Spatiotemporal expression of periostin during skin development and incisional wound healing: lessons for human fibrotic scar formation

Differentiation of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from embryonic through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the embryonic deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming growth factor beta 1 inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.     

Periostin localizes to cells in normal skin, but is associated with the extracellular matrix during wound repair

Epidermal tissue repair represents a complex series of temporal and dynamic events resulting in wound closure. Matricellular proteins, not normally expressed in quiescent adult tissues, play a pivotal role in wound repair and associated extracellular matrix remodeling by modulating the adhesion, migration, intracellular signaling, and gene expression of inflammatory cells, pericytes, fibroblasts and keratinocytes. Several matricellular proteins show temporal expression during dermal wound repair, but the expression pattern of the recently identified matricellular protein, periostin, has not yet been characterized. The primary aim of this study was to assess whether periostin protein is present in healthy human skin or in pathological remodeling (Nevus). The second aim was to determine if periostin is expressed during dermal wound repair. Using immunohistochemistry, periostin reactivity was detected in the keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin. In pathological nevus samples, periostin was present in the extracellular matrix. In excisional wounds in mice, periostin protein was first detected in the granulation tissue at day 3, with levels peaking at day 7. Periostin protein co-localized with α-smooth muscle actin-positive cells and keratinocytes, but not CD68 positive inflammatory cells. We conclude that periostin is normally expressed at the cellular level in human and murine skin, but additionally becomes extracellular during tissue remodeling. Periostin may represent a new therapeutic target for modulating the wound repair process.     

Animal models of skin regeneration

Cutaneous injury in the majority of vertebrate animals results in the formation of a scar in the post-injured area. Scar tissues, although beneficial for maintaining integrity of the post-wounded region often interferes with full recovery of injured tissues. The goal of wound-healing studies is to identify mechanisms to redirect reparative pathways from debilitating scar formation to regenerative pathways that lead to normal functionality. To perform such studies models of regeneration, which are rare in mammals, are required. In this review we discussed skin regenerative capabilities present in lower vertebrates and in models of skin scar-free healing in mammals, e.g. mammalian fetuses. However, we especially focused on the attributes of two unusual models of skin scar-free healing capabilities that occur in adult mammals, that is, those associated with nude, FOXN1-deficient mice and in wild-type African spiny mice.     

In vitro study of the impact of mechanical tension on the dermal fibroblast phenotype in the context of skin wound healing

Skin wound healing is finely regulated by both matrix synthesis and degradation which are governed by dermal fibroblast activity. Actually, fibroblasts synthesize numerous extracellular matrix proteins (i.e., collagens), remodeling enzymes and their inhibitors. Moreover, they differentiate into myofibroblasts and are able to develop endogenous forces at the wound site. Such forces are crucial during skin wound healing and have been widely investigated. However, few studies have focused on the effect of exogenous mechanical tension on the dermal fibroblast phenotype, which is the objective of the present paper. To this end, an exogenous, defined, cyclic and uniaxial mechanical strain was applied to fibroblasts cultured as scratch-wounded monolayers. Results showed that fibroblasts? response was characterized by both an increase in procollagen type-I and TIMP-1 synthesis, and a decrease in MMP-1 synthesis. The monitoring of scratch-wounded monolayers did not show any decrease in kinetics of the filling up when mechanical tension was applied. Additional results obtained with proliferating fibroblasts and confluent monolayer indicated that mechanical tension-induced response of fibroblasts depends on their culture conditions. In conclusion, mechanical tension leads to the differentiation of dermal fibroblasts and may increase their wound-healing capacities. So, the exogenous uniaxial and cyclic mechanical tension reported in the present study may be considered in order to improve skin wound healing.     

Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow

Myofibroblasts play a key role in wound closure but their origin is poorly understood. To investigate whether fibrocytes contribute to myofibroblast population, we examined the phenotype of fibrocytes and myofibroblasts present in the wounded skin of BALB/c mice. During wound healing, there was a marked increase in the number of cells expressing the myofibroblast marker alpha-smooth muscle actin in the granulation tissue. Between 4 and 7 days post-wounding, more than 50% of these cells also expressed the CD13 antigen. CD13(+)/collagen I+ fibrocytes could be isolated at an early stage of the healing process from digested fragments of wounded tissue by fluorescence-activated cell sorting. Like authentic fibrocytes, these cells were also CD45(+)/CD34(+)/CD14-. Between 4 and 7 days post-injury, 61.4% of the isolated fibrocytes were found to express alpha-smooth muscle actin gene and protein. We repeated similar experiments in female mice that had received a male whole bone marrow transplant after total body irradiation. By in situ hybridization, we identified the Y chromosome in the nuclei of the majority of fibrocytes isolated from the wounded tissue of these animals. Our data indicate that circulating fibrocytes contribute to the myofibroblast population in the wounded skin and that they originate from the bone marrow.     

Regional differences in mitotic activity due to injury in mouse skin

Summary Mitotic activity adjacent to a wound inflicted at different sites in the mouse skin was measured 24 h after injury. A regional difference in the epidermal mitotic activity due to injury was noted. Mitotic activity was high in the anterior parts of the body including the head, lower in the middle to posterior regions of the body and lowest in the posterior-most parts of the body. Regional differences in epidermal mitotic activity due to injury were demonstrated in both female and male mice. The existence of a cranio-caudal gradient in epidermal response to injury is suggested.     

Beneficial effect of gaseous nitric oxide on the healing of skin wounds

Intermittent daily exposures (60 s) to NO-containing gas flow (NO dose of 500 ppm) generated by air-plasma unit "Plason" improves healing of skin wounds in rats. The gas flow treatment shortened the recovery time of both aseptic and purulent wounds (300 mm2 area) by nearly a third. The treatment allows to achieve a marked improvement in the histological, histochemical, and electron-microscopic characteristics of the affected tissue. The mechanism of this phenomenon was studied by spin trapping method. The NO status of the wound tissue was investigated with EPR by following the formation of paramagnetic mononitrosyl complexes with iron-diethyldithiocarbamate, or with the heme groups in hemoglobin or myoglobin. For the first 5 min after a gas treatment with the exposure of 60s, detectable NO levels in the affected tissue were slightly lowered with respect to untreated controls. At subsequent times, treated tissues showed the formation of large quantities of nitroso-iron complexes: At 30-40 min after gas exposure, their levels were nearly two orders of magnitude higher than soon after (15 s-5 min) the exposure. The data demonstrate that the accumulation of nitrosyl-iron complexes reflects a sharp rise in endogenous NO production inside the affected tissue. Paradoxically, the beneficial effect of gaseous NO treatment can be mediated by the formation of limited quantities of peroxynitrite due to the reaction between exogenous NO and superoxide anions generated in high amount in wound tissue. This peroxynitrite has a strong prooxidant effect and can activate various antioxidant systems which diminish the amount of superoxide anions in wound tissue. The reduced superoxide levels allow to increase the contents of endogenous NO in gas-treated tissues. Therefore, the beneficial action of the treatment is attributed to enhanced NO bioavailability.     

外泌体在皮肤修复与再生中作用的研究进展

外泌体是直径在30-100 nm左右的囊泡结构。作为一种活细胞分泌的亚细胞成分,外泌体广泛参与细胞之间的交流,并可以作为干细胞的旁分泌因子来发挥生物学效应。研究发现外泌体可以参与皮肤组织修复与再生的各个过程,通过促进皮肤细胞的增殖迁移,促进血管新生,调节免疫反应来促进创伤愈合与皮肤组织再生,为进一步实现无细胞治疗提供了新的实现途径。对于某些慢性创面,例如糖尿病性皮肤溃疡等也有较好的治疗效果。本文就外泌体在皮肤修复与再生中作用的研究进展做一综述。     

Sus Scrofa immune tissues as a new source of bioactive substances for skin wound healing

