Simultaneous transfer of vascular endothelial growth factor and hepatocyte growth factor genes effectively promotes liver regeneration after hepatectomy in cirrhotic rats

BACKGROUND/AIMS: Liver regeneration in a cirrhotic liver is unsatisfactory. In the course of liver regeneration, non-parenchymal cells such as sinusoidal endothelial cells as well as hepatocytes increase in number while the liver structure and physiological functions are maintained. The aim of this study was to examine whether sufficient liver regeneration could be obtained by the simultaneous, preoperative injection of recombinant adenoviral vectors encoding human vascular endothelial growth factor (VEGF), a potent mitogen for sinusoidal endothelial cells, (pAxCAVEGF) and rat hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, (pAxCAHGF) in 70% hepatectomized cirrhotic rats. METHODOLOGY: Forty-eight hours before 70% hepatectomy, dimethylnitrosamine-induced cirrhotic rats were infused intravenously with pAxCAVEGF or with pAxCAVEGF and pAxCAHGF, or with a control virus encoding Escherichia coli beta-galactosidase (pAxCALacZ). RESULTS: Strong VEGF mRNA expressions were shown in the livers of VEGF and VEGF/HGF-treated animals. The plasma HGF concentrations in the VEGF/HGF-treated rats were elevated compared with the other groups. Proliferating cell nuclear antigen immunostaining showed increased labeling indices of hepatocytes in the VEGF/HGF-treated rats at 24 and 48 h after hepatectomy. PCNA labeling indices of SECs were increased in the VEGF and VEGF/HGF-treated rats compared with the control animals at 24 and 48 h after hepatectomy. Moreover, the hepatic regeneration rate after hepatectomy was significantly augmented by the VEGF and VEGF/HGF treatment. CONCLUSIONS: Simultaneous preoperative injection of recombinant adenoviral vectors encoding VEGF and HGF effectively stimulates liver regeneration in cirrhotic rats.     

Hepatocyte growth factor as well as vascular endothelial growth factor gene induction effectively promotes liver regeneration after hepatectomy in Solt-Farber rats

BACKGROUND/AIMS: Hepatic oval cells play an important role in liver regeneration when proliferation of mature hepatocytes is inhibited. The aim of this study was to examine the effect of hepatocyte growth factor (HGF), or vascular endothelial growth factor (VEGF) on proliferation of oval cells in the Solt-Farber rat model. METHODOLOGY: One hour after 70% partial hepatectomy, 2-acetyl-aminofluorene-induced damaged rats were infected intravenously with recombinant adenoviral vectors, encoding rat HGF or human VEGF, or Escherichia coli beta-galactosidase as a control. RESULTS: The plasma HGF concentrations in the HGF-transferred rats were elevated compared with the other groups at 4 and 7 days after hepatectomy. Oval cells were confirmed by positive staining of both cytokeratin-19 and alpha-fetoprotein. Oval cells around the portal tracts in the HGF or VEGF-transferred rats increased in number compared with the control rats at 7 and 9 days after hepatectomy. The proliferating cell nuclear antigen labeling indices of oval cells and the hepatic regeneration rate after hepatectomy were significantly augmented by the HGF or VEGF treatment. Moreover, cyclin E expression was elevated in the HGF-treated rats. CONCLUSIONS: In the Solt-Farber rat model, HGF or VEGF gene injection effectively promoted liver regeneration after hepatectomy mainly with increased proliferation of hepatic oval cells.     

