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. Received for publication on Sept. 6, 1997; accepted on April 8, 1998     

Radiation-enhanced hepatocyte growth factor secretion in malignant glioma cell lines

BACKGROUND: Postoperative radiotherapy is the standard treatment for patients with a malignant glioma. However, a malignant glioma is radioresistant and almost always recurs, even after a high dose of radiation. A malignant glioma is characterized by its proliferation, invasion and neoangiogenesis, which can be attributed to the high levels of HGF. The scope of this study is to investigate HGF secretion by malignant glioma cells with different radiosensitivity after irradiation. METHODS: Three human malignant glioma cell lines (U251, U251-NG2, and BT325) were irradiated with single doses of 0, 5, 10, and 20 grays of gamma-rays from a (137)Cs source. Hepatocyte growth factor levels in medium were measured by ELISA at 24, 48, and 72 hours after radiation. Cell survival was measured by the proliferation-based assay (XTT assay) 7 days after irradiation. RESULTS: After a single dose radiation, the HGF levels showed a dose-dependent increase in U251, U251-NG2, and BT325 glioma cells. Both baseline and radiation-enhanced HGF levels were about 10-fold higher in BT325 compared to U251 and U251-NG2 cells. In addition, in the XTT assay, the BT325 was more radioresistant than both U251 and U251-NG2 cell lines (dose modifying factor = 1.5 and 1.6, respectively). CONCLUSION: Irradiation-enhanced HGF secretion in all 3 tested glioma cell lines (up to 7 times basal levels). It is tempting to associate the radiation-enhanced HGF secretion with an increased angiogenic potential of the tumor, which may be a factor in radioresistance.     

肝细胞生长因子对大鼠颌下腺细胞增殖的影响

目的　研究肝细胞生长因子 (HGF)对体外培养大鼠颌下腺细胞增殖的剂量 效应及时间 效应。方法　采用体外颌下腺细胞培养在含 1 0 %胎牛血清的合成培养液 (DMEM) ,实验组分别加入不同浓度的HGF ,利用噻唑蓝 (MTT)比色法检测细胞的增殖能力 ,研究HGF对颌下腺细胞增殖的影响。结果　在 1 0 %胎牛血清配制的DMEM含HGF(1 .0～ 1 0 0 0 .0 μg/L)可促进培养细胞的增殖 (P <0 .0 1 ) ,且在一定浓度范围内HGF(1 .0、5 .0、1 0 .0、50 .0、1 0 0 .0 μg/L)呈剂量 效应关系 ,最大效应剂量为 1 0 0 .0 μg/L。加入HGF第 3天开始显示出明显的促增殖效应。 结论　HGF在 1 0 %胎牛血清DMEM培养条件下能够促进颌下腺细胞的增殖和分化 ,经体外条件培养的颌下腺细胞可以作为组织工程学研究中的细胞来源     

Altered expression of hepatocyte growth factor in cardiac allografts of nonhuman primates

Hepatocyte growth factor (HGF) plays a critical role in transplant rejection. Herein we addressed whether HGF expression could be used for accurate and early diagnosis of acute and chronic rejection in cardiac transplantation. We used a heterotopic cardiac transplantation model using nonhuman primates (Macaca fuscata, n = 7). The grafts were harvested on days 1, 7, 22, 28, 40, 41, and 95 for histology and immunohistochemistry. Histopathologically, HGF was expressed in the spindle-shaped cells of the acutely rejecting myocardium. The expression of HGF was enhanced in both thickened intima and media of the coronary arteries. Altered HGF expression is a sensitive indicator for acute and chronic cardiac rejection.     

Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma

Objectives. Scatter factor (hepatocyte growth factor) (SF/HGF) is a multifunctional polypeptide growth factor that has been implicated in tumor proliferation, angiogenesis, invasiveness, and metastasis. Little is known of the expression of SF/HGF in human prostatic carcinoma. The aims of this investigation were to quantitate the level of SF/HGF expression in benign versus malignant human prostatic tissues and to assess regulation of SF/HGF expression by human prostatic stromal myofibroblasts.Methods. We determined the level of SF/HGF expression in 10 human prostatic tissue samples (5 benign, 5 carcinoma) by Western blot analysis. Five purified growth factors—basic fibroblast growth factor (bFGF), interleukin-1beta (IL-1β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and endothelial growth factor (EGF)—were tested for their capacity to induce SF/HGF expression by a human prostatic stromal myofibroblastic cell line, as assessed by enzyme-linked immunosorbent assay. Supernatant from the normal PrEC prostatic epithelial cell line and the DU 145 carcinoma cell line were assayed for SF/HGF-inducing activity.Results. SF/HGF exhibited a mean fourfold overexpression in carcinoma tissues compared with benign prostatic tissue. Significant stimulation of SF/HGF expression by prostatic stromal myofibroblasts was detected for IL-1β (8.1-fold), PDGF (6.2-fold), bFGF (4.0-fold), VEGF (3.7-fold), and EGF (2.9-fold). DU 145-conditioned media, but not the PrEC-conditioned media, contained SF/HGF-inducing activity, which was determined to include IL-1β, bFGF, and PDGF by antibody-blocking experiments.Conclusions. SF/HGF is overexpressed in human prostatic carcinoma tissues. Prostatic carcinoma cell stimulation of SF/HGF expression by adjacent benign myofibroblastic cells as a type of epithelial–stromal paracrine interaction could potentially influence prostatic carcinoma cell behaviors.     

The clinical significance of hepatocyte growth factor for non–small cell lung cancer

Background. Hepatocyte growth factor (HGF) is a cytokine that is released after injury. It is a paracrine factor that is produced by mesenchymal cells; epithelial and endothelial cells respond to HGF through its receptor, the c-met protein. Hepatocyte growth factor induces cell growth and cell movement and is also highly angiogenic. Evidence from breast cancer patients suggests that HGF is a negative prognostic indicator for breast cancer and is associated with invasive disease.

Methods. We measured the HGF content in tumor tissue from 56 non–small cell lung cancer patients using the Western blot technique. The amount of HGF in tumor extracts was quantitated by densitometry after transfer of proteins to nitrocellulose and exposure to antibodies. Survival curves were generated based on clinical information obtained for each patient.

Results. Our data indicate that HGF is also a negative prognostic indicator in lung cancer. As in the study of breast cancer patients, HGF was associated with recurrence and poor survival; the relative risk was seen to increase with increasing HGF tumor content. At levels of HGF greater than 100 units, the relative risk was 10, compared with that in patients with an HGF level of 1 unit. Node-negative patients with an elevated HGF tumor content had a significantly poorer outcome than node-positive patients with a low HGF tumor content. The same relationship was observed if the patients were stratified by stage: elevated HGF was associated with stage I patients whose disease recurred and who died of their disease, and stage I patients with elevated HGF had a worse survival than higher stage patients with a low level of HGF.

Conclusions. These results suggest that elevated HGF may predict a more aggressive biology in non–small cell lung cancer patients. The level of HGF may be useful as an indicator of high risk in early stage lung cancer patients.     

转染HGF基因对肝细胞的生物学效应

目的探讨肝细胞生长因子(hepatocyte growth factor，HGF)基因对肝细胞生长增殖能力的影响。方法通过脂质体介导法，将HGF基因导入肝细胞中。用荧光显微镜以及原位杂交观察到HGF基因表达。采用检测细胞生长曲线、Ki-67蛋白和嗜银蛋白免疫组化观察肝细胞生长增殖能力及DNA合成能力的变化。结果荧光显微镜观察可见到绿色荧光蛋白的表达，用原位杂交方法进一步证实了HGF蛋白在细胞中的表达。细胞生长曲线显示，转染HGF基因的肝细胞增殖速度明显增快，Ki-67蛋白和嗜银蛋白表达明显增多，提示转导HGF基因使肝细胞增殖活性增加。结论本实验显示转染的HGF可表达并有促细胞分裂活性。为进一步了解HGF分子生物学特性及治疗应用提供了理论基础。     

A possible mechanism for prevention of intestinal programmed cell death after ischemia-reperfusion injury by hepatocyte growth factor pretreatment

