Previous studies of tissue repair have revealed osteopontin (OPN) to be up-regulated in association with the wound inflammatory response. We hypothesize that OPN may contribute to inflammation-associated fibrosis. In a series of in vitro and in vivo studies, we analyze the effects of blocking OPN expression at the wound, and determine which inflammatory cells, and which paracrine factors from these cells, may be responsible for triggering OPN expression in wound fibroblasts. Delivery of OPN antisense oligodeoxynucleotides into mouse skin wounds by release from Pluronic gel decreases OPN protein levels at the wound and results in accelerated healing and reduced granulation tissue formation and scarring. To identify which leukocytic lineages may be responsible for OPN expression, we cultured fibroblasts in macrophage-, neutrophil-, or mast cell–conditioned media (CM), and found that macrophage- and mast cell–secreted factors, specifically platelet-derived growth factor (PDGF), induced fibroblast OPN expression. Correspondingly, Gleevec, which blocks PDGF receptor signaling, and PDGF-Rβ–neutralizing antibodies, inhibited OPN induction by macrophage-CM. These studies indicate that inflammation-triggered expression of OPN both hinders the rate of repair and contributes to wound fibrosis. Thus, OPN and PDGF are potential targets for therapeutic modulation of skin repair to improve healing rate and quality.

Experimental downregulation of connexin43 (Cx43) expression at skin wound sites appears to markedly improve the rate and quality of healing, but the underlying mechanisms are currently unknown. Here, we have compared physiological and cell biological aspects of the repair process with and without Cx43 antisense oligodeoxynucleotide treatment. Treated wounds exhibited accelerated skin healing with significantly increased keratinocyte and fibroblast proliferation and migration. In vitro knockdown of Cx43 in a fibroblast wound-healing model also resulted in significantly faster healing, associated with increased mRNA for TGF-β1, and collagen α1 and general collagen content at the wound site. Treated wounds showed enhanced formation of granulation tissue and maturation with more rapid angiogenesis, myofibroblast differentiation and wound contraction appeared to be advanced by 2-3 days. Recruitment of both neutrophils and macrophages was markedly reduced within treated wounds, concomitant with reduced leukocyte infiltration. In turn, mRNA levels of CC chemokine ligand 2 and TNF-α were reduced in the treated wound. These data suggest that, by reducing Cx43 protein with Cx43-specific antisense oligodeoxynucleotides at wound sites early in the skin healing process repair is enhanced, at least in part, by accelerating cell migration and proliferation, and by attenuating inflammation and the additional damage it can cause.

Forkhead box O (Foxo) transcription factors may be involved in the salutary effect of dietary restriction (DR). This study examined the role of Foxo3 in lifespan extension and cancer suppression in DR mice. Wild-type (WT) and Foxo3-knockout heterozygous ((+/-) ) and homozygous ((-/-) ) mice were subjected to a 30% DR regimen initiated at 12 weeks of age. Control mice were fed ad libitum (AL) throughout the study. In contrast to WT mice, DR did not significantly extend the lifespan of Foxo3(+/-) or Foxo3(-/-) mice. However, DR reduced the prevalence of tumors at death in WT, Foxo3(+/-) , and Foxo3(-/-) mice. These results indicate the necessity of Foxo3 for lifespan extension but not cancer suppression by DR. The findings in Foxo3(+/-) mice contrast with those in Foxo1(+/-) mice reported previously by our laboratory suggest differential regulation of cancer and lifespan by DR via Foxo1 and Foxo3.

Cell-surface carbohydrate chains are known to contribute to cell migration, interactions, and proliferation, but their roles in skin wound healing have not been evaluated. We examined the biological roles of beta4-galactosylated carbohydrate chains in skin wound healing using mutant mice that lack beta-1,4-galactosyltransferase-I (beta4GalT-I), which is responsible for the biosynthesis of the type 2 chain in N-glycans and the core 2 branch in O-glycans. beta4GalT-I-deficient mice showed significantly delayed wound healing with reduced re-epithelialization, collagen synthesis, and angiogenesis, compared with control mice. Neutrophil and macrophage recruitment at wound sites was also impaired in these mice probably because of selectin-ligand deficiency. In accordance with the reduced leukocyte infiltration, the expression levels of macrophage-derived chemokines, transforming growth factor-beta1, and vascular endothelial growth factor were all reduced in beta4GalT-I(-/-) mice. These results demonstrate that beta4-galactosylated carbohydrate chains play a critical role in skin wound healing by mediating leukocyte infiltration and epidermal cell growth, which affects the production of chemokines and growth factors. This study introduces a suitable mouse model for investigating the molecular mechanisms of skin wound healing and is the first report showing that carbohydrate chains have a strong influence on skin wound healing.