Levels of vascular endothelial growth factor and hepatocyte growth factor in sera of patients with rheumatic diseases

 Angiogenesis plays an important role in the progression of rheumatic disease. We measured the levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in sera from patients with rheumatic diseases and investigated whether these angiogenic factors would be useful in the evaluation of rheumatic diseases. Serum VEGF and HGF levels were determined using ELISA in 128 patients with rheumatic diseases and in 11 healthy controls. Serum VEGF and HGF levels were significantly higher in patients with rheumatic diseases compared to healthy controls [VEGF, 312 ± 20 pg/ml versus 61 ± 8 pg/ml (mean ± SE), P < 0.001; HGF, 935 ± 36 pg/ml versus 413 ± 49 pg/ml, P < 0.01]. Serum VEGF and HGF levels were significantly elevated in patients with adult Still's disease (VEGF, 1021 ± 258 pg/ml; HGF, 1500 ± 295 pg/ml) and were relatively increased in patients with active rheumatoid arthritis (RA) (VEGF, 359 ± 94 pg/ml) and systemic sclerosis (SSc) (VEGF, 356 ± 43 pg/ml; HGF, 1294 ± 224 pg/ml). HGF levels correlated with the clinical course and disease severity in rheumatic disease patients. VEGF levels correlated with the presence of Raynaud's phenomenon (P < 0.05), interstitial lung disease (ILD) (P < 0.05), and serum KL-6 levels (P < 0.01), whereas HGF levels correlated with cryoglobulinemia (P < 0.05), ILD (P < 0.05), serum C-reactive protein (CRP) (P < 0.05), thrombomodulin (P < 0.05), and KL-6 levels (P < 0.05) in rheumatic disease patients. VEGF levels correlated with the skin scores and KL-6 levels in SSc patients and also correlated with the disease activity of RA patients. These data suggest that serum VEGF and HGF levels are related to rheumatic disease activity and the presence of complications. Analysis of VEGF and HGF may be useful in the clinical evaluation of rheumatic disease patients. Received: February 19, 2002 / Accepted: August 13, 2002 Acknowledgment We are grateful to Ms. Aki Nomura for assistance with the ELISA of VEGF and HGF.     

Levels of vascular endothelial growth factor and hepatocyte growth factor in sera of patients with rheumatic diseases

Abstract

Angiogenesis plays an important role in the progression of rheumatic disease. We measured the levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in sera from patients with rheumatic diseases and investigated whether these angiogenic factors would be useful in the evaluation of rheumatic diseases. Serum VEGF and HGF levels were determined using ELISA in 128 patients with rheumatic diseases and in 11 healthy controls. Serum VEGF and HGF levels were significantly higher in patients with rheumatic diseases compared to healthy controls [VEGF, 312 ± 20?pg/ml versus 61 ± 8?pg/ml (mean ± SE), P < 0.001; HGF, 935 ± 36?pg/ml versus 413 ± 49?pg/ml, P < 0.01]. Serum VEGF and HGF levels were significantly elevated in patients with adult Still's disease (VEGF, 1021 ± 258?pg/ml; HGF, 1500 ± 295?pg/ml) and were relatively increased in patients with active rheumatoid arthritis (RA) (VEGF, 359 ± 94?pg/ml) and systemic sclerosis (SSc) (VEGF, 356 ± 43?pg/ml; HGF, 1294 ± 224?pg/ml). HGF levels correlated with the clinical course and disease severity in rheumatic disease patients. VEGF levels correlated with the presence of Raynaud's phenomenon (P < 0.05), interstitial lung disease (ILD) (P < 0.05), and serum KL-6 levels (P < 0.01), whereas HGF levels correlated with cryoglobulinemia (P < 0.05), ILD (P < 0.05), serum C-reactive protein (CRP) (P < 0.05), thrombomodulin (P < 0.05), and KL-6 levels (P < 0.05) in rheumatic disease patients. VEGF levels correlated with the skin scores and KL-6 levels in SSc patients and also correlated with the disease activity of RA patients. These data suggest that serum VEGF and HGF levels are related to rheumatic disease activity and the presence of complications. Analysis of VEGF and HGF may be useful in the clinical evaluation of rheumatic disease patients.     

