Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.

Recent work has suggested a possible role of nitric oxide, a free radical gas, during the wound healing process. In this study we investigated the regulation of inducible nitric oxide synthase (iNOS) and GTP-cyclohydrolase I (GTP-CH I), the rate-limiting enzyme in the biosynthesis of the iNOS cofactor (6R) 5,6,7,8-tetrahydrobiopterin (6-BH4), during the repair process. We found a similar time course of induction of iNOS and GTP-CH I expression, whereas absolute expression levels were different for both genes. Immunohistochemical analysis revealed colocalization of iNOS and GTP-CH I proteins in the wound. Systemic treatment with glucocorticoids significantly altered the expression levels of iNOS and GTP-CH I. Expression of iNOS and GTP-CH I was suppressed by glucocorticoids in normal, and to a much greater extent in wounded skin. Furthermore, a role of nitric oxide as a novel mediator of gene regulation during healing is suggested by the demonstration of nitric oxide-mediated induction of vascular endothelial growth factor expression in keratinocytes. These findings may provide an explanation for the beneficial effects of orally supplemented L-arginine on wound healing, and suggest that a disturbed induction of iNOS and GTP-CH I expression may at least partially underlie the wound healing defect seen in glucocorticoid-treated animals.

Differential expression of matrix metalloproteinases and their physiological inhibitors in acute murine skin wounds.

Metalloproteinase-mediated proteolysis plays an important role during all stages of wound repair. In acute murine wounds, murine collagenase, matrix metalloproteinase (MMP)-9, stromelysin-1, stromelysin-2 and tissue inhibitor of metalloproteinase (TIMP)-1 are strongly induced within 24 h after wounding. At the time of reepithelialization the expression of these gene transcripts has declined more or less completely. Each gene shows a unique spatial and temporal transcription pattern.

Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair.

During cutaneous wound healing a number of migratory and remodeling events occur that require the action of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs). In this study, we analyzed the temporal and spatial expression patterns of these molecules during the healing of murine excisional skin wounds. Our data imply that defined phases of repair rely on distinct repertoires of MMP activity and TIMP counterregulation. Reepithelialization was found to be associated with active production of collagenase, 92-kDa gelatinase, and stromelysins-1 and -2 by distinct subpopulations of keratinocytes at the migrating border. Notably, no TIMP transcripts were expressed in the epidermis, but TIMP-1 expression in the wound colocalized with expression of collagenase, 92-kDa gelatinase, and stromelysin-1, albeit in distinct cells. Concomitant with the formation of an extensive hyperproliferative epithelium, TIMP-1 transcripts accumulated at the mesenchymal/epidermal border of the granulation tissue. During later phases of wound repair, we observed an increase in 72-kDa gelatinase and MT1-MMP expression, whereby the transcripts of these colocalizing MMPs were detected exclusively and at high levels in the granulation tissue. At completion of reepithelialization, the expression levels of the MMPs and TIMP-1 seen in epidermal and dermal compartments declined to near-basal levels, whereas the macrophage-specific metalloelastase (MME) reached maximum expression. In reepithelialized wound tissue, MME transcripts were detected in deep layers of reconstituted dermis and seemed to cluster around vascular structures. Systemic glucocorticoid treatment, which is known to result in impaired wound healing, led to a nearly complete shut-off of MME expression. These observations imply an additional role of macrophage-related proteolysis, independent of its classical roles during earlier, inflammatory phases of cutaneous wound repair.

cDNA cloning and expression of the gene encoding murine stromelysin-2 (MMP-10).

Recently, we demonstrated a biphasic induction of the epithelial broad-spectrum matrix metalloproteinase (MMP) stromelysin-2 during cutaneous wound healing. Now we have generated a murine wound cDNA libary and have used it to isolate the putative cDNA of this murine matrix metalloproteinase. The predicted sequence of the protein shows 76 and 89% identity with its human and rat analogues, respectively. Stromelysin-2 and stromelysin-1 transcripts were both detected at very low levels in the lung and the heart of adult Balb/c mice, whereas stromelysin-2 mRNA expression alone was found at comparatively high levels in the small intestine, a tissue characterized by continuous epithelial renewal. Recombinant forms of murine stromelysin-1 and -2 produced in transfected COS cells were secreted and could be induced to undergo autocatalytic processing by addition of the organomercurial salt 4-aminophenylmercuric acetate (APMA).

Regulation of the expression of stromelysin-2 by growth factors in keratinocytes: implications for normal and impaired wound healing.

