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ABSTRACT: Matrix metalloproteinases (MMPs) degrade extracellular matrix components. Increased MMP-9 content in diabetic skin contributes to skin vulnerability and refractory foot ulcers. To identify ways to decrease MMP-9 levels in skin, inhibition of MMP-9 expression in dermal fibroblasts using small interfering RNA was investigated in vitro.
A full-thickness wound was created on the midback of streptozotocin-induced diabetic rats; skin biopsies were performed 3 days later. Skin MMP-9 expression was observed by immunohistochemical analysis. Dermal fibroblasts from 1-day-old normal Sprague Dawley rats cultured with high glucose and homocysteine concentrations were transfected with small interfering RNA complexes. Cells were collected 30, 48, and 72 hours after transfection, and reverse transcription-polymerase chain reaction, Western blot analysis, and gelatin zymography for MMP-9 were performed.
Expression of MMP-9 was increased in diabetic rat skin, especially around wounds. After 30-, 48-, and 72-hour transfection with each MMP-9-specific small interfering RNA, reverse transcription-polymerase chain reaction showed markedly decreased MMP-9 messenger RNA expression, protein abundance, and activity. Of four MMP-9 small interfering RNAs, one sequence had a stable high inhibition rate (>70% at 30 and 48 hours after transfection).
Expression of MMP-9 was increased in diabetic rat skin, especially around wounds, and was markedly inhibited after MMP-9 small interfering RNA transfection in vitro (P < .05). These findings may provide new treatments for diabetic skin wounds.
Journal of the American Podiatric Medical Association 07/2012; 102(4):299-308. · 0.57 Impact Factor
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ABSTRACT: To explore the effects of matrix metallopeptidase 9 (MMP-9) on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts.
Lipopolysaccharide (LPS) at 0.1 and 1.0 µg/ml was used to stimulate fibroblasts to up-regulate the expression of MMP-9 for 18 and 48 h. And then RNA interference was employed to inhibit the high expression of MMP-9. Cell proliferation was tested by CCK-8, cell apoptosis by flow cytometry and type I and III procollagen expressions by quantitative reverse transcriptase PCR (qRT-PCR).
The MMP-9 expression of fibroblasts increased after the stimulation of LPS. And the 1.0 µg/ml LPS stimulation of 48 hours was 14.25 times of mRNA expression and 2.31 times of protein expression versus that of the normal group (both P < 0.05). The RNA interference obviously inhibited the high expression of MMP-9. The mRNA expression was 1/8 and protein expression 1/3 (both P < 0.05) as compared with the control group. Cell proliferation decreased with the rising expression of MMP-9 to some extent [(1.08 ± 0.08) vs (1.18 ± 0.09), P < 0.05] and improved after the inhibition of high expression of MMP-9 [(1.78 ± 0.17) vs (1.53 ± 0.15), P < 0.01]. There was no change of apoptosis accompanied with high expression of MMP-9 (P > 0.05). Apoptosis decreased after the inhibition of high expression of MMP-9 for 48 hours (3.53% ± 0.22% vs 4.47% ± 0.46%, P < 0.05). The synthesis of type I procollagen was the same no matter up-regulation or down-regulation of MMP-9 expression (P > 0.05). As the expression of MMP-9 increased, the synthesis of type III procollagen decreased (P < 0.01), but not increased by 1.02 folds after the inhibited expression of MMP-9 (P > 0.05).
MMP-9 can affect the biological behaviors of rat dermal fibroblasts.
Zhonghua yi xue za zhi 02/2012; 92(8):564-7.
