Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP‐1, IL‐8, IL‐18 and IL‐23 levels have a strong correlation with HS (P < .010‐0.004; AUC = 0.790‐0.883). Notably, combinations of two or three cytokines (TNF‐a, MCP‐1 and IL‐23; AUC: 0.942, Nagelkerke R²: 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.     

Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneous scars

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.     

Nicotine affects cutaneous wound healing in stressed mice

Stress is an important condition of modern life. Nicotine addiction can modulate the physiological response to stress. Cutaneous healing is a complex process resulting in scar formation, which can be delayed by stress. Therefore, the aim of this study was to investigate the effects of nicotine administration on cutaneous wound healing in chronically stressed mice. Male mice were submitted to rotational stress, whereas control animals were not subjected to stress. These stressed and control animals were treated with a transdermal nicotine patch that was changed every day. A full‐thickness excisional lesion was also generated, and 14 days later, lesions had recovered. However, the Stress + Nicotine group presented a delay in wound contraction. These wounds showed a decrease in inflammatory cell infiltration and lower expression of transforming growth factor‐β (TGF‐β), whereas there was an increase in angiogenesis and tumor necrosis factor‐α (TNF‐α) expression. In vitro fibroblast migration was also impaired by the nicotine treatment of stressed‐stimulated cells. In conclusion, nicotine administration potentiates the delay in wound closure observed in mice submitted to stress.     

强功率UVA1照射对增生性瘢痕动物模型瘢痕形成的影响

目的 探讨不同剂量UVA1对全层皮肤缺损诱导的兔耳增生性瘢痕模型的影响情况。方法18只新西兰白兔双耳腹面手术切除2 cm × 5 cm全层皮肤至筋膜,建立兔耳增生性瘢痕模型后,随机分成3组,每组6只兔,将每只兔左耳分别于伤后即刻、1个月、2个月开始用不同剂量大功率UVA1照射,右耳为非照射组。各照射组又分为两个剂量照射组,兔耳分别每次照射UVA1 60 J/cm2、110 J/cm2,连续30次。结果 创伤建模1个月、2个月后开始照射UVA1组,与照射前比较,高剂量组照射后瘢痕处真表皮厚度（282.32 ± 58.60;336.50 ± 98.34）和真皮胶原含量（24.91 ± 16.88;34.47 ± 8.90）均显著降低（P < 0.05）;照射组与非照射组在UVA1照射前后差值的比较,高剂量组照射后瘢痕处表真皮厚度差值（-143.52 ± 42.91;-142.44 ± 49.96）和真皮胶原含量差值（-56.39 ± 15.04;-48.35 ± 10.44）的差异有统计学意义（P < 0.05）;各照射组UVA1对瘢痕皮肤厚度（811.68 ± 79.03;659.08 ± 178.98）和胶原含量（67.80 ± 9.06;61.35 ± 12.91）的影响均存在剂量依赖性（P < 0.05）。而创伤建模的同时照射UVA1组,两种剂量的UVA1照射后瘢痕处皮肤厚度和胶原含量较非照射耳均显著增加（P < 0.05）。结论 上皮化后开始UVA1照射可使瘢痕变软,皮肤变薄,胶原含量降低。创伤同时照射UVA1不仅不能阻止瘢痕模型的建立,反而加重瘢痕。     

Laser-Assisted Skin Healing (LASH) in hypertrophic scar revision

Abstract

Laser-Assisted Skin Healing (LASH) is based on the therapeutic effects of controlled thermal post-conditioning. The authors have previously demonstrated on humans that an 810-nm diode-laser system could assist wound closure leading to an improvement of wound healing with a resulting indiscernible scar. A 47-year-old woman (skin type II), who developed systematically hypertrophic scars after surgery, was enrolled for a hypertrophic scar revision. Excess scar tissue was removed. Immediately after the conventional closure of the incision, laser irradiation (120 J/cm²) using a 0.8 cm² spot size (rectangular spot, length = 20 mm, width = 4 mm) was applied. Topical silicone gel sheeting (Cerederm®) was applied for 2 months afterwards to prevent a thick scar from reforming. No complications occurred during the course of this study. No recurrence of hypertrophic scarring was noticed 6 months after scar revision. This study reports, for the first time, the possibility of improving the appearance of hypertrophic scarring in scar revision by altering through thermal stress the wound-healing process. Since the appropriate initial management of wounds is of importance, the LASH technique could be offered as a new approach to prevent hypertrophic scarring.     

