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Abrogation of MMP-9 gene protects against the development of retinopathy in diabetic mice by preventing mitochondrial damage.

Kowluru RA, Mohammad G, dos Santos JM, Zhong Q - Diabetes (2011)

Bottom Line: The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage.Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.

View Article: PubMed Central - PubMed

Affiliation: Kresge Eye Institute, Wayne State University, Detroit, Michigan, USA. rkowluru@med.wayne.edu

ABSTRACT

Objective: In the development of diabetic retinopathy, mitochondrial dysfunction is considered to play an important role in the apoptosis of retinal capillary cells. Diabetes activates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 becomes proapoptotic. The objective of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina.

Research design and methods: Using MMP-9 gene knockout (MMP-KO) mice, we investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis, development of retinopathy, mitochondrial dysfunction and ultrastructure, and mitochondrial DNA (mtDNA) damage. To understand how diabetes increases mitochondrial accumulation of MMP-9, interactions between MMP-9 and chaperone proteins (heat shock protein [Hsp] 70 and Hsp60) were evaluated. The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).

Results: Retinal microvasculature of MMP-KO mice, diabetic for ∼7 months, did not show increased apoptosis and pathology characteristic of retinopathy. In the same MMP-KO diabetic mice, activation of MMP-9 and dysfunction of the mitochondria were prevented, and electron microscopy of the retinal microvasculature region revealed normal mitochondrial matrix and packed lamellar cristae. Damage to mtDNA was protected, and the binding of MMP-9 with Hsp70 or Hsp60 was also normal. As in the retina from wild-type diabetic mice, activation of mitochondrial MMP-9 and alterations in the binding of MMP-9 with chaperone proteins were also observed in the retina from donors with diabetic retinopathy. In endothelial cells transfected with MMP-9 siRNA, high glucose-induced damage to the mitochondria and the chaperone machinery was ameliorated.

Conclusions: Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage. Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.

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Effect of high glucose on mitochondrial MMP-9 in retinal endothelial cells. A: Localization of MMP-9 in retinal endothelial cells was performed immunohistochemically. For mitochondria, MitoTracker (green) was used, and for MMP-9, Texas Red–conjugated antibody was used. The coverslips were mounted using Vecta Shield containing DAPI (blue, nucleus). The slides were examined under a Zeiss ApoTome using 40× magnification. The picture is representative of three to four different preparations. B: Gelatinase activity of MMP-9 was measured in the incubation medium by in situ zymography. The active form of MMP-9 (∼80 kD) was quantified and presented in the accompanying histogram. C: Active MMP-9 in the mitochondrial fraction was quantified by performing ELISA. D: Cell apoptosis was quantified by performing ELISA for cytoplasmic histone–associated DNA fragments using a commercially available kit from Roche Diagnostics. Each measurement was performed in three to four different cell preparations. The values obtained from the untransfected cells incubated in 5 mmol/L glucose are considered as 100% (control). 5 and 20, untransfected cells incubated in 5 or 20 mmol/L glucose for 4 days; MMP-si and SC, cells transfected with MMP-9 siRNA or with scramble RNA, respectively; Mann, cells incubated in 20 mmol/L mannitol for 4 days. *P < 0.05, compared with the values obtained from cells incubated in 5 mmol/L glucose; #P < 0.05, compared with values from cells in 20 mmol/L glucose for 4 days. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Effect of high glucose on mitochondrial MMP-9 in retinal endothelial cells. A: Localization of MMP-9 in retinal endothelial cells was performed immunohistochemically. For mitochondria, MitoTracker (green) was used, and for MMP-9, Texas Red–conjugated antibody was used. The coverslips were mounted using Vecta Shield containing DAPI (blue, nucleus). The slides were examined under a Zeiss ApoTome using 40× magnification. The picture is representative of three to four different preparations. B: Gelatinase activity of MMP-9 was measured in the incubation medium by in situ zymography. The active form of MMP-9 (∼80 kD) was quantified and presented in the accompanying histogram. C: Active MMP-9 in the mitochondrial fraction was quantified by performing ELISA. D: Cell apoptosis was quantified by performing ELISA for cytoplasmic histone–associated DNA fragments using a commercially available kit from Roche Diagnostics. Each measurement was performed in three to four different cell preparations. The values obtained from the untransfected cells incubated in 5 mmol/L glucose are considered as 100% (control). 5 and 20, untransfected cells incubated in 5 or 20 mmol/L glucose for 4 days; MMP-si and SC, cells transfected with MMP-9 siRNA or with scramble RNA, respectively; Mann, cells incubated in 20 mmol/L mannitol for 4 days. *P < 0.05, compared with the values obtained from cells incubated in 5 mmol/L glucose; #P < 0.05, compared with values from cells in 20 mmol/L glucose for 4 days. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Exposure of retinal endothelial cells, the site of histopathology characteristic of diabetic retinopathy, to high glucose increased the accumulation of MMP-9 in the mitochondria; cells incubated in high glucose had increased staining of MMP-9 (red) in the mitochondria compared with the cells exposed to normal glucose (Fig. 7A). In the same cell preparations, the active MMP-9 was ∼40% higher in the mitochondria from the cells exposed to high glucose as confirmed by both in situ zymography (Fig. 7B) and ELISA (Fig. 7C), and cell apoptosis was elevated by >75% (Fig. 7D). Consistent with the retina, high glucose decreased mitochondrial Hsp70 and Hsp60 expressions and increased the binding of MMP-9 with Hsp70 or Hsp60 as evidenced by their increased expression in the cells immunoprecipitated for MMP-9 (Fig. 8).


