Cutaneous Wound Healing In Mice Research Articles

A Synthetic Uric Acid Analog Accelerates Cutaneous Wound Healing in Mice

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Rotational Stress Impairs Cutaneous Wound Healing In Mice

ObjectiveThe aim of this study was to investigate the effects of the rotational stress on cutaneous wound healing in mice.MethodsMale mice were spun at 45rpm for 15minutes every hour from three days before wounding until euthanasia. Control animals were not submitted to stress. A full‐thickness lesion was created and measured. Fourteen days after, animals were killed and lesions were collected. Sections were stained with HE and immunostained against F4/80 and alpha‐smooth muscle actin to quantify macrophages and myofibroblasts, respectively. Data are expressed as mean±S.E.M and analyzed by Student's t test.ResultsFourteen days after wounding, wound area was greater in the stress group than in the control group (p<0.05), while re‐epithelialized wound area was smaller in the stress group than in the control group (p<0.05). Stressed mice presented less organized collagenous granulation tissue (rich in inflammatory cells) when compared with control mice. The macrophages number and myofibroblasts density was greater in the stress group than in the control group (p<0.05).ConclusionThe rotational stress delays the cutaneous wound healing in mice.

Factor VII deficiency impairs cutaneous wound healing in mice.

Skin keratinocytes express tissue factor (TF) and are highly associated with skin wound healing. Although it has been demonstrated that perivascular TF expression in granulation tissue formed after dermal injury is downregulated during healing, studies of the mechanism of factor (F) VII, a TF ligand, in skin wound healing are lacking. We reported the use of a dermal punch model to demonstrate that low-expressing FVII mice (approximately 1% of wild type [WT]) exhibited impaired skin wound healing compared with WT controls. These low-FVII mice showed defective reepithelialization and reduced inflammatory cell infiltration at wound sites. This attenuated reepithelialization was associated with diminished expression of the transcription factor early growth response 1 (Egr-1). In vitro, Egr-1 was shown to be essential for the FVIIa-induced regulation of keratinocyte migration and inflammation. Both Egr-1 upregulation and downstream inflammatory cytokine appearance in keratinocytes depended on FVIIa/TF/protease-activated receptor 2 (PAR-2)-induced signaling and did not require subsequent generation of FXa and thrombin. The participation of Egr-1 in FVIIa-mediated regulation of keratinocyte function was confirmed by use of Egr-1-deficient mice, wherein a significant delay in skin wound healing after injury was observed, relative to WT mice. The results from these studies demonstrate an in vivo mechanistic relationship between FVIIa, Egr-1 and the inflammatory response in keratinocyte function during the wound healing process.

Systemic Administration of IL-1 Receptor Antagonist Fails to Reduce Post Injury Fibrosis

Introduction: The ability to modulate excessive post injury fibrosis remains an elusive therapeutic target. Recent results reported by this laboratory demonstrate decreased fibrosis in deep tissue wounds in IL-1 receptor knockout mice. These findings were duplicated with direct administration of the recombinant human interleukin-1-receptor antagonist (rhIL-1Ra, Anakinra, Amgen) into the wounds of mice. The present study investigated whether systemic administration of rhIL-1Ra reduced fibrosis by examining the repair response in two different species using two different injury models: hepatic fibrosis in rats and cutaneous wound healing in mice.

MAPKAPK-2 Signaling Is Critical for Cutaneous Wound Healing

Cutaneous wound healing is a complex process, which is heavily dependent on successful inflammatory action. Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2 or MK2), a major substrate of p38 MAPK, has been shown to be a major player in multiple inflammatory diseases, but its role in cutaneous wound healing has not yet been explored. In this study, by comparing excisional wounds made on the backs of MK2 knockout (KO) and MK2 wild-type (WT) mice, we found that the kinetics of wound healing are significantly affected by the absence of MK2 (P=0.010 to P<0.001). Histological examination showed a higher level of acanthosis of the migrating wound keratinocyte layer as well as a higher level of collagen deposition in the granulation tissue of the wounds from MK2 WT mice compared with those from MK2 KO mice. Interestingly, although MK2 did not influence macrophage and neutrophil infiltration of the wounds, the expression of many cytokines and chemokines was significantly affected at different days post wounding. Furthermore, the delayed healing rate of wounds in MK2 KO mice can be significantly improved by passive transfer of macrophages with intact MK2. Overall, these results show a critical role for MK2 gene expression in macrophages participating in the process of cutaneous wound healing.

