OBJECTIVE -We studied the relationships of diabetic ulcer wound fluid matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and transforming growth factor-ß1 (TGF-ß1) with wound healing rate.RESEARCH DESIGN AND METHODS -The ulcers were cleansed to remove exudates, and wound fluids were collected for analysis of MMP-2 and -9, TIMP-1, and TGF-ß1.RESULTS -At presentation, MMP-9 and the MMP-9 -to-TIMP-1 ratio correlated inversely with the wound healing rate at 28 days (P Ͻ 0.001). MMP-9 and the MMP-9 -to-TIMP-1 ratio were lower in the 23 patients who achieved complete healing at 12 weeks versus the 39 who did not. The pro-MMP-9 concentration was predictive of healing within 12 weeks. Addition of cutoffs for TIMP-1 (Ͼ480 pg/ml) and TGF-ß (Ͼ115 pg/ml) further improved its predictive power (area under the curve 0.94).CONCLUSIONS -These findings suggest that a milieu with high MMP-9 may be indicative of inflammation and poor wound healing. Measurements of MMP-9, TIMP-1, and TGF-ß in wound fluid may help to identify ulcers at risk of poor healing.

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis , including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however , whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown , especially in the renal system. In this study , we show that transforming growth factor (TGF)-␤1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-␤1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of ␤-catenin , the transcriptional induction of Slug , and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-␤1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease. (Am J Pathol

Monocytes express many cell surface markers indicative of their inflammatory and activation status. Whether these markers are affected by diabetes and its complications is not known and was investigated in this study. Blood was obtained from 22 nondiabetic and 43 diabetic subjects with a duration of diabetes >10 years, including 25 without and 18 with clinically significant complications. The number of CD45+CD14+ monocytes and the percentage expressing the proinflammatory marker CD16 were determined by flow cytometry. Other markers of monocyte activation and expression of chemokine receptors were also examined. The relationship between monocyte CD16 and clinical data, selected cytokines, and chemokines was also investigated. Diabetes had no effect on total white cell number but increased monocyte number. Diabetes also significantly decreased the number of CD16+ monocytes but only in those with diabetic complications. Other markers of monocyte activation status and chemokine receptors were not affected by diabetes or complications status. Diabetes induced plasma proinflammatory cytokines and they were lower in diabetic subjects with complications compared to those without complications. These results suggest that the circulating monocyte phenotype is altered by diabetic complications status. These changes may be causally related to and could potentially be used to predict susceptibility to diabetic complications.

CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF-κB or addition of antibodies to either TNF-α or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy. Am J Physiol Renal Physiol 297: F1229 -F1237, 2009. First published September 9, 2009 doi:10.1152/ajprenal.00074.2009.-Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-␣, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-␣, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P Ͻ 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P Ͻ 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P Ͻ 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-␣, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P Ͻ 0.05), but only TNF-␣ and IL-6 increased MMP-9 expression (50-and 60-fold, respectively; P Ͻ 0.05). Our results show that mesangial cellsecreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy. matrix metalloproteinases; matrix degradation; cell adhesion and infiltration; inflammation response

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LPS induces an up-regulation of promatrix metalloproteinase-9 (proMMP9) gene expression in cells of the monocyte/macrophage lineage. We demonstrate here that LPS preparations are also able to activate proMMP9 made by human macrophages or THP-1 cells via LPS-associated proteinases, which cleave the N-terminal propeptide at a site or sites close to the one cleaved upon activation with organomercurial compounds. LPS-associated proteinases are serine proteinases that are able to cleave denatured collagens (gelatin) and the mammalian serine proteinase inhibitor, α1-proteinase inhibitor, thereby pushing the balance of extracellular matrix turnover even further toward degradation. A low molecular mass, low affinity inhibitor of MMP9, possibly derived from the propeptide, is generated during proMMP9 activation. However, inhibition of the LPS-associated proteinases had no effect on proMMP9 synthesis, indicating that their proteolytic activity was not required for signaling the up-regulation of the proMMP9 gene.

Aims/hypothesis We examined whether age of type 2 diabetes onset is related to mitochondrial DNA content in peripheral blood monocytes (PBMCs). Methods PBMCs were isolated from 65 patients with type 2 diabetes. To minimise age as a confounder, only patients aged ≥50 years were studied. Sample mitochondrial DNA (mtDNA) content was determined by amplification of the mitochondrial gene CYT-B (also known as MT-CYB) and adjusted for single-copy nuclear control genes (36B4 [also known as RPLPO] and GAPDH). Results Age of diabetes onset ranged from 25 to 69 years. There was a significant positive relationship between age of diabetes onset in quartiles and mtDNA content for the whole group (p= 0.02 for trend). When stratified by the presence of diabetes complications, a strong positive relationship was observed between age of diagnosis and mtDNA content for participants without diabetic complications (r = 0.7; p = 0.0002), but not for those with complications (r=−0.04; p=0.8). Multivariate analysis confirmed age of onset and complication status as independent determinants. There was co-linearity between age of onset and disease duration, with similar relationships also seen between duration and mtDNA content. Conclusions/interpretation An earlier age of type 2 diabetes onset is associated with a lower PBMC mtDNA content, but only in patients without diabetes complications. This may reflect a differing biology of PBMC mtDNA in those with early-onset diabetes and those who are prone to complications. PBMC mtDNA depletion may accelerate diabetes onset; however the independent effect of diabetes duration remains to be evaluated.