Meng Ren

Involvement of RAGE, MAPK and NF-κB pathways in AGEs-induced MMP-9 activation in HaCaT keratinocytes

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.

Association between habitual daytime napping and metabolic syndrome: a population-based study

OBJECTIVE Our objective was to evaluate the association between habitual daytime napping and the prevalence of metabolic syndrome. MATERIALS AND METHODS We conducted a population-based study of 8,547 subjects aged 40 years or older. Metabolic syndrome was defined according to a harmonized definition from a joint statement and the recommended thresholds for the Chinese population. Information about sleep duration was self-reported. RESULTS The prevalence of metabolic syndrome in the no daytime napping group, the 0 to 1 hour daytime napping group and the more than 1 hour daytime napping group were 35.0%, 36.0% and 44.5% among the females (P<0.0001). Increased daytime napping hours were positively associated with parameters of metabolic syndrome in the female subjects, including waist circumference, systolic blood pressure, triglycerides and fasting plasma glucose (P<0.05 for all). Multivariate adjusted logistic regression analysis revealed that, compared to the no habitual daytime napping females, napping for more than 1 hour was independently associated with an increased prevalence of metabolic syndrome (odds ratio 1.39, 95% confidence interval, 1.13-1.72). Compared to the female subjects in the no daytime napping group, those habitually napped for more than 1 hour exhibited 46% and 26% increases in the prevalence of central obesity and hypertriglyceridemia (all P<0.05). No statistically significant associations were detected between daytime napping hours and metabolic syndrome among the male subjects. CONCLUSION Daytime napping is associated with an increased prevalence of metabolic syndrome in middle-aged non-obese Chinese women.

Effects of sodium butyrate on the differentiation of pancreatic and hepatic progenitor cells from mouse embryonic stem cells.

Recently significant progress has been made in differentiating embryonic stem (ES) cells toward pancreatic cells. However, little is known about the generation and identification of pancreatic progenitor cells from ES cells. Here we explored the influence of sodium butyrate on pancreatic progenitor differentiation, and investigated the different effects of sodium butyrate on pancreatic and hepatic progenitor formation. Our results indicated that different concentration and exposure time of sodium butyrate led to different differentiating trends of ES cells. A relatively lower concentration of sodium butyrate with shorter exposure time induced more pancreatic progenitor cell formation. When stimulated by a higher concentration and longer exposure time of sodium butyrate, ES cells differentiated toward hepatic progenitor cells rather than pancreatic progenitor cells. These progenitor cells could further mature into pancreatic and hepatic cells with the supplement of exogenous inducing factors. The resulting pancreatic cells expressed specific markers such as insulin and C‐peptide, and were capable of insulin secretion in response to glucose stimulation. The differentiated hepatocytes were characterized by the expression of a number of liver‐associated genes and proteins, and had the capability of glycogen storage. Thus, the current study demonstrated that sodium butyrate played different roles in inducing ES cells toward pancreatic or hepatic progenitor cells. These progenitor cells could be further induced into mature pancreatic cells and hepatocytes. This finding may facilitate the understanding of pancreatic and hepatic cell differentiation from ES cells, and provide a potential source of transplantable cells for cell‐replacement therapies. J. Cell. Biochem. 109: 236–244, 2010.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

Background Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of β-cyclodextrin (β-CD) core and poly(amidoamine) dendron arms (β-CD-[D3]7) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. Methods The cytotoxicity of β-CD-(D3)7 was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of β-CD-(D3)7/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by β-CD-(D3)7/MMP-9-siRNA complexes. The β-CD-(D3)7/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. Results β-CD-(D3)7 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The β-CD-(D3)7/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by β-CD-(D3)7/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). Conclusion β-CD-(D3)7 may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.

Role of site-specific DNA demethylation in TNFα-induced MMP9 expression in keratinocytes

Inappropriately high expression of matrix metalloproteinase 9 (MMP9) in the late stage of diabetic foot ulcers suppresses wound healing. The underlying mechanisms are not completely understood. Site-specific demethylation was reported to function in the regulation of genes, causing persistent high expression of target genes. Therefore, this study was designed to determine whether site-specific DNA demethylation was a key regulatory component of MMP9 expression in diabetic wound healing, and to further verify the crucial CpG site(s). Human keratinocyte cell line (HaCaT) cells were exposed to tumor necrosis factor a (TNFα), and changes in MMP9 expression and DNA methylation status were detected. We found TNFα treatment increased endogenous MMP9 expression in HaCaT cells and decreased the DNA methylation percentage at the -36 bp promoter site in a time-dependent manner. Bisulfite sequencing PCR revealed differentially demethylated CpG sites in the human MMP9 promoter region, but only the change at the -36 bp site was statistically significant. Dual-luciferase reporter assays showed that the promoter with only the -36 bp site demethylated had slightly higher transcriptional activity than the promoter with all other sites except the -36 bp site demethylated. Our results demonstrate that site-specific DNA demethylation plays an important role in MMP9 expression in TNFα-stimulated keratinocytes. The -36 bp site in the MMP9 gene promoter is crucial to this effect, but other CpG sites may exert synergistic effects. Collectively, these data may contribute to the future development of novel therapeutic strategies to treat diabetic foot ulcers and prevent gangrene and amputation.