Role of vascular endothelial growth factor in angiodysplasia: an interventional study with thalidomide

Background and Aim: The pathogenesis of angiodysplasia is still not fully understood and effective therapy is not available. Thalidomide was reported to be effective in the treatment of angiodysplasia, but the mechanisms underlying its activity are, as yet, unknown. We aimed to investigate the expression of vascular endothelial growth factor (VEGF) in angiodysplasia tissues, and the role of hypoxia‐inducible factor‐1α (HIF‐1α) and basic fibroblast growth factor (bFGF) on VEGF expression in human umbilical vein endothelial cells (HUVEC). Additionally, we aimed to study the role of thalidomide in these parameters. Methods: Immunohistochemistry was performed to visualize VEGF in angiodysplasia lesions. HUVEC were incubated under hypoxic conditions or in the presence of bFGF. Effects of exposure to thalidomide were studied. Cell growth was assessed in methylthiazolyte‐trazolium assays. Enzyme‐linked immunosorbent assays and real‐time polymerase chain reaction were performed to assess the expression of VEGF at protein and mRNA levels. Western blot was performed to detect the expression of HIF‐1α under hypoxic conditions. Results: VEGF was strongly expressed in 75% of patients with angiodysplasia lesions, as compared to expression in patients without angiodysplasia lesions. VEGF was also induced in HUVEC under hypoxic conditions (P < 0.05). bFGF was found to stimulate the proliferation of HUVEC and enhance the expression of VEGF. Thalidomide suppressed bFGF‐induced proliferation significantly and decreased VEGF expression, both at the protein and mRNA levels. Thalidomide also inhibited HIF‐1α in a dose‐dependent manner (P < 0.05). Conclusions: VEGF may play an important role in the pathogenesis of angiodysplasia. Thalidomide can suppress VEGF, either induced by HIF‐1α or bFGF.     

血管内皮生长因子基因转染对肝细胞移植的影响

目的探讨VEGF基因转染对移植后血管组织形成的作用及对移植细胞增殖的影响.方法SD大鼠pcDNA3VEGF121转染肝细胞移植10d后取样,行微血管密度(MVD)计数及增殖细胞核抗原(PCNA)染色.结果在体外VEGF转染肝细胞能产生足够的转基因蛋白诱导内皮细胞的增殖.而体内可形成大量移植细胞克隆及再生肝组织.各组MVD计数差异并无显著性,P>005;在VEGF基因转染组PCNA指数较pcDNA3对照组与非转染组显著升高,为13.13±275、475±1.58和4.63±1.41,P<0.01.提示在体内移植血管组织形成存在多重因素的影响.而VEGF基因表达可促进移植细胞增殖和再生肝组织形成.结论VEGF间接体内基因转染是诱导移植肝再生一有效方法.     

人脑胶质瘤中血管内皮细胞生长因子表达及其与细胞增殖的关系

为探讨人脑胶质瘤组织中血管内皮细胞生长因子 (VEGF)表达及其与细胞增殖的关系 ,应用链霉菌抗生物素蛋白 -过氧化物酶连接法 (SABC)免疫组织化学技术检测了 6 7例人脑胶质瘤、8例正常脑组织中 VEGF表达及增殖细胞核抗原 (PCNA)标记指数 (PCNA L I)。结果 VEGF在人脑胶质瘤组织中的阳性表达率为 83.6 %,正常脑组织中无表达 (P<0 .0 0 5 ) ;肿瘤组织中 VEGF表达与 PCNA L I呈显著正相关 (P<0 .0 0 5 )。认为胶质瘤细胞能分泌 VEGF,VEGF表达在肿瘤细胞增殖中起重要作用。     

Serum levels of platelet-derived growth factor-BB and vascular endothelial growth factor as prognostic factors for patients with fulminant hepatic failure

