Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair.

Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only slightly immunoreactive for RI and RII until day 5 when, immediately under the wound, immunostaining for fibroblasts, connective tissue cells, and newly forming vasculature began to increase and remained intense until day 14. Consistent with the role for TGF-beta in scarring, numerous fibroblasts, ostensibly active in the production of extracellular matrix components, continued to be slightly immunoreactive for RI and RII at 21 days. The ALK-1 (TSR-1) type I receptor, which binds both activin and TGF-beta, showed slight immunostaining early in repair (days 1 to 7) that progressively became more intense later in repair after day 10 and through day 21. This suggests that there may be a switch to a different type I receptor, implying different functions for the ALK-1 and ALK-5 receptors. The concomitant expression of TGF-beta isoforms and their signal-transducing receptors denote potential spatial and temporal activity of TGF-beta. Thus, although TGF-beta ligand is present, TGF-beta would not function in wound repair until a later time when RI and RII appear. This information should aid in the development of receptor antagonists as a therapeutic approach to scarring and fibrosis. In addition, these studies underscore the importance of defining the expression of proteins in vivo to establish a basis for the analysis of mechanisms in vitro.     

Epidermal participation in post-burn hypertrophic scar development

 The reconstruction of epidermal architecture over time in normotrophic and hypertrophic scars in untransplanted, spontaneously healed partial-thickness burns has scarcely been studied, unlike the regeneration of epidermal grafts used to cover burn wounds and the regeneration of the dermis during hypertrophic scarring. The expression of markers of epidermal proliferation, differentiation and activation in normotrophic and hypertrophic scars in spontaneously healed partial-thickness burns was assessed and compared with the expression of these markers in normal control skin of healthy persons to determine whether hypertrophic scarring is associated with abnormalities in the phenotype of keratinocytes. Punch biopsies were taken both of partial-thickness burns after re-epithelialisation and of matched unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. The biopsies were analysed using immunostaining for markers of keratinocyte proliferation, differentiation and activation (keratins 5, 10, 16 and 17, filaggrin, transglutaminase and CD36). We observed a higher expression of markers for proliferation, differentiation and activation in the epidermis of scars at 1 month post-burn than in normal control skin of healthy persons. There was a striking difference between normotrophic and hypertrophic scars at 4 months post-burn. Keratinocytes in hypertrophic scars displayed a higher level of proliferation, differentiation and activation than did normotrophic scars. At 7 months post-burn all keratinocyte proliferation and differentiation markers showed normal expression, but the activation marker CD36 remained upregulated in both normotrophic and hypertrophic scars. Surprisingly, in matched unburned skin of burn patients, a state of hyperactivation was observed at 1 month. Our results suggest that keratinocytes may be involved in the pathogenesis of hypertrophic scarring. Received: 16 September 1998 / Accepted: 28 September 1998     

Skin flap-induced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression

Previous studies have shown that covering granulation tissue of a full-thickness skin wound by a vascularized skin flap induces tissue remodeling, with a rapid loss of granulation tissue cells by apoptosis. In the present study, in situ hybridization has been used to examine mRNA expression for several factors that may be implicated in the apoptosis seen in this tissue. Skin wounds were made on the dorsal skin of 8-week-old rats. Ten days after wounding, skin flaps were created surgically and sutured over the granulation tissue. Tissue sections of granulation tissue from various times after addition of the skin flap were hybridized with 33P-labelled cRNA probes for transforming growth factor-beta1 (TGF-beta1), beta-inducible gene H3 (beta-ig-h3), alpha1 (1) procollagen, alpha-smooth muscle actin, matrix metalloproteinase-13 (MMP-13) and -2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), and inducible nitric oxide synthase (iNOS). Control granulation tissue prior to addition of the skin flap showed high levels of TGF-beta1, beta-ig-h3, alpha1 (1) procollagen, alpha-smooth muscle actin, and TIMP-1 expression. MMP-13, MMP-2, and iNOS mRNA were low in 10-day granulation tissue. Addition of a skin flap resulted in a decrease in the expression of TGF-beta1, beta-ig-h3, alpha1 (I) procollagen, alpha-smooth muscle actin, and TIMP-1, but increased expression of MMP-13 and MMP-2. Similarly, an increase in iNOS mRNA expression was observed in the granulation tissue after addition of the skin flap. Addition of a vascularized skin flap may result in rapid remodelling of granulation tissue due to a decrease in expression of the trophic growth factor TGF-beta1 and increased degradation of extracellular matrix due to an alteration in the balance between MMPs and their inhibitor, TIMP-1. Additionally, increased iNOS expression may also favour apoptosis through the generation of free radicals. The additive effect of reduced growth factor expression, increased extracellular matrix turnover, and nitric oxide generation may result in the fibroblast and vascular cell apoptosis seen during the rapid remodelling of this tissue.     

