Serum Hepatocyte Growth Factor Levels Research Articles

Hepatocyte growth factor, a biomarker of macroangiopathy in diabetes mellitus.

Atherosclerotic involvements are an essential causal element of prospect in diabetes mellitus (DM), with carotid atherosclerosis (CA) being a common risk-factor for prospective crisis of coronary artery diseases (CAD) and/or cerebral infarction (CI) in DM subjects. From another point of view, several reports have supplied augmenting proof that hepatocyte growth factor (HGF) has a physiopathological part in DM involvements. HGF has been a mesenchymal-derived polyphenic factor which modulates development, motion, and morphosis of diverse cells, and has been regarded as a humor intermediator of epithelial-mesenchymal interplays. The serum concentrations of HGF have been elevated in subjects with CAD and CI, especially during the acute phase of both disturbances. In our study with 89 type 2 DM patients, the association between serum concentrations of HGF and risk-factors for macrovascular complications inclusive of CA were examined. The average of serum HGF levels in the subjects was more elevated than the reference interval. The serum HGF concentrations associated positively with both intimal-media thickness (IMT) (r = 0.24, P = 0.0248) and plaque score (r = 0.27, P = 0.0126), indicating a relationship between the elevated HGF concentrations and advancement of CA involvements. Multivariate statistical analysis accentuated that serum concentrations of HGF would be associated independently with IMT (standardized = 0.28, P = 0.0499). The review indicates what is presently known regarding serum HGF might be a new and meaningful biomarker of macroangiopathy in DM subjects.

Downregulation Of SPINT2 Expression In Stromal Cells From Myelodysplastic Syndrome Patients Increases HGF and SDF-1 Secretion and Promotes Cell-Cell Adhesion By Alteration In Integrin Expression

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34+, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34+, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r2=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34+, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq Disclosures:No relevant conflicts of interest to declare.

Clinical significance of serum hepatocyte growth factor (HGF) levels in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the commonest primary malignant cancer of the liver in the world. This study was conducted to investigate the serum levels of hepatocyte growth factor (HGF)in HCC patients and the relationship with tumor progression and known prognostic parameters. Fifty-four patients with HCC were investigated. Pretreatment HGF levels were employed the quantitative sandwich enzyme immunoassay technique (ELISA). Age and sex matched 20 healthy controls were included in the analysis. The median age of the patients was 60 years (range 36-77 years); where males consistituted of majority of the group (88.8%). All of patients had cirrhotic history. Fourty-six percent (n = 25) of patients had Child-Pugh Score A, 30% (n = 16) had Score B or C. All of the patients were treated with local therapies but none of them received sorafenib. The baseline serum HGF levels were significantly higher in patients with HCC than in the control group (p < 0.001). Male patients had higher serum HGF levels compared with female patients (p = 0.01). Serum HGF levels were significantly higher in the patients with elevated serum ALT levels than others with normal serum ALT levels (p = 0.05). Poor performance status (p < 0.001), viral etiology of cirrhosis (p = 0.03), larger tumor size (p = 0.01), lower serum hemogloblin levels (p = 0.03), and not be treated for HCC (p = 0.001) related to worse survival. However, serum HGF did not have significantly adverse effect on survival (p = 0.58). Despite serum HGF levels were found diagnostic value, serum HGF levels had no prognostic value in patients with HCC.

Serial Changes of Serum Growth Factor Levels and Liver Regeneration after Partial Hepatectomy in Healthy Humans

This study aimed to investigate the associations of the serial changes of serum levels of various growth factors with liver regeneration after hepatectomy in healthy liver donors. Sixteen healthy liver donors who underwent conventional liver resection were included. Serum levels of various growth factors before hepatectomy and on postoperative day (POD) 1, 3, 5 and 7 were measured. Liver volume data calculated by multi-detector computed tomography using workstation. The ratio of remnant liver volume on POD 0 to liver volume before the operation was 51% ± 20%. The ratio of liver volume on POD 14 to liver volume on POD 0 were inversely correlated with remnant liver volume on POD 0 (r = −0.91). The ratio of liver volume on POD 14 to liver volume on POD 0 were significantly correlated with serum hepatocyte growth factor (HGF) levels on POD 1 (r = 0.54), serum leptin levels on POD 1 (r = 0.54), and serum macrophage colony-stimulating factor (M-CSF) levels on POD 5 (r = 0.76) and POD 7 (r = 0.80). These results suggest that early-phase elevation of serum levels of HGF, leptin and M-CSF may be associated with the acceleration of liver regeneration after hepatectomy in humans.

