Expression of c-Met receptor and hepatocyte growth factor/scatter factor in synovial sarcoma and epithelioid sarcoma

 Overexpression of c-Met receptor/hepatocyte growth factor (scatter factor) system (c-Met/HGF/SF) as a physiologically paracrine cellular signaling system is thought to be involved in the progression of malignant tumours. In 26 synovial sarcomas and epithelioid sarcomas, c-Met and HGF/SF expression was analysed immunohistochemically. There were 10 biphasic synovial sarcomas, 7 of which showed moderate to strong c-Met expression in epithelial areas compared with the fibrous component, with corresponding expression of HGF/SF. Six of 9 monophasic fibrous synovial sarcomas showed only very faint c-Met and corresponding HGF/SF expression. In 7 epithelioid sarcomas strong expression of c-Met and HGF/SF was observed within epithelioid tumour cells. Non-radioactive in situ hybridization demonstrated the synthesis of c-Met receptor in tumor cells by detecting c-met-mRNA. This analysis shows that in synovial sarcomas and epithelioid sarcomas, tumour entities with epithelial and mesenchymal structures, c-Met and HGF/SF overexpression can be detected, indicating a role of this signaling system in these subtypes of sarcoma, and especially in the more epithelioid tumour phenotype. An autocrine interaction between overexpressed c-Met receptor and HGF/SF may be hypothesized. Received: 21 July 1997 / Accepted: 19 November 1997     

Hepatocyte growth factor/scatter factor—Met signaling in tumorigenicity and invasion/metastasis

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. While HGF/SF-Met signaling clearly plays a role in a variety of normal cellular processes, this signaling pathway has also been implicated in the generation and metastatic spread of tumors. This review discusses in detail several model systems that have been developed to investigate the role of HGF/SF-Met signaling in malignancy and describes additional data regarding the expression of these molecules in human tumors. Collectively the findings support a role for this receptor-ligand pair in human malignancy.Abbreviations ECM/BM Extracellular matrix/basement membrane - HGF/SF Hepatocyte growth factor/scatter factor - uPA Urokinase-type plasminogen activator - uPAR Urokinase-type plasminogen activator receptor     

肝细胞生长因子和表皮生长因子联合体外诱导大鼠肝卵圆细胞分化的研究

目的探求卵圆细胞向成熟肝细胞分化的演变规律,并对其分化调控的分子机制作初步探讨。方法联合应用肝细胞生长因子和表皮生长因子体外培养大鼠肝脏卵圆细胞OC3,运用电镜、免疫细胞化学、半定量逆转录-聚合酶链反应（RT—PCR）等技术,检测OC3在分化过程中形态及相关分子标记物的改变,并运用蛋白芯片技术观察细胞蛋白表达谱的变化。结果诱导10周后,卵圆细胞胞质变丰富,细胞器增多,表达谷胱甘肽-S-转移酶（GST—P）和丙酮酸激酶（M2-PK）减弱,而表达白蛋白（ALB）、CK18增强,同时诱导后期的细胞较之诱导前主要出现了8种表达差异蛋白。结论在肝细胞生长因子与表皮生长因子的共同作用下,体外培养的卵圆细胞可逐步分化为成熟肝细胞。8种主要的结构蛋白或功能蛋白可能参与了对该过程的调控。     

肝细胞生长因子和类胰岛素样生长因子对心肌干细胞的诱导作用

目的探讨肝细胞生长因子(HGF)和类胰岛素样生长因子(IGF1)在外能否诱导心肌干细胞(CSCs)发生增殖并向心肌细胞定向分化。方法组织贴块法分离培养心肌干细胞,免疫荧光技术鉴定c-kit和CD34表达,流式细胞仪分选纯化c-kit+细胞,CFDA SE荧光探针示踪培养检测细胞增殖特征。实验分为单纯心肌干细胞组和与心肌共培养组,分别用HGF和IGF1干预,倒置显微镜观察细胞不同时期数量与形态的变化,活细胞工作站观察CFDA SE示踪剂荧光强弱,采集图像,进行统计学分析。免疫荧光技术检测Nkx2.5、心肌肌钙蛋白T(cTnT)的表达。结果单纯心肌干细胞组,生长因子刺激后心肌干细胞数量与形态均无明显变化。与心肌共培养组,细胞均发生增殖与形态变化,Nkx2.5、cTnT阳性表达,有个别心肌干细胞分化为自发搏动的心肌细胞。结论心肌干细胞与心肌细胞共培养条件下,HGF和IGF1能够促进心肌干细胞增殖,联合作用能够诱导心肌干细胞向心肌细胞分化。     

Effect of hepatocyte growth factor on sperm motility

PROBLEM: Hepatocyte growth factor (HGF) exists abundantly in seminal plasma and its receptor, c-met, is expressed on spermatozoa. Considering its motogenic activity, we speculated that HGF might affect the movement ability of spermatozoa. METHODS: Recombinant HGF was added to washed spermatozoa and their movements were analyzed using a computer-assisted sperm analyzer. The concentration of HGF in the seminal plasma of infertile patients (n = 83) was measured by ELISA, and the data were compared with their hormonal profile and semen parameters. RESULTS: The HGF physiological concentration (1 ng/mL) maintained the motility of sperm after a long incubation, though the difference was not statistically significant. Recombinant HGF did not affect the linearity or frequency of movement, which suggested that it does not evoke the hyperactivation of spermatozoa. The concentration of HGF in seminal plasma did not correlate with any clinical parameter of the patients. CONCLUSIONS: These findings contradict the theory that HGF controls the movement of sperm. The main role of this axis in the male reproductive system might be maturation in the epididymis.     

Regulation of spreading and growth of colon cancer cells by hepatocyte growth factor

Hepatocyte growth factor (HGF), also known as scatter factor, regulates both cell motility and the growth of some cell types. We have determined the effects of HGF on the motility and growth of human colon cancer cell lines (HT115, HT29, HRT18 and HT55). Cell motility, as measured by dissociation from carrier beads or by scattering of cell colonies, was greatly increased in all cell lines. The effects were completely blocked by anti-HGF antibody. In contrast, cell growth of HT115, HT29 and HRT18 cells was inhibited by a wide range of concentrations of HGF. HT55 cell growth was also inhibited but needed a prolonged culture period (>5 days). The HGF receptor/Met protein is highly expressed in the membrane fraction of these cells as determined by Western blotting. It is concluded that HGF has an effect on both colon cancer cell motility and growth, which may be important in the control of the spread of colon cancer.     

HIL-6和HGF双基因重组腺病毒增强对慢加急性肝衰竭大鼠的治疗效果

 目的: 比较超级白细胞介素6(HIL-6)和肝细胞生长因子(HGF)双基因重组腺病毒(Ad-HGF-HIL-6)与HIL-6或HGF重组腺病毒(Ad-HIL-6或Ad-HGF)对慢加急性肝衰竭(ACLF)大鼠的治疗效果。方法: 将ACLF大鼠随机分为:未感染(model)组、空载体(Ad0)组、Ad-HGF组、Ad-HIL-6组和Ad-HGF-HIL-6组。收集血清及肝组织进行生物化学、病理学及分子生物学检测。结果: Ad-HGF、Ad-HIL-6或Ad-HGF-HIL-6组与Ad0组比较,凝血酶原时间(PT)、血清丙氨酸氨基转移酶(ALT)、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)、高迁移率族蛋白B1(HMGB1)、凋亡指数及Bax蛋白明显降低且肝组织病变减轻,Bcl-2和Ki67蛋白的表达明显增高;以上各项变化均以Ad-HGF-HIL-6组更显著。结论: Ad-HGF-HIL-6比Ad-HGF或Ad-HIL-6对ACLF大鼠有更显著的治疗效果且无明显毒副作用。     

肝细胞生长因子/c-Met系统在鼻咽癌中的表达及意义

目的　探讨肝细胞生长因子(HGF)及其受体c- Met蛋白在鼻咽癌组织中的表达水平,研究HGF/c- Met系统对鼻咽癌细胞侵袭转移的影响。方法　收集1999—2003年期间45例确诊的鼻咽原发癌活检组织标本,采用免疫组织化学(LSAB)法检测鼻咽癌组织中HGF α亚单位和c- Met的表达,并与患者的病理及临床资料相联系。采用流式细胞术检测鼻咽癌细胞株CNE- 2在HGF刺激前后c- Met阳性癌细胞百分率的改变;蛋白印迹法和逆转录PCR法分别用于检测癌细胞株c -Met蛋白表达和mRNA表达的变化。结果　在45例鼻咽原发癌组织中,癌细胞c- Met的阳性表达率为91. 1% (41 /45),但仅有1例鼻咽癌细胞表达HGF( 2 .2%, 1 /45 )。HGF主要在鼻咽癌间质中的淋巴细胞表达。癌细胞c Met的表达水平与淋巴结转移有关(P=0 .024 ),且与淋巴细胞表达HGF呈正相关(rs=0 .450,P=0 .002)。癌细胞c Met表达量在间质淋巴细胞高表达HGF的病例中明显高于淋巴细胞低表达HGF的癌组织(P=0 .009)。但癌细胞c Met的表达与患者性别、年龄、病理组织学分型以及临床分期均未显示相关性。鼻咽癌细胞株CNE 2在HGF诱导24h后,c Met阳性的癌细胞比例即有明显增加,由(46 .6±9 .02)%增加至(85. 8±6. 05)% (P=0 .003 ),癌细胞c Met蛋白表达相对强度和mRNA表达水平均有显著提高,     

Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [³H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy.     

肝细胞生长因子基因转移预防术后肠黏连的实验研究

 目的 观察质粒DNA在大鼠术后肠黏连模型中的表达效率及肝细胞生长因子（HGF）基因转移对术后肠黏连的预防作用。 方法 以 pcDNA3-HGF 转染 CHO 细胞 24 h后收集培养上清，用 ELISA 测定培养上清中 HGF 的浓度；取培养的大鼠腹膜间皮细胞作划痕试验，观察表达 HGF 的培养上清对间皮细胞迁移的影响。利用无菌干纱布擦伤盲肠的方法建立 Wistar 大鼠术后肠黏连模型，将模型大鼠随机分为 5 组，每组 10 只。其中 4 组分别于创面涂布 pcDNA3-HGF 10、50、100 和 200 g，1 组于创面涂布绿色荧光蛋白（GFP）表达质粒 pcDNA3-GFP 200 g 作为对照。术后第2、14 天分别解剖 pcDNA3-GFP 200 g 组大鼠各 2 只取腹膜组织，荧光显微镜下观察 GFP 的表达；术后第14天解剖各组动物，观察肠黏连的发生情况。 结果 pcDNA3-HGF 质粒转染CHO细胞后 24 h 培养上清的 HGF 浓度达 40 ng/ml，该上清能够促进大鼠腹膜间皮细胞的迁移。pcDNA3-GFP 200 g 组大鼠术后第 2 天腹膜组织中观察到 GFP 的表达，并持续到术后第 14 天。术后第14 天各组大鼠中均有发生不同程度肠黏连者，pcDNA3-HGF 10、50、100 和 200 g 组和 pcDNA3-GFP 200 g 组大鼠肠黏连发生率分别为 9/10、7/10、6/10、4/10和 9/10，其中重度（3 ~ 4度）肠黏连发生率分别为5/10、5/10、2/10、3/10、7/10，pcDNA3-HGF对术后肠黏连的预防效果呈一定的量效关系。 结论 质粒DNA能有效转染损伤腹膜并介导外源基因表达；质粒载体介导 HGF 基因转移是预防术后肠黏连的有效手段。     

肝细胞生长因子对重症急性胰腺炎小鼠肠黏膜屏障保护作用

目的探讨肝细胞生长因子（HGF）对重症急性胰腺炎（SAP）小鼠肠黏膜屏障功能障碍修复作用及机制,以及肠道干细胞在黏膜损伤修复中的作用。方法健康雄性昆明小鼠60只,按数字表法随机分成正常对照（NC）组10只、SAP组25只和HGF组25只。酶耦联紫外分光光度法检测血清D哥L酸水平,肠系膜淋巴结、胰腺、肝脏、脾脏及肺组织细菌培养,流式细胞仪检测肠上皮细胞增殖指数,免疫组化染色法检测小肠隐窝干细胞变化。结果SAP组血清D-乳酸值为（11．17±1．90）mg／L,较NC组（4．87±0．73）mg／L和HGF组（7．30±0．88）mg／L高,差异均有统计学意义（F=78．20,P值均〈0．01）。胰腺、肠系膜淋巴结、肝脏、脾脏、肺脏细菌培养器官移位率SAP组（50％,40／80）高与NC组（14％,7／50）和HGF组（29％,29／100）（χ^2=7．427,χ^2=4．112,P值均〈0．01）。肠黏膜上皮细胞增殖指数SAP组较NC组和HGF组明显下降（F=42．71,P值均〈0．05）,HGF组与NC组比较则明显升高（P〈0．01）。SAP组小肠隐窝干细胞数（1．26±0．87）个,低于NC组（2．16±0．90）个和HGF组（2．50±0．96）个,差异均有统计学意义（F=8．34,P值均〈0．01）。结论HGF可促进肠黏膜上皮细胞的增殖,降低肠道通透性及肠道细菌移位率,减轻急性胰腺炎小鼠肠黏膜的损伤。     

Effect of rectal administration of rebamipide on dextran sulfate sodium-induced colitis: Role of hepatocyte growth factor

Objective and design: Since rebamipide is effective for the treatment of ulcerative colitis (UC), we examined the involvement of hepatocyte growth factor (HGF) in the action of rebamipide. Materials: Fifty-five and forty female Balb/c mice, respectively, were used in Exp. 1 and 2. Treatment: 50 mg/kg/day rebamipide (Exp. 1) and 1 × 10⁷ pfu pAxCAHGF (the CAG promoter-driving HGF gene in adenovirus vector) (Exp. 2) were intrarectally introduced after induction of colitis by 4 % dextran sulfate sodium (DSS). Methods: Therapeutic effects were assessed by cell proliferation and apoptosis. Results: Rebamipide caused proliferation of epithelial cells at 10 days after treatment, and decreased apoptosis at 10, 14 and 21 days, compared with controls. Expression of HGF was greatly increased in rebamipide-treated mice. pAxCAHGF caused cell proliferation and apoptosis, which showed the same pattern as with rebamipide treatment. Conclusions: Rectal administration of rebamipide is effective for DSS-induced colitis in association with induction of HGF. Received 17 June 2006; returned for revision 23 August 2006; returned for final revision 29 October 2006; accepted by I. Ahnfelt-R?nne 14 December 2006     

携带肝细胞生长因子基因的减毒沙门氏菌对小鼠免疫功能的影响

目的:探讨携带肝细胞生长因子基因的减毒沙门氏菌对小鼠免疫功能的影响.方法:分别用3×108cfu 减毒沙门氏菌(Ty)、携带绿色荧光蛋白基因的减毒沙门氏菌(Typl-GFP)、携带肝细胞生长因子基因的减毒沙门氏菌(Typl-HGF)、10% NaHCO3 于第O、14、28 天灌胃免疫小鼠,末次免疫后7天,分别收集各组动物全血及血清,用流式细胞仪检测抗凝全血中CD4+ T、CD8+T 及 IgM、IgGl、lgA 水平,用 ELISA 法检测血清中免疫球蛋白 IgM、IgGl,MTT 法检测脾脏组织中脾淋巴细胞的增殖能力.结果:流式细胞仪检测结果表明,Typl-HGF 组 CD4+T、CD8+T、CD4+/CD8+ 比值与其它三组比较显著降低(P<0.01,P<0.05,P<0.01);其IgM、IgA 与 NaHCO3 对照组相比无统计学意义(P>0.05),但与Ty、Typl-GFP 组相比显著降低(P<0.01,P<0.05,P<0.01,P<0.05),而 IgGl 与 NaHCO3 对照组相比显著升高(P<0.01),ELISA 结果与流式细胞仪检测结果基本一致;Typl-HCF 组脾细胞的增殖活性明显低于其它实验组(P<0.05).结论:初步证明口服Typl-HGF 后对小鼠的细胞免疫和体液免疫均有抑制作用.     

Improving selection of alphaIIbbeta3-binding phage antibodies with increased reactivity derived from immunized donors

Although many studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have demonstrated the frequent development of Abs directed against the alphaIIbbeta3 integrin, little is known about the induced anti-alphaIIbbeta3 autoantibodies at the molecular level. Phage display is a powerful technology for selecting and engineering mAbs expressed on the surface of filamentous bacteriophage. Combinatorial libraries of single-chain IgG were constructed from splenocytes from two patients with AITP and one patient with GT. In a previous study, activated platelets or alphaIIbbeta3-expressing CHO cells selection was performed to isolate human IgG anti-alphaIIbbeta3 binding fragments using combinatorial libraries created from the B cells of a GT and an AITP patient. However, we have experienced practical problems such as enrichment of truncated antibodies during selection. We decided to test prolonged treatments with elution agents after screening on the purified form of the alphaIIbbeta3 integrin activated with the RGD peptide. We obtained a higher percentage of clones with full-size antibody fragments as well as an enrichment of more specific alphaIIbbeta3-binding phage-Abs. Some of them, recognizing the activated form of the integrin, would be interesting to further study as potential diagnostic or therapeutic agents in acute coronary syndromes. Sequencing of selected phage-Abs revealed that they used different VH and VL genes with, for the majority of them, a high level of extensive hypermutations in the complementarity determining regions, indicating the diversity of the antigen-driven immune response that occurred in GT and AITP patients.     