Angiotensin II regulates collagen metabolism through modulating tissue inhibitor of metalloproteinase-1 in diabetic skin tissues

We investigated the effect of angiotensin II (Ang II) on matrix metalloproteinase-1 (MMP-1)/tissue inhibitor of metalloproteinase-1 (TIMP-1) balance in regulating collagen metabolism of diabetic skin. Skin tissues from diabetic model were collected, and the primary cultured fibroblasts were treated with Ang II receptor inhibitors before Ang II treatment. The collagen type I (Coll I) and collagen type III (Coll III) were measured by histochemistry. The expressions of transforming growth factor-β (TGF-β), MMP-1, TIMP-1 and propeptides of types I and III procollagens in skin tissues and fibroblasts were quantified using polymerase chain reaction (PCR), Western blot or enzyme-linked immunosorbent assay (ELISA). Collagen dysfunction was documented by changed collagen I/III ratio in streptozotocin (STZ)-injected mice compared with controls. This was accompanied by increased expression of TGF-β, TIMP-1 and propeptides of types I and III procollagens in diabetic skin tissues. In primary cultured fibroblasts, Ang II prompted collagen synthesis accompanied by increases in the expressions of TGF-β, TIMP-1 and types I and III procollagens, and these increases were inhibited by losartan, an Ang II type 1 (AT1) receptor blocker, but not affected by PD123319, an Ang II type 2 (AT2) receptor antagonist. These findings present evidence that Ang-II-mediated changes in the productions of MMP-1 and TIMP-1 occur via AT1 receptors and a TGF-β-dependent mechanism.

Activation of skin renin-angiotensin system in diabetic rats.

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.

Serum Gamma - Glutamyltransferase Is Associated with Albuminuria: A Population-Based Study

Background Serum γ - glutamyltransferase (GGT) is implicated in the pathogenesis of endothelial dysfunction and atherosclerosis. Albuminuria is a marker of endothelial damage and correlated with structural and functional integrity of the vasculature. Our objective was to evaluate the association between serum GGT level and prevalence of albuminuria in a Chinese population. Materials and Methods We conducted a population-based cross-sectional study in 9,702 subjects aged 40 years or older. Increased urinary albumin excretion was defined according to the urinary albumin-to-creatinine ratio (ACR) ranges greater or equal than 30 mg/g. Low-grade albuminuria was defined according to the highest quartile of ACR in participants without increased urinary albumin excretion. Results The prevalence of low-grade albuminuria and increased urinary albumin excretion were respectively 23.4% and 6.6% in this population and gradually increased across the sex-specific serum GGT quartiles (all P for trend <0.05). In logistic regression analysis, compared with subjects in the lowest quartile of serum GGT level, the adjusted odds ratios (ORs) in the highest quartile was 1.22 [95% confidence interval (CI), 1.04–1.43] for low-grade albuminuria and 1.55 (95% CI, 1.18–2.04) for increased urinary albumin excretion. In subgroup analysis, significant relationship of serum GGT level with both low-grade albuminuria and increased urinary albumin excretion were detected in women, younger subjects, overweight subjects and in those with hypertension or glomerular filtration rate greater than 90 (all P <0.05). Conclusion Serum GGT level is associated with urinary albumin excretion in middle-aged and elderly Chinese.

Effect of Intensive Nursing Education on the Prevention of Diabetic Foot Ulceration Among Patients with High-Risk Diabetic Foot: A Follow-Up Analysis

The aim of the study was to discuss the effect of intensive nursing education on the prevention of diabetic foot ulceration among patients at high risk for diabetic foot. One hundred eighty-five diabetes patients at high risk for foot diseases were enrolled in this study and provided with intensive nursing education, including individualized education about diabetes mellitus and diabetic foot diseases, instruction in podiatric care (the right way of washing the foot, the care of foot skin, appropriate choice of shoes and socks, intense examinations and records of feet by patients themselves every day, and the assistant management of calluses). Study subjects were followed up for 2 years. Once the foot ulceration developed, the inducing factors of foot ulceration were inquired about, the ulcers were evaluated, and the incidence of foot ulceration was analyzed before and after the intensive nursing education according to self-paired data. Results showed there were highly statistically significant improvements in the intensive treatment group compared with the control group in plasma glucose, blood pressure, and high-density lipoprotein cholesterol levels. More important is that intensive nursing education helps to prevent diabetic foot ulceration and to decrease the rate of amputation among patients at high risk for diabetic foot.

AGE‐induced keratinocyte MMP‐9 expression is linked to TET2‐mediated CpG demethylation

Studies have documented that unusually high expression of matrix metalloproteinase‐9 (MMP‐9) suppresses wound healing during the late stages of diabetic foot ulcers. Recently, it has been reported that the presence of advanced glycation end products‐bovine serum albumin (AGE‐BSA) resulted in a higher expression of MMP‐9 in skin primary keratinocytes. The aim of the present study was to elucidate the molecular machinery that is responsible for the inappropriately high AGE‐BSA–induced expression of MMP‐9. It has been demonstrated that site‐specific DNA demethylation played an important role in MMP‐9 expression in AGE‐BSA–stimulated keratinocytes. Ten–eleven translocation‐2 (TET2) was up‐regulated, whereas the percentage of methylation in the MMP‐9 promoter was reduced. Furthermore, TET2 directly bound to a fragment surrounding the transcriptional start site in the MMP‐9 promoter region, contributing to the regulation of MMP‐9 expression. In addition, evidence indicated that TET2 affected the migration and proliferation in vitro of cultured skin primary keratinocytes. These findings indicated that TET2 directly interacted with the promoter region of MMP‐9 in diabetic tissues and may be a novel master regulator of wound healing.