Influence of a new protein-peptide complex on promoting skin wound healing in male BALB/c mice was studied. Protein-peptide complex, extracted from Sus scrofa immune organs, was percutaneously administered using two methods: by lecithin gel-like liquid crystals and by liquid microemulsion. On the fifth day, wound closure in mice with a linear wound model become faster in group (less 2 days comparison to other ones), which was treated with lecithin liquid crystals carrying the protein-peptide complex. This promoting healing can be caused by resorption of bioactive high-molecular compounds the animal skin. In mice with the linear wound model, the tensile strength of the scars were respectively higher both in mice, treated using lecithin liquid crystals with protein-peptide complex, and in mice, treated using microemulsion containing protein-peptide complex, by 215.4% and 161.5% relative to the animals, which did not receive bioactive substances for wound treatment. It was associated with the regeneratory effects of tissue- and species-specific protein-peptide complexes, including α-thymosin Sus scrofa (C3VVV8_PIG, m/z 3802.8) and other factors, which were described as parts of the new extracted complex. This reveals that percutaneous administration of the complex reliably activates local regenerative processes in animals.     

蝇蛆油促进大鼠皮肤机械损伤愈合作用及机制研究

目的:本文拟研究蝇蛆油对皮肤机械损伤的治疗作用及作用机制。方法:SD大鼠随机分为正常组、模型组、蝇蛆油组和重组牛碱性成纤维细胞生长因子(rb-bFGF)凝胶组,制作大鼠皮肤机械性损伤模型,在不同时间点采集样本,检测创面愈合率、创面愈合时间、血管内皮生长因子(VEGF)含量、丙二醛(MDA)和超氧化物歧化酶(SOD)水平,并检测组织中胶原蛋白Ⅰ、胶原蛋白Ⅲ、血小板衍生因子(PDGF)和细胞角蛋白19(CK19)表达情况。结果:模型组大鼠创面愈合时间为29.5±2.6 d,而蝇蛆油创面愈合时间为22.4±2.8 d,存在显著差异(P0.01)。VEDF含量在模型组和给药组均随着创面愈合时间增加,给药组在第1 d即显著高于模型组(P0.01),并在第21 d达到最大值(2051.5±148.2 ng/L)。模型组大鼠创面组织中MDA含量为6.47±0.92 nmo L/mg,SOD含量为7.52±3.21 U/mg,而蝇蛆油给药组大鼠创面组织中MDA含量为3.42±0.83 nmo L/mg,SOD含量为21.32±2.94 U/mg,存在显著差异(P0.01)。进一步研究发现,与模型组比较,蝇蛆油给药组大鼠创面组织中胶原蛋白Ⅰ、胶原蛋白Ⅲ、PDGF和CK19含量均显著升高(P0.01)。结论:蝇蛆油能够促进机械性损伤皮肤创面愈合,其作用机制可能是通过促进血管生成,抗氧化损伤,促进胶原生成,诱导干细胞形成,从而促进创面愈合。     

Adenovirus-mediated RNA interference against collagen-specific molecular chaperone 47-KDa heat shock protein suppresses scar formation on mouse wounds

