Proinflammatory Cytokines and Skin Wound Healing in Mice

Molecular Biology - Skin wound healing is subject to an intricate regulation, involves many cell populations and molecular mediators, and is one of the key mechanisms that ensures the barrier...     

Mice Deficient in Urokinase-Type Plasminogen Activator Have Delayed Healing of Tympanic Membrane Perforations

Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA^−/−) or tPA (tPA^−/−) after TM perforation. Delayed healing of TM perforations was observed in uPA^−/− mice but not tPA^−/− mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA^−/− mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA^−/− mice are most likely due to the reduced generation of plasmin.     

Impaired Abdominal Wall Development and Deficient Wound Healing in Mice Lacking Aortic Carboxypeptidase-Like Protein

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.     

Leptin Promotes Wound Healing in the Skin

Introduction

Leptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.

Methods

Immunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.

Results

Ob-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.

Conclusion

Topically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR^−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR^−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR^+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of ¹²⁵I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR^−/− and u-PAR^+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR^+/+ cells, whereas it was present in the pericellular space around u-PAR^−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.     

Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2^LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2^LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2^LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.     

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 2^nd and 3^rd 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.     

Lactobacillus rhamnosus CGMCC 1.3724 (LPR) Improves Skin Wound Healing and Reduces Scar Formation in Mice

Probiotics and Antimicrobial Proteins - Skin wounds are an important clinical problem which affects millions of people worldwide. The search for new therapeutic approaches to improve wound healing...     

微小RNA miR-21在皮肤创伤愈合中的研究进展

微小RNA是一类真核细胞中广泛存在的内源性转录后调控分子,其在细胞的增殖、分化、凋亡、迁移等过程中发挥了重要的调控作用。皮肤创伤修复涉及复杂的细胞与分子的相互作用网络。近年来研究表明micro RNAs在皮肤创伤修复中发挥调控作用,引人关注。miR-21作为重要的癌基因是目前研究的最多的miRNAs分子之一,其在皮肤创伤修复中的作用研究也越来越受到重视。研究表明miR-21参与了细胞增殖与迁移、炎症反应、血管生成和细胞外基质合成等重要修复相关事件的调控。因此,阐明miR-21分子在正常皮肤创伤愈合中的作用,厘清miR-21表达失调在修复不足和修复过度中的功能,将深化我们对于皮肤创伤愈合基本理论的认识,并为促进创面愈合与防治修复不足和过度提供潜在的治疗靶点。本文就miR-21分子在正常皮肤创伤修复、慢性难愈性创面和增生性瘢痕中作用的研究进展进行综述展望。     

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

Background

Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds.

Methodology/Principal Findings

To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure.

Conclusions/Significance

Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.     

Wnt信号通路与皮肤创面愈合的关系

创面愈合是由炎性细胞、细胞因子等多种因素共同参与,涉及组织修复、再生、重建的一个复杂有序的病理生理过程。皮肤慢性创面的愈合仍然是临床研究的重点与热点,随着分子生物学的发展,对皮肤创面愈合机制的认识也逐渐深入。Wnt信号通路是一条由Wnt蛋白及其受体、调节蛋白等组成的高度保守的信号通路,参与细胞增殖、凋亡、分化等多种生物学过程。Wnt信号通路作为参与皮肤愈合的信号通路之一,被认为具有调控皮肤及其附属器的发育、诱导皮肤附件的形态发生、调节毛囊的周期生长、促进创面血管新生及上皮重塑等多方面的功能。因此本文试从炎性细胞、成纤维细胞、干细胞、血管新生、表皮新生与毛囊新生等方面对Wnt信号通路与皮肤创面愈合的关系作一综述。     

Wound Healing Improvement with PHD-2 Silenced Fibroblasts in Diabetic Mice

Background

Hypoxia-inducible factor 1α is the central regulator of the hypoxia-induced response which results in the up-regulation of angiogenic factors. Its activity is under precise regulation of prolyl-hydroxylase domain 2. We hypothesized that PHD2 silenced fibroblasts would increase the expression of angiogenic factors, which might contribute to the improvement of the diabetic wound healing.

Materials and Methods

50 dB/db mice were employed and randomly assigned into five groups with 10 mice in each: group 1 (untreated cell), group 2 (PHD2 silenced cell), group 3 (L-mimosine treated cells), group 4 (nontargeting siRNA treated cells) and group 5 (sham control). Fibroblasts were cultivated from the dermis of mice in each group and treated with PHD2 targeting siRNA, L-mimosine and non-targeting siRNA respectively. A fraction of the fibroblasts were employed to verify the silencing rate of PHD2 after 48 hours. The autologous fibroblasts (treated and untreated) labeled with adenovirus-GFP were implanted around the wound (Φ6mm), which was created on the dorsum of each mouse. The status of wounds was recorded periodically. Ten days postoperatively, 3 mice from each group were sacrificed and wound tissues were harvested. Molecular biological examinations were performed to evaluate the expressions of cytokines. 28 days postoperatively, the remaining mice were sacrificed. Histological examinations were performed to evaluate the densities of GFP+ cells and capillaries.

Results

The expression of PHD2 reduced to 12.5%, and the expressions of HIF-1α and VEGFa increased significantly after PHD2 siRNA treatment. With the increasing expressions of HIF-1α and VEGFa, the time to wound closure in group 2 was less than 2 weeks. Increased numbers of GFP+ cells and capillaries were observed in group 2.

Conclusion

PHD2 siRNA treatment not only increased the expression of HIF1α and VEGFa, but also improved the fibroblast proliferation. These effects might contribute to the improvement of the diabetic wound healing.     

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.     

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

Purpose

Macrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages.

Methods

Autologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl₂MDP-LIP). The presence of CD11b⁺ F4/80⁺ macrophages, α-smooth muscle actin⁺ (α-SMA⁺) myofibroblasts, CD31⁺ vascular endothelial cells and NG₂ ⁺ pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation.

Results

Sub-conjunctival Cl₂MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl₂MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion.

Conclusions

Macrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.