Hepatocyte Growth Factor Antibody Research Articles

Hepatocyte Growth Factor Secreted from Human Adipose-Derived Stem Cells Inhibits Fibrosis in Hypertrophic Scar Fibroblasts.

To study the effect of Adipose-derived stem cells (ADSCs) on fibrosis of hypertrophic scar-derived fibroblasts (HSFs) and its concrete mechanism. ADSCs have been reported to reduce collagen production and fibroblast proliferation in co-culture experiments. Conditioned medium from adipose-derived stem cells (ADSCs-CM) has successfully inhibited fibrosis by decreasing the expression of collagen type І (Col1) and α-smooth muscle actin (α-SMA) in rabbit ear scar models. Hepatocyte growth factor (HGF), the primary growth factor in ADSCs-CM, has been shown to reverse fibrosis in various fibrotic diseases. To test the hypothesis that ADSCs inhibit fibrosis of HSFs through the secretion of HGF. HSFs were treated with DMEM containing 0%, 10%, 50% and 100% concentration of ADSCs-CM. The effect of ADSCs-CM on the viability was determined by cell viability assay, and the collagen production in HSFs was examined by Sirius red staining. Expression and secretion of fibrosis and degradation proteins were detected separately. After measuring the concentration of HGF in ADSCs-CM, the same number of HSFs were treated with 50% ADSCs-CM or HGF. HGF activity in ADSCs-CM was neutralized with a goat anti-human HGF antibody. The results demonstrated that ADSCs-CM dose-dependently decreased cell viability, expression of fibrosis molecules, and tissue inhibitor of metalloproteinases-1 (TIMP-1), and significantly increased matrix metalloproteinase-1 (MMP-1) expression in HSFs. Collagen production and the ratio of collagen type І and type III (Col1/Col3) were also suppressed by ADSCs-CM in a dose-dependent manner. When HSFs were cultured with either 50% ADSCs-CM or HGF (1 ng/ml), a similar trend was observed in gene expression and protein secretion. Adding an HGF antibody to both groups returned protein expression and secretion to basal levels but did not significantly affect the fibrosis factors in the control group. Our findings revealed that adipose-derived stem cell-secreted HGF effectively inhibits fibrosis-related factors and regulates extracellular matrix (ECM) remodeling in hypertrophic scar fibroblasts.

Phase Ib study of gemcitabine, nab-paclitaxel, and ficlatuzumab in patients with advanced pancreatic cancer.

693 Background: Paired-related homeodomain transcription factor 1 (Prrx1) isoforms are involved in pancreatic development, pancreatitis, and carcinogenesis. Hepatocyte growth factor (HGF) is a transcriptional target of Prrx1b. Ficlatuzumab is a recombinant humanized HGF antibody, that neutralizes HGF/c-Met binding and HGF-induced c-Met phosphorylation. In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of Prrx1b-HGF signaling using ficlatuzumab and gemcitabine reduced primary tumor volume and eliminated metastatic disease. Methods: Patients (pts) with previously-untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3+3 design and two dose cohorts of ficlatuzumab (10mg/kg and 20mg/kg) administered intravenously every other week with gemcitabine (G; 1000mg/m2) and nab-paclitaxel (A; 125mg/m2) given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose (MTD) of the combination. Results: 24 pts (sex, 12M:12F; median age, 69 years [range, 51-82 years]) were enrolled. No dose-limiting toxicities were identified in the phase 1b (N = 6 pts) and ficlatuzumab at 20mg/kg with GA was advanced to the expansion phase (N = 18 pts). By RECISTv1.1 in the full study population, 7 (29%) pts had partial response, 15 (63%) had stable disease, and 2 (8%) could not be evaluated. Median progression-free survival was 8 months (range, 3-16 months), 4 pts are still on study treatment. The primary toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 21%; any grade, 91%) and edema (grade 3, 8%; any grade, 91%). Nine (38%) of the 24 pts discontinued study treatment due to these toxicities prior to disease progression. Conclusions: The combination of ficlatuzumab with gemcitabine and nab-paclitaxel is associated with durable treatment responses but also significant hypoalbuminemia and edema that may impair treatment tolerability. Serial blood samples were collected for circulating HGF measurements, and mandatory pretreatment biopsies were collected for tumor c-MET pathway activation and 3D organoid culture drug sensitivity testing. Clinical trial information: NCT03316599.

