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 serum concentrations of activated hepatocyte growth factor activator in patients with multiple myeloma

Objectives: Hepatocyte growth factor (HGF) is a potential key factor in multiple myeloma. Conversion of pro‐HGF to its active form is a critical limiting step for its biological effects. We aimed to examine the levels of the most potent activator, the hepatocyte growth factor activator (HGFA), in serum and bone marrow plasma of patients with multiple myeloma. Methods: The activated form of HGFA was measured by an enzyme‐linked immunosorbent assay in serum (n = 49) and bone marrow plasma (n = 16) from multiple myeloma patients, and in serum from healthy controls (n = 24). Results: The median concentrations of activated HGFA in myeloma and control sera were 39.7 (range 6.2–450.0) and 17.6 ng/mL (range 4.8–280.6), respectively. The difference was statistically significant (P = 0.037). The median concentration of activated HGFA in bone marrow plasma was 6.1 ng/mL (range 3.5–30.0). Conclusion: We here show for the first time that the activated form of HGFA is present at high levels in serum and bone marrow of myeloma patients, thus providing a necessary prerequisite for the activation of HGF.     

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.     

Elevated serum concentration of hepatocyte growth factor in patients with multiple myeloma: correlation with markers of disease activity

Hepatocyte growth factor (HGF) has been shown to be involved in angiogenesis, epithelial cell proliferation, and osteoclast activation. HGF and its receptor are expressed on myeloma cell lines and could be involved in the pathogenesis of bone destruction in multiple myeloma (MM). The aim of this study was to examine serum levels of HGF in untreated MM patients and its correlation with bone turnover indices and markers of disease activity. Forty-seven newly diagnosed MM patients and 25 controls were included: 12 patients were of stage I, 13 of stage II, and 22 of stage III (Durie-Salmon classification). Bone lesions were scored from 0 to 3, according to X-ray findings. Serum osteocalcin (OC), interleukin-6 (IL-6), TNF-alpha, beta(2)-microglobulin (beta(2)M), CRP, calcium, and 24-hr urine N-telopeptide cross-links of collagen breakdown (NTx) were determined. HGF levels were significantly higher at stage III compared to stages II and I (medians: 1,990.4 vs. 1,743.8 and 1,432.4 pg/mL, respectively, P < 0.05). Similarly, NTx, IL-6, TNF-alpha, CRP, beta(2)M, and calcium increased significantly with advancing stage (P < 0.01). OC was higher at stage I in comparison to stages II and III (P < 0.01). All parameters were significantly higher in patients than controls. HGF showed a strong correlation with IL-6 and TNF-alpha and less with beta(2)M, CRP, NTx, and OC. We conclude that serum HGF levels are increased in advanced stages of MM disease and extended bone lesions. HGF correlates with IL-6 and TNF-alpha, which are cytokines involved in osteoclast stimulation in MM. However, an independent association of HGF with bone turnover markers was not shown in this study, thus its role in MM bone disease needs to be further clarified.     

Hepatocyte growth factor in myeloma patients treated with high-dose chemotherapy

Hepatocyte growth factor (HGF) is a cytokine produced by myeloma cells. We examined serum HGF levels in a population of young myeloma patients (median age 52 years) treated with high-dose chemotherapy. Sera from 128 myeloma patients at diagnosis and serial samples from 16 patients were analysed. Compared with 62 healthy controls, HGF was elevated at diagnosis in 25% of patients (median 0.48 and 1.08 ng/ml respectively; P < 0.0001). The 95 patients who completed therapy were analysed for the impact of HGF on survival. Median survival was not reached after 77 months in the patient group with normal HGF values (< 1.7 ng/ml, n = 69). In the group with elevated HGF (>/= 1.7 ng/ml, n = 26), median survival was 63 months (P = 0.08). In 16 patients, serum was drawn at diagnosis and at the time of expected disease remission (6 weeks to 3 months after chemotherapy). HGF values declined after treatment in 14 of these patients, from a median of 0.9 ng/ml (0.49-1.65) to 0.42 ng/ml (0.32-0.73) (P = 0.005). Our results show that in young myeloma patients HGF is elevated, and that patients with higher levels had a trend towards poorer prognosis. Treatment with high-dose chemotherapy reduced HGF in the serum of the majority of patients.     

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.     

