Animal model of sclerotic skin. V: Increased expression of alpha-smooth muscle actin in fibroblastic cells in bleomycin-induced scleroderma.

Scleroderma is a connective tissue disorder with unknown etiology. Myofibroblasts appear during fibrotic processes such as scleroderma, hypertrophic scarring, and wound healing. We previously established a mouse model for scleroderma by local injections of bleomycin. To determine the phenotype of the fibroblasts in sclerotic skin after bleomycin treatment, we examined the expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in lesional skin as well as in fibrous lung in this model. Dermal sclerosis was induced by daily local injections of bleomycin (100 microg/ml) for 3 weeks in C3H mice. Immunohistochemical examination showed that alpha-SMA-reactive cells were detectable on fibroblastic cells in bleomycin-injected skin at 1 week. There was a significant increase in the immunoreactive fibroblastic cells for alpha-SMA in lesional skin in parallel with the induction of dermal sclerosis. After 3 weeks' treatment with bleomycin, the number of alpha-SMA-reactive fibroblasts showed an 11-fold increase compared with that in control PBS-treated mice. alpha-SMA-positive cells were also detected in lung parenchyma after bleomycin treatment. Following concomitant treatment with anti-transforming growth factor-beta (TGF-beta) antibody with bleomycin, the number of alpha-SMA-positive fibroblastic cells was significantly reduced up to 50%, along with the reduction of dermal sclerosis. To confirm the protein level of alpha-SMA, immunoblotting was carried out. Results showed an increase of alpha-SMA expression in lesional skin at 3 weeks of bleomycin treatment, which was reduced following anti-TGF-beta antibody treatment. These data suggest that fibroblastic cells are phenotypically altered into myofibroblasts during the fibrotic process in the experimental model of bleomycin-induced scleroderma, which was considered mediated, for the most part, by TGF-beta. Blockade of TGF-beta may be a therapeutic intervention for scleroderma.     

Antibodies to membranes of endothelial cells and fibroblasts in scleroderma

Anti-endothelial and other cell membrane-reactive antibodies in scleroderma were characterized by immunoblotting sera with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), dermal fibroblasts and a T cell lymphoma HUT78. Antibodies reactive with HUVEC membranes were found in 17 of 20 patients with scleroderma (33 bands) in contrast to only two of 20 controls (two bands; P < 0.01) and three of 11 patients with myocardial infarction (four bands). Eleven of the 20 patients possessed antibodies that were specific for HUVEC membrane and did not cross-react with other cell lines. Analysis of patient subgroups showed that HUVEC membrane antibodies were present in nine of 11 patients with systemic sclerosis and in all nine with the CREST syndrome, and were HUVEC-specific in five and six of these cases, respectively. Although considerable heterogeneity was seen, antibodies to an 18–19-kD membrane epitope were found in 11 of the 20 patients but in none of the controls (P < 0.01). This antibody which reacted particularly with HUVEC (n = 9) and HUT78 membranes (n = 9) was associated with CREST syndrome rather than systemic sclerosis (9/9 versus 1/11; P < 0.01), and after elution was shown to possess anticentromere activity. In addition, antibodies reactive with both fibroblast (n = 11; 18 bands) and HUT78 membranes (n = 18; 42 bands) were detected and were specific for either fibroblast or HUT78 membranes in nine and 14 patients, respectively. There was no significant difference in the incidence of these fibroblasts and HUT78 membrane antibodies in the two patient subgroups. These findings support the concept that membrane-reactive antibodies, including anticentromeric antibodies, may play a central role in the pathogenesis of scleroderma, through their ability to react with endothelial cells.     

The safety and efficacy of light emitting diodes-based ultraviolet A1 phototherapy in bleomycin-induced scleroderma in mice

Purpose

To define the efficacy and safety of narrowband ultraviolet A1 (UVA1) for the treatment of dermal fibrosis in bleomycin-induced mouse model of scleroderma.

Antifibrotic and anthelminthic effect of casticin on Schistosoma mansoni-infected BALB/c mice

Background/PurposeSchistosomiasis is an important tropical disease caused by Schistosoma. Although the pathogenesis of liver fibrosis has been intensively studied, the choice of effective treatment is still inadequate. In this study, we aimed to investigate the potential of using Casticin to treat Schistosoma mansoni-induced liver fibrosis.MethodsBALB/c mice were divided into three groups – control, infection, and treatment group. The infection and treatment group were percutaneously infected with 100–120 cercariae. Mice from the treatment group were treated with 20 mg/kg/day Casticin for 14 consecutive days to investigate the potential protective effects of Casticin. Mice were sacrificed and were used for histological, RNA, protein, and parasite burden analysis.ResultsOur results showed that hepatic fibrosis was significantly attenuated, as indicated by histology and reduction of fibrotic markers such as collagen AI, transforming growth factor β (TGF-β), and α-smooth muscle actin (α-SMA). Furthermore, Casticin treatment significantly reduced worm burden. Anthelmintic effect of Casticin was also observed by scanning electron microscopy.ConclusionCollectively, our study suggested that Casticin may be a beneficial candidate in treating S. mansoni infection.     

人皮肤成纤维前体细胞的分离培养及其功能鉴定

目的以人的皮肤为材料,拟在体外成功分离培养成纤维前体细胞并研究其除皱整形能力。方法组织块法分离人的皮肤成纤维前体细胞,并通过RT-PCR、流式细胞仪、软琼脂克隆等实验技术对分离的皮肤成纤维前体细胞进行鉴定,同时进行免疫荧光、动物实验检测其在治疗真皮损伤以及除皱整形方面的作用。结果成功分离并扩增人的皮肤成纤维前体细胞,优化了原代细胞的分离方法,RT-PCR和流式细胞仪检测结果显示该细胞表达部分干细胞表面标记HLA-I、HLA-DR、CD90以及CD105,软琼脂克隆实验表明该细胞不具有致瘤性。免疫荧光结果显示,注射该细胞的裸鼠与对照组相比,真皮层产生更多的I型胶原蛋白,皮肤厚度增加。结论一次取材,体外能够成功持续获得人的原代皮肤成纤维前体细胞,优化了原代细胞分离方法。并且该细胞注射后可以在一定程度上分化为成纤维细胞,产生胶原蛋白,修复受损皮肤。     

Hepatocyte giant mitochondria: An almost constant lesion in systemic scleroderma

Summary Liver electron microscopic studies were performed in 14 patients with systemic scleroderma. In 13 of these patients, giant mitochondria were demonstrated in the hepatocytes. This ultrastructal abnormality was present whatever the type and duration of the disease and was also present even when the liver was histologically normal. The mechanism of formation of giant mitochondria in systemic scleroderma is unknown.     

纤维粘连蛋白对培养的自发性高血压大鼠心肌成纤维细胞增殖及胶原合成的影响

目的:观察纤维粘连蛋白(FN)对培养的SHR和WKY大鼠心肌成纤维细胞(CFb, CFb_SHR, CFb_WKY)增殖及胶原合成的影响。方法:CFb取自12周的SHR和WKY大鼠, 采用组织块贴壁法培养, 以直接细胞计数法和 [³H]-TdR掺入率反映细胞增殖, 以[³H]-脯氨酸([³H]-proline)掺入率反映胶原合成。使用FN(5 μg/cm²)预先处理24孔培养板。 结果: 与0.4% FCS对照组相比, 经72 h孵育FN明显促进CFb_SHR和CFb_WKY细胞数增多, 分别为对照组的163.75%(CFb_SHR)和170.42%(CFb_WKY)。FN促进CFb_SHR和CFb_WKY [³H]-TdR掺入增加。FN促进CFb_SHR和CFb_WKY[³H]-proline掺入增加。 结论:FN促进SHR和WKY大鼠的CFb增殖及胶原合成。     

三氧化二砷对皮肤成纤维细胞、中性粒细胞MMPs活性，TIMP-1及TGF-β1表达的影响

目的:砒石是化腐生肌的常用中药,其主要成分是三氧化二砷(As2O3)。本研究通过观察As2O3对基质金属蛋白酶(MMPs)活性、基质金属蛋白酶组织抑制因子-1(TIMP-1)及转化生长因子β1(TGF-β1)表达影响,探讨化腐中药能否调节胶原代谢,从而治疗慢性皮肤溃疡。方法:明胶酶谱法检测大鼠中性粒细胞(PMNs)来源的MMP-9活性、人成纤维细胞(hFb)分泌的MMP-1、MMP-2的活性,免疫细胞化学法检测hFb TIMP-1、TGF-β1的表达。结果:As2O3浓度在50mg/L时可以提高大鼠PMNs来源的MMP-9的活性(P<0.01);在0.8mg/L可以提高hFb分泌的MMP-1、MMP-2的活性(分别P<0.01);同时As2O3作用于hFb6h、12h、18h后,TIMP-1、TGF-β1表达持续降低(P<0.01)。结论:As2O3在一定范围内可提高PMNs来源的MMP-9的活性;也可提高hFb分泌的MMP-1、MMP-2的活性,同时抑制hFbTIMP-1、TGF-β1的表达。提示砷类制剂可通过提高多种MMPs的活性,降低TIMP-1的表达从而发挥化腐作用。     

血小板源生长因子对人血管成纤维细胞DNA及胶原蛋白合成的影响

目的:观察血小板源生长因子对培养的人血管成纤维细胞DNA及胶原蛋白合成的影响。方法:采用培养的人血管成纤维细胞,应用 [³H]-TdR和 [³H]-脯氨酸掺入的方法,观察血小板源生长因子-BB对人血管成纤维细胞DNA合成以及胶原蛋白合成的影响。结果:血小板源生长因子-BB可促进静止状态的人血管成纤维细胞DNA及胶原蛋白的合成,在 30μg/L浓度时DNA及胶原蛋白的合成达到高峰,DNA及胶原蛋白分别于 2 4h和 36h合成最为显著。结论:血小板源生长因子-BB可明显促进培养的人血管成纤维细胞DNA及胶原蛋白的合成     

三氧化二砷对皮肤成纤维细胞、中性粒细胞MMPs活性,TIMP-1及TGF-β1表达的影响

目的：砒石是化腐生肌的常用中药，其主要成分是三氧化二砷（As₂O₃）。本研究通过观察As₂O₃对基质金属蛋白酶（MMPs）活性、基质金属蛋白酶组织抑制因子-1（TIMP-1）及转化生长因子β₁（TGF-β₁）表达影响，探讨化腐中药能否调节胶原代谢，从而治疗慢性皮肤溃疡。方法:明胶酶谱法检测大鼠中性粒细胞（PMNs）来源的MMP-9活性、人成纤维细胞（hFb）分泌的MMP-1、MMP-2的活性，免疫细胞化学法检测hFb TIMP-1、TGF-β₁的表达。结果:As₂O₃浓度在50 mg/L时可以提高大鼠PMNs来源的MMP-9的活性（P<0.01）；在0.8 mg/L可以提高hFb分泌的MMP-1、MMP-2的活性（分别P<0.01）；同时As₂O₃作用于hFb 6 h、12 h、18 h后，TIMP-1、TGF-β₁表达持续降低（P<0.01）。结论:As₂O₃在一定范围内可提高PMNs来源的MMP-9的活性；也可提高hFb分泌的MMP-1、MMP-2的活性，同时抑制hFbTIMP-1、TGF-β₁的表达。提示砷类制剂可通过提高多种MMPs的活性，降低TIMP-1的表达从而发挥化腐作用。     