It has been shown in many investigations that the abnormally increasing production and deposition of collagen is one of the important mechanisms of pathological scars and other fibrotic diseases [Wang Z, Inokuchi T, Nemoto TK, Uehara M, Baba TT. Antisense oligonucleotide against collagen-specific molecular chaperone 47-kDa heat shock protein suppresses scar formation in rat wounds. Plast Reconstr Surg 2003 May; 111(6):1980-7; Obayashi K, Akamatsu H, Okano Y, Matsunage K, Masaki H. Exogenous nitric oxide enhances the synthesis of type I collagen and heat shock protein 47 by normal human dermal fibroblasts. J Dermatol Sci 2006 Feb; 41(2): 121-6 [e pub. 2005 Sep 19]; Kakugawa T, Mukae H, Hayashi T, Ishii H, Nakayama S, Sakamoto N, et-al. Expression of HSP47 in usual interstitial pneumonia and nonspecific interstitial pneumonia. Respir Res 2005 Jun;14(6): 57; Razzaque MS, Taguchi T. The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999 Oct; 14(4): 1199-1212; Sharp PA. RNA interference - 2001. Genes Dev 2001 Mar 1; 15(5): 485-90; Ohashi S, Abe H, Takahashi T, Yamamoto Y, Takeuchi M, Arai H, et-al. Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy. J Biol Chem 2004 May 7; 279(19): 19816-23 [epub 2004 Mar 5]]. RNA interference is the process that double-stranded RNA induces the homology-dependent degradation of cognate mRNA mediated by 21-23 nucleotide short interfering RNA (siRNA). In this study, we investigated the effect of HSP47-specific siRNA on the fibroblast cells, and then constructed adenovirus containing siRNA against HSP47 to inhibit the formation of scar in animal model. In this pilot study, HSP47 was targeted by this vector. Our results showed that the HSP47-specific siRNA could inhibit the expression of HSP47 at the level of mRNA and protein. Furthermore, adenovirus-mediated transfer of siRNA against HSP47 could inhibit the expression of type I collagen and the formation of scar tissue in animal model. It is likely that the inhibition of HSP47 by RNA interference (RNAi) could be developed as a powerful approach to prevent the scar formation.     

Insulin-like growth factor-I in wound healing of rat skin

Endothelin-1 is involved in physiology and pathophysiology of the alimentary tract. The peptide modulates blood flow in the gastrointestinal microvasculature and regulates contractility of smooth muscles and, when present in excess, may be an important factor contributing to pathogenesis of various forms of mucosal injury and peristaltic disorders. Mechanisms that regulate endothelin concentration in the gastrointestinal tissues are unknown. Therefore, the aim of our study was to identify and characterize endothelin inactivating peptidases in the rat gastrointestinal mucosa and smooth muscle cells. We have found three high affinity and efficient endothelin-1 inactivating peptidases. The acidic (pH optimum 5.5), membrane-bound, thiorphan- (ED(50) 1.2+/-0.2 nM) and phosphoramidon (ED(50) 150+/-25 pM) sensitive, endothelin-1 inactivating peptidase (K(M) 0.12+/-0.03 microM) was present in the mucosal cells of duodenum and small intestine. The enzyme exhibited high molecular weight (>100 kDa) and characteristics similar to that of the rat and human kidney, acidic metalloendopeptidase that was recently described. Two forms of the unique, low molecular weight (100>MW>30 kDa), alkaline (pH optimum 8.5), specific (K(M) 0.5+/-0.2 microM), thiorphan- and phosphoramidon insensitive, 1,10 phenanthroline inhibitable (ED(50) 0.65+/-0.20 mM, mean+/-S.E.M.) endothelin-1 inactivating peptidase were present exclusively in the duodenal mucosal cells; soluble form in cytosol and membrane-bound form exhibiting an abundance ratio 5:1, respectively. Mucosa of the stomach and large intestine, and gastrointestinal smooth muscle cells do not contain the specific endothelin-1 inactivating peptidases. The enzymes may play a crucial role in regulation of endothelin concentration in the gastrointestinal tissues. Whether impairment of activity of the mucosal endothelin inactivating peptidases, resulting in the increase of concentration of endothelin peptides in gastrointestinal tissues, occurs in various pathological conditions is actually studied in our laboratory.     

Predicting the effect of relative humidity on skin temperature and skin wettedness

The purpose of this research is to investigate the relative humidity effects on skin temperature and skin wettedness for different operative temperatures. For this aim, thermal interactions between human body and environment are simulated. In this simulation, Gagge 2-node model is used but includes some significant modifications. The simulation is to apply the Gagge 2-node model to individual segments rather than to whole body. Also, the results of the simulation are compared with present measurements, and available experimental data and simulated results in the literature in order to present reliability of the 16 segments-Gagge 2-node model. It is determined that the simulation results are in good agreement with measured results obtained from present experiments, and simulated results and experimental data in the literature.     

