Akt1 controls insulin-driven VEGF biosynthesis from keratinocytes: implications for normal and diabetes-impaired skin repair in mice.

Here we investigated the potential role of protein kinase B (Akt) in normal or diabetes-impaired wound healing in mice. Interestingly, Akt1 was predominant in skin, wound tissue, and human keratinocytes cell line. Acute skin repair was characterized by an increase of Akt1 phosphorylation in wound margin keratinocytes. By contrast, phosphorylated Akt1 was nearly completely absent and paralleled by a poor phosphorylation of the eucaryotic initiation factor 4E-binding protein 1 (4E-BP1) and reduced levels of vascular endothelial growth factor (VEGF) protein in chronic wounds of diabetic ob/ob mice. Inhibition of the phosphatidyl-inositol-3 kinase/Akt pathway by wortmannin and specific abrogation of Akt1 protein using small-interfering RNA revealed a regulatory function of Akt1 in insulin-mediated VEGF biosynthesis in keratinocytes. Insulin-induced VEGF protein biosynthesis in keratinocytes was mediated by Akt1 from a constitutive VEGF-encoding mRNA pool at the posttranscriptional level through a downstream phosphorylation 4E-BP1. Moreover, transfection experiments introducing a constitutively active mutant of Akt1 into keratinocytes revealed the mammalian target of rapamycin kinase as a downstream mediator of Akt1-linked 4E-BP1 phosphorylation and translational control. Our data suggest that the endocrine hormone insulin contributes to VEGF release in skin wounds through an Akt1-mediated posttranscriptional mechanism in keratinocytes.

Keratinocyte-derived vascular endothelial growth factor biosynthesis represents a pleiotropic side effect of peroxisome proliferator-activated receptor-gamma agonist troglitazone but not rosiglitazone and involves activation of p38 mitogen-activated protein kinase: implications for diabetes-impaired skin repair.

The peroxisome proliferator-activated receptors (PPARs) represent pharmacological target molecules to improve insulin resistance in type 2 diabetes mellitus. Here we assessed a functional connection between pharmacological activation of PPAR and vascular endothelial growth factor (VEGF) expression in keratinocytes and during diabetes-impaired acute skin repair in obese/obese (ob/ob) mice. PPARbeta/delta agonist 4-[3-[4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid (L165,041) and PPARgamma agonists ciglitazone and troglitazone, but not rosiglitazone, potently induced VEGF mRNA and protein expression from cultured keratinocytes. Inhibitor studies revealed a strong functional dependence of troglitazone- and L165,041-induced VEGF expression on p38 and p42/44 mitogen-activated protein kinase (MAPK) activation in keratinocytes. Rosiglitazone also induced activation of p38 MAPK but failed to mediate the activation of p42/44 MAPK in the cells. Functional ablation of PPARbeta/delta and PPARgamma from keratinocytes by small interfering RNA did not abrogate L165,041- and troglitazone-induced VEGF biosynthesis and suggested VEGF induction as a pleiotropic, PPAR-independent effect of both drugs in the cells. In accordance with the in vitro situation, we found activated p38 MAPK in wound keratinocytes from acute wounds of rosiglitazone- and troglitazone-treated diabetic obese/obese mice, whereas keratinocyte-specific VEGF protein signals were only prominent upon troglitazone treatment. In summary, our data from cell culture and wound healing experiments suggested p38 MAPK activation as a side effect of thiazolidinediones; however, only troglitazone, but not rosiglitazone, seemed to translate p38 MAPK activation into a PPARgamma-independent induction of VEGF from keratinocytes.

Biphasic regulation of HMG-CoA reductase expression and activity during wound healing and its functional role in the control of keratinocyte angiogenic and proliferative responses.