Losartan Supports Liver Regrowth via Distinct Boost of Portal Vein Pressure in Rodents with 90 % Portal Branch Ligation

The study used a model of 90 % portal branch ligation (PBL) in rats to study the effect of losartan on portal vein pressure (PVP) and liver regeneration in rats after PBL. A total of 144 male Sprague-Dawley rats were arbitrarily designated into three treatment method groups: a sham operation group (Sham), a PBL treatment group (PBL), and a PBL plus losartan treatment group (PBL + L). Losartan (2 mg/day) was intragastrically gavaged 3 days before the PBL or sham operation to time points of study. Both the PBL and PBL + L groups showed an intense surge in PVP after PBL treatment, peaking at 12 h postsurgery, then lessening progressively afterwards. PVP was substantially greater in these two groups compared with the Sham group at 6-72 h postsurgery (p < 0.01). Compared with the PBL group, the PBL + L group showed a noticeable reduction in PVP 6-48 h postsurgery (p < 0.05); the PBL group showed considerably raised levels of plasma ALT and AST 6-72 h postsurgery (p < 0.01). Compared to the PBL group, the PBL + L group showed drastically reduced plasma ALT and AST levels 12-72 h postsurgery (p < 0.05). Losartan supports liver regeneration in 90 % of rats that underwent PBL. The mechanism may be related to losartan's ability to regulate PVP and increase serum hepatocyte growth factor levels.

Hepatocyte growth factor as a long‑term predictor for total and cardiovascular mortality in patients on peritoneal dialysis

Hepatocyte growth factor (HGF) is a regenerative protein involved in tissue protection and endothelial repair in response to injury. The aim of the study was to assess the effect of increased HGF levels on total and cardiovascular (CV) mortality in patients on peritoneal dialysis (PD) during 6‑year follow‑up. The study included 55 patients (mean age, 53 years; median duration of PD, 24 months). The ejection fraction (EF) and calcium score (CaSc) were measured. White blood cell (WBC) count and albumin, calcium (Ca), phosphorus (Pi), intact parathormone (iPTH), and high‑sensitivity C‑reactive protein (hsCRP) levels were measured. Serum levels of HGF, interleukin (IL) 6, and IL‑18 were determined using enzyme‑linked immunosorbent assays. Of all patients, 45% died within 6 years, including 80% from CV complications. HGF significantly correlated with the total (hazard ratio [HR], 1.97; P = 0.03) and CV (HR, 2.04; P = 0.04) mortality in a univariate Cox regression model. This was confirmed by a multiple model including age, dialysis duration, residual renal function (RRF), albumin, Ca × Pi, EF, and CaSc (HR, 2.24; P = 0.02 and HR, 2.58; P = 0.02 for total and CV mortality, respectively). Factors negatively affecting patients' survival included WBC count, hsCRP, IL‑6, and CaSc, while higher albumin levels and EF were associated with longer overall survival. HGF positively correlated with WBC count (r = 0.30, P = 0.01), hsCRP (r = 0.25, P = 0.04), IL‑6 (r = 0.46, P = 0.0004), CaSc (r = 0.29, P = 0.03), and mean arterial pressure (r = 0.25; P = 0.04) and negatively with RRF (r = -0.31, P = 0.02). Increased concentrations of proinflammatory cytokines and an association between HGF levels and CaSc may indicate higher total and CV mortality in patients on PD.

Abstract 5624: Anti-c-Met Nanobody®: A potential new drug in cancer treatment.