Decreased expression of TGF-beta type 2 receptor in primary B-cell lymphomas of the stomach

TGF-beta insensitivity has been reported in some malignant lymphomas showing loss of TGF-beta receptor expression. This loss of TGF-beta sensitivity is thought to have removed the immunosuppressive properties of TGF-beta, thus enhancing cell proliferation and resulting in the development of malignant lymphoma. In this study, we performed immunohistochemical stains for TGF-beta1, TGF-beta RI and TGF-beta RII in primary gastric B-cell lymphomas in order to ascertain their possible roles in lymphomagenesis. A total of twenty cases of gastric lymphoma were included. All cases of low- and high-grade lymphomas were negative or weakly positive for TGF-beta1. Reactive lymphoid cells, including the germinal center, were also negative for TGF-beta1. In contrast, reactive germinal centers showed moderate to strong cytoplasmic or membranous staining for TGF-beta RI and TGF-beta RII. In malignant lymphomas, TGF-beta RI expression was maintained in all cases of low- and high-grade lymphomas. In contrast, TGF-beta RII expression was decreased in all low- and high-grade lymphoma cells. These findings suggest that the loss of TGF-beta RII expression may be involved in the lymphomagenesis of the stomach.     

Abrogation of TGF-beta by antisense oligonucleotides modulates expression of VEGF and increases angiogenic potential in isolated fibroblasts from radiated skin

The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer good prospects for the modulation of wound healing, specifically those targeting TGF-beta. The aim of this study was to analyze the effect of TGF-beta targeting on the expression of angiogenic vascular endothelial growth factor (VEGF), a key regulator of angiogenesis and in vitro angiogenic activity in fibroblasts isolated from radiation-induced chronic dermal wounds. The expression of angiogenic VEGF in tissue samples from radiation-induced chronic dermal wounds was investigated by immunohistochemistry and microarray technique. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of VEGF in isolated fibroblasts was analyzed by ELISA and multiplex RT-PCR. Human endothelial cells (ECs) were grown in conditioned medium produced from the treated fibroblasts. EC migration was measured using a modified Boyden chamber; EC tube formation was analyzed under a light microscope. Immunohistochemical investigation and microarray analysis demonstrated a decreased expression of VEGF protein and mRNA in tissue samples from radiation-induced chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment significantly up-regulated VEGF secretion in vitro. Addition of conditioned medium from TGF-beta antisense-treated fibroblasts resulted in an increase in EC cell migration and tube formation. In conclusion, our results demonstrate that TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for stimulation of angiogenesis in radiation-induced dermal wounds.     

Activated keratinocytes in the epidermis of hypertrophic scars.

The etiology of hypertrophic scarring, a pathological end point of wound healing, is unknown. The scars most commonly occur when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds. Hypertrophic scars are conventionally described as a dermal pathology in which the epidermis has only a passive role. In this study, the expression of keratin intermediate filament proteins and filaggrin has been investigated in the epidermis of hypertrophic scars and site-matched controls from the same patients. Hypertrophic scar epidermis was found to express the hyperproliferative keratins K6 and K16 in interfollicular epidermis in association with K17 and precocious expression of filaggrin. K16 mRNA was localized by in situ hybridization using a highly specific cRNA probe. In contrast to the immunohistochemical location of K16 protein, the K16 mRNA was found to be expressed in the basal cell layer of normal skin. In hypertrophic scars the mRNA distribution corroborated the abnormal K16 protein distribution. These results suggest the keratinocytes in hypertrophic scar epidermis have entered an alternative differentiation pathway and are expressing an activated phenotype. Activated keratinocytes are a feature of the early stages of wound healing producing growth factors that influence fibroblasts, endothelial cells, and the inflammatory response. We propose that cellular mechanisms in the pathogenesis of hypertrophic scarring are more complex than isolated dermal phenomena. The persistence of activated keratinocytes in hypertrophic scar epidermis implicates abnormal epidermal-mesenchymal interactions.     