Treatment response to bortezomib in multiple myeloma correlates with plasma hepatocyte growth factor concentration and bone marrow thrombospondin concentration

Multiple myeloma (MM) is associated with increased rate of bone marrow angiogenesis. Increased concentration of hepatocyte growth factor (HGF) is associated with poor prognosis in patients treated with conventional chemotherapy or thalidomide. We have shown previously that decreased level of thrombospondin, an angiogenesis inhibitor, correlates with poor response to high‐dose chemotherapy. The aim of our current study was to evaluate association between therapeutic response to bortezomib and thrombospondin and HGF levels. Peripheral blood plasma concentration of HGF and bone marrow plasma concentration of thrombospondin were measured in patients with MM prior to the initiation of bortezomib therapy. Overall, 58 patients were enrolled, 44/58 (76%) of them with relapsed disease. Treatment outcomes were analyzed for possible associations with pretreatment HGF and thrombospondin levels. Patients who achieved complete response had significantly higher pretreatment HGF levels and lower pretreatment thrombospondin levels than others. More than 70% of patients with low pretreatment HGF and high pretreatment thrombospondin concentrations achieved very good partial response or complete response, in contrast to only 20% of patients with high HGF and low thrombospondin levels. High pretreatment thrombospondin and low pretreatment HGF concentrations are associated with therapeutic response to bortezomib in patients with MM.     

Novel therapeutic strategies targeting growth factor signalling cascades in multiple myeloma

Multiple myeloma (MM) remains largely incurable despite conventional and high-dose therapies, and novel biologically based treatment approaches are urgently required. Recent studies demonstrate that various growth factors including interleukin (IL)-6, insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), the tumour necrosis factor (TNF) family proteins, Wnt, and Notch family members play an important role in MM pathogenesis, and mediate tumour cell proliferation, drug resistance and migration in the bone marrow (BM) milieu. Targeting growth factors, therefore, represents a promising therapeutic strategy in MM. Novel agents inhibiting growth factor signalling cascades can target ligands, receptors, and/or downstream signalling cascade proteins in MM cells and the BM microenvironment. Combinations of these novel agents with conventional therapies may not only enhance cytotoxicity, but also avoid drug resistance and thereby improve patient outcome in MM.     

Activation of hepatocyte growth factor in monkey stomach following gastric mucosal injury

Background Hepatocyte growth factor (HGF) is involved in the proliferation and migration of various types of epithelial cells. HGF is produced as a single-chain precursor (pro-HGF) and functions after activation by HGF activator (HGFA). In this study, we aimed to examine the activation of pro-HGF to mature HGF and the expressions of HGF-related molecules, such as HGFA and HGFA inhibitor type 1 (HAI-1), in a monkey model of gastric mucosal injury.Methods Gastric mucosal injury was induced in Japanese monkeys by administration of HCl using nasal-gastric tubes. HGF activation was evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA), and the expression of HGF, HGFA, HAI-1, and c-Met were examined by Northern blotting and immunohistochemistry.Results Pro-HGF, but not mature HGF, was detected in normal gastric mucosa. When gastric mucosal injuries were induced, the expression of HGF significantly increased, and immunostaining of HGF was detected in fibroblasts in the gastric mucosa. In addition, conversion to mature HGF was observed in the injured gastric tissues, and mature HGF continued to increase for 48h. HGFA was stably expressed in monkey livers, regardless of HCl administration. HAI-1 expression was detected in normal gastric mucosa, and its levels decreased after the induction of gastric mucosal injury.Conclusions These results indicate that pro-HGF is stored in normal gastric tissues, and suggest that its conversion to mature HGF, associated with HGFA and HAI-1, is important for the repair of gastric mucosal injury.     

Soluble c-Met in serum of patients with multiple myeloma: correlation with clinical parameters

Objectives: The receptor tyrosine kinase c‐Met and its ligand, hepatocyte growth factor (HGF), play key roles in tumour genesis and metastasis and contribute in multiple myeloma pathogenesis. Substantial data support that a soluble extracellular fragment of c‐Met may function as a decoy receptor that downregulates the biological effects of HGF and c‐Met. We examined serum levels of soluble c‐Met in patients with myeloma and healthy individuals and investigated a possible relationship with clinical disease parameters and survival. Methods: The concentration of c‐Met and HGF was measured by enzyme‐linked immunosorbent assay in serum (n = 49) and bone marrow plasma (n = 16) from patients with multiple myeloma and in serum from healthy controls (n = 26). Results: The median serum concentration of soluble c‐Met was 186 ng/mL (range 22–562) in patients with multiple myeloma and 189 ng/mL (range 124–397) in healthy individuals. There was a significant negative correlation between serum c‐Met levels and disease stage, bone marrow plasma cell percentage and serum concentration of M‐protein. Conclusion: We have for the first time examined the concentration of soluble c‐Met in serum from patients with myeloma and found equal median levels in patients with myeloma as a group and healthy individuals. Still, serum levels of soluble c‐Met correlated negatively with parameters of disease burden in patients with myeloma. We suggest that a possible role for the c‐Met ectodomain as a negative regulator of HGF/c‐Met activity should be examined in multiple myeloma.     

