Bacterial load and bacteria type affect expression and activation of wound fluid matrix metalloproteinase-9: Possible role in promoting healing of Diabetic foot ulcers
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Authors: Mclennan,S. V.;Liu,Y.;Min,D.;Twigg,S. M.;Yue,D. K.
Results: Our previous studies have shown that high wound fluid (WF) concentrations of matrix metalloproteinase-9 (MMP-9) and bacterial load predict poor healing of foot ulcers in diabetes. Whether the increased WF MMP-9 is causally related to bacterial load is not known and was investigated. Diabetic patients with foot ulcers (Texas Grading 0-2 and stage A or B; initial ulcer area:315Â±345mm2 ) were studied at presentation and subsequent follow up. Sixty two patients (M/F 48/15, age 60.5Â±10.1yrs, diabetes duration 14.5Â±7.5yrs) were enrolled. The ulcers were debrided and exudates removed by flushing with saline. WF was collected by absorption onto a sterile 1 cm2 filter paper and recovered by immersion in PBS. The active and pro forms of MMP-9 were analyzed by zymography. The bacterial type and load (Colony Forming Unit, CFU) were determined by standard microbiological techniques. The relationship between bacterial type and MMP-9 was examined by incubation of endotoxins from pure cultures of E.coli or S.aureus isolated from WF with monocyte/macrophages (THP1 cells). At presentation the active MMP-9 (actMMP-9) and CFU in WFs of the 23 patients who achieved complete healing at week 12 were lower than those which failed to heal (actMMP-9: 1.2Â±0.2vs2.9Â±0.6ng/ml, CFU: 6.2Â±0.2vs7.1Â±0.2×106, both p<0.05) and decreased at a faster rate (actMMP-9: 0.31vs0.16ng/wk,CFU:2.45vs0.09×106/wk). The majority of WFs (75%) contained 2 or more bacterial types. The initial WF CFU correlated with actMMP-9 (r=0.51, p<0.01) but not with the proform. No MMP-9 was present in endotoxin from E.coli or S.aureus. In 3 experiments addition of endotoxin from E.coli and S.aureus increased MMP-9 in THP1 cells in a dose-dependent fashion but only the E.coli endotoxin increased actMMP-9. These observations provide a mechanism to explain how high bacterial load can increase MMP-9 and how certain type of bacteria can selectively increase activation of this enzyme. These relationships could have important implications in developing optimal strategy to promote wound healing of diabetic foot ulcers.