Effect of rat salivary glands extracts on the proliferation of cultured skin cells - a wound healing model

Objective: Salivary gland secretions play an important role in promotion of wound healing. The healing of intra- or extra-oral wounds is delayed in desalivated rats. However, the specific role of each salivary gland in promoting wound healing is unknown. This study was aimed to investigate the effect of crude extracts of rat salivary glands on a simplified in vitro wound healing model. Design/methods: Cultured human keratinocytes (HaCat) and murine fibroblasts (3T3) were subjected to 48 h serum starvation, and were later activated by extracts of rat salivary glands, 1–10 μg protein/ml of each gland. The resultant cellular metabolic activity of the activated cells was determined 24 h later, measuring reduction of XTT by mitochondrial enzymes, and calculated relatively to positive controls [optimal supplementation of 10% fetal calf serum (FCS)], and negative controls (starved non-supplemented cells). Results: The relative stimulatory effect of parotid (P) extract on the cells was significantly lower than either submandibular (SM) or sublingual (SL) extracts. Under the assumption that physiologically, the cells are exposed to the combined effect of saliva secreted from all the glands, different combinations of the extracts were presented to the cells. The relative stimulation was maximal following treatment with the three glands extracts (P + SM + SL) and exceeded the effect of 10% FCS. Conclusion: The results suggest that each salivary gland has a specific effect on wound healing and the combination of the three extracts has an additive effect but no the sum of all individual glands. This model might be useful to study the wound healing effect of salivary glands. In partial fulfillment of the requirement for MD thesis, The Joyce and Irving Goldman School of Medicine, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.     

活性氧在创伤愈合中作用的研究

：创伤愈合是一个复杂的生物学过程,包括出血与凝血、炎症渗出、血管和肉芽组织的形成、再上皮化、纤维化和瘢痕改建等,在这一系列的生物学活动过程中都需要能量支持;高等动物使用氧气作为终端氧化剂,通过对碳水化合物的氧化作用为愈合过程中的各种生命活动提供能量,但该过程却可以产生大量的活性氧,这些活性氧在创伤愈合的过程中扮演着重要的角色,在低浓度情况下可以促进伤口的愈合,而在高浓度时会抑制伤口愈合,而活性量浓度的过高过低都会影响创口的正常愈合过程。     

The plasmid encoding HSP47 enhances collagen expression and promotes skin wound healing in an alloxan-induced diabetic model

The objective of this work was initially to investigate the effects on skin wound healing process by local injection of HSP47 recombinant plasmid in an alloxan-induced diabetic rat model and assess the possibility and utility of gene therapy based on HSP47 plasmid to improve the diabetic skin wound healing. Rats were injected intraperitoneally with alloxan (120 mg/kg) to induce diabetes. The fragment containing the rat 47 kDa heat shock protein (HSP47) gene lacking its own promoter was cloned into plasmids containing a promoter and green fluorescent protein (GFP). The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo after inducing wounds with alloxan in diabetic rats. Immunohistochemistry, quantitative fluorescent RT-PCR, and Western blotting 3-5 days after plasmid injection were performed to measure the expression changes of HSP47 and collagen I. The results demonstrate an increase of HSP47 levels in vitro in 3T3 fibroblast cells and in vivo in diabetic rat after treatment with plasmids expressing HSP47. The level of collagen I around the wound during the repair process was higher in the treated group than that in the control group, indicating that the constructs may have use in human gene therapy in cases of impaired skin wound healing in diabetes.     

Keratinocyte growth factor: effects on keratinocytes and mechanisms of action

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, and it can protect these cells from various insults. Due to these properties, it is of particular importance for the repair of injured epithelial tissues, and it is currently therapeutically explored for the treatment of radiation- and chemotherapy-induced mucosal epithelial damage in cancer patients. In this review we summarize the current knowledge on the role of KGF in tissue repair and cytoprotection, and we report on its mechanisms of action in keratinocytes.