MET dysregulation is a hallmark of aggressive disease in multiple myeloma patients

Abnormal activation of MET/HGF (Hepatocyte Growth Factor) pathway has been described in several tumours and increased HGF plasmatic levels have been detected in patients with aggressive multiple myeloma (MM). MET and HGF mRNA expression was investigated in 105 samples of purified plasma cells derived from newly diagnosed MM patients treated with bortezomib‐based induction therapy. Gene expression was compared with response to therapy and clinical outcome. MET gene copy number was also evaluated. MET mRNA expression was higher in CD138⁺ than in CD138^? cells (median 76·90 vs. 11·24; P = 0·0009). Low MET mRNA expression characterized patients with better response (complete response or very good partial response) compared to other patients (median 56·10 vs. 134·83; P = 0·0006). After a median follow‐up of 50 months, patients with high MET mRNA expression displayed a worse progression‐free survival (PFS; P = 0·0029) and overall survival (OS; P = 0·0023) compared to those with low MET mRNA levels. Patients with both high MET mRNA expression and high β2‐microglobulin level (>5·5 mg/l) had further worse median PFS (P < 0·0001) and OS (P < 0·0001). Patients carrying 4 MET gene copies (8 out of 82, 9·8%) also had a short PFS. High MET mRNA expression identifies patients with dismal PFS and OS and the combination with high β2‐microglobulin further characterizes patients with worse outcome.     

Serum pleiotrophin levels are elevated in multiple myeloma patients and correlate with disease status

Pleiotrophin (PTN), a tightly regulated angiogenic and mitogenic heparin-binding protein, is markedly elevated in a variety of aggressive solid tumours. The role of PTN in haematological malignancies, however, has not been previously evaluated. This study demonstrated that PTN serum levels were elevated in multiple myeloma (MM) patients when compared with healthy subjects (P < 0.0001). Serum levels of this protein significantly increased during progression of disease, and decreased during response to anti-MM therapy (P < 0.001). These results suggest that serum PTN may be a new biomarker for monitoring the disease status and therapeutic response of MM patients.     

Prognostic significance of vascular endothelial growth factor immunoexpression in the context of adverse standard prognostic factors in multiple myeloma

OBJECTIVES: Vascular endothelial growth factor (VEGF) acts in several steps of multiple myeloma (MM) pathogenesis and it is an important mediator of tumor angiogenesis. The aim of this study was to examine the prognostic significance of VEGF immunoexpression in the context of standard prognostic factors present in a cohort of advanced MM patients. METHODS: Fifty untreated MM patients were enrolled from May 2000 to December 2002. Bone marrow sections were subjected to morphologic assessment and immunohistochemical studies with antibodies against CD34 and VEGF. Angiogenesis was measured by microvessel density (MVD) and stratified into high (MVD > or = 20) and low angiogenesis status (MVD < 20). VEGF immunoreactivity was examined on the basis of intensity and percentage of positive plasma cells (PC). RESULTS: Ninety-four percent of patients presented advanced disease at diagnosis. Median PC marrow infiltration was 80%. Twelve percent of patients presented plasmablastic morphology. Low angiogenesis was present in 27% of patients, while high angiogenesis was present in 73%. Twenty-nine percent of patients had VEGF < 10% and 71% had VEGF > or = 10%. Weak-intensity VEGF was observed in 34% of cases, while 37% had moderate/strong VEGF intensity. Although VEGF had prognostic impact on overall survival (OS) and event-free survival (EFS) in univariate analysis, multivariate analysis identified only plasmablastic morphology and elevated serum lactate dehydrogenase (LDH) level as independent prognostic factors to predict OS (P = 0.04 and P = 0.02, respectively). With regard to EFS, although VEGF showed statistical trend to influence survival (P = 0.08), the parameters of independent prognostic value were also plasmablastic morphology (P = 0.01) and elevated LDH level (P = 0.01). CONCLUSION: Our findings underline the frequent expression of VEGF in advanced-stage MM and the greater prognostic information of simple and readily available factors, namely plasmablastic morphology and elevated LDH. Moreover, despite the absence of prognostic importance in multivariate analysis, VEGF and its receptors remain promising therapeutic targets in MM.     