Comparison of bone morphogenetic protein receptors expression in the fetal and adult skin.

Wounds on fetal skin can be repaired without leaving scars until the second trimester, but after this period, skin wounds leave scars as in adults. It's known that certain growth factors such as TGF-beta, and bFGF are present at a very low levels during wound repair in fetal skin. These low levels of growth factors minimize inflammatory response and fibroblast proliferation at the wound site, which in turn inhibit collagen synthesis, and thus, allows scarless wound healing. Recently bone morphogenetic proteins (BMPs), one of the TGF-beta superfamily members, have been studied in the wound healing process. According to several studies, BMPs are related to the differentiation and growth of epithelial and mesenchymal cells, but the precise functions of BMPs and of BMP receptors on skin wound healing have not been elucidated. In this study, we investigated the expression pattern of BMP receptors in fetal skin during the second trimester and in adult skin by immunohistochemical staining and RT-PCR. BMP receptors were detected on the suprabasal epithelial cells and in the hair follicles in adult skin, but were not defected in the fetal skin except for the hair follicles. This was confirmed by confirming mRNA levels of BMP receptors by RT-PCR in both adult and fetal skins. In conclusion, BMPs and BMP receptors seem to be related to fetal and adult wound healing, and low levels of BMPs and BMP receptors during the second trimester seem to contribute to scarless wound healing in the fetus, as is TGF-beta during the second trimester.     

Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/-) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/- and -/-) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 post-wounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.     

TGF-beta 1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits.

TGF-beta modulates events of normal wound healing through multiple pathways that influence cell infiltration, proliferation, angiogenesis, extracellular matrix synthesis and remodeling. The effects of topically applied TGF-beta 1 on wound healing in two models of healing were evaluated when the healing response was impaired by the administration of methylprednisolone to rats or rabbits. TGF-beta 1 increased the healing of linear incision wounds on rats, as measured by breaking strength, to that of normal rats. Full thickness open wounds were also created on the inner ears of rabbits to simulate a non-contracting wound with limited blood supply. Healing was further impaired by the administration of methylprednisolone. The single application of TGF-beta 1 improved the healing of open wounds. TGF-beta 1 stimulated increased granulation tissue formation, as well as reepithelialization. The amount of granulation tissue and epithelialization were similar to wounds from normal-healing control rabbits. The delayed healing caused by methylprednisolone permitted the evaluation of multiple applications of TGF-beta 1 to wounds. Two applications of TGF-beta 1 spaced 7 days apart further improved the healing response when compared to a single application. Thus, single or multiple topical applications of TGF-beta 1 reversed impaired healing conditions secondary to methylprednisolone when used on incisional or open wounds. These observations support the hypothesis that growth factors, such as TGF-beta 1, may be useful as accelerators of wound repair in patients with impaired healing conditions.     

Matrix metalloproteinases in wound repair (review)

Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of growth factors from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and fibroblasts deposit and remodel the granulation tissue. Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue, all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example hypertrophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs. In this review, the role of MMPs in normal wound repair as well as in chronic ulcers is discussed. In addition, the role of signaling pathways, in particular, mitogen-activated protein kinases (MAPKs) in regulating MMP expression is discussed as possible therapeutical targets for wound healing disorders.     

