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-␣ 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. Journal of Cell Science 5194 the wound and lay down collagen matrix. We observed a decrease in neutrophil infiltration and a concomitant reduction in CC chemokine ligand 2 (Ccl2) and cytokine tumor necrosis factor ␣ (TNF-␣) mRNA. Subsequently, we saw a reduced recruitment of macrophages perhaps as a consequence of damping down of the initial inflammatory response, which is known to have downstream effects on the ensuing healing process. Together these modified responses resulted in significantly improved wound healing. ResultsDownregulation of Cx43 at wound sites with Cx43-asODN As previously reported, Cx43 was found to be predominantly expressed in the lower and middle spinous cell layers of the epidermis and in fibroblasts, blood vessels and dermal appendages of intact skin. Six hours after the injury Cx43 was expressed in hyperproliferative epidermis but began to be downregulated in the leading edge keratinocytes (Goliger and Paul, 1995;Coutinho et al., 2003). Delivery of Cx43-asODN from the time of injury markedly reduced protein levels of Cx43 in the epidermis and dermis within 2 hours of treatment (as revealed by immunohistochemistry) (Qiu et al., 2003). Such a rapid knockdown is possible because Cx43 protein is turned over rapidly, sometimes within 1.5-2 hours (Laird et al., 1991; Gaitta et al., 2002). To quantify the extent of Cx43 protein and mRNA knockdown and recovery after asODN treatment more precisely, we compared expression levels of Cx43 mRNA at treated and untreated wound sites by real-time PCR (RT-PCR; Fig. 1). One day after injury, expression of Cx43 mRNA at Cx43-asODN-treated wounds was significantly reduced by comparison with control sODN-treated wounds (2.95 versus 4.7 units, respectively, a 37% reduction; P<0.05). By 7 days after the injury, however, expression levels were similar in the two wound regimes (4.6 versus 5.2 units for asODN-and control sODN-treated, respectively). Immunostaining of wounds for Cx43 at 1 day, 2 days and 7 days after wounding revealed very low levels of Cx43 protein in the epidermis and dermis of the Cx43-asODN-treated wound edge at day 1 compared with controls ( Fig. 1). By day 2, some Cx43 staining had returned to the dermis of the Cx43-asODN-treated wound but the level was still very low in the epidermis. By day 7, in agreement with the RT-PCR findings, there was no obvious difference in Cx43 staining between treated and untreated wounds. T...

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.

To clarify biological roles of tumor necrosis factor receptor p55 (TNF-Rp55) -mediated signals in wound healing, skin excisions were prepared in BALB/c (WT) and TNF-Rp55-deficient (KO) mice. In WT mice, the wound area was reduced to 50% of the original area 6 days after injury, with angiogenesis and collagen accumulation. Histopathologically, reepithelialization rate was approximately 80% 6 days. Myeloperoxidase activity and macrophage recruitment were the most evident 1 and 6 days after injury, respectively. Gene expression of adhesion molecules, interleukin 1alpha (IL-1alpha), IL-1beta, monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-2, transforming growth factor beta1 (TGF-beta1) connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), Flt-1, and Flk-1 was enhanced at the wound site. In KO mice, an enhancement in angiogenesis, collagen content, and reepithelialization was accelerated with the increased gene expression of TGF-beta1, CTGF, VEGF, Flt-1, and Flk-1 at the wound sites, resulting in accelerated wound healing compared with WT mice. In contrast, leukocyte infiltration, mRNA expression of adhesion molecules, and cytokines were significantly reduced in KO mice. These observations suggest that TNF-Rp55-mediated signals have some role in promoting leukocyte infiltration at the wound site and negatively affect wound healing, probably by reducing angiogenesis and collagen accumulation.

The healing of a skin wound is a complex process involving many cell lineages. In adult tissues, repair is always accompanied by a robust inflammatory response, which is necessary to counter the potential for infection at any site where the skin barrier is breached. Unlike embryonic tissues that can repair perfectly without a remnant scar at the wound site, adult tissue repair always leads to formation of a fibrotic scar where the wound has healed. In recent years, it has become clear that the wound inflammatory response may be, at least in part, responsible for fibrosis at sites of tissue repair. In this review, we consider the beneficial vs the detrimental functions of inflammatory cells during the repair response and compare data from other tissues, the lung, and liver, where fibrosis and its resolution may be related to a damage-triggered inflammatory response. We also consider how it may be possible to molecularly disentangle the potentially good from the bad influences of inflammatory cells during tissue repair and how fundamental studies in inflammatory cell biology may prove the way forward for development of drug targets in this respect.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further de®ne the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines diering only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM 7/7 DT40 cells displayed retarded cellular proliferation, defective G 2 /M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM 7/7 DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but signi®cant reduction in targeted integration frequency was observed in ATM 7/7 DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM de®cient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.