Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury

Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.     

p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression

Nitric oxide (NO) represents a short lived mediator that pivotally drives keratinocyte movements during cutaneous wound healing. In this study, we have identified p68 DEAD box RNA helicase (p68) from an NO-induced differential keratinocyte cDNA library. Subsequently, we have analyzed regulation of p68 by wound-associated mediators in human and murine keratinocytes. NO, serum, growth factors, and pro-inflammatory cytokines were potent inducers of p68 expression in the cells. p68 was constitutively expressed in the epithelial compartment of murine skin. Upon injury, we found a transient down-regulation of overall p68 protein in wound tissue. However, p68 did not completely disappear during early wound repair, as we found an expression of p68 protein in isolated wound margin tissue 24 h after wounding. Moreover, immunohistochemistry and cell fractionation analysis revealed a restricted localization of p68 in keratinocyte nuclei of the developing epithelium. Accordingly, cultured keratinocytes also showed a nuclear localization of the helicase. Moreover, confocal microscopy revealed a strong localization of p68 protein within the nucleoli of the cells. Functional analyses demonstrated that p68 strongly participated in keratinocyte proliferation and gene expression. Keratinocytes that constitutively overexpressed p68 protein were characterized by a marked increase in serum-induced proliferation and vascular endothelial growth factor expression, whereas down-regulation of endogenous p68 using small interfering RNA markedly attenuated serum-induced proliferation and vascular endothelial growth factor expression. Altogether, our results suggest a tightly controlled expression and nucleolar localization of p68 in keratinocytes in vitro and during skin repair in vivo that functionally contributes to keratinocyte proliferation and gene expression.     

The Mobilization and Recruitment of C-Kit+ Cells Contribute to Wound Healing after Surgery

Delayed wound healing is a serious clinical problem in patients after surgery. A recent study has demonstrated that bone marrow-derived c-kit-positive (c-kit⁺) cells play important roles in repairing and regenerating various tissues and organs. To examine the hypothesis that surgical injury induces the mobilization and recruitment of c-kit+ cells to accelerate wound healing. Mice were subjected to a left pneumonectomy. The mobilization of c-kit+ cells was monitored after surgery. Using green fluorescent protein (GFP⁺) bone marrow-transplanted chimera mice, we investigated further whether the mobilized c-kit+ cells were recruited to effect wound healing in a skin puncture model. The group with left pneumonectomies increased the c-kit⁺ and CD34⁺ stem cells in peripheral blood 24 h after surgery. At 3 days after surgery, the skin wound size was observed to be significantly smaller, and the number of bone marrow-derived GFP⁺ cells and GFP⁺/c-kit+ cells in the wound tissue was significantly greater in mice that had received pneumonectomies, as compared with those that had received a sham operation. Furthermore, some of these GFP⁺ cells were positively expressed specific markers of macrophages (F4/80), endothelial cells (CD31), and myofibroblasts (αSMA). The administration of AMD3100, an antagonist of a stromal-cell derived factor (SDF)-1/CXCR4 signaling pathway, reduced the number of GFP⁺ cells in wound tissue and completely negated the accelerated wound healing. Surgical injury induces the mobilization and recruitment of c-kit+ cells to contribute to wound healing. Regulating c-kit+ cells may provide a new approach that accelerates wound healing after surgery.     

Effects on neurite outgrowth and cell survival of a secreted fibroblast growth factor binding protein upregulated during spinal cord injury

