The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE − / − mice before the development of manifest atherosclerotic lesions. Platelet–endothelial cell interaction involved both platelet glycoprotein (GP)Ibα and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE − / − mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.

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.

Wound-healing disorders are major complications of diabetes mellitus. Here, we investigated insulin-mediated signaling in nonwounded skin and in cutaneous tissue regeneration of healthy C57BL/6 and diabetesimpaired leptin-deficient obese/obese (ob/ob) mice. The insulin receptor (InsR) was abundantly expressed in wound margins and granulation tissue during acute healing in healthy mice. Remarkably, active signaling from the InsR, as assessed by phosphorylation of downstream targets such as protein tyrosine phosphatase-1B, glycogen synthase (GS), and GS kinase, was nearly absent in nonwounded and acutely healing skin from ob/ob mice. Systemic leptin administration to ob/ob mice reverted the diabetic phenotype and improved tissue regeneration as well as the impaired expression of InsR, insulin receptor substrate-1 and insulin receptor substrate-2, and downstream signaling (phosphorylation of GS kinase and GS) in late wounds and nonwounded skin of ob/ob mice. Importantly, tumor necrosis factor (TNF)-␣ was a mediator of insulin resistance in keratinocytes in vitro and in ob/ob wound tissue in vivo. Systemic administration of a monoclonal anti-TNF-␣ antibody (V1q) in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialization, and restored InsR expression and signaling in wound tissue of ob/ob mice. These data suggest that InsR signaling in diabetes-impaired wounds is sensitive to inflammatory conditions and that anti-inflammatory approaches, such as anti-TNF-␣ strategies, improve diabetic wound healing.

In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.

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.