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ABSTRACT: Advanced glycation end products (AGEs) exert divergent effects on the pathogenesis of diabetes complications. Excessive expression of matrix metalloproteinases-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. However, the effect of AGEs on MMP-9 induction in skin cells and the exact molecular mechanisms involved are still poorly understood. In this study, we investigated the effect of AGEs on the production of MMP-9 in HaCaT keratinocytes and characterized the signal transduction pathways activated by AGEs that are involved in MMP-9 regulation. We showed that AGE-BSA increased MMP-9 expression in HaCaT cells at both the protein and mRNA levels. The stimulatory effect of AGE-BSA on MMP-9 was attenuated by inhibitors of extracellular-signal-regulated kinase (ERK1/2, U0126), p38 mitogen-activated protein kinase (MAPK, SB203580) and NF-κB, but not c-Jun N-terminal kinase. Furthermore, receptor for advanced glycation end products (RAGE) was expressed in keratinocytes, and incubation with AGE-BSA resulted in a significant upregulation of RAGE expression in a dose-dependent manner. Silencing of the RAGE gene prevented AGE-BSA-induced MMP-9 activation and the phosphorylation of ERK1/2 and p38 MAPK. We also observed the involvement of NF-κB in AGE-BSA-induced MMP-9 activation, which was not blocked by U0126 and SB203580. These results suggest that AGEs may play an important role in the impairment of diabetic wound healing by upregulating MMP-9 expression in keratinocytes via the RAGE, ERK1/2 and p38 MAPK pathways; activation of NF-κB is also involved in this process. These pathways may represent potential targets for drug interventions to improve diabetic wound healing, a process in which MMP-9 plays a critical role.
Experimental Dermatology 02/2012; 21(2):123-9. · 3.54 Impact Factor
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ABSTRACT: The renin-angiotensin system (RAS) is reportedly involved in chronic diabetic complications such as diabetic nephropathy, but changes of the RAS in diabetic skin remain unknown. The aim of this study was to investigate the expression of angiotensin (Ang) II and its type 1 (AT1) and type 2 (AT2) receptors in diabetic skin tissues, and explore the relationship between the local RAS and pathological changes of diabetic skin. Our results showed that thinning of epidermis, degeneration of collagen, fracture of dermal layer, and atrophy/disappearance of subcutaneous fat were observed in diabetic skin. The expression level of AngII was increased in diabetic skin tissues compared to that in controls. mRNA and protein expression of AT1 receptor were also increased while the level of AT2 receptor decreased; the relative expression of AT1 to AT2 receptors was approximately threefold higher in diabetes than in controls. Furthermore, in the culture medium of primary cultured fibroblasts from diabetic skin, the concentration of AngII was significantly higher than that of normal control. The mRNA and protein expression of AT1 receptor was also increased in fibroblasts of diabetic skin compared to controls, while the protein expression of AT2 receptor was decreased. Taken together, our results suggest that the local RAS system is activated in diabetic skin and AngII receptor is likely to mediate the pathological changes of diabetic skin.
Endocrine 06/2011; 39(3):242-50. · 1.42 Impact Factor
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ABSTRACT: The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48 h of incubation with 50 μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72 h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.
Archives for Dermatological Research 12/2010; 303(5):339-50. · 2.28 Impact Factor
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ABSTRACT: We investigated the mechanism of delayed would healing caused by diabetes and measured the dynamic changes in matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels. We noted differences in the ratio of MMP-9 to TIMP-1 in the wounds of diabetic and nondiabetic rats.
Forty-two Sprague-Dawley rats weighing 250 g were randomly assigned to either the control group or the streptozotocin-induced diabetes group. Then, full-thickness excision wounds were created on the middle of the back of each animal. Skin biopsy specimens were obtained on days 0, 3, 7, and 14 after incision. The content of collagen was quantified by Masson's staining and the macrophage marker, and CD68 was detected by immunohistochemical analysis. Messenger RNA and protein expression of MMP-9 and TIMP-1 was measured by reverse transcriptase-polymerase chain reaction and Western blot, respectively.
Diabetic rats exhibited slower wound healing than control animals (P < .05). On days 3, 7, and 14 after incision, higher levels of MMP-9 messenger RNA and protein expression were detected in the diabetic group compared with the control group (P < .05), and expression of TIMP-1 messenger RNA and protein was significantly decreased. In addition, the ratio of MMP-9 to TIMP-1 was stable in controls, whereas there was a marked increase in the ratio in diabetic skin wounds.
The balance between MMP-9 and its inhibitor, TIMP-1, is disturbed in diabetic skin tissue after injury, which may lead to histologic abnormality of diabetic skin and delayed wound healing.
Journal of the American Podiatric Medical Association 99(6):489-96. · 0.57 Impact Factor