The fibroblast growth factor binding protein (FGF-BP; GenBank accession no. NP_005121) is a secreted protein that mobilizes FGFs from the extracellular matrix, protects them from degradation, and enhances their biological activity. Several previous studies reported that FGF-BP is an early response gene upregulated during tissue repair processes including wound healing and atherogenesis. In this study we analyzed whether FGF-BP expression was impacted by spinal cord injury and could have an effect on neuronal cell viability. Immunohistochemical and in situ hybridization studies revealed a dramatic upregulation of FGF-BP protein and mRNA levels following unilateral hemisection and contusion injury of adult rat spinal cord. In spinal cord sections of laminectomized rats, increased FGF-BP expression was observed in the fibers and cell bodies ipsilateral to the lesion site but was absent in the uninjured spinal cord tissue contralateral to the lesion. Increased expression of FGF-BP was observed at all postinjury time points, examined with peak levels occurring at day 4, a time when injury-induced increased levels of FGF2 have also been reported to be maximal. Moreover, using PC12 cells as a neuronal model, we observed that exogenous FGF-BP increased the capacity of FGF2 to stimulate neurite outgrowth and to increase cell survival. At the molecular level, FGF-BP enhanced FGF2-induced protein tyrosine phosphorylation and AKT/PKB activation. Collectively, these results suggest that FGF-BP is an early response gene after spinal cord injury and that its upregulation in regenerating spinal cord tissue may provide a molecular mechanism for enhancing the initial FGF2-mediated neurotrophic effects occurring after such tissue damage.     

The healing skin wound: a novel site of action of the chemokine C10

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. For this purpose we generated a subtractive cDNA library from normal mouse back skin and 1-day full-thickness excisional wounds. One of the differentially expressed genes encodes the chemokine C10. Using Northern blotting, RNase protection assay and Western blotting, we confirmed the injury-induced expression of C10 at the mRNA and protein level. Maximal levels of C10 mRNA and protein were seen at day 1 after wounding, and expression levels subsequently declined. In situ hybridization and immunohistochemistry revealed expression of C10 in macrophages of the clot and the granulation tissue as well as in keratinocytes of the epidermis and the hair follicles at the wound edge. Since C10 is a potent chemoattractant for macrophages, our results suggest that this chemokine contributes to the strong macrophage influx observed in the healing skin wound.     

Deficiency of macrophage migration inhibitory factor gene delays healing of the medial collateral ligament: a biomechanical and biological study

The role played by macrophage migration inhibitory factor (MIF) in the process of wound healing is controversial. Besides, there have been no reports that investigated the expression or the role of MIF in the repair process after ligament injury. In this study, we hypothesized that the deficiency in MIF gene might delay ligament healing in mice. The aim of this study was to clarify this hypothesis using MIF gene-deficient mice (MIFKO) and murine model of injury to the medial collateral ligament (MCL). Biomechanical testing showed that the levels of mechanical properties were significantly lower in MIFKO than in wild-type mice (WT) on day 28 after injury. Levels of matrix metalloproteinase (MMP)-2 and -13 mRNA in the healing tissue were significantly lower in MIFKO than in WT on day 28 and on day 7, respectively. Histologically, healing tissues in MIFKO exhibited prolonged hypertrophy, poor vascularity, and prolonged increase in cell number compared with those in WT. Taken together, it was suggested that MIFKO exhibited delayed healing of the MCL, which might be caused by lower mRNA expression of MMP-2 and -13.     

Adipose mesenchymal stem cell-derived exosomes promote cell proliferation,migration, and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing

Cutaneous wounds, a type of soft tissue injury, are difficult to heal in aging. Differentiation, migration, proliferation, and apoptosis of skin cells are identified as key factors during wound healing processes. Mesenchymal stem cells have been documented as possible candidates for wound healing treatment because their use could augment the regenerative capacity of many tissues. However, the effects of exosomes derived from adipose-derived stem cell (ADSC-exos) on cutaneous wound healing remain to be carefully elucidated. In this present study, HaCaT cells were exposed to hydrogen peroxide (H₂O ₂) for the establishment of the skin lesion model. Cell Counting Kit-8 assay, migration assay, and flow cytometry assay were conducted to detect the biological function of ADSC-exos in skin lesion model. Finally, the possible mechanism was further investigated using Western blot assay. The successful construction of the skin lesion model was confirmed by results of the enhanced cell apoptosis of HaCaT cells induced by H ₂O ₂, the increased Bax expression and decreased Bcl-2 expression. CD9 and CD63 expression evidenced the existence of ADSC-exos. The results of functional experiments demonstrated that ADSC-exos could prompt cell proliferation and migration of HaCaT cells, and repress cell apoptosis of HaCaT cells. In addition, the activation of Wnt/β-catenin signaling was confirmed by the enhanced expression of β-catenin at the protein level. Collectively, our findings suggest that ADSC-exos play a positive role in cutaneous wound healing possibly via Wnt/β-catenin signaling. Our study may provide new insights into the therapeutic target for cutaneous wound healing.     

