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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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MMP-9 siRNA regulates glucose-induced decrease in the mitochondrial Hsp70 and Hsp60, and binding of MMP-9 with these chaperone proteins. Expressions of (A) Hsp70 and (B) Hsp60 were quantified in mitochondria prepared from BRECs transfected with MMP-9 siRNA (MMP-si) or scramble RNA (SC), and incubated in 20 mmol/L glucose for 4 days. For binding, MMP-9 was immunoprecipitated from cell homogenate using anti–MMP-9, and the relative abundance of (C) Hsp70 and (D) Hsp60 was determined by Western blotting using IgG as the loading control. Results are expressed as mean ± SD of three or more experiments. The values obtained from the cells incubated in 5 mmol/L glucose are considered as 100%. *P < 0.05, compared with 5 mmol/L glucose; #P < 0.05, compared with 20 mmol/L glucose.
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Figure 8: MMP-9 siRNA regulates glucose-induced decrease in the mitochondrial Hsp70 and Hsp60, and binding of MMP-9 with these chaperone proteins. Expressions of (A) Hsp70 and (B) Hsp60 were quantified in mitochondria prepared from BRECs transfected with MMP-9 siRNA (MMP-si) or scramble RNA (SC), and incubated in 20 mmol/L glucose for 4 days. For binding, MMP-9 was immunoprecipitated from cell homogenate using anti–MMP-9, and the relative abundance of (C) Hsp70 and (D) Hsp60 was determined by Western blotting using IgG as the loading control. Results are expressed as mean ± SD of three or more experiments. The values obtained from the cells incubated in 5 mmol/L glucose are considered as 100%. *P < 0.05, compared with 5 mmol/L glucose; #P < 0.05, compared with 20 mmol/L glucose.

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)

MMP-9 siRNA regulates glucose-induced decrease in the mitochondrial Hsp70 and Hsp60, and binding of MMP-9 with these chaperone proteins. Expressions of (A) Hsp70 and (B) Hsp60 were quantified in mitochondria prepared from BRECs transfected with MMP-9 siRNA (MMP-si) or scramble RNA (SC), and incubated in 20 mmol/L glucose for 4 days. For binding, MMP-9 was immunoprecipitated from cell homogenate using anti–MMP-9, and the relative abundance of (C) Hsp70 and (D) Hsp60 was determined by Western blotting using IgG as the loading control. Results are expressed as mean ± SD of three or more experiments. The values obtained from the cells incubated in 5 mmol/L glucose are considered as 100%. *P < 0.05, compared with 5 mmol/L glucose; #P < 0.05, compared with 20 mmol/L glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: MMP-9 siRNA regulates glucose-induced decrease in the mitochondrial Hsp70 and Hsp60, and binding of MMP-9 with these chaperone proteins. Expressions of (A) Hsp70 and (B) Hsp60 were quantified in mitochondria prepared from BRECs transfected with MMP-9 siRNA (MMP-si) or scramble RNA (SC), and incubated in 20 mmol/L glucose for 4 days. For binding, MMP-9 was immunoprecipitated from cell homogenate using anti–MMP-9, and the relative abundance of (C) Hsp70 and (D) Hsp60 was determined by Western blotting using IgG as the loading control. Results are expressed as mean ± SD of three or more experiments. The values obtained from the cells incubated in 5 mmol/L glucose are considered as 100%. *P < 0.05, compared with 5 mmol/L glucose; #P < 0.05, compared with 20 mmol/L glucose.
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