Abstract c-Met is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-Met signaling is involved in a wide variety of human malignancies including colon, gastric, bladder, breast, ovarian, pancreatic, kidney, liver, lung, head and neck, thyroid, and prostate cancers, as well as sarcomas, hematological malignancies, melanoma and central nervous system tumors. In recent years, multiple agents have been developed directed to interfere at different levels in the HGF-c-Met signaling pathway, and some are currently being tested in Phase II and/or III clinical trials. Nanobodies® are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, occurring in the Camelidae family. Importantly, they retain the full antigen-binding capacity of the original heavy chain only antibodies and are highly stable. In this study, we examined the anti-cancer effects of an anti-c-Met Nanobody in vitro using human multiple myeloma as a model system. The anti-c-Met Nanobody comprises of two Nanobody moieties, one targeting c-Met and one binding to human serum albumin for half-life extension. HGF is thought to contribute to the pathogenesis of multiple myeloma in different ways, as HGF is a pivotal growth and pro-migratory factor and inhibits osteoblastogenesis in vitro suggesting that HGF may contribute to the development of myeloma bone disease. Elevated levels of HGF in serum of multiple myeloma patients are correlated with a poor prognosis. We show that the anti-c-Met Nanobody effectively inhibited the proliferation of ANBL-6 human multiple myeloma cells via inhibition of an HGF autocrine growth loop, as well as the proliferation of INA-6 cells induced by exogenously added HGF. In addition, the HGF-induced migration of INA-6 cells was completely and specifically blocked following treatment with the Nanobody at a concentration of 1 μM. The Nanobody also inhibited the HGF-induced adhesion of multiple myeloma cells to fibronectin, but did not affect stromal cell-derived factor-1 alpha-induced adhesion. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced ALP-activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced the HGF mediated phosphorylation of the c-Met Tyrosine (Y) residues Y1349, Y1234/1235 and Y1003 and the phosphorylation of the downstream proteins MAPK and Akt in INA-6 cells. In conclusion, the anti-c-Met Nanobody inhibited c-Met signaling with high specificity and potency resulting in inhibition of multiple myeloma cell migration, proliferation and adhesion, and in blocking of the HGF mediated inhibition of osteoblastogenesis. Given the potential of Nanobodies to surpass drawbacks of antibodies, this anti-c-Met Nanobody might represent a potential novel therapeutic agent in the treatment of multiple myeloma and other cancers driven by HGF-c-Met signaling. Citation Format: Tobias S. Slørdahl, Tinneke Denayer, Siv Helen Moen, Therese Standal, Magne Børset, Cedric Ververken, Torstein B. Rø. Anti-c-Met Nanobody®: A potential new drug in cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5624. doi:10.1158/1538-7445.AM2013-5624

Abstract LB-316: The anti-c-Met Nanobody®, a novel and promising anti-cancer therapeutic.

Abstract The hepatocyte growth factor (HGF) and its receptor, c-Met, are implicated in many human cancers to promote tumor proliferation, migration, invasion and angiogenesis. Dysregulation of the HGF/c-Met pathway is known to correlate with poor prognosis. In recent years, multiple anti-cancer agents have been developed interfering at different levels in the HGF/c-Met pathway. Here, we investigated the in vitro and in vivo efficacy of a novel Nanobody effectively antagonizing c-Met. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, naturally occurring in Camelidae. The Nanobody platform allows designing modular drugs, including multivalent, biparatopic and bifunctional molecules. The anti-c-Met Nanobody is a bispecific Nanobody that comprises two building blocks, one targeting c-Met and one binding human serum albumin for half-life extension purposes. In vitro characterization revealed that the Nanobody is able to specifically and completely inhibit HGF/c-Met interaction with high potency in an ELISA-based assay. In addition, the anti-c-Met Nanobody was able to completely block the HGF-dependent c-Met-phosphorylation in the A549 non-small cell lung cancer cell line. Importantly, no agonistic activity was observed in absence of HGF, as the Nanobody was designed to have a monovalent interaction with c-Met. Next, the in vivo effect was translated towards a potent in vivo effect. The anti-tumor efficacy of the anti-c-Met Nanobody was assessed in xenograft mouse models in which autocrine U87MG (HGF+, c-Met+) glioblastoma cells, or autocrine KP4 (HGF+, c-Met+) pancreatic carcinoma cells were subcutaneously injected. In the U87MG xenograft model, Nanobody treatment (10 mg/kg i.p 3x/week) resulted in significant tumor growth inhibition compared to vehicle and sustained tumor growth suppression after cessation of the treatment. Treatment with the anti-c-Met Nanobody in the KP4 xenograft model (10 mg/kg i.p 3x/week) led to a significant tumor growth inhibition and even tumor regression. In the latter model, serum levels of soluble c-Met, IL-8 and HGF were shown to be significantly reduced upon Nanobody treatment, which could make these markers attractive as potential translational pharmacodynamics biomarkers. Furthermore, the ratio of tyrosine phosphorylated c-Met over total c-Met levels was significantly lower in KP4 tumors treated with Nanobody versus vehicle. It could be postulated based on literature data that the anti-c-Met Nanobody, by possessing an anti-albumin Nanobody building block, has the potential to show superior tumor penetration than c-Met targeting antibodies. Hence, the anti-c-Met Nanobody might be a valuable novel biological for the treatment of cancer driven by HGF-c-Met signaling. Citation Format: Tinneke Denayer, Thomas Stöhr, Gerald Beste, Ann Brige, Cedric Ververken, Josefin-Beate Holz. The anti-c-Met Nanobody®, a novel and promising anti-cancer therapeutic. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-316. doi:10.1158/1538-7445.AM2013-LB-316