Rotational stress-induced increase in epinephrine levels delays cutaneous wound healing in mice

Stress impairs wound healing of cutaneous lesions; however, the mechanism is still unclear. The aim of this study was to evaluate the effects of rotational stress on cutaneous wound healing in mice and propose a mechanism. Male mice were spun at 45rpm for 15min every hour beginning 3days before wounding until euthanasia. Control animals were not subjected to stress. To confirm that catecholamines participate in stress-induced delay of wound healing, mice were treated daily with propranolol. An excisional lesion was created and measured. Seven and 14days later, animals were killed and lesions collected. Sections were stained with hematoxylin–eosin and immunostained for α-smooth muscle actin and proliferating cell nuclear antigen. Matrix metalloproteinase (MMP)-2 and -9 activity, nitrite levels, and tumor necrosis factor-α (TNF-α) expression were measured in the wounds. In addition, murine skin fibroblast cultures were treated with high levels of epinephrine and fibroblast activity was evaluated. Stressed mice exhibited reduced locomotor activity and increased normetanephrine plasma levels. Rotational stress was associated with decreased wound contraction, reduced re-epithelialization, reduced MMP-2 and MMP-9 activation, but with strongly increased nitrite levels. Furthermore, inflammatory cell infiltration, TNF-α expression, myofibroblastic differentiation, and angiogenesis were all delayed in the stress group. Propranolol administration reversed the deleterious effects of stress on wound contraction and re-epithelialization. High epinephrine concentrations increased murine skin fibroblast proliferation and nitric oxide synthesis, and strongly inhibited skin fibroblast migration and both pro- and active MMP-2. In conclusion, rotational stress impairs cutaneous wound healing due to epinephrine increased levels.

Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-α expression

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.

CD19, a Response Regulator of B Lymphocytes, Regulates Wound Healing through Hyaluronan-Induced TLR4 Signaling

Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-beta. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-beta through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing.

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

Impaired cutaneous wound healing in mice lacking tetranectin

Tetranectin was originally purified from human serum on the basis of plasminogen kringle 4-binding properties. Tetranectin enhances plasminogen activation by a tissue-type plasminogen activator so that it has been suggested to play a role in tissue remodeling. We have generated mice with a targeted disruption of the tetranectin gene to elucidate the biological function of tetranectin. In this study, we showed that wound healing was markedly delayed in tetranectin-null mice compared with wild-type mice. A single full-thickness incision was made in the dorsal skin. By 14 days after the incision, the wounds fully healed in all wild-type mice based on the macroscopic closure; in contrast, the progress of wound healing in the tetranectin null mice appeared to be impaired. In histological analysis, wounds of wild-type mice showed complete reepithelialization and healed by 14 days after the incision. However, those of tetranectin-null mice never showed complete reepithelialization at 14 days. At 21 days after the injury, the wound healed and was covered with an epidermis. These results supported the fact that tetranectin may play a role in the wound healing process.

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII ( Col18a1 −/− ). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1 −/− mice, and the vascularisation rate was accelerated in the Col18a1 −/− mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half ( n = 10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal–dermal junction in the presence of an excess of endostatin.

Methanol Extracts of Stewartia koreana enhance angiogenesis and accelerate cutaneous wound healing in mice

Methanol Extracts of <i>Stewartia koreana</i> enhance angiogenesis and accelerate cutaneous wound healing in mice

Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor

Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this "stop and return" process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (-/-) mice. Even at 90 days, long after wound closure, wounds in the CXCR3(-/-) mice remained hypercellular and presented immature matrix components. The CXCR3(-/-) mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair.

Delayed Cutaneous Wound Healing in Mice Lacking Solute Carrier 11a1 (Formerly Nramp1): Correlation with Decreased Expression of Secretory Leukocyte Protease Inhibitor

Control of macrophage functions by natural resistance-associated macrophage protein 1 (NRAMP1) has proven to be important for murine resistance to several intracellular pathogens, including Mycobacterium bovis BCG and Salmonella typhimurium, although the exact molecular mechanism of its action remains unknown. We identified secretory leukocyte protease inhibitor (SLPI) as a novel candidate gene whose expression is dependent on Nramp1 gene expression using B10A.Nramp1+/+ and B10A.Nramp1-/- macrophage cell lines in vitro, as well as mice bearing the resistance alleles (wild type (WT)) of the Nramp1 and mice with an ablated Nramp1 gene (knockout (KO)). We established that B10A.Nramp1+/+ cells spontaneously expressed a 10-fold higher level of SLPI messenger RNA (mRNA) compared to B10A.Nramp1-/- expression. Similarly, protein secretion was detected only in supernatants from B10A.Nramp1+/+ macrophages. Induction of SLPI in excisional cutaneous wounds and, most importantly, in macrophages infiltrating these wounds was significantly higher in Nramp1 WT mice compared to KO mice. These differences in SLPI expression in vivo correlated with a significant delay in the kinetics of wound healing in Nramp1 KO mice compared to WT controls. Taken together, these results suggest for the first time that Nramp1 controls macrophage SLPI mRNA and protein expression, and can also have an important effect on the kinetics of wound healing.

