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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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Binding of MMP-9 with the chaperones and TIM44. Retinal homogenate was immunoprecipitated with anti–MMP-9. Relative abundance of Hsp70, Hsp60, or TIM44 was determined by Western blotting using their respective antibodies. IgG was used as the loading control. Results are expressed as mean ± SD of five to seven mice in each group. The values obtained from mice in the WT-N group are considered as 100%. *P < 0.05 and #P < 0.05, compared with WT-N and WT-D, respectively.
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Figure 5: Binding of MMP-9 with the chaperones and TIM44. Retinal homogenate was immunoprecipitated with anti–MMP-9. Relative abundance of Hsp70, Hsp60, or TIM44 was determined by Western blotting using their respective antibodies. IgG was used as the loading control. Results are expressed as mean ± SD of five to seven mice in each group. The values obtained from mice in the WT-N group are considered as 100%. *P < 0.05 and #P < 0.05, compared with WT-N and WT-D, respectively.

Mentions: Diabetes of ∼7 months in WT mice, as expected, significantly increased the apoptosis of capillary cells. The number of TUNEL-positive cells in the trypsin-digested retinal microvasculature was about twofold higher compared with the WT-nondiabetic (WT-N) mice (Fig. 1A). In the same retinal vasculature, the number of degenerative capillaries was also significantly increased (Fig. 1B). Consistent with the increase in active MMP-9 in the retina in diabetes (7,8), retinal mitochondria also had an ∼25% increase in active MMP-9, as confirmed by both ELISA and in situ zymography assays (Fig. 2A and B). In the same WT-diabetic (WT-D) mice, mitochondrial superoxide levels were elevated by >100%, mtDNA was damaged (40% reduction in the amplification of the long fragment of mtDNA), mitochondrial permeability was increased, and the translocation of Bax into the mitochondria was increased by ∼50% compared with the values from age-matched WT-N mice (Fig. 2C–F). A significant number of mitochondria in WT-D mice in the endothelium region of the microvasculature were elongated and swollen with electron-lucent matrix and partial cristolysis compared with the intact and tightly packed lamellar cristae in WT-N mice (Fig. 3A). The average area and length of the mitochondria in the endothelial region were ∼40% larger in WT-D mice compared with WT-N mice (Fig. 3B and C). The chaperone proteins (Hsp70 and Hsp60) and the translocators (TOM34 and TIM44) were significantly decreased in the retinal mitochondria (Fig. 4A–D), but the binding of MMP-9 with Hsp70, Hsp60, or TIM44 was increased by 40–80% (Fig. 5).


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)

Binding of MMP-9 with the chaperones and TIM44. Retinal homogenate was immunoprecipitated with anti–MMP-9. Relative abundance of Hsp70, Hsp60, or TIM44 was determined by Western blotting using their respective antibodies. IgG was used as the loading control. Results are expressed as mean ± SD of five to seven mice in each group. The values obtained from mice in the WT-N group are considered as 100%. *P < 0.05 and #P < 0.05, compared with WT-N and WT-D, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Binding of MMP-9 with the chaperones and TIM44. Retinal homogenate was immunoprecipitated with anti–MMP-9. Relative abundance of Hsp70, Hsp60, or TIM44 was determined by Western blotting using their respective antibodies. IgG was used as the loading control. Results are expressed as mean ± SD of five to seven mice in each group. The values obtained from mice in the WT-N group are considered as 100%. *P < 0.05 and #P < 0.05, compared with WT-N and WT-D, respectively.
Mentions: Diabetes of ∼7 months in WT mice, as expected, significantly increased the apoptosis of capillary cells. The number of TUNEL-positive cells in the trypsin-digested retinal microvasculature was about twofold higher compared with the WT-nondiabetic (WT-N) mice (Fig. 1A). In the same retinal vasculature, the number of degenerative capillaries was also significantly increased (Fig. 1B). Consistent with the increase in active MMP-9 in the retina in diabetes (7,8), retinal mitochondria also had an ∼25% increase in active MMP-9, as confirmed by both ELISA and in situ zymography assays (Fig. 2A and B). In the same WT-diabetic (WT-D) mice, mitochondrial superoxide levels were elevated by >100%, mtDNA was damaged (40% reduction in the amplification of the long fragment of mtDNA), mitochondrial permeability was increased, and the translocation of Bax into the mitochondria was increased by ∼50% compared with the values from age-matched WT-N mice (Fig. 2C–F). A significant number of mitochondria in WT-D mice in the endothelium region of the microvasculature were elongated and swollen with electron-lucent matrix and partial cristolysis compared with the intact and tightly packed lamellar cristae in WT-N mice (Fig. 3A). The average area and length of the mitochondria in the endothelial region were ∼40% larger in WT-D mice compared with WT-N mice (Fig. 3B and C). The chaperone proteins (Hsp70 and Hsp60) and the translocators (TOM34 and TIM44) were significantly decreased in the retinal mitochondria (Fig. 4A–D), but the binding of MMP-9 with Hsp70, Hsp60, or TIM44 was increased by 40–80% (Fig. 5).

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