Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.     

外源性肝细胞生长因子抗肝细胞凋亡机制的研究

目的通过小鼠尾静脉高压注射的方法在肝细胞中建立持续、高效表达外源性肝细胞生长因子(HGF)的体系,探讨其在肝细胞凋亡中的作用机制。方法通过尾部静脉注射pCMV-HGF质粒,检测肝组织中HGF表达的高峰。实验动物分为模型组、pcDNA3保护组、pCMV-HGF质粒保护组和生理盐水对照组,Western blot检测tBid、细胞色素C在各组肝细胞质及线粒体中的表达情况。结果在小鼠尾静脉快速大量注射质粒4h后肝脏中即有外源性HGF表达,8h达高峰。D-GalN/LPS可诱导明显的细胞凋亡,pCMV-HGF保护组tBid及细胞色素C的表达明显减少。结论通过小鼠尾静脉高压注射的方法在肝细胞中建立持续表达外源性HGF的体系,通过抑制Bid的活性片段tBid表达和细胞色素C释放来减轻凋亡蛋白的激活。     

Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy

Impairment of cardiac function in cardiomyopathy has been postulated to be related to decreased blood blow and increased collagen synthesis. Therefore, a therapeutic approach to alter the blood flow or fibrosis directly by means of growth factors may open a new therapeutic concept in dilated cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with antifibrosis and angiogenesis effects. Using the hereditary cardiomyopathic Syrian hamster as a model of genetically determined cardiomyopathy and heart failure, the effects of overexpression of HGF on fibrosis and microvascular dysfunction were examined. HGF gene or control vector was injected by the Hemagglutinating Virus of Japan-liposome method into the anterior heart of cardiomyopathic hamsters (Bio 14.6) under echocardiography once a week, from 12 to 20 weeks of age (total, 8 times). Blood flow, as assessed by a laser Doppler imager score, and the capillary density in hearts, as assessed by alkaline phosphatase staining, were significantly increased in hamsters transfected with HGF gene compared with control-vector-transfected hamsters (P<0.01). In contrast, the fibrotic area was significantly decreased in hamsters transfected with HGF gene compared with control (P<0.01). Overall, in vivo experiments demonstrated that transfection of HGF gene into the myocardium of cardiomyopathic hamsters stimulated blood flow through the induction of angiogenesis and reduction of fibrosis. These results suggest that HGF gene transfer may be useful to protect against myocardial injury in cardiomyopathy through its cardioprotective effects such as antifibrosis and angiogenesis actions.     

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.     

Sustained induction of hepatocyte growth factor by sensitization with freeze-thawed hepatic tissue

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, and has therapeutic potential for fibrotic/cirrhotic liver. Therefore, the induction of HGF in vivo is considered to be useful in the treatment of liver dysfunction caused by cirrhosis, chronic hepatitis, or extensive surgical resection. In this study, we examined the sustained induction of HGF by inoculation of freeze-thawed hepatic tissue (FTHT). Serum from rats inoculated with FTHT increased [³H]thymidine incorporation i.e., increased DNA synthesis, in primary cultured rat hepatocytes. The DNA synthesis was significantly promoted by the addition of the FTHT-sensitized serum, while this DNA synthesis was inhibited by neutralizing anti-rat HGF antibody. The concentration of HGF in the FTHT-sensitized serum was increased by day 3 after the inoculation. The time of HGF induction was dependent on the inoculated volume of FTHT, but peaks of HGF concentration were found on day 5 with different volumes of FTHT. Injurins, inducers of HGF, were also induced in the FTHT-sensitized rats, with their peak levels on day 3. The FTHT inoculated tissue showed inflammatory cell infiltration, which was gradually absorbed, and had completely disappeared by day 14 after the inoculation. Although mild inflammatory cell infiltration was observed in non-freeze-thawed inoculated hepatic tissue (NFHT) a tight capsule formed around the NFHT, and was scarcely phagocytized on day 14. These results suggest that FTHT inoculation induces HGF sustainedly through the increased synthesis of injurins, and that freeze-thawed tissue, which is easily phagocytized, is important for the sustained induction of HGF.     

Defect of hepatocyte growth factor secretion by fibroblasts in idiopathic pulmonary fibrosis

Hepatocyte growth factor (HGF) is a growth factor that protects alveolar epithelial cells from pulmonary fibrosis in various animal models. We compared in vitro HGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 8) and from control subjects (n = 6). Basal HGF secretion by IPF fibroblasts was decreased by 50% when compared with control fibroblasts (p < 0.05). HGF was secreted mainly in the cleaved mature form, both in IPF and control fibroblasts. HGF messenger RNA levels were reduced in IPF fibroblasts. Prostaglandin (PG) E2 secretion by IPF fibroblasts was low when compared with control subjects (p < 0.05). After the addition of PGE2 (10-6 M) or dibutyryl cyclic AMP (10-3 M), HGF secretion by IPF fibroblasts reached the level of control subjects. Inhibition of PGE2 synthesis with indomethacin reduced HGF secretion by control fibroblasts but had no effect on IPF fibroblasts. HGF secretion by control fibroblasts was also slightly inhibited by transforming growth factor (TGF)-beta1 and stimulated by anti-TGF-beta antibody, whereas both agents had no effect on IPF fibroblasts. Our results demonstrate a defect in HGF production by IPF fibroblasts that seems secondary to a defect in PGE2 secretion.     

Induction of hepatocyte growth factor in stromal cells by tumor-derived basic fibroblast growth factor enhances growth and invasion of endometrial cancer

Tumor progression is often regulated through interactions between carcinoma cells and host stromal cells. In this study of endometrial cancer, we investigated one mechanism potentially involved in hepatocyte growth factor (HGF)-mediated cancer-stromal interactions. Endometrial cancer cells (HEC-1 and ISHIKAWA) expressed the c-met receptor, but HGF did not. HGF, however, did stimulate the proliferation and invasion of these cells. The HGF gene was expressed in stromal cells, which had been separated from primary cultures of endometrial cancers, 6.4 times more than in isolated normal endometrial stromal cells. Immunohistochemical staining revealed immunoreactive HGF in cancer stromal cells, the staining intensity being more pronounced in cancer tissue than in normal endometrium. The conditioned medium from normal epithelial cells and cancer cell lines induced HGF production in normal stromal cells. We identified basic fibroblast growth factor as an HGF inducer derived from endometrial cancer cell lines. Basic fibroblast growth factor derived from tumor cells may induce HGF in endometrial stromal cells, whereas stromal cell-derived HGF leads to the invasive growth of carcinoma cells. These interactions, mediated by HGF and HGF inducers, may play a significant role in the progression of endometrial cancer.     

Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

AIM: To construct pEGFP-hepatocyte growth factor (HGF) expression vector, the to detect its expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocytes. METHODS: Human HGF cDNA was ligated to the pEGFP vector. Recombinant plasmid was transfected into human hepatocyte line QZG with liposome. Expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 h after transfection to detect the number of ((3)H)-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions. RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment was cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The ((3)H)-TdR uptake became 7 times as many as in the control group 96 h after transfection. After HGF transfection, the survival rate of hepatocytes poisoned by CCl(4) significantly increased (83% vs 61%, P<0.05), and the leakage of intracellular alanine transaminase and potassium ions decreased (586 nkat/L vs 1 089 nkat/L, P<0.01; and 5.59 mmol/L vs 6.02 mmol/L, P<0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning.     

Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma

Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis including tumour neovascularization. Serum levels of VEGF and HGF were measured in 52 patients with MM by enzyme-linked immunosorbent assay (ELISA). Serum levels of VEGF and HGF were elevated in MM patients compared with healthy controls (VEGF: mean 0.31 ng/ml and 0.08 ng/ml respectively, P < 0.01; HGF: mean 2.17 ng/ml and 0.45 ng/ml, respectively, P < 0.001). In serial samples taken after chemotherapy, serum VEGF and HGF levels were correlated with M-protein levels. Serum levels of VEGF were higher in patients with extramedullary plasmacytomas than in patients without them (P < 0.05). They were also significantly higher in a group of patients who showed poor response to chemotherapy (P < 0.01). Serum levels of HGF were higher in patients with complications such as anaemia, hypercalcaemia and amyloidosis than in patients without these complications (P < 0.01, P < 0.05, P < 0.05 respectively). Both serum VEGF and HGF levels were significant predictors of mortality (P = 0.01, P = 0.02, respectively, log-rank test). The present study demonstrated that serum levels of VEGF and HGF are significantly elevated and dependent on the severity of MM, suggesting that measurement of VEGF and HGF may be useful for assessing disease progression and for predicting the response to chemotherapy in MM patients.     

肝内表达的外源性肝细胞生长因子抑制肝细胞凋亡的机制

目的 通过体内基因转染方法在肝细胞中建立持续、高效表达外源性肝细胞生长因子(HGF)的体系,探讨其在肝细胞凋亡中的作用.方法 通过尾部静脉快速注射大量pCMV-HGF质粒,ELISA检测外周血和肝组织中HGF表达的高峰和持续时间.实验动物分为模型组、pcDNA3空质粒保护组、pCMV-HGF质粒保护组和0.9%氯化钠溶液对照组,每组10只,Western印迹检测Caspase-3,tBid、Bax、细胞色素C的表达.所有数据组间均数比较采用单因素方差分析,均数间的两两比较用q检验.结果 在小鼠尾静脉快速大量注射pCMV-HGF 4 h后就有外源性HGF表达,外周血和肝组织中分别于12 h和8 h达高峰,于注射后第6天仍可检测到HGF表达.D-氨基半乳糖胺/脂多糖可诱导明显的细胞凋亡,并导致tBid、Bax、Caspase-3及细胞色素C的表达明显增加.与模型组和pcDNA3空质粒保护组相比,pCMV-HGF保护组tBid、Bax、Caspase-3及细胞色素C的表达明显减少.结论 外源性HGF能抑制小鼠肝细胞凋亡,通过抑制Bid的活性片段tBid的表达,减少下游凋亡蛋白的激活.     

Involvement of growth factor receptor-bound protein-2 in rat hepatocyte growth

Growth factor receptor-bound protein-2 (GRB-2) is a protein linking receptor tyrosine kinase and Sos ( Son of Sevenless gene; Ras GDP/GTP exchange protein), leading to activation of the Ras-mitogen-activated protein kinase (MAPK) cascade. So far, it remains unclear how GRB-2 plays a role in signal transduction pathways evoked by hepatotrophic factors. This study was attempted to evaluate the involvement of GRB-2 in signalling in rat hepatocyte growth. Using rat cultured hepatocytes stimulated by hepatotrophic factors and regenerating livers after partial hepatectomy (PH) we examined GRB-2-mediated linkage of hepatotrophic factor receptors to signal transducing molecules such as Sos or dynamin-II by immunoprecipitation and western blot analysis. In primary cultured hepatocytes stimulated with hepatocyte growth factor (HGF) or epidermal growth factor (EGF), GRB-2 linked HGF receptor or EGF receptor, respectively, to Sos which activated the mitogen-activated protein kinase (MAPK) cascade. In contrast, in primary cultured hepatocytes stimulated with insulin, GRB-2 linked insulin receptor substrate-1 (IRS-1) to dynamin-II as well as Sos. In the early phase after PH, GRB-2 activated the Ras-MAPK cascade by linking HGF receptor, IRS-1, or EGF receptor to Sos. In the late phase after PH, a complex of IRS-1-GRB-2 associated with dynamin-II, indicating that GRB-2 may transduce signals from IRS-1 to dynamin-II. We conclude that GRB-2 may play a role in transmitting signals from hepatotrophic factors to not only MAPK but also to other signalling pathways in hepatocyte growth.     

Impaired response to hepatocyte growth factor in FRTL-5 rat thyroid cells expressing a functional hepatocyte growth factor receptor.

The FRTL-5 rat thyroid cell line is widely used for studies of thyrocyte function and growth. The objective of the present study was to investigate the effects of hepatocyte growth factor (HGF) on FRTL-5 cells. HGF has previously been known to be a potent regulator of thyrocyte growth and differentiation. Met, the receptor for HGF was expressed in FRTL-5 cells, as well as in primary cultures of porcine thyrocytes included in the study as control. On HGF stimulation Met was tyrosine phosphorylated in both porcine and FRTL-5 cells, indicating an activation of the receptor. Addition of HGF induced changes of cell shape, scattering and proliferation of the porcine thyrocytes, but not in the FRTL-5 cells; yet, a functional coupling of Met to the p85 subunit of the phosphatidylinositol-3'-kinase (PI3'-K) in coprecipitation experiments, formation of focal adhesions detected in immunofluorescence staining with an antivinculin antibody, and induction of c-fos mRNA in Northern blot analysis was observed in FRTL-5 cells, showing a transduction of the HGF/Met signal. In summary, despite the expression of apparently functional Met, the FRTL-5 cells are unable to properly respond to HGF.     

人肝细胞生长因子基因重组腺病毒载体的构建

目的:利用ADEASYTM系统,构建高效而又安全的肝细胞生长因子(HGF)基因腺病毒载体(AdHGF),以研究HGF基因在心脏内的表达。方法:从人胎肝中抽提总RNA,并以该肝细胞总RNA为模版,利用合成的1对引物,采用RTPCR技术获得HGF基因的cDNA,并引入酶切位点,先转入感受态质粒,再转化大肠杆菌进行扩增,筛选阳性菌落,经酶切及测序,证实目的HGF基因已经转入,再将该基因转入pUC18质粒,pUC18质粒与AdEASY质粒进行同源重组,用PacⅠ酶切成线性化DNA,利用脂质胺转染293细胞,经3轮扩増,制备高效表达并用绿色荧光标志AdHGF。并将该载体作实验组,与HGF组作比较,观察对VEC的抗凋亡作用。结果:在荧光显微镜发现293细胞发光率为100%,AdHGF的HGFcDNA经过测序鉴定与基因库序列一致。结论:AdHGF的成功构建,为冠心病转基因治疗的基础与临床研究奠定了基础。     

肝细胞生长因子与肝纤维化的研究进展

肝细胞生长因子是一种具有多种生物活性的细胞因子,其在促进肝细胞增殖、抑制肝星状细胞活化等方面有重要作用.肝纤维化进展过程中,各种因素引起肝细胞持续损伤,使损伤部位肝细胞再生,肝星状细胞激活,细胞外基质大量沉积,从而导致肝纤维化形成.此文就肝细胞生长因子的生物学特性及其在肝纤维化中发挥的作用作一综述.     

血清肝细胞生长因子在慢性肝病中的意义

目的 肝细胞生长因子(HGF)能促进上皮细胞增生、运动、变形，是肝再生的起始因子之一，近来发现其在肝硬化和肝肿瘤的发生发展中也有重要作用。现主要探讨血清HGF水平在慢性肝病中的意义。方法 检测197例个体血清HGF水平，包括肝细胞癌(HCC)80例，肝硬化(LC)57例，慢性肝炎(CH)22例，正常对照38例。ELISA法检测血清HGF水平，并描绘受试者工作曲线(ROC)，确定HCC和LC患者HGF水平的最佳临界点。运用Spearman相关分析HGF水平和ALT、AST、GGT、白蛋白、总胆红索、凝血时间、肝癌大小、病理分级的相关性。结果 HCC、LC、CH和正常对照组的血清HGF中位值分别是6．767、151．200、7．017和3．476pg／m1。其中HCC组(P＜0．05)和比组(P＜0．01)的血清HGF水平显著高于正常对照组。LC组根据Child分级分层发现，Child C级患者的HGF水平明显高于Child A、B级。肝硬化ROC曲线显示，14pg／m1为临界值时效率最高。血清HGF水平仅发现与凝血时间有相关性(r=0．45，P＜0．01)。在HCC组中，未发现血清HGF水平与肿瘤大小、病理分级有任何相关。结论 血清中HGF水平增高与LC程度有关。     

Production of hepatocyte growth factor during acute myocardial infarction

OBJECTIVE—To investigate the clinical significance of circulating hepatocyte growth factor (HGF) and the role of peripheral blood mononuclear cells (monocytes), which are a possible source of HGF, in patients with acute myocardial infarction.
DESIGN AND PATIENTS—37 patients with acute myocardial infarction and 13 normal control subjects were recruited. Peripheral venous blood samples were drawn from the infarct patients 1, 7, 14, and 21 days after onset. Monocytes were isolated from peripheral blood at those times. HGF concentrations in serum and in a culture medium of monocytes after incubation for 24 hours (monocyte HGF levels) were measured by enzyme linked immunosorbent assay.
RESULTS—Serum HGF and monocyte HGF values within seven days after onset of myocardial infarction were significantly higher than those of control subjects and decreased by day 14. There were significant positive correlations between serum HGF and monocyte HGF levels on day 7; between maximum plasma creatine phosphokinase levels and serum HGF levels on day 1; between maximum plasma C reactive protein and serum HGF levels; and between maximum C reactive protein and monocyte HGF levels. Monocyte HGF levels were raised in the patients with progression of ventricular enlargement in the course of acute myocardial infarction.
CONCLUSIONS—Early serum HGF concentrations reflect the extent of myocardial damage in acute myocardial infarction patients. Inflammation after acute myocardial infarction is supposed to be involved in enhanced HGF production. Monocytes may play an important role in ventricular remodelling after acute myocardial infarction by releasing the cardiovascular protective mitogen HGF.


Keywords: C reactive protein; cytokines; ischaemic heart disease; remodelling; hepatocyte growth factor