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.     

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.     

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.     

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...     

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.     

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.     

Carbonate Ion-Enriched Hot Spring Water Promotes Skin Wound Healing in Nude Rats

Hot spring or hot spa bathing (Onsen) is a traditional therapy for the treatment of certain ailments. There is a common belief that hot spring bathing has therapeutic effects for wound healing, yet the underlying molecular mechanisms remain unclear. To examine this hypothesis, we investigated the effects of Nagano hot spring water (rich in carbonate ion, 42°C) on the healing process of the skin using a nude rat skin wound model. We found that hot spring bathing led to an enhanced healing speed compared to both the unbathed and hot-water (42°C) control groups. Histologically, the hot spring water group showed increased vessel density and reduced inflammatory cells in the granulation tissue of the wound area. Real-time RT-PCR analysis along with zymography revealed that the wound area of the hot spring water group exhibited a higher expression of matrix metalloproteinases-2 and -9 compared to the two other control groups. Furthermore, we found that the enhanced wound healing process induced by the carbonate ion-enriched hot spring water was mediated by thermal insulation and moisture maintenance. Our results provide the evidence that carbonate ion-enriched hot spring water is beneficial for the treatment of skin wounds.     

烧伤膏对小鼠皮肤烫伤创面愈合的修复作用研究

目的:探讨烧伤膏对烫伤小鼠皮肤创面修复的可能作用机制。方法:将50只小鼠随机分为5组:正常组、模型组,烧伤膏高、中、低剂量组。除正常组和模型组外,烧伤膏实验组小鼠每天外涂烧伤膏治疗,连用15天。治疗结束后,采用Masson染色技术检测各组小鼠皮肤厚度及细胞病理形态;采用生化法检测皮肤中羟脯氨酸(Hyp)含量;采用ELISA技术检测皮肤中的TNF-α,IL-1β、Collagen I、EGF和TGF-β1的含量。结果:与正常组相比,模型组小鼠皮肤烫伤创面可见表皮细胞脱落,结构模糊,细胞变性坏死,表皮和真皮厚度明显减少,胶原纤维变薄,排列紊乱无序,呈断裂状;烧伤后创面局部组织中Hyp、TNF-α、IL-1β的含量显著升高(P0.01,P0.01,P0.01),而EGF、TGF-β1和Collagen I含量均明显减少(P0.01,P0.01,P0.01),而应用烧伤膏治疗15天后,上述各项实验指标明显得到改善。结论:烧伤膏抑制创面早期感染,减少胶原蛋白的分解和促进后期创面修复的作用可能与抑制炎性细胞因子和增加皮肤相关生长因子表达的作用机制有关。     

Impaired Hair Growth and Wound Healing in Mice Lacking Thyroid Hormone Receptors

Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies.