TGF-beta2 activates proliferative scar fibroblasts

BACKGROUND. Cytokines, such as the transforming growth factor beta (TGF-beta) isoforms, have been linked to the formation of proliferative scars. This study examines the stimulating effects of exogenous TGF-beta2 on cultured keloid, burn hypertrophic scar, and normal skin fibroblasts and whether such effects can be suppressed with TGF-beta2 antibody. METHODS. In vitro, the fibroblast-populated collagen lattice (FPCL) is used in the evaluation of fibroblast activation by measuring contraction of the lattice over time. Primary cultures of fibroblasts were grown from keloids, burn hypertrophic scars, and normal skin using standard cell culture techniques. TGF-beta2 (10 ng/ml) was added to each of the three types of cell cultures and placed on prefabricated FPCLs. Each was tested against their normal control counterparts. TGF-beta2 antibody (100 ng/ml) was then placed on the TGF-beta2-treated FPCLs. All lattices were allowed to contract and areas were measured for 5 days. RESULTS. Compared to controls, keloid fibroblasts were most affected by the addition of exogenous TGF-beta2. Normal skin fibroblasts did not show a significant increase in contraction early on, yet a significant difference was seen as time progressed. The addition of TGF-beta2 antibody inhibited the function of keloid and burn hypertrophic scar fibroblasts. It also reversed the increased contraction of the TFG-beta2-treated proliferative scar fibroblasts. CONCLUSION. By utilizing an in vitro model, we have demonstrated that TGF-beta2 antibody reverses the increased contraction of FPCLs by proliferative scar fibroblasts treated with TGF-beta2. This points to a possible treatment modality in patients afflicted with this disfiguring problem.     

血小板源性生长因子受体-β在瘢痕疙瘩成纤维细胞中的表达

目的 从细胞及组织水平检测血小板源性生长因子受体－β（ＰＤＧＦＲ－β）在正常皮肤及瘢痕疙瘩中的表达及分布,探讨ＰＤＧＦＲ－β在瘢痕疙瘩形成中的生物学作用。方法①应用免疫组织化学技术检测ＰＤＧＦＲ－β在正常和瘢痕疙瘩组织中的分布;②应用免疫荧光、流式细胞仪检测正常皮肤和瘢痕疙瘩成纤维细胞群体的ＰＤＧＦＲ－β的表达;③应用免疫荧光、激光共聚焦显微镜检测ＰＤＧＦＲ－β的亚细胞分布。结果 ①免疫组织化学正     

Suppression of in vitro proliferative scar fibroblast contraction by interferon alfa-2b

Increased fibroblast activity and collagen production have been observed frequently in proliferative scars. Previous studies have demonstrated that interferons suppress collagen production by means of normal, keloid, and hypertrophic scar-derived fibroblasts. The fibroblast-populated collagen lattice is an in vitro model used to study fibroblast function. We used fibroblast-populated collagen lattices to evaluate the effect of interferon on fibroblasts harvested from normal human skin, human keloid, and hypertrophic scar tissues. Human recombinant interferon alfa-2b (1000 IU/ml) was added to the culture media. The collagen gel, prepared from rat tail tendon bundles, was overlaid with 5 x 10(4) fibroblast cells. Keloid fibroblast-populated collagen lattices showed the highest contraction. Contraction in all the groups appeared suppressed by interferon alfa-2b during the first 72 hours of study (p < 0.05). The reduction in fibroblast-populated collagen lattice contraction by interferon alfa-2b was similar among the groups. The contractile properties of fibroblasts taken from normal human skin, keloids, and hypertrophic scars in this in vitro study were suppressed by interferon alfa-2b. This suggested that interferon alfa-2b may be beneficial for the treatment of proliferative scars.     

Recombinant human decorin inhibits TGF-β1-induced contraction of collagen lattice by hypertrophic scar fibroblasts

Decorin was reported to bind transforming growth factor-beta (TGF-β₁) and neutralise some of its activity as a key regulator of wound contraction and hypertrophic scar formation. In this study, we investigated whether recombinant human decorin affected TGF-β₁-induced fibroblast contractile activity, by using fibroblast-populated collagen lattice with decorin added to the collagen gel. Hypertrophic scar fibroblasts showed greater basal contraction of collagen gels than normal fibroblasts, and the addition of TGF-β₁ significantly enhanced this. Decorin inhibited both the basal and TGF-β₁-enhanced contraction of collagen gel by both normal and hypertrophic scar fibroblasts. Decorin also inhibited TGF-β₁-induced α-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1) protein and mRNA expressions in normal and hypertrophic scar fibroblasts. These results suggest that decorin may have therapeutic potential for excessive skin contraction as observed in hypertrophic scarring.     

Suppression of transforming growth factor beta/smad signaling in keloid-derived fibroblasts by quercetin: implications for the treatment of excessive scars

BACKGROUND: Keloids are characterized by abnormal proliferation and overproduction of extracellular matrix. Quercetin, a dietary compound, has strong antioxidant and anticancer properties. Previous studies by the authors have shown that quercetin inhibits fibroblast proliferation, collagen production, and contraction of keloid and hypertrophic scar-derived fibroblasts. Quercetin also blocks the signal transduction of insulin-like growth factor-1 in keloid fibroblasts. This study assessed the effects of quercetin on the transforming growth factor (TGF)-beta/Smad-signaling pathway in keloid-derived fibroblasts, which may be an important biologic mechanism of this proliferative scarring. METHODS: Keloid fibroblasts were isolated from keloid tissue specimens. Cells were treated with quercetin at different concentrations, then harvested, and subjected to immunoblotting analysis. RESULTS: Quercetin significantly inhibited the expression of TGF-beta receptors 1 and 2 in keloid fibroblasts at three concentrations (low, medium, and high). Quercetin also strongly suppressed the basal expression of Smad2, Smad3, and Smad4 as well as the phosphorylation of Smad2 and Smad3 and the formation of the Smad2-Smad3-Smad4 complex. CONCLUSIONS: Taken together, these data suggest that quercetin effectively blocks the TGF-beta/Smad-signaling pathway in keloid fibroblasts. These data indicate that quercetin-based therapies for keloids should be investigated further.     

Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB

Pathologic remodeling of mesangial matrix after glomerular injury is the central biologic feature of glomerular scarring (sclerosis). Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF)-BB have been implicated in the development of glomerular scarring in rat and human glomerulonephritis. To clarify molecular and cellular mechanisms involved in abnormal mesangial remodeling, this study focused on the role of alpha1beta1 integrin, a collagen/laminin receptor, in rat mesangial cells, using collagen gel contraction as an experimental model of in vivo collagen matrix remodeling and scar formation. In addition, the influence of TGF-beta and PDGF-BB on mesangial cell (MC)-mediated collagen gel contraction in association with the alpha1beta1 integrin expression was evaluated. Integrin function blocking studies using anti-alpha1, beta1 subunit antibodies indicated that MC-alpha1beta1 integrin is essentially required not only for collagen-dependent adhesion/migration, but also for gel contraction. Protein synthesis and mRNA analysis experiments demonstrated that TGF-beta, but not PDGF-BB, increases the expression of alpha1beta1 integrin in mesangial cells cultured on plastic surface and in collagen gels. The upregulation of alpha1beta1 integrin expression by TGF-beta correlated with increases in gel contraction and collagen-dependent adhesion but not migration of mesangial cells. On the other hand, PDGF-BB enhanced MC-mediated gel contraction and migration without affecting cell adhesion to collagen I. Growth factor-induced collagen-dependent adhesion, migration, and gel contraction were significantly attenuated by incubation with anti-alpha1, beta1 subunit antibodies. Thus, these data indicate that alpha1beta1 integrin-mediated collagen matrix remodeling can be modulated by TGF-beta and PDGF-BB via different mechanisms. Alpha1 integrin-mediated mesangial matrix remodeling induced by TGF-beta or PDGF-BB may be a pathogenic mechanism leading to glomerular scarring.     