In this study, we determined the regulation and potential function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) during skin repair in mice. Upon skin injury, healthy mice exhibited a biphasic increase in HMGR expression and activity with elevated levels at days 3 and 13 post-wounding. In situ hybridization revealed wound margin keratinocytes as a cellular source of HMGR expression. In vitro experiments using cultured HaCaT keratinocytes uncovered epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and insulin as potent co-inducers of HMGR activity and vascular endothelial growth factor (VEGF) in the cells. Insulin-, but not EGF-mediated VEGF protein expression was functionally connected to co-induced HMGR activity, as simvastatin restrictively interfered only with insulin-induced translation of VEGF mRNA by inhibition of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation. Functional ablation of insulin-induced sterol regulatory element-binding protein (SREBP)-2 by siRNA abolished HMGR expression and insulin-triggered VEGF protein release from keratinocytes. Simvastatin also blocked proliferation of cultured keratinocytes. The observed inhibitory effects of simvastatin on keratinocyte VEGF expression and proliferation could be reversed by mevalonate, the product of HMGR enzymatic activity. In accordance, simvastatin-mediated inhibition of HMGR activity in acutely regenerating tissue of wounded mice was paralleled by a marked loss of VEGF protein expression and disturbances of normal proliferation processes in wound margin keratinocytes during skin repair.

Systemic anti-TNFalpha treatment restores diabetes-impaired skin repair in ob/ob mice by inactivation of macrophages.

To date, diabetes-associated skin ulcerations represent a therapeutic problem of clinical importance. The insulin-resistant type II diabetic phenotype is functionally connected to obesity in rodent models of metabolic syndrome through the release of inflammatory mediators from adipose tissue. Here, we used the impaired wound-healing process in obese/obese (ob/ob) mice to investigate the impact of obesity-mediated systemic inflammation on cutaneous wound-healing processes. Systemic administration of neutralizing monoclonal antibodies against tumor necrosis factor (TNF)alpha (V1q) or monocyte/macrophage-expressed EGF-like module-containing mucin-like hormone receptor-like (Emr)-1 (F4/80) into wounded ob/ob mice at the end of acute wound inflammation initiated a rapid and complete neo-epidermal coverage of impaired wound tissue in the presence of a persisting diabetic phenotype. Wound closure in antibody-treated mice was paralleled by a marked attenuation of wound inflammation. Remarkably, anti-TNFalpha- and anti-F4/80-treated mice exhibited a strong reduction in circulating monocytic cells and reduced numbers of viable macrophages at the wound site. Our data provide strong evidence that anti-TNFalpha therapy, widely used in chronic inflammatory diseases in humans, might also exert effects by targeting "activated" TNFalpha-expressing macrophage subsets, and that inactivation or depletion of misbehaving macrophages from impaired wounds might be a novel therapeutic clue to improve healing of skin ulcers.

Oncostatin M expression is functionally connected to neutrophils in the early inflammatory phase of skin repair: implications for normal and diabetes-impaired wounds.

In this study, we investigated the role of the cytokine oncostatin M (OSM) for wound biology. OSM and its specific OSM receptor subunit beta (OSMRbeta) were induced upon injury. OSM induction paralleled the early influx of polymorphonuclear neutrophils (PMN) into the wound. OSM protein was localized in PMN in very early wounds, whereas OSMRbeta could be detected on macrophages, keratinocytes, and fibroblasts later in repair. To establish a functional connection between PMN and OSM expression in wounds, we depleted mice from circulating PMN by injecting an anti-PMN monoclonal antibody (Ly-6G). PMN-depleted wounds were characterized by a nearly complete loss of OSM but not OSMRbeta mRNA and protein expression within the initial 16-24 hours after injury. PMN-rich chronic wounds from diabetic ob/ob mice were characterized by a strongly elevated OSM mRNA and protein expression as compared to healthy animals. Moreover, a leptin-mediated improvement of chronic wounds in ob/ob mice was paralleled by a complete inhibition of PMN influx associated again with a dramatic loss of OSM expression at the wound site. These data constitute strong evidence that OSM expression during wound inflammation is functionally connected to PMN infiltration.