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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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Related in: MedlinePlus

MMP-9 is activated in retinal mitochondria from patients with diabetic retinopathy, and binding of MMP-9 with chaperones is increased. Mitochondria were prepared from the retina of donors with diabetic retinopathy and age-matched nondiabetic subjects by differential centrifugation. A: Active MMP-9 was quantified by ELISA technique. Expression of (B) Hsp70 and (C) Hsp60 was quantified in the mitochondria by Western blot technique, and binding of MMP-9 with (D) Hsp70 or (E) Hsp60 by immunoprecipitating MMP-9. Each measurement was performed in duplicate in the samples obtained from four nondiabetic donors and five donors with diabetic retinopathy. The values are represented as mean ± SD. *P < 0.05, compared with the values from nondiabetic donors.
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Figure 6: MMP-9 is activated in retinal mitochondria from patients with diabetic retinopathy, and binding of MMP-9 with chaperones is increased. Mitochondria were prepared from the retina of donors with diabetic retinopathy and age-matched nondiabetic subjects by differential centrifugation. A: Active MMP-9 was quantified by ELISA technique. Expression of (B) Hsp70 and (C) Hsp60 was quantified in the mitochondria by Western blot technique, and binding of MMP-9 with (D) Hsp70 or (E) Hsp60 by immunoprecipitating MMP-9. Each measurement was performed in duplicate in the samples obtained from four nondiabetic donors and five donors with diabetic retinopathy. The values are represented as mean ± SD. *P < 0.05, compared with the values from nondiabetic donors.

Mentions: To confirm our results obtained from the rodent model, major parameters were quantified in the retinal mitochondria prepared from human donors with diabetic retinopathy. Mitochondria from donors with diabetic retinopathy had 60–70% increase in active MMP-9 compared with age-matched, nondiabetic donors, and significantly decreased expression of Hsp70 and Hsp60. The binding of MMP-9 with Hsp70 or with Hsp60 was also significantly increased (Fig. 6).


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 is activated in retinal mitochondria from patients with diabetic retinopathy, and binding of MMP-9 with chaperones is increased. Mitochondria were prepared from the retina of donors with diabetic retinopathy and age-matched nondiabetic subjects by differential centrifugation. A: Active MMP-9 was quantified by ELISA technique. Expression of (B) Hsp70 and (C) Hsp60 was quantified in the mitochondria by Western blot technique, and binding of MMP-9 with (D) Hsp70 or (E) Hsp60 by immunoprecipitating MMP-9. Each measurement was performed in duplicate in the samples obtained from four nondiabetic donors and five donors with diabetic retinopathy. The values are represented as mean ± SD. *P < 0.05, compared with the values from nondiabetic donors.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198054&req=5

Figure 6: MMP-9 is activated in retinal mitochondria from patients with diabetic retinopathy, and binding of MMP-9 with chaperones is increased. Mitochondria were prepared from the retina of donors with diabetic retinopathy and age-matched nondiabetic subjects by differential centrifugation. A: Active MMP-9 was quantified by ELISA technique. Expression of (B) Hsp70 and (C) Hsp60 was quantified in the mitochondria by Western blot technique, and binding of MMP-9 with (D) Hsp70 or (E) Hsp60 by immunoprecipitating MMP-9. Each measurement was performed in duplicate in the samples obtained from four nondiabetic donors and five donors with diabetic retinopathy. The values are represented as mean ± SD. *P < 0.05, compared with the values from nondiabetic donors.
Mentions: To confirm our results obtained from the rodent model, major parameters were quantified in the retinal mitochondria prepared from human donors with diabetic retinopathy. Mitochondria from donors with diabetic retinopathy had 60–70% increase in active MMP-9 compared with age-matched, nondiabetic donors, and significantly decreased expression of Hsp70 and Hsp60. The binding of MMP-9 with Hsp70 or with Hsp60 was also significantly increased (Fig. 6).

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