Independent association of elevated serum hepatocyte growth factor levels with development of insulin resistance in a 10‐year prospective study

Hepatocyte growth factor (HGF) receptors form a hybrid complex with insulin receptors in the liver of mice, which lead to robust signalling to regulate glucose metabolism. Serum HGF levels are high in subjects with metabolic syndrome and/or obesity. Accordingly, we prospectively investigated the relationship between HGF and the development of insulin resistance (IR) in a general population without IR at baseline. A total of 1492 subjects received health examinations. After excluding subjects with diabetes and/or IR (n=402) at baseline, the remaining subjects (n=1090) were followed-up 10 years later. Complete data sets were available from 716 subjects for prospective analysis. Logistic regression was performed to determine factors associated with the development of IR after 10years. In subjects without diabetes at baseline, serum HGF levels were higher (0·26±0·10ng/ml, n=259) in subjects with IR than without it (0·22±0·09ng/ml, n=1090). After deleting subjects who developed liver disease during follow-up, 188 were found to have developed IR at 10years after the original screening. HGF (P<0·05), age (P<0·001), homoeostasis model assessment index (P<0·001), HDL-c (P<0·05; inversely) and hypertensive medication (P<0·05) were significantly associated with the development of IR by multivariate stepwise logistic regression analysis. A significant (P<0·05) relative risk [1·75 (95%CI: 1·01-3·12)] for the development of IR was observed in the highest (≥0·30ng/ml) vs the lowest categories (<0·15ng/ml) of HGF after adjustments for confounders. Our 10-year prospective study suggests that elevated serum HGF levels were significantly associated with the development of IR.

Serum from hepatectomized rats induces the differentiation of adipose tissue mesenchymal stem cells into hepatocyte-like cells and upregulates the expression of hepatocyte growth factor and interleukin-6 in vitro

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are an attractive alternative for clinical application due to their minimally invasive accessibility and availability in the body. However, the hepatic differentiation efficiency of AT-MSCs is insufficient for therapeutic application and the role of extrahepatic stem cells in liver regeneration remains poorly understood. This study was designed to investigate the effects of serum from rats subjected to 70% partial hepatectomy (PH) on the differentiation ability of rat AT-MSCs invitro, and to explore the potential role of AT-MSCs invivo following PH injury. Results showed that AT-MSCs treated with serum collected from rats 24h after 70% PH differen-tiated into hepatocyte-like cells, resembled hepatocyte-like cells with round or polygonal shape, expressed α-fetoprotein, secreted albumin, synthesized urea and acquired cytochrome P450 type3A4 enzyme activity, and upregulated the expression of interleukin (IL)-6 and hepatocyte growth factor (HGF) transiently invitro, although the hepatic differentiation efficiency was extremely low. AT-MSC transplantation after 70% PH ameliorated liver injury and promoted liver regeneration, but did not increase the serum levels of IL-6 and HGF invivo. This result suggests that the therapeutic effect of AT-MSCs invivo after 24h of 70% PH does not increase IL-6 and HGF expression.

Investigation of hepatocyte growth factor and proinflammatory cytokine levels in patients with febrile neutropenia

It has been thought that hepatocyte growth factor (HGF) levels increase during acute phases of infectious diseases. The aim of this study was to determine whether HGF, C-reactive protein (CRP), and proinflammatory cytokines have any diagnostic and prognostic value, and to assess HGF as a biomarker in patients with febrile neutropenia. Materials and methods: This study included 20 patients with febrile neutropenia as the study group and 20 healthy individuals as the control group. Serial measurements of serum HGF, CRP, and proinflammatory cytokine levels were performed by enzyme-linked immunosorbent assay (ELISA) at the baseline, after 48 h of treatment, and at posttreatment. Results: It was found that HGF levels at the baseline were significantly higher than those in the healthy controls (P = 0.001). It was also seen that HGF levels 48 h after treatment and at posttreatment were significantly lower than those at the baseline (P = 0.012). High-risk group patients had higher mean serum HGF levels compared with the low-risk group. It was detected that IL-6 and TNF-a levels at the baseline were significantly decreased after treatment (P = 0.02 and P = 0.005). Conclusion: Our findings suggested that serial measurement of serum HGF levels may be an important marker to identify risk levels of febrile neutropenia and to predict its prognosis.

Molecular targeting of hepatocyte growth factor by an antagonist, NK4, in the treatment of rheumatoid arthritis

IntroductionHepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.MethodsArthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4+ T cells stimulated with allogeneic spleen cells.ResultsThe intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4+ T cells stimulated with allogeneic spleen cells.ConclusionsThese results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4+ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.