Background and Aims: In animal models for acute liver injury, the administration of some angiogenic factors such as vascular endothelial growth factor (VEGF) and granulocyte‐colony stimulating factor (G‐CSF) are shown to reduce liver injury and improve liver proliferative capacity. The aim of the present study was to assess the role of angiogenic factors in fulminant hepatic failure (FHF). Methods: Serum levels of nine angiogenic factors (angiopoietin‐2, follistatin, G‐CSF, hepatocyte growth factor [HGF], interleukin‐8, leptin, platelet‐derived growth factor [PDGF]‐BB, platelet endothelial cell adhesion molecule‐1 and VEGF) were measured using the Bio‐Plex Protein Array System in 30 patients, 17 of whom were diagnosed with FHF, 13 with acute hepatitis (AH), and 20 controls. Results: Serum levels of PDGF‐BB and VEGF were lower in FHF patients than AH patients and controls (PDGF‐BB; 2050 ± 1572 pg/mL vs 4521 ± 2419 pg/mL vs 8506 ± 5500 pg/mL, VEGF; 39 ± 38 pg/mL vs 144 ± 122 pg/mL vs 205 ± 121 pg/mL). By using univariate logistic regression models, serum levels of PDGF‐BB and VEGF were associated with poor outcomes. Serum PDGF‐BB levels were strongly correlated with serum VEGF levels (r = 0.70). Furthermore, serum PDGF‐BB levels were significantly correlated with platelet counts (r = 0.79), PT activity (r = 0.37) and D.Bil/T.Bil ratio (r = 0.50), while serum VEGF levels were significantly correlated with platelet counts (r = 0.68) and PT activity (r = 0.38). Conclusions: We consider that serum levels of PDGF‐BB and VEGF are worth investigating as biomarkers for predicting outcomes of FHF patients.     

导入外源肝细胞生长因子基因对肝细胞增殖的影响

目的 利用重组腺病毒载体将外源人肝细胞生长因子(HGF)基因导入原代培养的大鼠肝细胞, 观察HGF表达对肝细胞增殖特性的影响。方法 同源重组构建表达HGF的复制缺陷型重组腺病毒AdHGF,用 其感染原代培养的肝细胞。逆转录聚合酶链反应检测肝细胞HGF和c—met(HGF受体)mRNA的表达;酶联免 疫吸附实验测定培养上清液中HGF水平。MTS测定感染前后细胞增殖情况;流式细胞仪测定细胞周期的变化; 细胞免疫荧光法检测HGF基因导入后增殖细胞核抗原(PCNA)表达。结果 同源重组获得约4×10~(10)efu/ml 滴度的AdHGF。AdHGF感染原代培养肝细胞后,肝细胞HGF和c-met mRNA表达均明显上调;细胞上清液 中HGF分泌水平显著增加,达到(5 939.00±414.39)pg/ml(F=13.661,P<0.01)。细胞增殖能力增强(F ≥15.158,P<0.01),细胞周期由G_0/G_1期向S期转化(X~2=41.616,P<0.01);细胞免疫荧光法提示HGF 基因导入后PCNA指数显著提高(F=42.122,P<0.01)。结论 通过重组腺病毒载体将外源HGF基因 导入肝细胞后可维持HGF高效表达并能促进肝细胞增殖,是基因修饰供体肝细胞、增强肝细胞移植治疗效 果的有效方法。     

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。     

Triiodothyronine and interleukin‐6 (IL‐6) induce expression of HGF in an immortalized rat hepatic stellate cell line

Abstract: Background/Aims: Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens. Methods: In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index. In vitro studies: a rat hepatic stellate cell line (HSC‐6T) was cultured with T3, IL‐6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL‐6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT‐PCR. Results: In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor α1, but not β1. Proliferation of stellate cells is not affected by T3, with or without IL‐6. T3 has no effect on secreted levels of IL‐6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL‐6 show significantly increased amounts of secreted HGF after 48 h in culture. Conclusion: T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL‐6.     

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.     