Keloid-derived fibroblasts are refractory to Fas-mediated apoptosis and neutralization of autocrine transforming growth factor-beta1 can abrogate this resistance

The pathogenesis of keloid remains poorly understood. As no effective therapy for keloid is as yet available, an insight into its pathogenesis may lead to novel approaches. Apoptosis has been found to mediate the decrease in cellularity during the transition between granulation tissue and scar. Here, we report that in contrast to hypertrophic scar-derived and normal skin-derived fibroblasts, keloid-derived fibroblasts are significantly resistant to both Fas-mediated and staurosporine-induced apoptosis. The caspases-3, -8, and -9 were not activated indicating that the block in the apoptotic pathway in keloid is upstream of the caspases. There were no significant differences in the level of expression of Fas, Bcl-2, and Bax between the three groups but addition of transforming growth factor (TGF)-beta1 significantly inhibited Fas-mediated apoptosis in hypertrophic scar-derived and normal skin-derived fibroblasts and neutralization of autocrine TGF-beta1 with anti-TGF-beta1 antibody abrogated the resistance of keloid-derived fibroblasts. Anti-apoptotic activity was not observed with TGF-beta2. This is the first study linking refractory Fas-mediated apoptosis to cellular phenotype in keloids and indicating a pivotal role for the anti-apoptotic effect of TGF-beta1 in this resistance. Hence, it becomes important to treat keloids as a separate entity different from hypertrophic scars and enhancement of Fas-sensitivity could be a promising therapeutic target.     

Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.     

Platelet-derived growth factor-BB and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds.

Recombinant platelet-derived growth factor (PDGF) and transforming growth factor beta 1 (TGF-beta 1) influence the rate of extracellular matrix formed in treated incisional wounds. Because incisional healing processes are difficult to quantify, a full-thickness excisional wound model in the rabbit ear was developed to permit detailed analyses of growth-factor-mediated tissue repair. In the present studies, quantitative and qualitative differences in acute inflammatory cell influx, glycosaminoglycan (GAG) deposition, collagen formation, and myofibroblast generation in PDGF-BB (BB homodimer)- and TGF-beta 1-treated wounds were detected when analyzed histochemically and ultrastructurally. Although both growth factors significantly augmented extracellular matrix formation and healing in 10-day wounds compared with controls (P less than 0.002). PDGF-BB markedly increased macrophage influx and GAG deposition, whereas TGF-beta 1 selectively induced significantly more mature collagen bundles at the leading edge of new granulation tissue (P = 0.007). Transforming growth factor-beta 1-treated wound fibroblasts demonstrated active collagen fibrillogenesis and accretion of subfibrils at the ultrastructural level. Myofibroblasts, phenotypically modified fibroblasts considered responsible for wound contraction, were observed in control, but were absent in early growth-factor-treated granulating wounds. These results provide important insights into the mechanisms of soft tissue repair and indicate that 1) PDGF-BB induces an inflammatory response and provisional matrix synthesis within wounds that is qualitatively similar but quantitatively increased compared with normal wounds; 2) TGF-beta 1 preferentially triggers synthesis and more rapid maturation of collagen within early wounds; and 3) both growth factors inhibit the differentiation of fibroblasts into myofibroblasts, perhaps because wound contraction is not required, due to increased extracellular matrix synthesis.     

磷脂多不饱和脂肪酸抑制兔耳增生性瘢痕

背景：多不饱和脂肪酸有抑制细胞炎症反应及免疫功能的作用,增生性瘢痕的形成与炎症、细胞免疫、细胞因子有着密切关系,但目前尚无应用多不饱和脂肪酸防治增生性瘢痕的实验研究。 目的：探讨磷脂多不饱和脂肪酸对兔耳增生性瘢痕的抑制作用。 方法：在9只新西兰大白兔兔耳腹侧做直径1 cm的圆形全层皮肤缺损创面,每侧6个,共108个,其中形成增生性瘢痕92个,瘢痕形成率为85%。实验分3组：每只兔耳靠前3个创面涂磷脂多不饱和脂肪酸霜,右耳靠后3个创面涂多磺酸黏多糖乳膏,创面上皮化后立即涂药,每日1次,左耳靠后3个创面自然愈合。分别在术后28,42,63,90 d,观察创面的愈合情况;显微镜下观察瘢痕组织的厚度、胶原纤维和成纤维细胞密度;免疫组织化学染色检测胶原纤维的表达。 结果与结论：涂抹磷脂多不饱和脂肪酸霜和多磺酸黏多糖乳膏可使增生性瘢痕体积缩小、厚度变薄、成纤维细胞密度减小、胶原纤维表达减少。尤以磷脂多不饱和脂肪酸霜的效果最为明显。说明磷脂多不饱和脂肪酸可抑制兔耳增生性瘢痕的形成,减轻瘢痕的增生程度。     

Anomalous expression of HLA class II molecules on keratinocytes and fibroblasts in hypertrophic scars consequent to thermal injury.

