Concentrations of hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor in pericardial fluid and plasma

Some angiogenic factors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), have been reported to promote angiogenesis and improve myocardial perfusion in experimental models of ischemic heart disease. These factors are produced in various tissues, including myocardium. We measured the concentrations of HGF, bFGF, and VEGF by enzyme-linked immunosorbent assay in plasma and in pericardial fluid sampled during open heart surgery (12 patients with ischemic heart disease and 17 with nonischemic heart disease). HGF levels were significantly higher in plasma than in pericardial fluid (12.0 +/- 1.8 versus 0.26 +/- 0.04 ng/mL, P < 0.0001). On the other hand, bFGF levels were significantly higher in pericardial fluid than in plasma (243.5 +/- 50.9 versus 49.6 +/- 7.8 pg/mL, P = 0.009). VEGF levels were not significantly different between pericardial fluid and plasma (47.2 +/- 17.6 versus 24.5 +/- 3.6 pg/mL, P = 0.23). Concentrations of angiogenic factors in pericardial fluid and in plasma were not significantly different between patients with ischemic and nonischemic heart disease. These results suggest that the production, secretion, and kinetics of HGF, bFGF, and VEGF are different. These angiogenic factors may have different pathophysiologic roles.     

Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor

OBJECTIVE: In the present study, we delivered vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene to a rabbit model of hind limb ischemia utilizing an ex vivo method of gene transfer, and evaluated the functional performance of the developed collateral vessels. METHOD: The left femoral artery of a male Japanese White rabbit was excised to induce limb ischemia, and a section of skin was resected for culture of auto-fibroblasts. Twenty days later, the VEGF gene, bFGF gene or beta-galactosidase gene (LacZ) was adenovirally transferred to the cultured auto-fibroblasts (5x10(6) cells), and the next day, a pair of specifically infected fibroblasts (total 1x10(7) cells) was injected via the left internal iliac artery of the same rabbit. Pairs of transferred genes into the fibroblasts were as follows: LacZ/LacZ (control group), VEGF/LacZ (VEGF group), bFGF/LacZ (FGF group) and VEGF/bFGF (combination group). Twenty-eight days after cell administration, collateral development and its function were evaluated. RESULTS: Calf blood pressure ratio, resting blood flow of the left iliac artery and capillary density of ischemic muscle showed similar degrees of angiogenic effects in the VEGF and FGF groups, which were significantly greater than those in the control group. On the contrary, angiographic score, collateral conductance and smooth muscle cell (SMC)-positive vessel density in the FGF group were significantly greater than those in the VEGF group. In the combination group, collateral conductance showed synergistic effects, and in vivo blood flow and smooth muscle cell-positive vessel density revealed additive effects of VEGF and bFGF. CONCLUSION: These findings suggested that bFGF-induced collateral development exceeded VEGF-induced collateral development in the induction of arteriogenesis, and that combined gene delivery of VEGF and bFGF produced additive or synergistic effects of collateral development as compared with the effects induced by transfer of each gene alone.     

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.     

Diaphragmatic angiogenic growth factor mRNA responses to increased ventilation caused by hypoxia and hypercapnia.

This study investigates the effect of increased ventilation on the expression of messenger ribonucleic acid (mRNA) levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) in the diaphragm of intact, awake, spontaneously breathing rats, compared with responses in paralysed, mechanically-ventilated animals at similar blood gas and ventilatory levels. Four groups of intact, rats were studied in a body box, each group breathing one of four gases: room air, 12% oxygen (O2), 5% carbon dioxide (CO2), or 12% O2+5% CO2 for 1 h. Another 4 groups of paralysed, mechanically-ventilated animals were matched for arterial blood gas and ventilatory level. The results showed that VEGF mRNA abundance was increased three-fold and that of bFGF 1.5-fold when 12% O2+5% CO2 were breathed, but TGF-beta1 did not change. A significant linear relationship of VEGF and bFGF mRNA to minute ventilation was observed in awake animals (r=0.98, p<0.02 and r=0.87, p<0.03, respectively). The paralysed, mechanically-ventilated animals showed no mRNA increases for any probe. Systemic hypoxia had no additional effect on VEGF or bFGF levels in the diaphragm. It was concluded that messenger ribonucleic acid for vascular endothelial growth factor and basic fibroblast growth factor in the diaphragm rises significantly as a result of active ventilation and not due to blood gas/pH changes or to passive muscle shortening per se.     