Hepatocyte growth factor protects against Fas‐mediated liver apoptosis in transgenic mice

Background: Apoptosis via the Fas/Fas ligand signalling system plays an important role in the development of various liver diseases. The administration of an agonistic anti‐Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors including hepatocyte growth factor (HGF) have been found to prevent apoptosis. Methods: In this study, we demonstrated the overexpression of HGF to have a protective effect on Fas‐mediated hepatic apoptosis using a transgenic mice (Tg mice) model. Results: In HGF Tg mice, the elevation of alanine aminotransferase was dramatically inhibited at 12 and 24 h after the administration of 0.15 mg/kg anti‐Fas antibody. HGF Tg mice showed a significantly lower number of apoptotic hepatocytes at 12 h compared with wild‐type (WT) mice. Furthermore, 85% (six of seven) HGF Tg mice were able to survive after the administration of 0.3 mg/kg anti‐Fas antibody, while none of the WT mice survived. The Bcl‐xL expression was increased in HGF Tg mice, while there was no difference in the expression of Bax, Bid, Mcl‐1 and bcl‐2 between WT mice and HGF Tg mice. In addition, the HGF Tg mice showed more Akt phosphorylation than the WT mice both before and after the anti‐Fas antibody injection. Conclusions: Taken together, our findings suggest that HGF protects against Fas‐mediated liver apoptosis in vivo, and the upregulation of Bcl‐xL via Akt activation may also play a role in the protective effects of HGF.     

溃疡性结肠炎患者结肠黏膜肝细胞生长因子的表达意义

目的探讨肝细胞生长因子（HGF）及其受体c-Met在活动性和非活动性溃疡性结肠炎（UC）患者结肠黏膜组织的表达意义。方法采用免疫组化SABC法检测活动性和非活动性UC患者以及对照组肠镜活检组织中HGF、c-Met表达；SP法检测增殖细胞核抗原（PCNA）表达。结果对照组、活动性UC组、非活动性UC组HGF阳性表达率分别为25％、88％、100％；c-Met阳性表达率分别为25％、92％、100％；组间比较有显著性差异，P均〈0．05；HGF、c-Met在UC结肠黏膜表达与PCNA过表达正相关（r分别为0．648、0．645，P均〈0．05）。结论HGF及其受体c-Met可能在UC结肠炎症黏膜修复中起作用。     

Treatment of experimental hepatic fibrosis by combinational delivery of urokinase-type plasminogen activator and hepatocyte growth factor genes.

PURPOSE: To investigate the effect of combinational delivery of urokinase-type plasminogen activator (uPA) and hepatocyte growth factor (HGF) genes on hepatic fibrosis. METHODS: Replication-deficient adenoviral vectors expressing either human HGF (AdHGF) or uPA (AduPA) were generated. HGF gene was transferred into primary cultured hepatocytes and uPA gene to hepatic stellate cell (HSC) to investigate the effect on the biological character of cells. Combinational adenoviruses were infused into hepatic fibrosis rats. Serum markers as well as histological and immunohistochemical examination were carried out to test the reversal of hepatic fibrosis. RESULTS: Transfection of exogenous HGF gene induced expression of c-met/HGF receptor and stimulated hepatocyte proliferation. uPA gene delivered into HSC decreased the amount of collagen types I and III accompanied with the increased expression of matrix metalloproteinase-2. In vivo, the area of extracellular matrix in the fibrotic liver decreased to 72% in AdHGF-treated rats (P<0.01), 64% in the AduPA-treated group (P<0.01), and 51% in bi-genes transfection (P<0.01), compared with that of the controls. Moreover, immunohistochemical staining of collagen types I and III revealed that combinational genes delivery exerted more effect on reversal of hepatic fibrosis than mono-gene transfection. CONCLUSIONS: Our study indicated that simultaneous delivery of two antifibrotic genes could confer synergistic effect on hepatic fibrosis.     

Role of circulating vascular endothelial growth factor and hepatocyte growth factor in patients with coronary artery disease

Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are thought to stimulate endothelial cell proliferation and induce angiogenesis in vivo. However, the precise mechanism responsible for VEGF and HGF release in patients with coronary artery disease is still unknown. We studied serum concentrations of VEGF and HGF in 20 patients with acute myocardial infarction (AMI), 20 patients with stable angina pectoris (AP) who had reversible perfusion defects on stress myocardial scintigraphy, and 16 patients with old myocardial infarction (OMI) who had no reversible defects on stress myocardial scintigraphy. The control group consisted of 20 patients with atypical chest pain who had angiographically normal coronary arteries. Serum VEGF and HGF concentrations were measured by enzyme-linked immunosorbent assay. Both the serum VEGF and HGF concentrations in the early stage of myocardial infarction in the patients with AMI were higher than those in the patients with AP and with OMI, and control patients. The VEGF concentration in the patients with AP was higher than in the patients with OMI, whereas the HGF concentration did not differ in the patients with AP and OMI. The VEGF concentration in AMI patients who had had preinfarction angina on admission was higher than that of patients who had had no preinfarction angina, whereas the HGF concentration did not differ between the two groups of patients. These results suggest that the serum VEGF concentration may reflect myocardial ischemia to a greater degree than the serum HGF concentration. Received June 9, 2000 / Accepted September 30, 2000     

Induction of focal angiogenesis through adenoviral vector mediated vascular endothelial cell growth factor gene transfer in the mature mouse brain

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and morphogen, which stimulates angiogenesis in a wide variety of tissues and lesions in vivo. In this study, we applied adenoviral vector delivered human VEGF165 cDNA to develop focal non-tumor angiogenesis in the mature mouse brain. Seventy-two adult CD-1 mice underwent Ad h VEGF, Ad lacZ, and saline injection for up to fourweeks. An adenoviral suspension containing 1 x 10(9) particles was injected stereotactically into the right hemisphere of the brain. The results showed that VEGF expression was increased in the Ad h VEGF transduced mice compared to Ad lacZ or saline injected mice ( P < 0.05). VEGF-positive cells were mainly located in the injection hemisphere of Ad h VEGF transduced mice. Quantitative vessel counting showed that microvessels in the Ad h VEGF transduced mice increased following 2 weeks of Ad h VEGF gene transfer compared to the other two groups (Ad h VEGF:241 +/- 19 vs. Ad lacZ :148 +/- 17 and Saline:150 +/- 14 vessels/mm2, P < 0.05). Morphology showed typical angiogenic changes. PCNA-positive staining confirmed these microvessels were actively proliferating. Our study demonstrates that Ad h VEGF-induced VEGF hyper-stimulation causes focal angiogenesis in the mature mouse brain. This novel method of inducing in vivo brain focal angiogenesis provides an opportunity to study the molecular mechanisms independent of the confounding effects of upstream inciting stimuli such as ischemia or tumor.     

Recombinant hepatocyte growth factor treatment in a canine model of congenital liver hypoplasia

Background: Although the liver has a large regenerative capacity, in many hepatopathies, these repair mechanisms fail. The therapeutic potential of hepatocyte growth factor (HGF) has been proven in numerous toxin‐induced liver failure models in rodents, but never in spontaneously occurring liver diseases in larger animal models. Aim: The aim of this study was to induce liver growth in a hypoplastic liver by the administration of exogenous recombinant HGF. The natural hypoplastic liver model used is the canine congenital portosystemic shunt (CPSS) characterized by strongly reduced liver growth and function. Methods: Recombinant HGF (rHGF), 200 μg/kg, was given twice daily during 3 weeks by an intravenous injection in six dogs with CPSS. Liver volumes were determined by computed tomography before and at 1, 2, 3 and 7 weeks after the initiation of treatment. Portosystemic shunting was evaluated with an ammonia tolerance test and liver portal perfusion was quantified with scintigraphy. Simultaneously, blood parameters for liver function were assayed and liver biopsies were taken for histology, immunohistochemistry and gene‐expression measurements. Results: During 3 weeks of HGF treatment, hepatocyte proliferation increased and an increase in liver volume up to 44% was seen, persisting in two dogs up to 4 weeks after the termination of treatment. Ki‐67 expression, gene expression of E2F1 and CDC6, phosphorylated‐c‐MET and phosphorylated‐ERK1/2 protein levels confirmed increased hepatocyte proliferation and HGF signalling. The aberrant portal perfusion did not change during treatment. Conclusions: Transient in vivo liver growth is shown using CPPS as a naturally occurring large animal model, indicating the therapeutic potential of HGF in liver disease.