大鼠肝卵圆细胞增殖过程中基质细胞衍生因子-1及其受体CXCR4的表达

近年来,有研究表明基质细胞衍生因子1(stromal cell-derived factor-1,SDF-1)和其受体CXCR4(SDF-1/CXCR4)轴在干细胞的增殖分化中起至关重要的作用[1].本研究采用2-乙酰氨基芴联合部分肝脏切除术(AAF/PH)成功诱导肝卵圆细胞增殖,同时给AAF/PH模型大鼠注射CXCR4特异性抑制剂AMD3100,通过检测SDF 1和其受体CXCR4在卵圆细胞增殖过程中表达的变化情况,进一步研究SDF-1/CXCR4轴在肝卵圆细胞增殖过程中的作用.     

Stromal cell–derived factor 1/CXCR4 signaling is critical for the recruitment of mesenchymal stem cells to the fracture site during skeletal repair in a mouse model

Objective

Stromal cell–derived factor 1 (SDF‐1; CXCL12/pre–B cell growth‐stimulating factor) is a dominant chemokine in bone marrow and is known to be involved in inflammatory diseases, including rheumatoid arthritis. However, its role in bone repair remains unknown. The purpose of this study was to investigate the role of SDF‐1 and its receptor, CXCR4, in bone healing.

Methods

The expression of SDF‐1 during the repair of a murine structural femoral bone graft was examined by real‐time polymerase chain reaction and immunohistochemical analysis. The bone graft model was treated with anti–SDF‐1 neutralizing antibody or TF14016, an antagonist for CXCR4, and evaluated by histomorphometry. The functional effect of SDF‐1 on primary mesenchymal stem cells was determined by in vitro and in vivo migration assays. New bone formation in an exchanging‐graft model was compared with that in the autograft models, using mice partially lacking SDF‐1 (SDF‐1^+/−) or CXCR4 (CXCR4^+/−).

Results

The expression of SDF1 messenger RNA was increased during the healing of live bone grafts but was not increased in dead grafts. High expression of SDF‐1 protein was observed in the periosteum of the live graft. New bone formation was inhibited by the administration of anti–SDF‐1 antibody or TF14016. SDF‐1 increased mesenchymal stem cell chemotaxis in vitro in a dose‐dependent manner. The in vivo migration study demonstrated that mesenchymal stem cells recruited by SDF‐1 participate in endochondral bone repair. Bone formation was decreased in SDF‐1^+/− and CXCR4^+/− mice and was restored by the graft bones from CXCR4^+/− mice transplanted into the SDF‐1^+/− femur, but not vice versa.

Conclusion

SDF‐1 is induced in the periosteum of injured bone and promotes endochondral bone repair by recruiting mesenchymal stem cells to the site of injury.

     

Insights into the pathogenesis of systemic sclerosis based on the gene expression profile of progenitor‐derived endothelial cells

Objective

To determine the gene expression profile of endothelial cells derived from the endothelial progenitor cells (EPCs) of patients with systemic sclerosis (SSc).

Methods

Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST Arrays in unstimulated and hypoxia‐stimulated EPC‐derived cells from patients with SSc and control subjects. Followup of the raised hypotheses was performed ex vivo by immunohistochemical analysis of skin tissue.

Results

Signals from 92 probe sets and 188 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with SSc compared with controls. Within the largest groups of genes related to cell–cell interaction and vascular remodeling, down‐regulation of tumor necrosis factor ligand superfamily member 10 (TNFSF10) and homeobox A9 (HOX‐A9) was confirmed by real‐time polymerase chain reaction and Western blots in EPC‐derived cells and by immunohistochemistry in SSc skin tissue. Signals from 221 and 307 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with diffuse cutaneous SSc compared with patients with limited cutaneous SSc. Within the largest group of genes related to the inflammatory response, differential expression of TNFα‐induced protein 3 and prostaglandin‐endoperoxide synthase 2 was observed in EPC‐derived cells and skin tissue from patients with SSc.

Conclusion

Our data revealed important gene expression changes in EPC‐derived endothelial cells from patients with SSc, characterized by a proadhesive, proinflammatory, and activated phenotype. Differential expression in lesional SSc skin tissue of new targets, such as TNF family members and HOX‐A9, may contribute to the pathogenesis of SSc and deserves more in‐depth exploration.

     

E-selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis

Objective. To assess endothelial cell activation in patients with systemic sclerosis (SSc). Methods. Concomitant study of salivary gland biopsy tissues and sera for expression of E-selectin and its potent activator tumor necrosis factor α (TNFα), using immunostaining and enzyme-linked immunosorbent essay. Results. E-selectin was overexpressed in SSc patients, but not in controls. TNFα was detected in mast cells. Conclusion. Mast cell–derived TNFα may contribute to endothelial cell activation in SSc.     