Changes in skin angiotensin II receptors in rats during wound healing.

Angiotensin (AII) is associated with increased vascular smooth muscle growth and we have found increased levels of tissue AII during healing of wounded skin. Here we have determined changes in skin AII receptors during wound healing in adult male Sprague-Dawley rats. An abdominal surgical incision was made under anesthesia and rats were sacrificed at different times after wounding. Specific binding of 125I-AII was significantly decreased at 12, 18 and 24 hours in the wounded tissue compared to control tissue from the same rat. By 3 days the binding had recovered to baseline levels. Receptors were mostly AT1, with a high and a low affinity site in the skin both in control and healing tissue. The Bmax of the high affinity site was significantly decreased in healing tissue but there was no significant change in Kd. Our results demonstrate that adult rat skin contains predominantly AT1 receptors and also that these receptors are downregulated for 12-24 hours after wounding.     

Zinc accumulation and metallothionein gene expression in the proliferating epidermis during wound healing in mouse skin

Metallothionein (MT), a low molecular weight metal-binding protein, has been related to zinc and copper metabolism, the acute-phase response, and cellular proliferation. In this study, we investigated changes in zinc metabolism and MT gene expression occurring in tissue damage and repair during wound healing in mouse skin. Northern blot analysis revealed that a significant increase of MT mRNA was observed in the liver for 18 h after wounding, and serum zinc downfall and hepatic zinc uptake were observed. In situ hybridization analysis showed that no significant expression of MT mRNA was detected within the first 9 h after wounding. However, it was expressed restrictively in the proliferating epidermis of the wound margin after 12 h. Zinc began to accumulate in wounded skin after MT gene expressed. Northern blotting and immunocytochemical staining revealed that MT has been synthesized actively during the growth phase compared with the stationary phase in normal human epidermal keratinocytes in vitro. Intracellular zinc accumulation was observed in the proliferating cells. We concluded that hepatic MT plays an important role as an acute phase protein against host damage, and epidermal MT contributes in the supply of zinc to wounded tissue and activates proliferation for the regeneration of epidermis. Accepted: 2 July 1999     

Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis

Recently, we demonstrated a strong upregulation of activin expression after skin injury. Furthermore, overexpression of this transforming growth factor beta family member in the skin of transgenic mice caused dermal fibrosis, epidermal hyperthickening and enhanced wound repair. However, the role of endogenous activin in wound healing has not been determined. To address this question we overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice. These animals were born with open eyes, and the adult mice had larger ears, longer tails and reduced body weight compared with non-transgenic littermates. Their skin was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was significantly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller than in control animals. These results implicate an important function of endogenous activin in the control of wound repair and scar formation.     

Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury

We have recently demonstrated that macrophage migration inhibitory factor (MIF) is a myocardial depressant protein and that MIF mediates late, prolonged cardiac dysfunction after endotoxin challenge in mice. Because many factors, including endotoxin, have been implicated in the pathogenesis of cardiac dysfunction after burn injury, we tested the hypothesis that MIF might also be the mediator of prolonged cardiac dysfunction in this model. At 4 h after 40% total body surface area burn in anesthetized mice, serum MIF levels increased significantly compared with baseline (2.2-fold). This increase was accompanied by a significant decrease in cardiac tissue MIF levels (2.1-fold decrease compared with controls). This pattern was consistent with MIF release from preformed cytoplasmic stores in the heart and other organs. To determine whether MIF mediates cardiac dysfunction after burn injury, mice were pretreated with anti-MIF neutralizing monoclonal antibodies or isotype control antibodies. Beginning 4 h after burn injury (and continuing through 48 h), burned mice demonstrated a significantly depressed left ventricular shortening fraction of 38.6 +/- 1.8%, compared with the normal controls (56.0 +/- 2.6%). Mice treated with anti-MIF displayed an initial depression of cardiac function similar to nontreated animals but then showed rapid restoration of cardiac function with complete recovery by 24 h, which persisted for the duration of the protocol. This study is the first to demonstrate that MIF mediates late, prolonged cardiac dysfunction after burn injury and suggests that MIF blockade should be considered a therapeutic target for the treatment of burn injury.     