Association between serum hepatocyte growth factor and survival in untreated hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) is a common hepatic malignancy worldwide. Its nature of rapid growth results in a grave prognosis. Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, responsible for their proliferation. The aim of the present study was to investigate the prognostic roles of serum HGF in untreated HCC patients.Methods Fifty-five patients with inoperable HCC were studied. The diagnosis of HCC was based on either liver histopathology or imaging evidence of a liver mass, together with elevated serum alpha-fetoprotein. Serum HGF levels of the patients, at the time of diagnosis, were compared to those of 28 healthy controls. All patients received only palliative treatments and were followed up until they died. Comparison of survival curves between patients with a serum HGF level of 1.0ng/ml or more and those with lower serum HGF was performed, using the log-rank test. Data values are expressed as means and SD.Results Fifty-one men and four women with inoperable HCC were recruited. The mean age was 54.15 ± 15.34 years. The serum HGF levels in the inoperable HCC patients were significantly higher than those in the controls (0.58 ± 0.43 vs 0.14 ± 0.04ng/ml; P < 0.001). The patients mean survival time was 5.28 ± 6.73 months (range, 0.1–33 months). Serum HGF levels exhibited a negative correlation with the survival time (P = 0.032). In addition, HCC patients with serum HGF levels of 1.0ng/ml or more had a shorter survival time than the other HCC patients (P = 0.0025).Conclusions Patients with inoperable HCC had higher levels of serum HGF than the healthy controls, and serum HGF was negatively correlated with the survival time. Serum HGF levels of 1.0ng/ml or more in HCC patients are suggestive of a grave prognosis, indicating that HGF plays important and active roles in the disease progression. The detailed mechanisms need to be further investigated.     

High levels of serum angiogenic growth factors in patients with AL amyloidosis: comparisons with normal individuals and multiple myeloma patients

Serum levels of five angiogenic cytokines were evaluated in 82 patients with primary systemic amyloidosis (AL). Angiopoietin‐1, vascular endothelial growth factor, basic fibroblast growth factor and angiogenin were higher in AL patients than in controls (n = 35) and newly‐diagnosed, symptomatic, myeloma patients (n = 35). Angiopoetin‐1/Angiopoetin‐2 ratio was lower in AL compared to controls but higher than in myeloma patients. Angiopoetin‐2 correlated with cardiac dysfunction indices; however, none of the angiogenic growth factors was prognostically significant. The increased angiogenic cytokine levels observed in AL seem to represent either a toxic effect of amyloid fibrils or light chains, or a compensatory response to organ dysfunction.     

Circulating levels of hepatocyte growth factor and left ventricular remodelling after acute myocardial infarction (from the REVE-2 study)

Aim As experimental studies suggest that hepatocyte growth factor (HGF) is cardioprotective after myocardial infarction (MI), this study sought to investigate relationships between circulating levels of HGF and left ventricular (LV) remodelling in patients after acute MI. METHODS AND RESULTS: This prospective multicentre study included 246 patients with a first anterior Q-wave MI. Serial echocardiographic studies were performed at hospital discharge and 3 and 12 months after MI; quantitative analysis was performed at a core echocardiography laboratory. Blood samples to measure HGF, brain natriuretic peptide (BNP), and C-reactive protein were obtained at discharge and at the 1, 3, and 12 month follow-up visits. Plasma HGF levels were high at baseline, decreased at 1 month, and remained stable thereafter. In the post-MI period (at 3 and 12 months), HGF levels were positively associated with LV volumes, wall motion systolic index, E/Ea, and BNP; and negatively with LV ejection fraction. High HGF levels were associated with higher C-reactive protein levels. Multivariate analysis showed that both BNP (P < 0.0001) and C-reactive protein (P < 0.0001) were independently associated with HGF levels at 3 and 12 months. Patients who died or were rehospitalized for heart failure during follow-up had higher HGF levels at 1 month (P = 0.0006), 3 months (P = 0.018), and 1 year (P = 0.006) after MI. CONCLUSIONS: Circulating HGF levels correlate with all markers of LV remodelling after MI and are associated with rehospitalization for heart failure.     

Response to thalidomide in progressive multiple myeloma is not mediated by inhibition of angiogenic cytokine secretion

Thalidomide (Thal) is a drug with anti-angiogenic properties. To explore whether the effect of Thal on angiogenesis is associated with a reduction of angiogenic cytokine levels in progressive multiple myeloma (MM), plasma levels of basic fibroblast growth factor, vascular endothelial growth factor, interleukin 6, tumour necrosis factor-alpha and hepatocyte growth factor (HGF) were measured in 51 patients at 0, 3 and 6 months of Thal therapy. After 6 months of treatment, 26 patients were considered to be responsive to Thal therapy, including 17 minimal responses, eight partial responses and one complete response. Only HGF (decreasing, P = 0.02) in the group of responsive patients showed a statistically significant change over a period of 6 months. Because HGF levels are known to correlate to MM tumour burden, we conclude that the mechanism of action of Thal in MM is not caused by a specific inhibition of angiogenic cytokine secretion.     

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.