576 The role of hepatocyte growth factor in human hair follicle – dermal white adipose tissue communication

Although human dermal white adipose tissue (DWAT) envelopes scalp hair follicles (HFs), their underlying bidirectional communication remains unexplored. Recently, we determined that culturing human HFs with surrounding DWAT promoted hair growth and pigmentation ex vivo. The current study aimed to further expand knowledge of the human DWAT-HF axis. Intriguingly, proteomic analysis identified hepatocyte growth factor (HGF) as the most abundantly-secreted DWAT product within long-term HF+DWAT ex vivo cultures. Therefore, we chose to stimulate HFs with HGF ex vivo. Both HFs cultured with DWAT, or HGF in the absence of DWAT showed a significantly increased pigment content, number of gp100+ melanocyte cells and melanocyte dendrites. These positive effects were partially ablated by a neutralizing HGF antibody added to HF+DWAT. As expected, HGF-treated HFs displayed increased proliferation and decreased apoptosis within matrix keratinocytes, effects mirrored by culturing HFs with surrounding DWAT. Transcriptomic analysis of HFs treated with HGF (6hr) revealed that HGF up-regulated PAX3 and SOX10, as well as VEGFA and WNT5B. Gene expression analysis on 24hr cultures coupled with IPA revealed that key Wnt pathway inhibitors are down-regulated in HFs treated with HGF, whereas Wnt agonists, WNT6 and WNT10B, show an upward trend for HF+HGF and HF+DWAT. Here we provide the first evidence that human DWAT communicates with adjacent scalp HFs by secreting HGF, one of the most potent promoters of hair growth and pigmentation. This study designates human perifollicular adipocytes and HGF-dependent DWAT-HF communication an intriguing novel target for therapeutic intervention in the future management of disorders of hair growth or pigmentation.

Abstract 2957: Sequential transcriptomic and phosphorylation landscape of acute myelogenous leukemia (AML) on the single-cell level

Abstract Background: Patients with AML refractory to induction therapy or relapse within one year have poor outcomes. Elevated serum hepatocyte growth factor (HGF) level is an adverse prognostic factor in AML. Preclinical models have shown that myeloid blasts produce HGF in an autocrine fashion and pharmacologic blockade of the HGF/c-Met axis sensitizes blasts to cell death. We initiated a phase I study to assess the safety and tolerability of the HGF antibody ficlatuzumab (Fi) combined with high-dose cytarabine (Cy) in patients with AML who are refractory to 7+3 or have relapsed within one year of induction. Fi is given in escalated dosing of 10, 15, or 20 mg/kg for 4 doses every 2 weeks, starting on day 0, and Cy at a fixed dose of 2 g/m2 on days 2-7, using a 3x3 design. PBMCs, BM and serum are collected at defined times. Preliminary results demonstrating the safety of this combination with a promising response rate of 44% in nine evaluable patients were previously presented. Here we report the correlatives using single-cell RNA sequencing (scRNAseq) and mass cytometry (Cy-TOF) on PBMC specimens collected longitudinally during treatment for twelve patients. Methods: scRNAseq was conducted using the 10x Genomics Chromium platform. Twelve genetically distinct samples were multiplexed per run and the resulting sequencing data were de-convoluted using individual SNP information from the OmniExpressExome54 Array and the novel DEMUXLET algorithm. The Cy-TOF antibody panel included markers for identification of myeloid blasts, T and B cells as well as intracellular phospho-signaling pathways downstream of the HGF/c-MET axis. Results: Sixty samples from twelve patients corresponding to different treatment time points were analyzed using scRNAseq and thirty samples were run using Cy-TOF. On average, each scRNA run captured 17-23K cells and 19-24K reads per cell, corresponding to approximately 1,000 unique gene trasncripts/cell. Cy-TOF identified surface phospho-MET activation and downstream pathways. Conclusions: scRNAseq combined with Cy-TOF identified changes in cellular populations corresponding to treatment. Effector signaling pathways were blunted with HGF blockade, suggesting on-target effect of the antibody. Each population also exhibited distinct transcription and phosphorylation profile in response to the drugs. Dose expansion is ongoing and future studies will apply these methods to analyze the bone marrow specimens to assess how the microenvironment evolves during the treatment course. Clinical trial information: NCT02109627 Citation Format: Victoria E. Wang, Graham Heimberg, Gregory K. Behbehani, Chun Ye, Charalambos Babis Andreadis. Sequential transcriptomic and phosphorylation landscape of acute myelogenous leukemia (AML) on the single-cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2957.

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269-78. ©2017 AACR.

HGF/Met Signaling in Head and Neck Cancer: Impact on the Tumor Microenvironment.

