Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.
ABSTRACT: Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-? during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1? and TNF-? between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-?1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-?1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression of wound closure.
Project description:Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO-1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO-2 deficient mice is impaired with exorbitant inflammation and absence of HO-1 expression. This study addresses the role of HO-2 in cutaneous excisional wound healing using HO-2 knockout (KO) mice. Here, we show that HO-2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO-2 KO mice compared to WT controls. Surprisingly, wound closure in HO-2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO-1 induction in HO-2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C-X-C) ligand-11 (CXCL-11) in wounds of HO-2 KO mice. Abnormal regulation of CXCL-11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL-11 expression in HO-2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.
Project description:Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.
Project description:Cutaneous wounds heal more slowly in elderly males than in elderly females, suggesting a role for sex hormones in the healing process. Indeed, androgen/androgen receptor (AR) signaling has been shown to inhibit cutaneous wound healing. AR is expressed in several cell types in healing skin, including keratinocytes, dermal fibroblasts, and infiltrating macrophages, but the exact role of androgen/AR signaling in these different cell types remains unclear. To address this question, we generated and studied cutaneous wound healing in cell-specific AR knockout (ARKO) mice. General and myeloid-specific ARKO mice exhibited accelerated wound healing compared with WT mice, whereas keratinocyte- and fibroblast-specific ARKO mice did not. Importantly, the rate of wound healing in the general ARKO mice was dependent on AR and not serum androgen levels. Interestingly, although dispensable for wound closure, keratinocyte AR promoted re-epithelialization, while fibroblast AR suppressed it. Further analysis indicated that AR suppressed wound healing by enhancing the inflammatory response through a localized increase in TNF-alpha expression. Furthermore, AR enhanced local TNF-alpha expression via multiple mechanisms, including increasing the inflammatory monocyte population, enhancing monocyte chemotaxis by upregulating CCR2 expression, and enhancing TNF-alpha expression in macrophages. Finally, targeting AR by topical application of a compound (ASC-J9) that degrades AR protein resulted in accelerated healing, suggesting a potential new therapeutic approach that may lead to better treatment of wound healing.
Project description:Damage to cutaneous nerves inhibits wound healing in patients. Results from animals on the nerve contributions to healing are various and sometimes contradictory. Here, we aim to clearly define the collective role of central, caudal, and rostral nerves in ear wound healing of mice, rats, and rabbits. These wounds heal with minimal contraction like wounds in humans. We resected central, caudal, and rostral nerves at the base of ear pinnae by microsurgery and created excisional full-thickness skin wounds in the pinnae neurologically downstream from the resections. Denervation in mice resulted in no closure for 14 days post-wounding (dpw) and led to only 17.2% closure at 21 dpw when the excisional wounds of non-denervated ear pinnae were completely closed. Compared to excisional wounds that were not denervated in sham surgery, wounds with denervation showed an increase of excisional wound areas for 5.0% by 7 dpw and a 43.7% reduction of wound closure at 12 dpw for rats. In rabbits, denervation attenuated wound closure for 14.2, 34.4, and 28.3% at 7, 14, and 18 dpw, respectively. Our histological analysis showed marked denervation impairment in pivotal healing processes, re-epithelialization and granulation tissue growth, suggesting denervation impairment in the regeneration of blood capillaries and/or connective tissue in wounds. These results reveal the critical contributions made by central, caudal, and rostral nerves in ear pinnae to minimal-contraction skin wound healing. Our study also provides small animal models of minimal-contraction wound healing of denervated ear skins that recapitulate human wound healing involving surgical or traumatic nerve damages.
Project description:Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.
Project description:Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5-2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8-3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.
Project description:Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit(W-sh/W-sh), WBB6F1-Kit(W/W-v), and Cpa3-Cre; Mcl-1(fl/fl) mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson's Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.
Project description:Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-? (TGF-?) modulation. Using an adult fmod-null (fmod(-/-)) mouse model, this study further elucidates the interplay between FMOD and TGF-? expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod(-/-) and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod(-/-) mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod(-/-) wounds exhibited early elevation (within 24? hours post-wounding) of type I and type II TGF-? receptors as well as unexpectedly high fibroblast expression of TGF-?3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-?3, fmod(-/-) fibroblasts were significantly less motile than WT fibroblasts. fmod(-/-) fibroblasts were also more susceptible to migration inhibition by TGF-?3, leading to profound delays in dermal cell migration. Increased scarring in fmod(-/-) mice indicates that TGF-?3's antimotility effects predominate over its antifibrotic effects when high TGF-?3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-? bioactivity.
Project description:BACKGROUND:When the deer antler is cast, it leaves a cutaneous wound that can achieve scarless healing due to the presence of antler stem cells (ASCs). This provides an opportunity to study regenerative wound healing. METHODS:In this study, we investigated the therapeutic effects and mechanism of antler stem cell-conditioned medium (ASC-CM) on cutaneous wound healing in rats. In vitro, we investigated the effects of the ASC-CM on proliferation of HUVEC and NIH-3T3 cell lines. In vivo, we evaluated the effects of ASC-CM on cutaneous wound healing using full-thickness skin punch-cut wounds in rats. RESULTS:The results showed that ASC-CM significantly stimulated proliferation of the HUVEC and NIH-3T3 cells in vitro. In vivo, completion of healing of the rat wounds treated with ASC-CM was on day 16 (±?3?days), 9?days (±?2?days) earlier than the control group (DMEM); the area of the wounds treated with ASC-CM was significantly smaller (p?<?0.05) than the two control groups. Further molecular characterization showed that the ratios of Col3A1/Col1A2, TGF-?3/TGF-?1, MMP1/TIMP1, and MMP3/TIMP1 significantly increased (p?<?0.01) in the healed tissue in the ASC-CM group. CONCLUSIONS:In conclusion, ASC-CM effectively accelerated the wound closure rate and enhanced the quality of healing, which might be through transforming wound dermal fibroblasts into the fetal counterparts. Therefore, the ASC-CM may have potential to be developed as a novel cell-free therapeutic for scarless wound healing.
Project description:Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2-deficient mice (CCR2-/- ) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2-/- and WT mice revealed a significant decrease in inflammatory, Ly6CHi recruited monocyte/macrophages in CCR2-/- wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2-/- wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2+/+ and CCR2-/- mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2+/+ and CCR2-/- mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2-deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.