Immunoperoxidase staining of skin sections obtained from 11 hypertrophic scars, six normotrophic scars and three samples of normal skin were performed using anti-HLA monoclonal antibodies (HLA-DR, -DQ, class I), anti-interleukin-2 receptor (IL-2R) and anti-CD1. Sections from all hypertrophic scars showed an anomalous expression of HLA-DR molecules on keratinocytes and fibroblasts. Moreover hypertrophic scars were characterized by dense infiltrates of IL-2R-positive cells and by the presence of abundant Langerhans (CD1+) cells in the epidermis and dermis. These results support the hypothesis that immunologic mechanisms play an important role in hypertrophic scarring and point to an involvement of cell-mediated immune phenomena.     

增生性瘢痕中时序变化的骨形态发生蛋白及其受体

背景：骨形态发生蛋白与瘢痕的形成和发展相关,而成纤维细胞与瘢痕的增殖、成熟关系密切,但骨形态发生蛋白在不同时期增生性瘢痕成纤维细胞中的表达变化尚少见报道。 目的：检测骨形态发生蛋白2/4、骨形态发生蛋白7、骨形态发生蛋白IA型受体在不同时期增生性瘢痕成纤维细胞中的表达水平。 方法：应用免疫组织化SP法检测来自2008年3月至2010年7月广西壮族自治区人民医院美容整形外科患者的增殖期增生性瘢痕和成熟期增生性瘢痕中成纤维细胞中骨形态发生蛋白2/4、骨形态发生蛋白7、骨形态发生蛋白IA型受体的表达。 结果与结论：增生性瘢痕增殖期成纤维细胞中骨形态发生蛋白2/4的表达水平显著高于成熟期(P < 0.05);但不同时期增生性瘢痕成纤维细胞中骨形态发生蛋白7和骨形态发生蛋白IA型受体的表达差异无显著性意义  (P > 0.05)。表明在增生性瘢痕由增生期至成熟期的发展过程中,其成纤维细胞中的骨形态发生蛋白2/4的表达下调,而骨形态发生蛋白7、骨形态发生蛋白IA型受体的表达无变化。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro‐apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)‐β1 in vitro. bFGF induced caspse‐3 activation and apoptosis in TGF‐β1‐pretreated granulation tissue‐derived fibroblasts (GF‐1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF‐1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF‐1 cells pretreated with TGF‐β1. Pretreatment of GF‐1 with TGF‐β1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF‐β1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF‐1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF‐injected skin wounds. These results led to the hypothesis that temporal activation of TGF‐β1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increased expression of beta6-integrin in skin leads to spontaneous development of chronic wounds

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.     

Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-β in human post-burn hypertrophic and mature scars

The distributions of the small proteoglycans, decorin and biglycan and the large proteoglycan, versican, in normal skin and post-burn hypertrophic and mature scars, were compared using monoclonal and polyclonal antibodies to the core proteins. Biglycan and verscan were virtually undetectable in normal dermis but readily seen in hypertrophic scars. Staining for decorin was strong throughout the dermis in normal skin but restricted to the deep dermis and a narrow zone under the epidermis in hypertrophic scar—areas which did not stain for versican. Decorin was absent or reduced in the nodules in these specimens. In mature post-burn scars, staining for all three proteoglycans demonstrated an intensity that was intermediate between that in normal dermis and that in the nodules of the hypertrophic scars. Transforming growth factor-β was present in the nodules of hypertrophic scars but the deep dermis of these specimens stained most intensely for this cytokine which was also found in the dermis of mature scars but was not detectable in normal dermis. The apparent co-distribution of decorin and transforming growth factor-β suggests that this proteoglycan may play an active role in the resolution of the scars. Changes in proteoglycan type and distribution could possibly account, at least in part, for the derangement of collagen and the altered physical properties of hypertrophic scar tissue.