Expression of hepatocyte growth factor and its receptor (c-met) in skin fibroblasts from patients with systemic sclerosis

OBJECTIVE: To determine the effect of excessive induction of hepatocyte growth factor (HGF)/c-met signaling in fibroblasts derived from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from skin of patients with SSc and healthy controls. The 2.4 kb interleukin 1alpha (IL-1alpha) cDNA was subcloned into pcDNA3 expression vector, which was stably transfected into normal fibroblasts by lipofection. HGF production in cultured fibroblasts was measured by ELISA. C-met protein (a receptor for HGF) in cultured fibroblasts was evaluated by immunocytochemistry using polyclonal anti-c-met antibody. Production of procollagen type I was estimated by an ELISA system using antibodies against procollagen type I C-peptide. RESULTS: Cultured skin fibroblasts expressed mRNA and protein of HGF constitutively in both SSc and control cultures. However, HGF production in SSc fibroblasts was significantly higher than in normal fibroblasts. In both SSc and normal fibroblasts, HGF production was dose dependently increased by the addition of recombinant IL-1alpha. Immunocytochemical staining revealed that c-met was spontaneously expressed in SSc fibroblasts, whereas no expression of c-met was detected in normal fibroblasts. C-met mRNA was expressed in normal fibroblasts transfected with the IL-1alpha gene. Addition of recombinant HGF (100 ng/ml) to cultured SSc fibroblasts significantly decreased procollagen type I production. CONCLUSION: High concentration of HGF inhibited collagen production in cultured fibroblasts derived from patients with SSc. Overexpression of HGF/c-met appears to be a biological feedback response to the fibrotic process of SSc, suggesting that the antifibrotic effect of HGF might be used as a novel strategy for treatment of SSc.     

Impairment of endothelial cell differentiation from bone marrow–derived mesenchymal stem cells: New insight into the pathogenesis of systemic sclerosis

Objective

Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair.

Methods

MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony‐forming unit–fibroblastoid colonies was determined. After culture in endothelial‐specific medium, the endothelial‐like MSC (EL‐MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed.

Results

MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR‐2+, CXCR4+, VEGFR‐2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL‐MSCs showed increased expression of VEGFR‐1, VEGFR‐2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell–derived factor 1 to cultured SSc EL‐MSCs increased their angiogenic potential less than that in controls.

Conclusion

Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.

     

Lack of evidence of stimulatory autoantibodies to platelet‐derived growth factor receptor in patients with systemic sclerosis

Objective

Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal organs. Recent findings suggested that the disease is driven by stimulatory autoantibodies to platelet‐derived growth factor receptor (PDGFR), which stimulate the production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues of research into the diagnosis and treatment of SSc. The present study was undertaken to confirm the presence of anti‐PDGFR antibodies in patients with SSc.

Methods

Immunoglobulins from 37 patients with SSc were purified by protein A/G chromatography. PDGFR activation was tested using 4 different sensitive bioassays, i.e., cell proliferation, ROS production, signal transduction, and receptor phosphorylation; the latter was also tested in a separate population of 7 patients with SSc from a different research center.

Results

Purified IgG samples from patients with SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFRα or PDGFRβ in any of the tests. Cell stimulation with PDGF itself consistently produced a strong signal.

Conclusion

The present results raise questions regarding the existence of agonistic autoantibodies to PDGFR in SSc.

     

血清血管内皮生长因子及可溶性血管内皮生长因子受体1在系统性红斑狼疮患者中的表达及其意义

目的 研究血清中血管内皮生长因子(VEGF)及可溶性血管内皮生长因子受体1(sFlt-1)在活动期系统性红斑狼疮(SLE)及不同肾脏病理类型的狼疮肾炎(LN)患者中表达的意义. 方法 采用双抗体夹心酶联免疫吸附法(ELISA)对60例SLE患者及30名健康人血清中VEGF及sFlt-1的水平同时进行检测,结合临床资料及肾脏病理进行相关分析. 结果 活动期SLE患者血清VEGF及sFlt-1水平均明显升高;血清中VEGF/sFlt-1的比值健康对照组较活动期SLE、非活动期SLE及LN组患者降低(P＜0.01),Ⅴ型LN组该比值较Ⅱ、Ⅲ、Ⅳ型LN组升高(P＜0.05);血清sFlt-1的浓度与尿蛋白呈正相关(rs=0.6244,P＜0.01),血清VEGF的浓度与尿蛋白无明显相关(rs=0.1807,P＞0.05);血清sFlt-1的浓度与ESR正相关(rs=0.4235,P＜0.01),血清VEGF的浓度与ESR无明显相关(rs=0.0532,P＞0.05);血清VEGF及sFlt-1浓度与SLE疾病活动指数(SLEDAI)均呈正相关(rs=0.5046,P＜0.01,rs=0.5152,P＜0.01);血清VEGF浓度与肾组织活动指数(RAI)呈正相父(r=0.3386,P＜0.05),血清sFlt-1浓度与RAI无明显相关(rs=0.0240,P＞0.05);SLE患者中VEGF、sFlt-1水平与血压、血肌酐、尿素氮、C3、C4、C反应蛋白(CRP)无明显相关. 结论 血清VEGF及sFlt-1的水平可作为SLE病情活动评价指标,VEGF的高表达可能与增殖性肾小球病变相关,sFlt-1的表达与蛋白尿关系密切.