Abrogation of MMP-9 gene protects against the development of retinopathy in diabetic mice by preventing mitochondrial damage.

Kowluru RA, Mohammad G, dos Santos JM, Zhong Q - Diabetes (2011)

Effect of high glucose on mitochondrial MMP-9 in retinal endothelial cells. A: Localization of MMP-9 in retinal endothelial cells was performed immunohistochemically. For mitochondria, MitoTracker (green) was used, and for MMP-9, Texas Red–conjugated antibody was used. The coverslips were mounted using Vecta Shield containing DAPI (blue, nucleus). The slides were examined under a Zeiss ApoTome using 40× magnification. The picture is representative of three to four different preparations. B: Gelatinase activity of MMP-9 was measured in the incubation medium by in situ zymography. The active form of MMP-9 (∼80 kD) was quantified and presented in the accompanying histogram. C: Active MMP-9 in the mitochondrial fraction was quantified by performing ELISA. D: Cell apoptosis was quantified by performing ELISA for cytoplasmic histone–associated DNA fragments using a commercially available kit from Roche Diagnostics. Each measurement was performed in three to four different cell preparations. The values obtained from the untransfected cells incubated in 5 mmol/L glucose are considered as 100% (control). 5 and 20, untransfected cells incubated in 5 or 20 mmol/L glucose for 4 days; MMP-si and SC, cells transfected with MMP-9 siRNA or with scramble RNA, respectively; Mann, cells incubated in 20 mmol/L mannitol for 4 days. *P < 0.05, compared with the values obtained from cells incubated in 5 mmol/L glucose; #P < 0.05, compared with values from cells in 20 mmol/L glucose for 4 days. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Effect of high glucose on mitochondrial MMP-9 in retinal endothelial cells. A: Localization of MMP-9 in retinal endothelial cells was performed immunohistochemically. For mitochondria, MitoTracker (green) was used, and for MMP-9, Texas Red–conjugated antibody was used. The coverslips were mounted using Vecta Shield containing DAPI (blue, nucleus). The slides were examined under a Zeiss ApoTome using 40× magnification. The picture is representative of three to four different preparations. B: Gelatinase activity of MMP-9 was measured in the incubation medium by in situ zymography. The active form of MMP-9 (∼80 kD) was quantified and presented in the accompanying histogram. C: Active MMP-9 in the mitochondrial fraction was quantified by performing ELISA. D: Cell apoptosis was quantified by performing ELISA for cytoplasmic histone–associated DNA fragments using a commercially available kit from Roche Diagnostics. Each measurement was performed in three to four different cell preparations. The values obtained from the untransfected cells incubated in 5 mmol/L glucose are considered as 100% (control). 5 and 20, untransfected cells incubated in 5 or 20 mmol/L glucose for 4 days; MMP-si and SC, cells transfected with MMP-9 siRNA or with scramble RNA, respectively; Mann, cells incubated in 20 mmol/L mannitol for 4 days. *P < 0.05, compared with the values obtained from cells incubated in 5 mmol/L glucose; #P < 0.05, compared with values from cells in 20 mmol/L glucose for 4 days. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Exposure of retinal endothelial cells, the site of histopathology characteristic of diabetic retinopathy, to high glucose increased the accumulation of MMP-9 in the mitochondria; cells incubated in high glucose had increased staining of MMP-9 (red) in the mitochondria compared with the cells exposed to normal glucose (Fig. 7A). In the same cell preparations, the active MMP-9 was ∼40% higher in the mitochondria from the cells exposed to high glucose as confirmed by both in situ zymography (Fig. 7B) and ELISA (Fig. 7C), and cell apoptosis was elevated by >75% (Fig. 7D). Consistent with the retina, high glucose decreased mitochondrial Hsp70 and Hsp60 expressions and increased the binding of MMP-9 with Hsp70 or Hsp60 as evidenced by their increased expression in the cells immunoprecipitated for MMP-9 (Fig. 8).

Bottom Line: The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage.Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.

View Article: PubMed Central - PubMed

Affiliation: Kresge Eye Institute, Wayne State University, Detroit, Michigan, USA. rkowluru@med.wayne.edu

ABSTRACT

Objective: In the development of diabetic retinopathy, mitochondrial dysfunction is considered to play an important role in the apoptosis of retinal capillary cells. Diabetes activates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 becomes proapoptotic. The objective of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina.

Research design and methods: Using MMP-9 gene knockout (MMP-KO) mice, we investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis, development of retinopathy, mitochondrial dysfunction and ultrastructure, and mitochondrial DNA (mtDNA) damage. To understand how diabetes increases mitochondrial accumulation of MMP-9, interactions between MMP-9 and chaperone proteins (heat shock protein [Hsp] 70 and Hsp60) were evaluated. The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).

Results: Retinal microvasculature of MMP-KO mice, diabetic for ∼7 months, did not show increased apoptosis and pathology characteristic of retinopathy. In the same MMP-KO diabetic mice, activation of MMP-9 and dysfunction of the mitochondria were prevented, and electron microscopy of the retinal microvasculature region revealed normal mitochondrial matrix and packed lamellar cristae. Damage to mtDNA was protected, and the binding of MMP-9 with Hsp70 or Hsp60 was also normal. As in the retina from wild-type diabetic mice, activation of mitochondrial MMP-9 and alterations in the binding of MMP-9 with chaperone proteins were also observed in the retina from donors with diabetic retinopathy. In endothelial cells transfected with MMP-9 siRNA, high glucose-induced damage to the mitochondria and the chaperone machinery was ameliorated.

Conclusions: Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage. Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.

Show MeSH
Related in: MedlinePlus