巨噬细胞移动抑制因子经Rho途径促进人肺成纤维细胞III型胶原合成

目的: 观察重组巨噬细胞移动抑制因子(rMIF)对人胚肺成纤维细胞(MRC-5)的作用,探讨哮喘气道重塑的发生机制。方法: 不同浓度的rMIF (25-100 μg/L)分别作用于MRC-5细胞48 h, RT-PCR法检测III型胶原mRNA表达,Western blotting检测III型胶原蛋白合成; 100 μg/L rMIF刺激MRC-5细胞48 h,刺激前0.5 h加入Rho激酶特异性抑制剂Y27632, RT-PCR法检测III型胶原mRNA表达,Western blotting法检测III型胶原蛋白表达。结果: 刺激MRC-5细胞48 h,rMIF可呈剂量依赖地诱导III型胶原mRNA(P<0.01)和蛋白(P<0.01)表达。Y27632预刺激后,显著抑制rMIF100μg/L诱导的III型胶原mRNA和蛋白表达(均P<0.01)。结论: MIF可能通过Rho途径刺激人肺成纤维细胞III型胶原合成,从而在哮喘气道重塑的发病机制中发挥重要作用。     

同型半胱氨酸对气道平滑肌细胞及成纤维细胞的影响

目的:探讨同型半胱氨酸(HCY)对气道平滑肌细胞及成纤维细胞的增殖和成纤维细胞胶原生成的影响。方法:在培养的兔气道平滑肌细胞和大鼠成纤维细胞上,用[³H]-胸腺嘧啶和[³H]-脯氨酸掺入方法观察不同浓度HCY对细胞增殖和成纤维细胞胶原生成的影响。 结果:HCY(0.1~1.0 mmol/L)呈浓度依赖性刺激气道平滑肌细胞和成纤维细胞增殖,并呈浓度依赖性刺激气道成纤维细胞的胶原合成与分泌。蛋白激酶C抑制剂H7及多粘菌素B可抑制HCY诱导的气道平滑肌细胞增殖。 结论:HCY刺激气道平滑肌 细胞及成纤维细胞增殖,并促进成纤维细胞的胶原合成和分泌。HCY刺激气道平滑肌细胞增殖可能通过PKC信号传导途径。     

压力下角质形成细胞与成纤维细胞共培养对细胞增殖及胶原合成的影响

目的研究压力下人角质形成细胞(human keratinocytes,HKC)和人成纤维细胞(human fibroblasts,HFB)的三维共培养对细胞增殖及胶原蛋白合成的影响。方法将HKC与HFB分别接种于壳聚糖-明胶支架2 d后,将气-液界面诱导分化1 d后的HKC-壳聚糖-明胶复合物与HFB-壳聚糖-明胶复合物共培养12 h,3.4 k Pa气体压力加载24 h,并以压力单独培养、无压力单独培养或无压力共培养作为对照。HE染色观察细胞在支架中的分布及生长情况,MTT法测定细胞增殖情况,羟脯氨酸试剂盒测定上清液中的胶原含量。结果 HE染色发现,HKC与HFB均可以在壳聚糖-明胶支架上正常增殖成片;3.4 k Pa压力或共培养均可以促进HKC增殖和胶原合成,抑制HFB增殖及胶原合成。结论压力及共培养是影响HKC与HFB增殖及胶原蛋白合成的重要因素,研究结果为手术切除瘢痕后移植组织工程表皮结合压力法治疗增生性瘢痕的可能机制提供参考。     