壳多糖-胶原-糖胺聚糖凝胶人工皮肤的初步研究

目的 研制一种新型的胶原凝胶类人工皮肤。方法 制备壳多糖—胶原—糖胺聚糖(GAGs)—成纤维细胞真皮替代物(DE)，观察成纤维细胞(FB)在凝胶中的生长情况和不同因素对凝胶收缩的影响，并绘制FB生长曲线和凝胶收缩曲线。了解不同含量壳多糖对FB和角质形成细胞(KC)生长的影响，不同含量壳多糖DE的抗感染能力。再于“成熟。的DE表面接种KC，先浸没培养，后行气液界面培养，构建完整的人工皮肤。对DE和人工皮肤行组织学和电镜分析。结果 DE收缩度与FB数量呈正比，终末收缩度与胶原蛋白浓度成反比；FB在凝胶中2—9天呈指数增生。DE基质配方对FB的生长无明显抑制作用，但可促进KC的生长，对金黄色葡萄球菌的抑制作用随壳多糖的含量增大而增强。扫描电镜示DE有丰富的微孔结构。人工皮肤组织学观察见类似于正常皮肤，有分化良好的表皮和致密的真皮。结论 壳多糖—胶原—GAGs胶原凝胶人工皮肤是一种新型、有一定抗感染能力的活人工皮肤。     

Recombinant human platelet-derived growth factor and transforming growth factor-β mediated contraction of human dermal fibroblast populated lattices is inhibited by Rho/GTPase inhibitor but does not require phosphatidylinositol-3'' kinase

Matrix reorganization and tissue contraction are essential for wound healing. However, the intracellular signals that mediate these processes are largely unknown. We investigated cytokine-induced signaling and its potential role in contraction of adult human dermal fibroblast populated collagen lattices. The results document that recombinant human platelet-derived growth factor-BB and transforming growth factor-1 individually stimulate contraction of fibroblast populated collagen lattices, while a combination of the two cytokines leads to increased contraction. Although recombinant human platelet-derived growth factor-BB promoted collagen contraction, it failed to stimulate phosphatidylinositol-3' kinase in the human dermal fibroblasts. An inhibitor for phosphatidylinositol 3' kinase, wortmannin, had no effect on the cytokine-mediated collagen contraction. In addition, this failed activation of phosphatidylinositol 3' kinase is consistent with absence of tyrosine phosphorylation of the platelet-derived growth factor receptor when the cells are in a collagen matrix. In contrast, tyrosine phosphorylation of the platelet-derived growth factor receptor was readily detected in the dermal fibroblasts in monolayers. We also probed the potential role of Rho/GTPase in the cytokine-mediated contraction of fibroblast populated collagen lattices. Toxin B from Clostridium difficile at picomolar concentrations blocked both recombinant human platelet-derived growth factor and transforming growth factor-5 induced contraction. Further, this inhibition was correlated with the inhibition of cell spreading in collagen, which suggests the formation of actin fibers inside the cells is essential for cytokine-mediated contraction of fibroblast populated collagen lattices. Taken together, these results imply that Rho/GTPase signaling but not phosphoinositol-3' kinase is involved in the cytokine-mediated contraction of fibroblasts populated collagen lattice. These findings suggest a potential mechanism for these signaling components during human wound contraction.     

反义结缔组织生长因子对人瘢痕疙瘩成纤维细胞的作用

目的：研究结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人瘢痕疙瘩成纤维细胞CTGFmRNA的表达和细胞增殖及胶原合成的影响,探讨瘢痕疙瘩的基因治疗。方法：体外分离、培养人正常皮肤成纤维细胞（NSF）和瘢痕疙瘩成纤维细胞（KF）,以脂质体介导方法将CTGFASODN转染KF中,用逆转录-聚合酶链式反应（RT-PCR）方法检测细胞中CTGFmRNA的表达;采用MTT方法测细胞增殖3;H-脯氨酸掺入法检测细胞的胶原合成量。结果：CTGFmRNA在NSF中几乎无表达,在KF中表达增高,CTGFASODN可以抑制其增殖和胶原合成（P〈0.05）。结论：CTGFASODN能够抑制成纤维细胞CTGFmRNA表达和细胞增殖及胶原合成,表明阻断CTGF可能是延缓瘢痕纤维化的有效手段。     

Stromelysin-1-deficient fibroblasts display impaired contraction in vitro.

Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in wound contraction. Therefore, we postulated that stromelysin-1 activity is responsible for initiating contraction. To test this hypothesis, we compared the contractile capacity of fibroblasts from stromelysin-1 knockout mice (strom-1 KO) with that of normal fibroblasts using a collagen gel contraction model. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice, then transferred to the surface of collagen gels. The extent of contraction was determined by measuring greatest gel diameter. Results demonstrated that (1) all fibroblasts contracted collagen gels in a uniform concentric fashion, (2) skin fibroblasts from both sets of mice exhibited greater gel contraction than did lung fibroblasts, and (3) strom-1 KO fibroblasts demonstrated significantly less contraction (21-23%) than wild-type fibroblasts. These data support the hypothesis that absence of stromelysin-1 results in defective fibroblast contraction that may contribute to delayed wound healing.     