Background/Purpose: Intestinal ischemia-reperfusion (IR) injury produces necrosis and apoptosis resulting in tissue loss. The authors have observed previously that pretreatment with hepatocyte growth factor (HGF) attenuates enterocyte apoptosis after IR. This study investigated the effects of HGF on tissue levels of caspase-8, an apoptosis initiator, and caspase-3, an apoptosis effector, in intestinal mucosa after IR. Methods: Thirty rats underwent placement of jugular venous catheters connected to osmotic pumps; 15 rats received vehicle, and 15 rats received HGF (150 [mu ]g/kg/d). After a 48-hour infusion, 5 rats from each group underwent either 35 minutes of superior mesenteric artery occlusion alone, or ischemia followed by 2 or 6 hours of reperfusion. Mucosal protein was analyzed for caspase-8 and caspase-3 activity. DNA fragmentation was used to measure the presence of apoptosis. Statistical significance was determined using analysis of variance. Results: After 6 hours of reperfusion, caspase-3 activity was increased significantly in control animals (P [lt ] .05). In HGF-pretreated animals, caspase-8 and caspase-3 activities were significantly reduced at 6 hours compared with control animals (P [lt ] .05). Conclusion: By preventing the activation of enterocyte caspase enzymes, and, thus, reducing apoptosis, HGF may enhance preservation of the intestine after IR injury.     

转肝细胞生长因子基因肝细胞模型的建立

目的：利用脂质体介导法在体外建立转肝细胞生长因子（HGF）基因的人肝细胞模型。方法：建立HGF真核细胞表达载体，利用脂质体介导法在体外将HGF基因转染入人肝细胞，利用荧光显微镜观察、免疫组织化学、原位杂交方法检测HGF真核细胞表达载体的转录和表达情况。结果：以阳离子脂质体LipofectAMINE为载体将HGF基因转染人肝细胞后，经400mg/L的G418筛选后可形成抗性克隆；Neo基因原位杂交结果显示转染基因的细胞有阳性表达；荧光显微镜下观察到有绿色荧光；免疫组织化学证实转染HGF基因的肝细胞有HGF蛋白的表达。结论：HGF基因可被成功转染入人肝细胞并能有效表达，这可能为肝病的基因治疗提供一种新途径。     

肝细胞生长因子在环孢素A肾病大鼠肾组织中的表达意义

目的　探讨肝细胞生长因子 (HGF)在环孢素A(CsA)肾病大鼠肾组织中的表达及意义。　方法　 2 8只SD大鼠低盐饮食饲养 7d后 ,随机分为对照组、CsA组、CsA加维拉帕米组、CsA加依那普利组。每组 7只。除对照组外 ,其余 3组大鼠皮下注射CsA 15mg·kg-1·d-1,采用放射免疫法检测各组动物血管紧张素Ⅱ (AngⅡ )水平 ;Northernblot、原位杂交检测肾组织HGFmRNA的表达。对各组动物肾小管损伤进行半定量计分。　结果　CsA处理组肾组织AngⅡ水平均明显高于对照组 :(2 9.8± 6.0 )vs (8.7± 1.7)ng/g肾组织 ,P <0 .0 0 1,HGFmRNA表达也明显减少。依那普利组肾组织AngⅡ水平明显降低 ,HGFmRNA表达增加 ,P <0 .0 5。依那普利能明显减轻CsA引起的肾小管损伤 ,异博定对肾小管损伤无显著改善。肾组织HGFmRNA表达与肾小管损伤呈负相关 ,P <0 .0 5。　结论　肾素血管紧张素系统 (RAS)的激活使HGFmRNA表达下调 ,阻断RAS可以使HGFmRNA表达增加 ,减轻CsA引起的肾小管损伤     

Increased gene expression of scatter factor—hepatocyte growth factor and basic fibroblast growth factor in granulation tissue in the rat

Scatter factor-hepatocyte growth factor is a protein secreted by fibroblasts which disperses colonies of epithelial cells and keratinocytes in culture. The factor is also a patent mitogen for hepatocytes, synthesized in the liver. Basic fibroblast growth factor, another heparin-binding factor, is most abundant in the brain but also plays a role in wound healing. Using a solution hybridization/RNAase protection assay, we have measured the abundance of messenger RNA for scatter factor-hepatocyte growth factor and basic fibroblast growth factor in granulation tissue obtained from subcutaneously Hunt-Schilling wound cylinders. The levels of scatter factor-hepatocyte growth factor messenger RNA increased after weeks 2 through 4 to a twofold higher level in weeks 5 through 7 after implantation of the cylinders, whereas no changes in basic fibroblast growth factor messenger RNA levels were noticed. At week 3 after implantation of the cylinders, scatter factor-hepatocyte growth factor messenger RNA levels in granulation tissue were more than threefold higher than in skin dermis fibroblasts but markedly lower than in the liver. The abundance of basic fibroblast growth factor messenger RNA was also significantly increased in granulation tissue compared with dermis but, as expected, markedly lower than in the brain. In conclusion, the gene expression of the scatter factor-hepatocyte growth factor, as well as basic fibroblast growth factor, is increased in granulation tissue. Because there was a time-dependent increase in the expression of scatter factor-hepatocyte growth factor, it is hypothesized that scatter factor-hepatocyte growth factor acts as a signal from fully developed granulation tissue to stimulate skin epithelial cells to scatter over the wound.     