Regenerative healing,scar‐free healing and scar formation across the species: current concepts and future perspectives

All species have evolved mechanisms of repair to restore tissue function following injury. Skin scarring is an inevitable and permanent endpoint for many postnatal organisms except for non‐amniote vertebrates such as amphibians, which are capable of tissue regeneration. Furthermore, mammalian foetuses through mid‐gestation are capable of rapid wound repair in the absence of scar formation. Notably, excessive cutaneous scar formation, such as hypertrophic and keloid scars, is a species limited clinical entity as it occurs only in humans, although wounds on the distal limbs of horses are also prone to heal with fibroproliferative pathology known as equine exuberant granulation tissue. Currently, there are no reliable treatment options to eradicate or prevent scarring in humans and vertebrates. The limited number of vertebrate models for either hypertrophic or keloid scarring has been an impediment to mechanistic studies of these diseases and the development of therapies. In this viewpoint essay, we highlight the current concepts of regenerative, scar‐free and scar‐forming healing compared across a number of species and speculate on areas for future research. Furthermore, in‐depth investigative research into the mechanisms of scarless repair may allow for the development of improved animal models and novel targets for scar prevention. As the ability to heal in both a scarless manner and propensity for healing with excessive scar formation is highly species dependent, understanding similarities and differences in healing across species as it relates to the regenerative process may hold the key to improve scarring and guide translational wound‐healing studies.     

Suppressed inflammatory gene expression during human hypertrophic scar compared to normotrophic scar formation

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar (HTscar) and not to normotrophic scar (NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix (ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52 weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes (TNFα, IL‐1α, IL‐1RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL‐10) and an extended increased expression of ECM‐related genes (PLAU, Col3A1, TGFβ3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti‐inflammatory scar therapies.     

Biphasic expression of stromal cell-derived factor-1 during human wound healing

BACKGROUND: Chemokines tightly regulate the spatial and temporal infiltration of invading leucocyte subsets during wound healing. Stromal cell-derived factor-1 (SDF-1/CXCL12) is a homeostatic chemokine with multiple functions; its role during cutaneous wound healing, however, needs to be explored. OBJECTIVES: To elucidate expression of the multifunctional CXC chemokine SDF-1/CXCL12 during human wound healing. METHODS: Skin biopsies were obtained from 14 volunteers between 1 and 21 days after incisional wounding and processed for in situ hybridization and immunohistochemistry. RESULTS: We analysed the spatial and temporal distribution of SDF-1/CXCL12 after artificial wounding and detected a complete downregulation at both the mRNA and the protein level within the fibrous stroma that replaces the initial wound defect. However, increased levels of SDF-1/CXCL12 were observed at the wound margins. Focusing on mediators regulating SDF-1/CXCL12 expression in vitro we realized that both tumour necrosis factor-alpha and interferon-gamma downregulated its expression in human dermal microvascular endothelial cells and fibroblasts. CONCLUSIONS: Our data suggest that SDF-1/CXCL12 is tightly regulated during wound repair. Increased expression at the wound margin may contribute to the accumulation of endothelial progenitor cells, thus accelerating neovascularization.     

Human dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength

Endogenous electric fields are generated lateral to skin wounds, with the cathodal pole of the field residing in the center of the wound. These fields are thought to be an important mechanism in guiding the migration of keratinocytes and other cells into wounds to effect healing. In this work, human dermal fibroblasts were exposed to direct current electric fields of physiological strength, and their migrational behavior was quantitated. Only random migration of human dermal fibroblasts was observed in direct-current electric fields under conditions that support the directional migration of human epidermal keratinocytes. Additionally, neither the presence of serum nor serum plus additional Mg++ in the experimental medium supported directional migration. Migratory rates of fibroblasts varied depending on the experimental medium used: in serum-containing medium the average velocity was as low as 0.23 micro m/min, while in serum-free keratinocyte medium the average velocity was as high as 0.36 micro m/min. These studies suggest that dermal fibroblasts do not respond to the endogenous electric field of a wound, and use other migratory cues to direct their movement into the wound bed.     

