < 0.05 and #P < 0.05 compared with the values obtained from the cells incubated in 5 mM glucose (5 G) and 20 mM glucose (20 G), respectively.

Figure 6.

Regulation of H-Ras activation prevents MMP-9 activation. Retinal endothelial cells from the third to the sixth passages were incubated in 5 mM glucose or 20 mM glucose medium for 4 days in the presence or absence of 25 μM FTI-277 or 10 μM manumycin. For siRNA experiments, the cells were transfected with H-Ras-siRNA (Ras-siR) or scrambled siRNA (Scramb) or H-Ras mutant that is dominant negative (N17) or wild-type followed by incubation in 5 mM or 20 mM glucose for 4 days. Controls were also run using the cells incubated with the transfection reagents alone before exposure to 5 mM or 20 mM glucose for 4 days (Mock). At the end of the incubation, the gelatinase activity of MMP-9 was quantified in the medium. Each measurement was made in duplicate in at least three different experiments. Values obtained from the untransfected cells incubated in 5 mM glucose are considered as 100% (control). *P < 0.05 compared with the values obtained from the cells incubated in 5 mM glucose.