Human tissue inhibitor of metalloproteinases‐1 improved wound healing in diabetes through its anti‐apoptotic effect

Impaired wound healing accompanies severe cell apoptosis in diabetic patients. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) was known to have effects on promoting growth and anti‐apoptosis for cells. We aimed to determine the actual levels of TIMP‐1 and cell apoptosis in: (i) the biopsies of diabetic and non‐diabetic foot tissue and (ii) the human fibroblasts with or without treatments of advanced glycation end‐products (AGEs). Next, we aimed to determine the improved levels of cell apoptosis and wound healing after the treatments of either active protein of TIMP‐1 or in vivo expression of gene therapy vector‐mediated TIMP‐1 in both the human fibroblasts and the animal model of diabetic rats. The levels of TIMP‐1 were significantly reduced in diabetic skin tissues and in AGEs‐treated fibroblasts. Both AGEs‐treated cells were effectively protected from apoptosis by active protein of TIMP‐1 at appropriate dose level. So did the induced in vivo TIMP‐1 expression after gene delivery. Similar effects were also found on the significant improvement of impaired wound healing in diabetic rats. We concluded that TIMP‐1 improved wound healing through its anti‐apoptotic effect. Treatments with either active protein TIMP‐1 or TIMP‐1 gene therapy delivered in local wound sites may be used as a strategy for accelerating diabetic wound healing.     

Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.     

Effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on migration of epidermal keratinocytes in vitro and wound healing in vivo

Tissue inhibitors of metalloproteinases (TIMP), common inhibitors of matrix proteinases, have cell-promoting activity. We studied the effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on the migration of normal human epidermal keratinocytes (NHEK). An in vitro migration assay revealed that rh-TIMP-2 enhanced random migration (up to 170%, p<0.05) in a dose-dependent manner. When we applied rh-TIMP-2 solution (20 microg/20 microl/wound) daily to full-thickness wounds made with an 8-mm punch on the backs of healthy (n=8), aged (n=9), and diabetic (n=15) rodents, we observed faster wound closure (p<0.05) than in vehicle-treated controls. Accelerated wound closure was dose-dependent (0-20 microg/wound) in diabetic mice (n=6), and the optimal concentration was 10-20 microg of rh-TIMP-2/wound. Histological examinations performed on days 0, 5, 10, 15, and 20 in diabetic mice revealed faster migration of epidermal keratinocytes from wound edges. These results suggest that rh-TIMP-2 plays an important role in wound healing.