Aberrant Notch signalling contributes to hypertrophic scar formation by modulating the phenotype of keratinocytes

Hypertrophic scar (HS) is characterized by fibroblast hyperproliferation and excessive matrix deposition. Aberrant keratinocyte differentiation and their abnormal cytokine secretion are said to contribute to HS by activating fibroblasts. However, the signalling pathway causing the aberrant keratinocytes in HS has remained unidentified thus far. Given that Notch signalling is crucial in initiating keratinocyte differentiation, we hypothesized that Notch signalling contributes to HS by modulating the phenotype of keratinocytes. We found that Notch1, Notch intracellular domain, Jagged1 and Hes‐1 were overexpressed in the epidermis of patients with HS. Supernatants from recombinant‐Jagged1–treated keratinocyte cultures could accelerate dermal fibroblast proliferation and collagen production. Furthermore, Jagged1 induced keratinocyte differentiation and upregulated the expression of fibrotic factors, including transforming growth factors β₁ and β₂, insulin‐like growth factor‐1, connective tissue growth factor, vascular endothelial growth factor and epidermal growth factor, while DAPT (a Notch inhibitor) significantly suppressed these processes. In a rabbit ear model of HS, local application of DAPT downregulated the production of fibrotic factors in keratinocytes, together with ameliorated scar hyperplasia. Our findings suggest that Notch signalling contributes to HS by modulating keratinocyte phenotype. These results provide new insights into the pathogenesis of HS and indicate a potential therapeutic target.     

Differential expression of collagen integrin receptor on fetal vs. adult skin fibroblasts: implication in wound contraction during healing

BACKGROUND: Fetal skin wound healing is characterized by an absence of contraction and scar formation, two important observations associated with adult healing often leading to pathological problems. OBJECTIVES: We have studied the capacity of adult and fetal human skin fibroblasts to contract collagen gels, collagen being the major structural component of dermal matrix. METHODS: In parallel with collagen gel contraction studies, we have used fluorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of fibroblasts derived from fetal or adult skin samples. RESULTS: Strong differences were detected between freshly isolated fetal and adult fibroblasts. Fetal fibroblasts had a very low capacity to contract collagen gel, whereas adult cells significantly contracted gels in the same conditions. The expression of alpha1, alpha2 and alpha3 integrin subunits was also significantly different depending of the donor age: alpha1 and alpha3 integrin subunit expression was lower in fetal cells compared with adult cells, whereas alpha2 integrin subunit expression was higher. When grown in monolayers, adult cells showed rapid changes in their contractile capacity and integrin expression while fetal cells were only affected after several passages. CONCLUSIONS: These observations indicate that intrinsic differences between fetal and adult fibroblasts can strongly influence the quality of wound repair.     

The expression of cytokines, growth factors and ICAM-1 in the healing of human cutaneous xenografts on nude mice

Abstract We postulate that wound healing is an orderly process mediated by a programmed expression of cytokines and growth factors. We suggest that these factors are produced in a consistent sequence, in regulated quantities and eliminated when their function is complete. We report here the results of studies on several cytokines, growth factors and the intercellular adhesion molecule expressed during the healing of human skin grafted onto athymic nude mice. Signs of healing of grafts were visible clinically around 3–5 days post-graft and were completed by 4 weeks post-graft. During the Ist 2 weeks, we observed the following, (i) K-14 keratin was prominent throughout the entire epidermis. Thereafter it was limited to basal cell layers, (ii) Langerhans cells were not detectable wilh anti-human CDIa antibodies during the first week of healing but were clearly detectable 2 weeks post-graft, (iii) DOPA (dihydroxy phenyl-alanine) positive melanocytes gradually increased with time. The epidermis 21 to 28 days post-graft clinically and histologically seemed to be morphologically intact. Interleukin-l (IL-I) was clearly detected in some basal cells of the epidermis, especially in melanoeytes and some keratinocytes during the early stage of healing. Transforming growth factor-α: (TGF-(α) was detected in epidermis first in melanocytes and some kera-tinocytes shortly after grafting and again in the late stage of healing. It was also found in some dermal cells. Its expression coincided wilh kera-tinocyte proliferation and melanocyte migration. TGF-β was strongly ex-pressed in the epidermis and dermis after the first week post graft, (iv) ICAM-1 was transiently expressed only at the onset of healing. We previously reported that pro-opiomelanocortin and its derivatives MSH/ACTH are expressed strongly during the healing of human xenografts. The 4 additional molecules which are the subject of this report all are ex-pressed in healing human skin in a predictable sequence and quantity (intensity of stain). Together these data support our hypothesis that healing is a highly regulated process mediated by numerous cytokines.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

Spatial and temporal expression of basic fibroblast growth factor protein during wound healing of rat skin