Triamcinolone Stimulates bFGF Production and Inhibits TGF-β1 Production by Human Dermal Fibroblasts

BACKGROUND: Triamcinolone acetonide has been shown to decrease both cellular proliferation and collagen production by dermal fibroblasts. An alteration of cytokine levels may mediate these effects. OBJECTIVE: To delineate the effect of triamcinolone acetonide on both cellular proliferation and the production of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1) by human fibroblasts grown in a serum-free in vitro model. METHODS: Human normal and keloid dermal fibroblasts were propagated in a serum-free in vitro model with exposure to 0, 5, 10, or 20 microm triamcinolone acetonide for 0, 24, 72, or 96 hours. Cell counts were determined by phase contrast microscopy. Levels of bFGF and TGF-beta1 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In our study, 20 microm triamcinolone acetonide caused statistically significant increases in the peak levels of bFGF for normal and keloid fibroblast cell lines (P < 0.05). It also caused statistically significant decreases in the level of TGF-beta1 for normal and keloid fibroblast cell lines. For the keloid fibroblasts, 10 microm triamcinolone acetonide also caused a statistically significant decrease in the level of TGF-beta1. CONCLUSION: We conclude from these results that triamcinolone acetonide increases the production of bFGF and decreases production of TGF-beta1 by human dermal fibroblasts.     

Resveratrol inhibits fibrogenesis and induces apoptosis in keloid fibroblasts

Keloids are benign dermal fibrotic tumors arising during the wound healing process. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Resveratrol, a dietary compound, has anticancer properties and, from recent studies, it has been suggested that resveratrol may have an antifibrogenic effect on organs such as the liver and kidney. Based on this idea, we investigated its effect on the regulation of extracellular matrix expression, proliferation, and apoptosis of keloid fibroblasts. Type I collagen, α‐smooth muscle actin, and heat shock protein 47 expression decreased in resveratrol‐treated keloid fibroblasts in a dose‐dependent manner. In addition, resveratrol diminished transforming growth factor‐β1 production by keloid fibroblasts. We also demonstrated that it suppressed their proliferation and induced apoptosis of the fibroblasts. Conversely, resveratrol did not decrease type I collagen, α‐smooth muscle actin, and heat shock protein 47 mRNA expression in normal skin fibroblasts and barely suppressed cell proliferation. Our data indicate that resveratrol may have an antifibrogenic effect on keloid fibroblasts without any adversely effects on normal skin fibroblasts, suggesting the potential application of resveratrol for the treatment of keloids.     

重组人核心蛋白多糖固相拮抗转化生长因子β1对瘢痕成纤维细胞的刺激作用

目的模拟人体内核心蛋白多糖和转化生长因子β1(TGF-β1)接触方式,观察核心蛋白多糖固相拮抗TGF-β1刺激瘢痕成纤维细胞的效果.方法制备成纤维细胞胶原网格(FPCL)体外三维培养模型,将其分为4组.对照组向FPCL中加入培养液;核心蛋白多糖组向FPCL中混入终浓度2 mg/L的重组人核心蛋白多糖,再加入培养液;TGF-β1组向FPCL中加入含5 μg/L TGF-β1的培养液;TGF-β1+核心蛋白多糖组向FPCL中混入终浓度2 mg/L的重组人核心蛋白多糖,然后加入含5μg/L TGF-β1的培养液.在培养12、24、48、72、96 h时观察各组FPCL的收缩情况,并用蛋白质印迹法与逆转录聚合酶链反应分别检测FPCL中瘢痕成纤维细胞Ⅰ型纤溶酶原激活物抑制因子1(PAI-1)、α平滑肌肌动蛋白(α-SMA)的蛋白及mRNA表达水平.结果各培养时相点下,TGF-β1组FPCL收缩比对照组明显增强,核心蛋白多糖组FPCL收缩则比对照组明显减弱.TGF-β1组的PAI-1、α-SMA的蛋白及相应mRNA表达水平(3 482±211、4 320±272;0.89±0.15、0.56±0.11)显著高于对照组(1 764±147、1 699±146;0.29±0.06、0.21±0.06,P＜0.01);其余两组相应检测指标与对照组比较差异无统计学意义(P＞0.05).结论重组人核心蛋白多糖混入胶原凝胶,可显著抑制TGF-β1对瘢痕成纤维细胞的刺激作用,表明在体外核心蛋白多糖具有拮抗TGF-β1的作用.提示皮肤组织损伤后,由于创面机械性缺少核心蛋白多糖,TGF-β1活性上调,可能是瘢痕增生的一个重要因素.     

Characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived from keloid

Phenotypic alterations of keloid-derived fibroblasts were characterized by comparison with the phenotypes of normal fibroblasts from the same patient. Explant cultures of keloids showed unique features. Keloid explants contracted considerably and reduced their size during culture, whereas the size of normal skin explants remained unchanged. Enlarged cells were found among fibroblasts which had grown out of all the explants and were morphologically distinct from fibroblasts; however, keloid explants produced many more of them than did the normal tissues. The growth rate of fibroblast colonies formed from normal explants was five times higher than keloid explants. Keloid fibroblasts which had been serially cultivated contracted lattices of collagen gels at a rate similar to normal fibroblasts. Proteins extracted from serially cultivated fibroblasts were mapped on polyacrylamide two-dimensional electrophoretic gels. No significant qualitative alterations in protein expression in keloid cells were found as compared with normal fibroblasts. However, some quantitative changes were found between the two. A computer-assisted image analyzer detected 151 polypeptide spots--50 spots (33%) of which increased their amounts in keloid cells, whereas 34 spots (22.5%) decreased in comparison with normal fibroblasts. Sixteen major polypeptides were identified as known proteins with the aid of time-of-flight mass spectrometry. The level of expression of these identified proteins was similar between normal and keloid cells, except stathmin whose expression was suppressed in keloid fibroblasts.     

Different wound healing properties of dermis,adipose, and gingiva mesenchymal stromal cells

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis‐, adipose‐, and gingiva‐derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin‐derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.     

RNA干扰技术对瘢痕疙瘩中结缔组织生长因子表达及胶原代谢的影响

目的利用RNA干扰（RNA inteferenc,RNAi）技术探讨结缔组织生长因子（connective tissue growth factor,CTGF）对人瘢痕疙瘩（keloid,KD）中胶原代谢的影响。方法将针对人CTGF基因设计合成的3对特异性小干扰RNA（siRNA）-CTGF分别转染体外培养的人瘢痕疙瘩成纤维细胞（keloid fibroblasts,KFB）,通过逆转录-聚合酶链反应（RT-PCR）筛选出干扰人KFB CTGF基因表达最佳的siRNA,而后构建质粒,采用RNAi技术,通过RT-PCR和Western印迹检测此质粒转染KFB后瘢痕疙瘩组织中CTGF表达量的变化及胶原含量的变化,并与对照组比较。结果第3对（C3）siRNA-CTGF转染人KFB后,CTGF的基因表达及胶原蛋白表达水平显著受抑,其抑制率为86．8％及65．6％。结论质粒siRNA—CTGF转染瘢痕疙瘩成纤维细胞后有效沉默CTGF的表达,进而使胶原合成降低;CTGF对瘢痕疙瘩形成过程中胶原蛋白的合成有促进作用。     

Inhibition of wound contraction by papaverine: in vitro analysis with a submucosal tissue model.

We studied the effect of topical application of papaverine, a smooth-muscle relaxant, on the contraction of open, dorsal skin wounds in rats. Wound contraction was inhibited significantly in papaverine-applied wounds compared with saline-dressed control wounds. The in vitro effect of papaverine on wound contraction was studied by using human oral fibroblasts cultured three-dimensionally in the hydrated collagen gels containing papaverine. Papaverine inhibited collagen gel contraction in a dose-dependent manner. Any change in actin filament organization in fibroblasts cultured three-dimensionally in the collagen gels was observed by staining the filaments with fluorescent dye-conjugated phalloidin. In the control cultures, well-organized stress fibers were formed in the cell projections; in papaverine-treated fibroblasts, during the early stages of the treatment, the stress fibers were disrupted and actin filament aggregation was observed. In addition, papaverine induced a delay in the formation of the processes in fibroblasts cultured in collagen gels. These results indicate that wound contraction inhibition by papaverine is mediated by its effects on the organization of actin filaments of fibroblasts.