The relationship between soluble receptor of interleukin-6 with angiogenic cytokines and proliferation markers in multiple myeloma

Soluble interleukin-6 receptor (sIL-6R) is part of IL-6 receptor that may stimulate cells that do not express the whole molecule. It may enhance myeloma cell proliferation and furthermore angiogenesis. The aim of the study was to evaluate the clinical significance and the relationship between serum levels of sIL-6R, with various stimulators of angiogenesis, such as hepatocyte growth factor (HGF) and interleukin-18 (IL-18) and with markers of proliferation, such as beta-2 microglobulin (B2M) levels and plasma cell Ki-67 proliferation index in the bone marrow, in patients with multiple myeloma (MM). We studied 45 newly diagnosed MM patients. Serum levels of sIL-6R, HGF, IL-18, and B2M and Ki-67 proliferation index (Ki-67 PI) in bone marrow's plasma cells were determined. The mean concentrations of sIL-6R, HGF, IL-18, and B2M and the value of Ki-67 were significantly higher in the patients compared to controls and with increasing disease stage. sIL-6R was strongly positively correlated with HGF, IL-18, B2M, and Ki-67 PI. There is a positive correlation between plasma cell growth, as determined by Ki-67 PI, and different angiogenic cytokines, such as HGF and IL-18, with sIL-6R. This relationship suggests the significant role of these cytokines in the proliferation and disease activity in MM patients.

Abstract B65: Biomarker effects of an oral green tea extract, Polyphenon E, in women with a history of hormone receptor-negative breast cancer

Abstract Background: Observational studies have correlated green tea intake with reduced breast cancer risk. In preclinical studies, the main polyphenol of green tea, epigallocatechin-3-gallate (EGCG), inhibits growth factors, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which influence tumor cell growth, migration, and invasion. We conducted an ancillary study using archived serum and urine from a completed phase IB randomized, placebo-controlled dose escalation trial of an oral green tea extract, Polyphenon E (Poly E), in women with hormone receptor (HR)-negative breast cancer. The objective was to understand the effects of Poly E on potential biomarkers of breast cancer risk. Methods: Forty women with stage I-III HR-negative breast cancer who completed adjuvant treatment were randomized using an adaptive trial design to Poly E 400 mg (n = 16), 600 mg (n = 11), 800 mg (n = 3) twice daily or matching placebo (n = 10) for 6 months. Blood and urine collection occurred at baseline, 2, 4, and 6 months. Biologic endpoints included oxidative damage (urine 8-oxoG and isoprostane, plasma carbonyls), inflammatory (serum C-reactive protein [CRP], urine prostaglandin E2 metabolite [PGE-M]), growth factor (serum VEGF, HGF), and lipid (total cholesterol, triglycerides) biomarkers, as well as total urinary tea polyphenols. Results: From July 2007 to Sept 2009, 40 women were enrolled from 4 sites. During the intervention, 5 women developed a dose-limiting toxicity which required stopping study drug, 4 were lost to follow-up, and complete biomarker data was available for 34 participants (26 Poly E, 8 placebo). Concentration of total urinary tea polyphenol metabolites increased from baseline by an average of 10-fold in the Poly E group (all dose levels combined) compared to placebo (p = 0.001). Within the Poly E group, the mean decrease from baseline in serum VEGF was statistically significant at months 2 (11.5%, p = 0.02) and 4 (13.9%, p = 0.04). However, these changes were not significant when compared to placebo. At 2 months, mean serum HGF levels decreased by 12.6% for Poly E compared to a 6.3% increase for placebo (p = 0.04). This trend persisted at 4 and 6 months, but was no longer statistically significant. With Poly E treatment, there was a non-significant trend toward a decrease from baseline in serum cholesterol at 2, 4, and 6 months. No significant differences were observed in the biomarkers of oxidative damage (urine 8-oxoG and isoprostane, plasma carbonyls) or inflammation (serum CRP, urine PGE-M) after treatment with Poly E compared to placebo. Discussion: We demonstrated a significant reduction in serum HGF and a favorable decline in serum VEGF and total cholesterol during a 6-month intervention of Poly E in women with breast cancer. Given the small sample size and lack of adjustment for multiple comparisons, these results need to be confirmed in a larger study. Nevertheless, these findings are consistent with published results from short-term intervention trials of Poly E. This suggests potential mechanisms of action of green tea in angiogenesis, growth factor signaling, and metabolic pathways. Citation Format: Katherine D. Crew, Kimberly A. Ho, Powel Brown, Heather Greenlee, Therese B. Bevers, Banu Arun, Clifford Hudis, Heather L. McArthur, Jenny Chang, Mothaffar Rimawi, Lana Vornik, Terri L. Cornelison, James Cardelli, Regina M. Santella, Antai Wang, Scott M. Lippman, Dawn L. Hershman. Biomarker effects of an oral green tea extract, Polyphenon E, in women with a history of hormone receptor-negative breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B65.

Improvement of metabolic syndrome by irbesartan via the PPARγ/HGF pathway in apolipoprotein E knockout mice

Irbesartan, a partial agonist of peroxisome proliferators activated receptor-γ (PPARγ), has been reported to improve insulin resistance and lipid profile in patients with diabetes mellitus or metabolic syndrome (MS). However, the down effectors of PPARγ have yet to be elucidated. Thus, in this study, we focused on the role of the hepatocyte growth factor (HGF) in the anti-metabolic effects of irbesartan, using apolipoprotein E (ApoE) knockout (KO) mice. ApoE KO mice placed on a high-fat diet (HFD) for 12 weeks were divided into four groups: i) the control (HFD only), ii) the HFD + irbesartan (5 mg/kg/day), iii) the HFD + irbesartan + GW9662, a PPARγ antagonist (0.5 mg/kg/day) and iv) the HFD + irbesartan + anti-HGF neutralizing antibody (200 μg/week). The liver and epididymal adipose tissues were evaluated histologically. Serum adiponectin and HGF levels were also measured by ELISA. Fatty liver (as detected by oil-red O staining) and macrophage infiltration were markedly reduced by irbesartan. Irbesartan treatment also reduced macrophage infiltration into epididymal adipose tissue and hypertrophy of adipocytes. However, these effects of irbesartan were attenuated by GW9662 as well as by anti-HGF neutralizing antibody. Serum and hepatic HGF levels were also markedly increased by irbesartan, whereas GW9662 decreased the HGF level. In conclusion, irbesartan, an angiotensin (Ang) receptor blocker (ARB) and partial agonist of PPARγ (metabosartan), demonstrated a reduction in fatty liver and chronic inflammation, such as macrophage infiltration, beyond its blood pressure-lowering effect. These favorable characteristics of irbesartan might be due to local HGF activation through its partial PPARγ agonistic action, in addition to Ang II blockade. Upregulation of local HGF by irbesartan might provide a novel advantage in a strategy for the prevention and treatment of cardiovascular diseases (CVDs).

Endothelial Vulnerability and Endothelial Damage Are Associated with Risk of Graft-versus-Host Disease and Response to Steroid Treatment

There is accumulating evidence indicating that endothelial factors are involved in the pathogenesis of GVHD. We have recently shown that steroid-refractory, but not sensitive, GVHD is characterized by higher pretransplantation serum levels of angiopoetin-2 (ANG2), a hormone mediating endothelial vulnerability. To evaluate whether endothelial vulnerability is a risk factor for GVHD per se or becomes important only when noticeable GVHD is established, we measured ANG2 along with additional serum markers of endothelial stress, including soluble thrombomodulin (sTM), IL-8 (CXCL8), and hepatocyte growth factor (HGF), in patients with no, low-grade, or severe GVHD. Patients with refractory GVHD exhibited elevated serum levels of ANG2, sTM, HGF, and IL-8 posttransplantation compared with patients with sensitive GVHD and patients without GVHD. Pretransplantation ANG2 was the only growth factor correlated with the risk of refractoriness and mortality, and then only within the subset of patients who developed grade III-IV GVHD. In contrast, ANG2 was not predictive of GVHD or nonrelapse mortality (NRM) in patients with no GVHD or low-grade GVHD. These findings provide evidence that endothelial function plays an important role in the pathogenesis of steroid refractoriness in ongoing GVHD; however, endothelial vulnerability does not predict incidence of GVHD.