Gene expression analysis in burn wounds of rats

The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.     

D‐dopachrome tautomerase in adipose tissue inflammation and wound repair

D‐dopachrome tautomerase (D‐DT/MIF‐2) is a member of the macrophage migration inhibitory factor (MIF) cytokine superfamily, and a close structural homolog of MIF. MIF and D‐DT have been reported to be involved in obesity, but there is little known about the regulation of D‐DT in adipose tissue inflammation and wound healing. Subcutaneous adipose tissue was collected from 54 healthy donors and 28 donors with acutely inflamed wounds undergoing wound debridement. In addition, epididymal fat pads of mice were injected with lipopolysaccharide to study receptor expression and cell migration in vivo. D‐DT protein levels and mRNA expression were significantly decreased in subcutaneous adipose tissue adjacent to acutely inflamed wounds. D‐DT improved fibroblast viability and increased proliferation in vitro. While D‐DT alone did not have a significant effect on in vitro fibroblast wound healing, simultaneous addition of neutralizing MIF antibody resulted in a significant improvement of fibroblast wound healing. Interestingly, expression of the MIF and D‐DT receptor CD74 was down‐regulated while the MIF receptors CXCR2 and CXCR4 were up‐regulated primarily on macrophages indicating that the MIF‐CXCR2/4 axis may promote recruitment of inflammatory cells into adipose tissue. Our results describe a reciprocal role of D‐DT to MIF in inflamed adipose tissue, and indicate that D‐DT may be beneficial in wound repair by improving fibroblast survival and proliferation.     

Changes in albumin precursor and heat shock protein 70 expression and their potential role in response to corneal epithelial wound repair

Many proteins displayed differential expression (either up- or down-regulation) when proteome of migrating and non-migrating epithelium was assessed using 2-DE and ESI-Q-TOF MS/MS. From the up-regulated set, we have identified for the first time a 69-kDa albumin precursor protein with four peptides sequences and 70-kDa heat shock protein (hsp70) with one peptide in the active phase of cell migration (48 h) during the healing process. Western blot analysis was used to further characterize these proteins at different phases (24, 48 and 72 h) of healing. An increase in the mRNA expression (measured using RT-PCR) in the active migration phase (48 h) for albumin precursor and hsp70 was also observed. Furthermore, co-immunoprecipitation studies with anti-albumin precursor and anti-hsp70 antibodies, followed by immunoblotting with anti-fibronectin antibody demonstrated a novel and biologically relevant interaction between albumin precursor protein and fibronectin in corneal epithelial wound healing but not with hsp70. The increased gene and protein expression of albumin and hsp70 during the active phase of cell migration (48 h) in the corneal epithelium suggests their possible role in corneal wound healing. These findings may have broader implications for developing therapeutic strategies for treating wound healing disorders.     

Stimulation of growth factor synthesis in skin wounds using tissue extract (G-90) from the earthworm Eissenia foetida

Growth factors are biologically-active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. In the case of wound healing, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate cell proliferation and stimulate the healing process. In this study we examined the efficacy of a glycolipoprotein tissue homogenate extract from Eisenia foetida (G-90) to activate signal transduction pathways, leading to wound healing. We measured the activation of EGF and FGF in healthy skin, in wounds with physiological healing and in wounds treated with G-90. The activation of EGF and FGF was measured during the first 24 h of wound healing under both physiological conditions and treatment with G-90. In both cases an increased concentration of EGF and FGF was observed 6 h after wounding. In comparison with healthy skin, the concentration of EGF increased 10-fold and FGF five-fold in wounds treated with G-90 (10 ng ml(-1)). Healing in physiological conditions resulted in a two-fold increase of EGF and 1.5-fold of FGF.