The Effects of ZD6474, an Inhibitor of VEGF Signaling, on Cutaneous Wound Healing in Mice 1

ZD6474 is an inhibitor of the VEGFR-2 receptor tyrosine kinase with additional activity against EGFR-1 receptor tyrosine kinases that has been shown to inhibit tumor growth and wound-induced neovascularization in pre-clinical studies and phase I clinical trials. The purpose of this study was to determine the effects of ZD6474 on breaking strength in a murine model of cutaneous wound healing. Balb/C mice were given ZD6474 (50 or 100 mg/kg p.o.) or vehicle starting 7 days before wounding. Two full-thickness incisions were made in each mouse and closed using suture. On post-wounding day 7 or 28, laser Doppler blood flow measurements were made, and the breaking strength of the wounded skin was determined. Microvessel density measurements were performed using computer image analysis of CD31-stained sections. Compared with controls, mice treated with ZD6474 showed a significantly reduced dose-dependent decline in breaking strength, both at POD 7 (P < 0.001) and at POD 28 (P < 0.005). Histologically, the ZD6474-treated mice showed a qualitative reduction in the degree of fibrosis and epithelial proliferation at the wound site, but no significant difference was noted between the 50 mg/kg and 100 mg/kg ZD6474-treated groups. Also, microvessel density measurements demonstrated no significant difference between groups. In a murine model of wound healing, ZD6474 treatment did not prevent wound healing, but was associated with a reduced skin breaking strength compared with vehicle-treated controls at both 7 and 28 days post-wounding. These observations may have clinical relevance for the perioperative management of patients treated with inhibitors of angiogenesis.

Possible involvement of mast cells in collagen remodeling in the late phase of cutaneous wound healing in mice

We examined possible roles of mast cells in cutaneous wound healing using mast cell deficient (W/Wv) mice and their normal littermates (+/+). A round full-thickness wound was made on the back skin of these mice. The wounds closed completely within 20 days, and there was no difference in wound contraction between +/+ and W/Wv mice during the wound healing. While either chymase or tryptase activities were hardly detectable in W/Wv mice, chymase activities decreased at the impaired sites and recovered to the control level within 20 days in +/+ mice. Tryptase activities were higher than the control level on day 15 and day 20 in +/+ mice. Histological observations on day 15 and day 20 in +/+ mice revealed that mast cells were abundant at the wound edges but absent at the center. The latent and the active forms of MMP-2 and MMP-9 increased on day 10 and day 15 but recovered nearly to control levels on day 20 in both mice groups. The hydroxyproline contents in W/Wv mice were significantly higher than those in +/+ mice on day 15 and day 20. Furthermore, histological observations revealed that the collagen aggregation at the wound edges was tighter and less interwoven in W/Wv mice compared with +/+ mice. These results suggest that mast cells accumulated at the wound edge may participate in tissue remodeling in the late phase of wound healing.

Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.

Temporally controlled expression of genes in transgenic mice has advanced our understanding of many physiological processes. One of the more common modes of acutely altering gene levels involves the doxycycline-regulated "tet-on/tet-off" systems. There has been concern that the administration of doxycycline in itself might compromise many aspects of wound repair. Here we report that oral ad libitum administration of doxycycline (2 mg/ml in drinking water) to mice does not alter dermal or epidermal wound healing. The healing of both full- and partial-thickness skin wounds proceeded similarly regardless of doxycycline administration; in fact, collagen organization and tensile strength development appeared to accelerate in the presence of doxycycline. These data suggest that wound healing studies incorporating transgene expression can utilize tet-on/tet-off regulation of gene expression without interference from doxycycline.

Restraint Stress Slows Cutaneous Wound Healing in Mice

The impact of stress on cutaneous wound healing was assessed in a murine model. Female, hairless SKH-1 mice, 6–8 weeks of age were subjected to restraint stress (RST) 3 days before and for 5 days following dorsal application of a 3.5-mm sterile punch wound. Control mice were wounded, but not restrained. Using photography and image analysis, the rate of wound healing was compared between the two groups. Wounds on control mice healed on average 3.10 days sooner than RST-treated mice. In addition, cross-sectional, morphometric analysis of the dermal and epidermal layers revealed reduced inflammation surrounding wounds from RST mice at 1, 3, and 5 days after wounding. In the RST group, serum corticosterone levels averaged 162.5 ng/ml compared to 35.7 ng/ml in the controls. Treatment of RST-stressed animals with the glucocorticoid receptor antagonist RU40555 resulted in healing rates comparable to those of control animals. Thus, the reduction in inflammation and delayed healing correlated with serum corticosterone levels and suggest that disruption of neuroendocrine homeostasis modulates wound healing.