The levels of hepatocyte growth factor in serum and follicular fluid and the expression of c-Met in granulosa cells in patients with polycystic ovary syndrome

To evaluate the levels of hepatocyte growth factor (HGF) in follicular fluid (FF) and the expression of c-Met in granulosa cells (GCs) with respect to the quality of the oocyte and embryo both in patients with polycystic ovary syndrome (PCOS) and in the normal ovary during controlled ovarian hyperstimulation cycles. Prospective controlled study. University hospital. Fifty-nine women undergoing IVF treatment (of whom 21 had PCOS and 38 were in the control group). A total of 168 FF samples were collected at the time of oocyte retrieval. The HGF levels were measured by ELISA, and the mRNA expression of c-Met in GCs was detected by real-time polymerase chain reaction. The predictive values of HGF levels in serum and FF and the mRNA expression of c-Met in GCs for successful fertilization and oocyte-embryo quality. The levels of HGF in serum and FF and the c-Met expression in GCs were similar between the PCOS and control groups. Granulosa cells of fertilized oocytes (2PN) had a significantly higher level of c-Met expression than that in oocytes that failed to fertilize. The mean HGF level in FF was significantly higher in the grade 1 embryos than in the grades 2-4 embryos. This study suggests that HGF/c-Met signaling may be a crucial determinant of fertilization success.

The levels and clinical significances of hepatocyte growth factor in the hepatic tissue and serum of hepatocellular carcinoma

Objective To investigate hepatocyte growth factor (HGF) levels in the tissue and serum of patients with chronic hepatitis, cirrhosis or hepatocellular carcinoma (HCC), and analyze the clinical significances of HGF for HCC. Method Surgical specimens from 97 patients were collected during Dec.2003 to Aug.2008 in the Third Central Hospital.The patients were prospectively enrolled and categorized into four groups:normal subjects (n=11), chronic hepatitis B or C (n=6), cirrhosis (n=20) and HCC (n= 60) including well-differentiated (n= 21), moderately differentiated (n= 23), poorly differentiated (n= 16) specimens.N0 (n=24), N1 (n=21), N2 (n=54) and N3 (n=43) were tissues respectively removed from liver at 0, 1, 2 or 3 cm beyond the margin of tumor.HGF mRNA expression in liver tissues was determined by real-time quantitative reverse transcription- (RT)-PCR.Serum HGF levels in the other cases of normal subjects (n=20), chronic hepatitis B or C (n=20), cirrhosis (n=20) and HCC (n=57) were measured by ELISA.The Kaplan-Meier method with log-rank test was employed for survival analysis.Univariate and multivariate analyses were performed to identify prognostic factors in each group. Results The HGF mRNA in normal subjects, chronic hepatitis, cirrhosis, N3, N2, N1, N0 and HCC were 0.99(0.78-1.66), 2.15(1.06-3.40), 1.78(1.18-2.73), 4.59(2.67-8.63), 3.86(2.25-6.45)，3.12(1.59-5.74), 2.92(0.88-5.99) and 0.48(0.19-1.06) respectively.The serum concentration of HGF in the normal subjects, chronic hepatitis, cirrhosis and HCC patients were (0.31±0.05), (0.65±0.07), (1.27±0.30) and （2.06±0.66） μg/L respectively.The highest level of HGF mRNA was found in N3, while the HGF mRNA expression in HCC was ［（2.14±0.52）μg/L］ lower than that not only in the non-tumor tissues, but also in the normal control (U=196.50, P=0.03).The serum concentration of HGF was significantly higher in patients with chronic hepatitis, cirrhosis or HCC than in normal subjects.The serum HGF level of HCC was bounced after hepatectomy (t=2.70, P＜0.01).On the logistic regression analysis，the tumor numbers and Child-pugh were related with the levels of the tissue HGF mRNA and serum HGF of HCC, OR were 0.15 (95%CI：0.03-0.72, P<0.05) and 0.13 (95%CI：0.27-0.89, P<0.05), respectively.Univariate analysis using the Cox proportional hazards model in the complication groups revealed that the levels of the tissue HGF mRNA and serum HGF were significant risk factors of death for HCC, OR were 0.02 (95%CI:0.00-0.52, P<0.05) and 10.01 (95%CI:1.16-86.23, P< 0.05), respectively.On the Log-rank analysis, no statistically difference in the cumulative survival was found between the two groups categorized by median (0.49) of tissue HGF mRNA 2-ΔΔCT (χ2=0.13，P=0.72). While the HCC patients were dichotomized by their the median(0.69 μg/L) of serum HGF concentration, the death risk for the patients with higher levels of HGF was increased 2.84 fold than those with lower levels (95%CI:1.03-7.92，P<0.05). Conclusion HGF mRNA expression is decreased in tumor tissues, while its level in tumor adjacent live and serum is significantly elevated and is in association with shortened postoperative survival of HCC patients.（Chin J Lab Med,2012,35:598-603） Key words: Liver neoplasms; Carcinoma，hepatocellular; Hepatocyte growth factor; Reverse transcription-quantitative polymerase chain; Enzyme-linked immunosorbent assay

Serum hepatocyte growth factor and interleukin-6 as prognostic markers for stage III non-small cell lung cancer.

