Danqing Min

Increased Matrix Metalloproteinase-9 Predicts Poor Wound Healing in Diabetic Foot Ulcers

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 < 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 (>480 pg/ml) and TGF-ß (>115 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.

Disruption of E-Cadherin by Matrix Metalloproteinase Directly Mediates Epithelial-Mesenchymal Transition Downstream of Transforming Growth Factor-β1 in Renal Tubular Epithelial Cells

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)-beta1-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-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-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-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy

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-alpha, 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-alpha, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 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 < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-alpha, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF-alpha and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted 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.

Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in 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.

Activation of Macrophage Promatrix Metalloproteinase-9 by Lipopolysaccharide-Associated Proteinases

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.

Alterations in Monocyte CD16 in Association with Diabetes Complications

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.

Increased Matrix Metalloproteinase-9 Predicts Poor Wound Healing in Diabetic Foot Ulcers Response to Muller et al.

We thank the authors for their comment (1) on our study (2) and we agree that the pathophysiology of diabetic foot ulcers is complex and not well understood. We recognize that we are not the first to establish a potential role for matrix metalloproteinase-9 (MMP-9) in chronic wound healing. However, we report that its measurement …

The interaction of metal ions and Marimastat with matrix metalloproteinase 9

The effect of a range of metal ions on the ability of Marimastat to inhibit matrix metalloproteinase 9 (MMP-9) was examined in a fluorescence based proteolytic assay. Whilst none of the metals examined significantly affected the inhibitory ability of Marimastat, several metal ions did have a significant effect on MMP-9 activity itself. In the absence of Marimastat, Zn(II) and Fe(II) significantly inhibited MMP-9 activity at metal ion concentrations of 10 and 100 microM, respectively. In both the absence and presence of Marimastat, Cd(II) significantly inhibited MMP-9 at 100 microM. In contrast, 1 mM Co(II) significantly upregulated MMP-9 proteolytic activity.

Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.

Monocyte CD163 is altered in association with diabetic complications: possible protective role

The scavenger receptor CD163 is exclusively expressed by monocyte/macrophages and is shed by matrix metalloproteinases (MMPs) and neutrophil elastase (ELA2) as soluble CD163 (sCD163). Monocyte phenotype is altered in diabetes, but the relationship among monocyte CD163, sCD163, and diabetic complications is not known and was investigated in this study. Blood was obtained from patients with diabetes for >10 yr and mice with diabetes for ≤20 wk. Blood from people and mice without diabetes acted as controls. The percentage of CD163+ monocytes and monocyte CD163 mRNA was determined by flow cytometry and qRT‐PCR, respectively. Plasma sCD163, MMPs, and ELA2 were measured by ELISA. The ability of glucocorticoids to stimulate isolated monocyte CD163 expression was also investigated. The percentage of CD163+ monocytes was significantly decreased and sCD163 significantly increased (both P < 0.05) in patients with diabetes with complications compared to those without complications. Plasma ELA2 and MMP‐3 were also increased (P < 0.05), but CD163 mRNA was unaltered. sCD163 correlated with worsening renal function, as determined by eGFR (r = −0.48, P < 0.05). In diabetic mice, increased sCD163 at wk 5 and decreased percentage of CD163+ monocytes at wk 10 preceded alteration in kidney collagen IV mRNA at wk 20 (all P < 0.05). In vitro incubation of monocytes in anti‐inflammatory glucocorticoid increased the percentage of CD163+ monocytes (P < 0.05). In people, higher sCD163 and decreased percentage of CD163+ monocytes were consistent with increased monocyte activation and shedding. The murine data indicated that these changes preceded the development of diabetic complications. Taken together, these results suggest that higher circulating percentage of CD163+ monocytes may have anti‐inflammatory effects and may protect from development of diabetic complications.