BACKGROUND: Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key part in wound healing. OBJECTIVES: To determine the spatial and temporal expression of bFGF protein during wound healing after burning of rat skin. METHODS: Immunohistochemical methods were used. RESULTS: The immunostaining for bFGF in the normal epidermis was faint and sporadic in the basal cell layer. However, significant staining for bFGF was found in four locations: regenerated epidermis, a band-like zone near the regenerated epidermis, renewed capillaries, and cells infiltrating into the granulation tissue at the inflammatory to proliferative stages after the burn. The intensity of immunostaining of regenerated epidermis, the band-like zone and renewed capillaries was maximal during the proliferative stage and decreased to normal levels or disappeared simultaneously with wound closure. Immunopositive macrophage-like cell numbers in the granulation tissue increased during the proliferative stage and promptly decreased after wound closure, but such cells were only poorly visible in the scar tissue until 42 days postburn. CONCLUSIONS: bFGF may affect the proliferation, differentiation and migration of regenerated keratinocytes and the recruitment of inflammatory cells, as well as neovascularization in granulation tissue during wound healing. Macrophages may play a pivotal role in cutaneous wound repair by producing bFGF not only during the inflammatory or proliferative stages but also during the remodelling stage.     

Are melanocortin peptides future therapeutics for cutaneous wound healing?

Cutaneous wound healing is a complex process divided into different phases, that is an inflammatory, proliferative and remodelling phase. During these phases, a variety of resident skin cell types but also cells of the immune system orchestrate the healing process. In the last year, it has been shown that the majority of cutaneous cell types express the melanocortin 1 receptor (MC1R) that binds α‐melanocyte‐stimulating hormone (α‐MSH) with high affinity and elicits pleiotropic biological effects, for example modulation of inflammation and immune responses, cytoprotection, antioxidative defense and collagen turnover. Truncated α‐MSH peptides such as Lys‐Pro‐Val (KPV) as well as derivatives like Lys‐d ‐Pro‐Thr (KdPT), the latter containing the amino acid sequence 193‐195 of interleukin‐1β, have been found to possess anti‐inflammatory effects but to lack the pigment‐inducing activity of α‐MSH. We propose here that such peptides are promising future candidates for the treatment of cutaneous wounds and skin ulcers. Experimental approaches in silico, in vitro, ex vivo and in animal models are outlined. This is followed by an unbiased discussion of the pro and contra arguments of such peptides as future candidates for the therapeutic management of cutaneous wounds and a review of the so‐far available data on melanocortin peptides and derivatives in wound healing.     

Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.     

The regenerative potential of fibroblasts in a new diabetes‐induced delayed humanised wound healing model

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human‐skin‐engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell‐based wound therapy consisting of the application of plasma‐derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re‐epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.     

Angiopoietin‐1, angiopoietin‐2 and Tie‐2 receptor expression in human dermal wound repair and scarring

Background The angiopoietin (Ang)/Tie‐2 ligand/receptor system is known to interact with the vascular endothelial growth factor (VEGF) pathway to determine the fate of blood vessels during angiogenesis. However, the precise contribution of this system to angiogenesis and the mechanisms of vascular maturation and remodelling in human tissue repair have yet to be elucidated. Objectives To examine the spatial and temporal expression of Ang‐1, Ang‐2, Tie‐2 and VEGF in relation to angiogenesis in human surgical wounds. Methods Punch biopsies were taken either from normal unwounded skin (controls) during surgery or from mastectomy scars between 3 days and 2 years postsurgery. Ang‐1, Ang‐2, Tie‐2 and VEGF fibroblast/myofibroblast and endothelial expression were characterized by immunohistochemistry, analysed semiquantitatively and correlated with microvessel density (MVD) and scar age. Results The expression of VEGF, Ang‐1, Ang‐2 and Tie‐2 in fibroblasts/myofibroblasts was increased significantly in early scars, decreased in older scars and was related to scar age (P < 0·001) and MVD (P < 0·0004), with strong correlations between all factors. In contrast, vascular expression of Ang‐1 was decreased slightly in early scars, vascular Ang‐2 remained constant and Tie‐2 vascular expression increased, although there were no correlations with scar age or MVD. Conclusions These data demonstrate that angiopoietins and their receptor, Tie‐2, are expressed in both fibroblasts/myofibroblasts and endothelial cells in healing human wounds. Fibroblast/myofibroblast expression correlates with angiogenesis and VEGF expression, suggesting a role for the angiopoietin/Tie‐2 system in normal wound repair and scarring.     

NADPH oxidase‐2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor‐beta1 (TGF‐β1) exerts pro‐oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF‐β1‐induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real‐time Q‐PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock‐down analysis. Hydrogen peroxide production and 2′,7′‐dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non‐phagocytic NADPH oxidase mRNA. TGF‐β1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF‐β1 phosphorylated MAPKs and increased activator protein‐1 (AP‐1) in a redox‐sensitive manner, and NOX2 suppression inhibited baseline and TGF‐β1‐mediated stimulation of Smad2 phosphorylation. Moreover, TGF‐β1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI‐1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.