10542 Background: We surveyed prognostic biomarkers for resectable non-small cell lung cancer (NSCLC). Methods: We obtained preoperative serum from 109 patients admitted to our facility, and measured the levels of hepatocyte growth factor (HGF), interleukin-6 (IL-6), and nicotinamide N–methytransferase (NNMT) in the sera by the ELISA method. We also examined the clinical backgrounds of the patients. Results: The median HGF and IL-6 contents in sera from 109 patients were 860 pg/ml and 2.7 pg/ml, respectively. Analysis of survival curves indicated that HGF or IL-6 levels higher than the median were associated with poor overall survival (HGF, P = 0.019; IL-6, P = 0.002). On the other hand, carcinoembryonic antigen (CEA), a known tumor marker, and NNMT, which we found to be a candidate tumor marker, exhibited no correlation with the prognosis. The tumor status (pT factor) and stage were strong prognostic indicators (P &lt; 0.001). In addition, we analyzed stage III lung cancer alone. Higher HGF or IL-6 levels were associated with poor overall survival (HGF, P = 0.016; IL-6, P = 0.013). Disease-free survival was also affected by these cytokine contents, but not statistically significantly. The prognosis of patients with adenocarcinomas (ADC) was better than that of patients with other histological types. However, the pT factor and nodal status (pN factor) were not associated with the survival of stage III patients. Conclusions: The levels of HGF and IL-6 in serum could be useful prognostic indicator of the survival of stage III NSCLC patients undergoing surgery and chemotherapy.

Abstract 5061: Genomic and functional analysis of Myxofibrosarcoma identifies novel prognostic markers and promising therapeutic targets

Abstract Introduction: Myxofibrosarcoma (MXF), which primarily affects the limbs of older patients, has few effective systemic therapies. Little is known about the MXF genome or the genes that drive tumorigenesis. We sought to identify subgroups and to identify genes that associate with outcome and could serve as therapeutic targets. Methods: Copy number alterations (CNAs) and gene expression profiles were measured in 73 MXF, 11 normal muscle, and 22 normal fat samples using Agilent 1M CGH and Affymetrix U133A arrays. Profiles were analyzed by unsupervised clustering and correlated with disease-specific survival (DSS) and distant recurrence-free survival (DRFS). Associations with disease-specific survival (DSS) and distant recurrence-free survival (DRFS) were analyzed for the 68 patients with primary disease; this cohort included 21 patients who had distant recurrence and 15 who died of disease during follow-up (mean, 2.7 years). [ss1] Genes of interest were assayed using shRNA knockdown (for ITGA10) or serum ELISA levels (for HGF). Results: MXF samples had highly complex CNAs, some spanning entire chromosome arms. Unsupervised clustering based on CGH divided samples into 4 main groups driven by a loss at 1q and a gain at 5p. Unsupervised clustering based on U133A profiles divided samples into 3 main groups, 2 of which had a 3-year DSS of 85% while the third had a DSS of 31% (p&amp;lt;0.02). This third group was defined in part by high expression of integrin alpha-10 (ITGA10) and hepatocyte growth factor (HGF). Expression of ITGA10 was associated with worse DRFS (HR=2.7, p=0.03), and particularly strongly associated with worse DSS (HR=8.2, p=0.006). HGF expression was also associated with worse outcomes for both DRFS (HR=3.2, p=0.01) and DSS (HR=3.5, p=0.01). Knockdown of ITGA10 inhibited proliferation and induced apoptosis in an MXF cell line, but not in a normal fat cell line. Patients with high expression of both HGF and its receptor MET had a 3-year DRFS of 25%, vs approximately 60% for those with low expression of one of these genes and 85% for those with low expression of both genes (p=0.0016). Serum HGF was elevated in preoperative patients with MXF (n=38) compared to healthy volunteers (n=8): mean ± SD 1626 pg/mL ± 725 vs. 771 ± 110 (p=0.002). Patients with low serum HGF (≤mean +3 SD of normal [1101 pg/mL], n=10) tended to have better DSS (100% vs. 64%, p=0.14) and better DRFS (83% vs. 55%, p=0.08). Conclusions: MXF is genomically complex and diverse, but gene expression profiles cluster patients into groups associated with outcome. Levels of ITGA10, HGF and MET associate with outcome, and ITGA10 overexpression in a MXF cell line contributes to cell proliferation and survival. All three of these genes may be useful as therapeutic targets. Preoperative serum HGF levels may be a useful prognostic indicator in patients with MXF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5061. doi:1538-7445.AM2012-5061