血管内皮生长因子基因在原代培养大鼠肝细胞的转染表达

目的　研究血管内皮生长因子基因 (VEGF12 1)在原代培养大鼠肝细胞中转染表达。方法　构建 pIRES EYFP /VEGF12 1质粒 ,逆转录 聚合酶链反应 (RT PCR)、WesternBlot和荧光显微镜观察VEGF基因在原代培养大鼠肝细胞中的转染表达。结果　pIRES EYFP/VEGF12 1质粒成功构建并转染原代培养大鼠肝细胞 ,RT PCR观察到 2 5 8bp的VEGF特异性条带 ,WesternBlot检测到相对分子质量 42× 10 3 的特异性条带 ,荧光显微镜观察到转染阳性肝细胞发出黄绿色荧光。结论　pIRES EYFP/VEGF12 1质粒在原代培养大鼠肝细胞转染并表达 ,为VEGF基因修饰肝细胞移植和基因治疗奠定基础。     

Clinical significance of lymphocytes hepatocyte growth factor mRNA expression in patients after liver transplantation

Hepatocyte growth factor (HGF) plays a key role in the regulation of liver regeneration after hepatocyte damage. Changes in HGF gene expression reflect the status of the regeneration process. AIM: The aim of this study was to ascertain the clinical significance of the expression of HGF among liver transplant patients. METHODS: Expression of the mRNA of HGF among peripheral blood lymphocytes were measured prior to as well as at 1, 2, 6, and 10 days after liver transplantation in a group of 30 liver recipients. RESULTS: In first 24 hours after reperfusion, the patients with compromised graft function (group 1) showed persistently higher HGF gene expression after reperfusion compared with patients displaying well-functioning grafts (group 0; P = .0189). Between postoperative days 1 and 10, there was a rapid decrease in gene expression among group 0 compared with group 1 (P = .0155). The significant decrease observed in the both groups reached a certain plateau after 48 hours postoperatively. There was no statistical difference in aminotransaminase levels over the days after liver transplantation. The decreased mRNA HGF expression in lymphocytes preceded the decrease in aminotransferase levels. CONCLUSIONS: HGF was more sensitive to predict early graft function than prothrombin time, aspartate aminotransferase, and alanine aminotransferase levels. The determination of HGF expression level in lymphocytes after liver transplantation may yield valuable information for evaluation of early graft function.     

高糖对大鼠系膜细胞肝细胞生长因子受体表达的影响及其机制研究

目的 观察高糖作用下大鼠系膜细胞(MsC)肝细胞生长因子（HGF）受体c-Met的表达，并探讨其机制和意义。 方法 用RT-PCR和Western 印迹方法检测高糖作用大鼠MsC的不同时间点（0、12、24、48、96 h）c-Met的表达。分别用光辉霉素A（mithramycin A）和SU11274抑制转录因子Sp1的DNA结合活性和阻断c-Met。用电泳迁移率改变实验(EMSA)观察Sp1与c-Met基因启动子的结合活性。以荧光探剂二氯双氢荧光素二乙酸酯(DCFH-DA)捕获细胞内活性氧。 结果 大鼠MsC的c-Met表达在高糖作用12、24和48 h都明显上升，96 h开始下降。光辉霉素A呈浓度依赖性抑制高糖作用下大鼠MsC的c-Met表达上调。大鼠MsC内Sp1与c-Met基因启动子的结合活性在高糖作用下明显增强。HGF及c-Met显著抑制高糖诱导的大鼠MsC内活性氧的增多。 结论 高糖作用下大鼠MsC的c-Met表达增强，其机制可能是通过Sp1介导。HGF-c-Met信号通路激活能抑制高糖所致大鼠MsC内的氧化应激反应。     

Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor.

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.     

Molecular basis of mammalian cell invasion by Trypanosoma cruzi

Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT) and mammalian tissue culture trypomastigotes (TCT). During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.