小鼠真皮间充质干细胞对硬皮病成纤维细胞胶原分泌和TGF-β1表达的影响

目的:研究体外小鼠真皮间充质干细胞(mdMSCs)与硬皮病患者皮损区成纤维细胞(hsFb)共培养对其胶原成分分泌和转化生长因子β1(TGF-β1)表达的影响,探讨MSCs对硬皮病治疗的可能性。方法:采用低血清培养基,消化-贴壁-传代法体外培养、鉴定mdMSCs,并与体外分离培养的hsFb或正常人成纤维细胞(hnFb)于Transwell培养体系中共培养,样本碱水解法和ELISA法分别检测第4、8 d培养上清液中羟脯氨酸(Hyp)和TGF-β1的变化。结果:第8 d单独培养的hsFb上清液Hyp、TGF-β1含量较hnFb高(P<0.05);经各细胞密度mdMSCs处理的hsFb培养上清液中Hpy含量与单独培养时无显著差异;经mdMSCs 2.5×104处理的hsFb培养组,在共培养第4 d上清液TGF-β1含量较单独培养时明显增高(P<0.05),第8 d时与单独培养比较无明显差异(P>0.05)。共培养组Hyp含量与TGF-β1水平无明显相关(r=0.221,P>0.05);单独培养组Hyp含量与TGF-β1水平有正相关关系(r=0.682,P<0.01)。结论:体外mdMSCs与hsFb共培养未能有效减少Hyp和TGF-β1的分泌,提示mdMSCs不能通过对hsFb的作用达到治疗硬皮病的目的。     

Biased cell migration of fibroblasts exhibiting contact guidance in oriented collagen gels

We present here the first quantitative correlation for cell contact guidance in an oriented fibrillar network in terms of biased cell migration. The correlation is between the anisotropic cell diffusion parameter,D _A=D_x/D_y, and the collagen gel birefringence, Δn, a measure of axially biased collagen fibril orientation in thex-direction. The cell diffusion coefficients,D _x andD _y, measure the dispersal of cells in the directions coincident with and normal to the axis of fibril orientation, respectively. Three essential methodological components are involved: (i) exploiting the orienting effect of a magnetic field on collagen fibrils during fibrillogenesis to systematically prepare uniform axially oriented collagen gels; (ii) using a microscope/image analysis workstation with precise, computer-controlled rotating and translating stages to automate birefringence measurement and, along with rapid “coarse optical sectioning” via digital image processing, to enable 3-D cell tracking of many cells in multiple samples simultaneously; and (iii) employing a rigorous statistical analysis of the cell tracks to estimate the magnitude and precision of the direction-dependent cell diffusion coefficients,D _x andD _y, that defineD _A. We find that this measure of biased migration in contact guidance (D _A) increases with increasing collagen fibril orientation (Δn) due mainly to a rapid enhancement of migration along the axis of fibril orientation at low levels of fibril orientation, and to a continued suppression of migration normal to the axis of fibril orientation at high levels of fibril orientation.     

Histological evidence for enhanced anal fistula repair using autologous fibroblasts in a dermal collagen matrix

An anal fistula has a primary track passing from an internal anal or rectal opening to an external opening in the perianal area. Surgery aims to eradicate sepsis whilst preserving faecal continence. Fistulotomy, when all tissue caudal to the primary fistulous track is opened, provides the surest method of cure but may diminish patient continence. An alternative sphincter-preserving procedure is to instill a sealant into the track. An experimental porcine model of fistula-in-ano has been developed in the Surgical Research Department at Northwick Park Institute for Medical Research. This allows histological assessment of fistula tracks after novel, sphincter-preserving surgery and treatments have been applied. Under general anaesthetic, 24 anal fistulae were created and treated, three in each of eight adult Large White/Landrace crossbred pigs. Under the same general anaesthetic, a split skin graft was taken from which to culture fibroblasts for future treatment. All tracks were treated at 4 weeks post-track induction when the tracks were established and very similar in clinical appearance to human tracks. All tracks were prepared for treatment using an instrument designed to remove granulation tissue from the wall of the track. Five control tracks were not infilled but simply had their internal and external openings closed with a Vicryl suture. Nine tracks were treated by infill using an acellular porcine dermal collagen matrix. Ten tracks were treated using a mixture of this matrix and autologous cultured fibroblasts. Histological examination of six tracks was carried out at 2 weeks, nine tracks at 2 months and nine tracks at 3 months. Histological assessment demonstrated persistent fistula tracks in only two fistulae, both of which were control tracks. All treated tracks were closed and cured at all times of examination. When autologous fibroblasts were added to the infill material, cellular integration and vascularization were improved. Using this pre-clinical model, provided fistulous tracks were prepared using a new, in-house developed instrument; treatment with acellular collagen matrix alone healed all tracks. Adding autologous fibroblasts improved the quality of wound healing. A pilot study using this treatment in human fistula patients is in progress. This paper was first presented at the European Histology Forum Annual Conference on 24–26 April 2006.     

Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma

Accumulative data have demonstrated that plasminogen activator inhibitor-1 (PAI-1) plays an important role in the extracellular matrix metabolism; however, the involvement of PAI-1 in scleroderma has not been fully elucidated. In this study, we investigated the role of PAI-1 in bleomycin-induced murine scleroderma. 100 microg of bleomycin was injected subcutaneously to the back skin of C3H/HeJ mice on alternate day for 4 weeks. Histopathological findings revealed that PAI-1 was positive in macrophage-like cells and fibroblastic cells in the dermis, in parallel with the induction of dermal sclerosis. PAI-1 mRNA expression in the whole skin was up-regulated at 1 and 4 weeks. The production of active PAI-1 protein in the lesional skin was significantly increased 3 and 4 weeks after bleomycin treatment. Next, we examined whether dermal sclerosis is induced by bleomycin in PAI-1-deficient (PAI-1-/-) mice. 10 microg of bleomycin was subcutaneously injected to PAI-1-/- and wild type (WT) mice 5 days per week for 4 weeks. Histological examination revealed that dermal sclerosis was similarly induced even in PAI-1-/- as well as WT mice. Dermal thickness and collagen contents in the skin were significantly increased by bleomycin injection in both PAI-1-/- and WT mice, and the rate of increase was similar. These data suggest that PAI-1 plays an important role, possibly via TGF-beta pathway activation. However, the fact that PAI-1 deficiency did not ameliorate skin sclerosis suggest that PAI-1 is not the essential factor in the development of bleomycin-induced scleroderma, and more complex biochemical effects other than PA/plasmin system are greatly suspected.     

Immunological characterization of heterochromatin protein p25β autoantibodies and relationship with centromere autoantibodies and pulmonary fibrosis in systemic scleroderma

 Anticentromere antibodies (ACA) are immunological markers for the subset of systemic scleroderma with the symptoms calcinosis cutis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (CREST). In western blotting, some ACA-positive sera also recognize a doublet of 23 kDa (p23) and 25 kDa (p25) in addition to centromere protein antigens A (17 kDa), B (80 kDa), and C (140 kDa). Two forms of p25 have been shown to be human homologues of Drosophila heterochromatin-associated protein HP1. One form of p25 (p25β) which was recently cloned in this laboratory was used to evaluate anti-p25β antibody response in scleroderma sera. Of 318 scleroderma sera 42 had ACA (13.2%), and 16 of the 42 sera (38%) had anti-p25β antibodies. On the other hand, 5 of 276 ACA-negative sera (1.8%) showed anti-p25β antibody response, demonstrating that anti-p25β antibody is significantly associated with the ACA response (P<10^–8). Clinically the anti-p25β response was significantly associated with the CREST syndrome. Fourteen (36.8%) of 38 CREST patients compared to seven (2.5%) of 280 patients with other forms of scleroderma were anti-p25β antibody positive (P<10^–8). The 14 CREST patients with anti-p25β antibodies had significantly more interstitial lung disease than those without anti-p25β antibodies (P<0.003). There was also a tendency to increased liver involvement. Two dominant autoepitopes in p25β were determined by western blotting using p25β recombinant fragments. In immunofluorescence C-terminal specific antibodies showed staining of heterochromatin, but N-terminal specific antibodies showed no staining. Interestingly, the majority of sera reacted preferentially with one or the other of the two dominant autoepitopes. Received: 6 March 1997 / Accepted: 21 July 1997