TFE3-renal carcinoma in an adult patient: a case with strong expression of phosphorylated hepatocyte growth factor (HGFR)/Met

TFE3-renal carcinoma is rare in adults. Patients with this disease often have a poor prognosis, because it has reached an advanced stage at presentation, and there is lack of an effective therapy. The exact mechanism of its malignant behavior is still unclear. In recent years, a significant relationship between TFE3 fusion protein and hepatocyte growth factor receptor (HGFR)/Met tyrosine kinase activity was reported in several malignancies. We previously reported that phosphorylation of HGFR/Met was associated with malignant aggressiveness and survival in patients with conventional RCC. Here, using immunohistochemical techniques, we examined two types of phosphorylated HGFR/Met (pY1234/1235 and pY1349) in a specimen of a 29-year-old man with TFE3-renal carcinoma. Strong expression of both proteins was detected in carcinoma cells, but not in normal kidney tissues. In addition, they were expressed more strongly in TFE3-renal carcinoma than in conventional RCC. Although tumor was diagnosed at T1N0M0 and the patient received radical nephrectomy, the tumor metastasized to multiple organs, and he died 2 years after surgery. We speculate that upregulated phosphorylation of HGFR/Met could be partly associated with the malignant aggressiveness and poor survival of this patient.     

Effect of antibodies to nerve growth factor and serum albumin on the development and behavior of mice

We studied physical development, behavioral characteristics, and learning capacity in the off-spring of mice immunized with nerve growth factor and bovine serum albumin. High titer of antibodies to these factors in the blood of pregnant females determines high levels of these antibodies in the blood of their pups. These changes modulate physical development, behavior, and learning capacity of rat pups. The effects of these antibodies differed in the strength and directionality. Antibodies to nerve growth factor more markedly retarded physical development, reduced learning capacity, and considerably increased pain thresholds in animals. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 7, pp. 98–100, July, 2004     

Effect of anticardiolipin antibodies on prolactin and insulin-like growth factor binding protein-1 production by human decidual cells.

PROBLEM: The effect of anticardiolipin antibodies (ACAs) on basal- and growth factor-stimulated prolactin and insulin-like growth factor (IGF) binding protein (BP)-1 production by cultured human decidual cells was investigated. METHOD OF THE STUDY: Decidual cells were cultured for 24, 48, or 96 hr in medium supplemented with 5% ACA-containing or 5% control serum and increasing concentrations of insulin (1-10 micrograms/mL) or IGF-1 (10-100 ng/mL). RESULTS: No significant increase in prolactin production was observed after addition of increasing doses of insulin and IGF-I in the presence of ACA-containing serum, while a dose-dependent stimulation was seen with control serum. Time-dependent prolactin accumulation was also reduced when cells were cultured in the former conditions. IGF BP-1 release was not affected by insulin and IGF-I in the presence of both sera. However, lower IGF BP-1 levels and a less pronounced time-dependent accumulation were observed in the presence of ACA-positive serum. CONCLUSIONS: Our data suggest that ACAs affect cellular transduction mechanisms regulating critical events, such as decidual cell differentiation. These cellular dysfunctions might be relevant in the induction of some obstetric disorders typical of this syndrome.     

Interleukin-6- and tumour necrosis factor alpha-mediated expression of hepatocyte growth factor by stromal cells and its involvement in the growth of endometriosis

BACKGROUND: We investigated the expression of the hepatocyte growth factor (HGF) gene and protein by the stromal cells derived from women with or without endometriosis and its regulation by interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha). METHODS: Stromal cells immunoreactive to vimentin were isolated from the eutopic and ectopic endometrium of 18 infertile women with endometriosis and 12 women without endometriosis. The production of HGF in the culture media of basal and IL-6- or TNFalpha-stimulated stromal cells was examined with an enzyme-linked immunosorbent assay. The mRNA expression of HGF and its receptor c-Met in the stroma was investigated by RT-PCR. The localization of HGF and c-Met in isolated stromal cells and in intact tissue was examined by immunohistochemistry. The effect of HGF on the growth of stromal cells alone or in combination with IL-6 or TNFalpha was examined in a bromodeoxyuridine (BrdU) incorporation study. RESULTS: The production of HGF in the culture medium of stromal cells was significantly increased after single or combined treatment with either IL-6 or TNFalpha when compared with non-treated cells. The production of HGF by stromal cells derived from the eutopic endometrium of women with endometriosis was significantly higher than that of cells from women without endometriosis. This effect was paralleled by increased expression of HGF and c-Met mRNA, as demonstrated by RT-PCR. The BrdU incorporation study indicated that the addition of HGF enhanced the growth of endometrial and endometriotic stroma alone or in combination with IL-6 or TNFalpha. CONCLUSION: IL-6 and TNFalpha are involved in the production of HGF by endometrial stromal cells and may be involved in the growth of endometriosis by an autocrine mechanism.