Keratinocyte growth factor (KGF) has been implicated in wound re-epithelialization and branching morphogenesis of several organs. To determine whether KGF induces these effects via induction of matrix metalloproteinase expression we have analysed the effect of KGF on the expression of stromelysin-2 in cultured HaCaT keratinocytes. Here we show a strong induction of stromelysin-2 mRNA within 5-8 h of stimulation of these cells with KGF. The degree of induction was similar to that achieved by treatment with epidermal growth factor or tumour necrosis factor alpha, whereas the stimulatory effect of transforming growth factor beta 1 was even stronger. To determine whether the induction of stromelysin-2 expression by growth factors and cytokines might be important for wound healing, we analysed the expression of this gene during the healing process of full-thickness excisional wounds in mice. Whereas stromelysin-2 mRNA could hardly be detected in unwounded skin, a biphasic induction was seen after injury and highest levels were found at days 1 and 5 after wounding. Hybridization in situ revealed the presence of stromelysin-2 mRNA in basal keratinocytes at the wound edge but not in the underlying mesenchymal tissue. During impaired wound healing as seen in glucocorticoid-treated mice, stromelysin-2 expression was significantly increased compared with untreated control mice. Taken together, these results suggest that correct regulation of this broad-spectrum metalloproteinase might be important for normal repair.

Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.

Recently we demonstrated an important function of keratinocyte growth factor (KGF) in wound re-epithelialization. As KGF is mitogenic for various epithelial cells, we speculated about a role of KGF in epithelial repair processes of other organs as seen in a variety of inflammatory diseases. Here we demonstrate a strikingly increased expression of KGF in surgical specimens from patients suffering from Crohn's disease and ulcerative colitis. The levels of KGF expression strongly correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue and expression analysis of the pro-inflammatory cytokine interleukin-1 beta. The highest levels of KGF mRNA and protein were found in mesenchymal cells of the lamina propria, particularly in highly inflamed areas. As the KGF receptor is expressed in intestinal epithelial cells, KGF seems to act in a paracrine manner to stimulate proliferation of these cells. These data suggest a crucial role of KGF in epithelial repair after injury caused by inflammatory processes.

Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.

It has long been speculated that pro-inflammatory cytokines play an important role in wound repair. However, little is known about the temporal and spatial expression pattern of these cytokines during normal and impaired wound healing. In this study we show a strong and early induction of interleukins 1 alpha and beta (IL-alpha and beta) and of tumour necrosis factor alpha (TNF-alpha) expression after cutaneous injury. Highest levels of these cytokines were seen as early as 12-24 h after wounding. After completion of the proliferative phase of wound healing, mRNA levels of these cytokines returned to the basal level. During the early phase of wound repair, proinflammatory cytokines were predominantly expressed in polymorphonuclear leukocytes, suggesting a novel function of these cells in the initiation of wound healing. At later stages of the repair process, expression of IL-1 alpha, IL-1 beta and TNF-alpha was also seen in macrophages. Furthermore, TNF-alpha was detected in the hyperproliferative epithelium at the wound edge and IL-1 alpha was found in keratinocytes of the hair follicles. Induction of these cytokines after injury was significantly reduced during wound repair in healing-impaired glucocorticoid-treated mice. This finding demonstrates that wound healing defects are associated with impaired cytokine expression and suggests that the early induction of these genes is important for normal repair.

Transforming growth factors beta1, beta2, and beta3 and their receptors are differentially regulated during normal and impaired wound healing.

A series of studies has shown that application of transforming growth factor beta (TGF-beta) to a wound has a beneficial effect, especially in animals with wound healing disorders. In this study we have investigated the regulation of TGF-beta1, beta2, and beta3 and their receptors during the repair process. We found a large induction of all three TGF-beta isoforms and also of TGF-beta types I and II receptors, although the time course of induction and the absolute expression levels were different for these genes. Furthermore, each TGF-beta isoform had distinct sites of expression in the wound. Systemic treatment with glucocorticoids significantly altered the expression levels of TGF-betas and TGF-beta receptors. Whereas expression of TGF-beta1, TGF-beta2, and TGF-beta type II receptor was suppressed by glucocorticoids in normal and wounded skin, expression of TGF-beta3 and TGF-beta receptor type I mRNA was stimulated. These findings provide an explanation for the beneficial effect of exogenous TGF-beta in the treatment of impaired wound healing in glucocorticoid-treated animals. Furthermore, they suggest that a disturbed balance between the levels of the three TGF- beta isoforms and their receptors might underlie the wound healing defect seen in glucocorticoid-treated animals.