培哚普利在糖尿病大鼠肢体缺血后血管再生中的作用

目的探讨培哚普利在1型糖尿病（T1DM）大鼠肢体缺血后血管再生中的作用及相关机制。方法对T1DM大鼠模型予单侧肢体股动脉离断。分为对照（DM）组、培哚普利治疗（DMA）组和培哚普利＋内皮型一氧化氮合酶（eNOS）阻断剂治疗（DML）组。治疗4周后比较各组缺血肢体血管再生情况以及eNOS、血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）的mRNA和蛋白表达水平。结果与正常对照（Nc）组相比,DM组的缺血肢体微血管密度（MVD）、eNOS、VEGF和bFGF的mRNA和蛋白表达均下降,DMA组上述指标明显上调（P均〈0．05）。DML组的MVD、eNOS和bFGF的mRNA和蛋白表达降低（P〈0．05）,而VEGF表达不受影响（P〉0．05）。结论T1DM大鼠的缺血性血管再生受损,与之有关的生长因子表达下调。培哚普利能促进T1DM大鼠缺血肢体的血管再生,其机制与eNOS、VEGF和bFGF的表达上调有关。     

Elevation of serum hepatocyte growth factor during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

We examined serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. Granulocyte colony-stimulating factor (G-CSF) (filgrastim 400 microg/m2/d) was administered to 23 donors for 5 d and aphereses were performed on days 4 and 5. Although bFGF remained at similar levels after G-CSF treatment, serum VEGF and HGF levels increased 1.5-fold (n = 13; P = 0.02) and 6.8-fold (n = 23; P < 0.0001) respectively. The serum HGF level before G-CSF administration on day 1 correlated inversely with mobilized CD34+ cell numbers. Time course kinetics of HGF showed that on the day after G-CSF administration (day 2), serum HGF levels increased to 3678 pg/ml. For auto PBSC mobilization with chemotherapy and G-CSF 200 microg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose-dependent on the G-CSF administered.     

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.     

Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in multiple myeloma

Angiogenesis is a crucial process in growth and progression of cancer and there is growing evidence that neovascularisation is important in hematological malignancies. Since an increased angiogenic potential has been identified in multiple myeloma, we simultaneously measured circulating serum levels of the cytokines bFGF, VEGF, HGF and IL-6 by ELISA in 67 patients with multiple myeloma or monoclonal gammopathies of undetermined significance (MGUS) and in 20 controls. Median values of bFGF were 4.7 pg/ml in healthy volunteers, 6.2 in MGUS, 6.3 in myeloma stage I, 13.4 in stage II and 21.7 in stage III. Myeloma patients had significantly higher bFGF serum levels than controls (p<0.001). Pretreatment bFGF levels differed significantly in the Salmon and Durie stages I-III (p=0.02) and were significantly elevated in stage II-III compared to stage I myeloma (p=0.02). In patients responding to chemotherapy according to the CLMTF criteria, a significant decrease in serum bFGF, VEGF and HGF levels occurred (median pretreatment values for bFGF 23.9 pg/ml, post-treatment 6.5 pg/ml; p<0.001, for VEGF 223 pg/ml versus 105 pg/ml; p=0.02 and for HGF 1429 pg/ml versus 1077 pg/ml; p=0.02, respectively). In 11 patients who did not achieve a remission, there was no significant decrease in bFGF, VEGF and HGF levels. These data show that myeloma in stages II and III is associated with an increase in serum bFGF concentrations and give the first report that effective chemo-therapy is accompanied by a significant decrease in the angiogenic factors bFGF, VEGF and HGF, while no decrease of these factors could be found in nonresponders.     

Elevated circulating levels of basic fibroblast growth factor and vascular endothelial growth factor in patients with acute myocardial infarction

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have both shown strong angiogenetic effects in ischemic animal models and it has been reported that these growth factors were increased after acute myocardial ischemia. However, there have been few reports on the serum levels of bFGF and VEGF after acute myocardial infarction (AMI), in particular there has not been a comparative study of bFGF and VEGF in human subjects. The time course of circulating levels of bFGF and VEGF was examined in 36 patients with AMI who were within 24h of the onset of the AMI. The serum bFGF and VEGF levels of 50 age- and sex-matched healthy volunteers served as the baseline value. All the patients had undergone coronary angiography on the day of admission (Day 0), but prior to that the serum bFGF and VEGF levels were examined by enzyme-linked immunoassay. The serum bFGF and VEGF levels were also evaluated on Days 7, 14 and 28. Creatine kinase, myosin light chain I and troponin-T were measured subsequently and radionuclide examinations were performed during the early phase of AMI to determine the infarct size. The serum bFGF levels were significantly increased at Day 0 and were maintained until Days 7 and 14. Although serum VEGF levels at Day 0 were similar to the baseline values, they showed a remarkable increase by Days 7 and 14. A high serum level of bFGF was detected in the acute phase of AMI, and a later increase in VEGF was determined in the sub-acute phase, which suggest that these 2 growth factors play an important role at different time points of the reconstructing process of infarcted myocardial tissue.     

Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy

BACKGROUND: Impaired regeneration and dysfunction of the cirrhotic liver following partial hepatectomy (PHx) are the most serious risk factors for postoperative liver failure. AIMS: Using naked hepatocyte growth factor (HGF) plasmid by the electroporation (EP) in vivo method, we investigated HGF for its role and mechanism of proliferation and restoration of liver mass in cirrhotic mice following PHx. ANIMALS: Eight week old female mice were used. METHODS: HGF plasmid 50 micro g was injected intramuscularly and transferred by EP in vivo once a week for three weeks. After establishment of carbon tetrachloride induced cirrhosis, mice underwent PHx. The HGF treated group was given naked HGF plasmid four days before PHx, and additional HGF was given once a week until they were killed, while a control group was given only empty plasmid. Mice were killed 2, 4, 10, and 14 days after PHx. Morphological and functional restoration of the liver were examined, as well as activation of mitogen activated protein kinase (MAPK) and mRNA levels of HGF activator (HGFA). RESULTS: The HGF treated group demonstrated a continuous threefold increase in HGF levels in plasma. Therapy with HGF in cirrhotic PHx resulted in effective liver regeneration via restoration of HGFA and activation of MAPK p44/p42, accelerated normalisation of liver function, and increased collagen degradation. CONCLUSIONS: HGF gene therapy by in vivo EP may be useful for hepatic resection in cirrhotic livers by stimulating liver proliferative and collagenolytic capacities, as well as accelerating functional recovery.     

Synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in murine hepatocellular carcinoma

The growth of any solid tumor depends on angiogenesis. Among the known angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), are potent and representative factors involved in tumor development. It has been reported that bFGF and VEGF showed a synergistic effect in both in vitro and in vivo angiogenesis. However, the interaction of these factors on tumor development and angiogenesis, including hepatocellular carcinoma (HCC), has not yet been elucidated. In this study, we examined the combined effect of bFGF and VEGF overexpression by means of a combination of a retroviral tetracycline (tet)-regulated (Retro-Tet) gene expression system, which can manipulate the gene expression in vivo by providing tet in the drinking water, and a conventional plasmid gene expression system. In an allograft study, bFGF and VEGF overexpression synergistically increased tumor growth and angiogenesis in the murine HCC cells. This synergistic effect also was found in established tumors. VEGF messenger RNA (mRNA) expression in the tumor was increased 3.1-fold by bFGF-overexpression, and the bFGF-induced tumor development was significantly attenuated by treatment with KDR/Flk-1 neutralizing monoclonal antibody. In conclusion, these results suggest that bFGF synergistically augments VEGF-mediated HCC development and angiogenesis at least partly by induction of VEGF through KDR/Flk-1.     

Vascular endothelial growth factor and basic fibroblast growth factor differentially modulate early postnatal coronary angiogenesis

The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 microm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 microm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.     

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

BACKGROUND: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood. METHODS: NTs and their receptors were investigated by RT-PCR and immunostaining in regenerating livers after two-thirds hepatectomy (PH) and in hepatocytes and hepatic stellate cells (HSCs) isolated from regenerating livers in mice. Induction of apoptosis after treatment with NGF and the expression of alpha-smooth muscle actin (SMA), interleukin 6 (IL-6) and hepatocyte growth factor (HGF) were also investigated in regenerating HSCs. RESULTS: Nerve growth factor (NGF) and p75 NT receptor (p75NTR) mRNA were elevated after PH, while other NTs and NT receptors showed no remarkable change. NGF was detected in regenerating hepatocytes, but not in normal hepatocytes. Regenerating HSCs expressed increased p75NTR and SMA in vivo and showed an activated phenotype and the high expression of HGF and IL-6 in vitro. Enhanced cell death was seen in HSCs, both from normal and regenerating liver, after treatment with NGF. CONCLUSIONS: Although activated HSCs may produce the factors that regulate liver regeneration, the de novo NGF production by regenerating hepatocytes may induce the death of activated HSCs via p75NTR, leading to termination of hepatic regeneration.