Abrogation of MMP-9 Gene Protects Against the Development of Retinopathy in Diabetic Mice by Preventing Mitochondrial Damage

Abstract

OBJECTIVEIn 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 METHODSUsing 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).RESULTSRetinal 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.CONCLUSIONSRegulation 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.

Interleukin-1β and mitochondria damage, and the development of diabetic retinopathy

“…Mice were sacrificed by carbon dioxide asphyxiation. The retina from one eye was used to quantify biochemical and molecular parameters, and the other eye was incubated immediately in 10% buffered formalin for apoptosis/histopathology [18][19][20]. The treatment of the animals conformed to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research, and the Institutional Guidelines.…”

Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy

“…Transgenic MMP-9 Knockout mice showed enhanced retinal protection against oxidative stress and reduced apoptosis after diabetes. Supporting this, high glucoseincubated retinal endothelial cells treated with MMP-2 and -9 inhibitors or knock down by siRNA showed reduced levels of apoptosis; such reduction in total cell death may contribute in prevention of pathogenesis of diabetic retinopathy [24].…”

“…In general, mitochondrial targeting signals are recognized by outer membrane import receptors, translocase of the outer membrane (TOM), and the translocase of the inner membrane (TIM) import is further assisted by Hsp70 complex in pulling these proteins into the matrix [65]. In diabetes, MMP-9 translocates to mitochondria via TOM/TIM44 complex and induces modulation in the mitochondrial redox state, leading to mitochondria dysfunction [24]. The mechanisms responsible for mitochondria dysfunction by MMPs in diabetes might be that the mitochondria is a major source of superoxide generation and MMPs residing in the mitochondria are sensitive to superoxide.…”

“…We, along with others, have shown that retinal cells incubated in high glucose upregulate the MMP-2 and -9 expression/activation, and inhibition of MMP-2 and -9 is able to prevent high glucose-induced retinal capillary cell apoptosis. Furthermore, we have also shown that high glucose translocate MMP-2 and -9 into the mitochondria and induce mitochondria dysfunction, and inhibition of MMP-2 and -9 ameliorate the high glucose-induced mitochondria dysfunction [23,24,26,35,36]. Hyperglycemia can lead to the activation of MMP-2 and -9 by oxidative stress, MAP kinase pathways [23,26,37].…”

“…Understanding on such mechanistic pathways might help to develop future adjuvant therapies for diabetic retinopathy. Results of a recent research indicate that MMPs and tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in the pathogenesis of diabetic retinopathy [24][25][26][27].…”