Studies to date have revealed several major molecular alterations that contribute to head and neck squamous cell carcinoma (HNSCC) initiation, progression, metastatic spread, and therapeutic failure. The EGFR is the only FDA-approved therapeutic target, yet responses to cetuximab have been limited. Activation and cross-talk of cellular receptors and consequent activation of different signaling pathways contribute to limited activity of blockade of a single pathway. The hepatocyte growth factor (HGF) receptor, Met, has been implicated in HNSCC tumorigenesis and EGFR inhibitor resistance. HGF, the sole ligand of Met, is overexpressed in the tumor microenvironment. The role of HGF/Met signaling in proliferation, metastasis, and angiogenesis has been investigated in HNSCC, leading to clinical trials with various Met inhibitors and HGF antibodies. However, the role of the HGF/Met signaling axis in mediating the tumor microenvironment has been relatively understudied in HNSCC. In this review, we discuss the functional roles of Met and HGF in HNSCC with a focus on the tumor microenvironment and the immune system. Clin Cancer Res; 22(16); 4005-13. ©2016 AACR.

Hepatocyte growth factor reduces sensitivity to the epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib, in lung adenocarcinoma cells harboring wild-type EGFR.

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is an option for lung cancers harboring wild-type EGFR when chemotherapeutic reagents have failed. In this study, we found that the EGFR-TKI, gefitinib, modestly suppressed proliferation of the lung cancer cell lines, A549 and H358, which both harbor wild-type EGFR. Treatment with hepatocyte growth factor (HGF) reduced the sensitivity to gefitinib, whereas sensitivity was restored by treatment with an HGF antibody, a MET inhibitor, or depletion of MET but not ErbB3 gene. Moreover, both PI3K/mTOR inhibitors and MEK inhibitors suppressed proliferation of A549 cells, whereas only PI3K/mTOR inhibitors effectively suppressed cell viability of EGFR mutant PC-9 cells. Our findings suggest that HGF reduced the gefitinib sensitivity through MET and downstream PI3K and MAPK pathways. Combined use of EGFR-TKI and MET inhibitors or inhibition of downstream signaling molecules might be a better second or third line choice for a group of patients with advanced lung cancer harboring wild-type EGFR.

Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway.

BackgroundMesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood.MethodsWe introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence.ResultsMSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF antibody or with both HGF and VEGF antibodies. Additionally, the effects of MSC-CM and VEGF/HGF on reducing endothelial paracellular and transcellular permeability were weakened when HPMECs were pretreated with the Rac1 inhibitor NSC23766.ConclusionHGF secreted by MSCs protects the endothelial barrier function; however, VEGF secreted by MSCs may synergize with HGF to stabilise endothelial cell barrier function. Rac1 is the pathway by which MSC-secreted VEGF and HGF regulate endothelial permeability.

Mitigation of Tumor-Associated Fibroblast-Facilitated Head and Neck Cancer Progression With Anti-Hepatocyte Growth Factor Antibody Ficlatuzumab.

Ficlatuzumab can be used to treat head and neck squamous cell carcinoma (HNSCC) by inhibiting c-Met receptor-mediated cell proliferation, migration, and invasion. To understand the effect of ficlatuzumab on HNSCC proliferation, migration, and invasion. The effects of ficlatuzumab on HNSCC proliferation, invasion, and migration were tested. Mitigation of c-Met and downstream signaling was assessed by immunoblotting. The tumor microenvironment has emerged as an important factor in HNSCC tumor progression. The most abundant stromal cells in HNSCC tumor microenvironment are tumor-associated fibroblasts (TAFs). We previously reported that TAFs facilitate HNSCC growth and metastasis. Furthermore, activation of the c-Met tyrosine kinase receptor by TAF-secreted hepatocyte growth factor (HGF) facilitates tumor invasion. Ficlatuzumab is a humanized monoclonal antibody that sequesters HGF, preventing it from binding to and activating c-Met. We hypothesized that targeting the c-Met pathway with ficlatuzumab will mitigate TAF-mediated HNSCC proliferation, migration, and invasion. Representative HNSCC cell lines HN5, UM-SCC-1, and OSC-19 were used in these studies. The HNSCC cell lines were treated with ficlatuzumab, 0 to 100 µg/mL, for 24 to 72 hours. Ficlatuzumab inhibited HNSCC progression through c-Met and mitogen-activated protein kinase (MAPK) signaling pathway. Ficlatuzumab significantly reduced TAF-facilitated HNSCC cell proliferation (HN5, P < .001; UM-SCC-1, P < .001), migration (HN5, P = .002; UM-SCC-1, P = .01; and OSC-19, P = .04), and invasion (HN5, P = .047; UM-SCC-1, P = .03; and OSC-19, P = .04) through a 3-dimensional peptide-based hydrogel (PGmatrix). In addition, ficlatuzumab also inhibited the phosphorylation of c-Met at Tyr1234/1235 and p44/42 MAPK in HNSCC cells exposed to recombinant HGF. We demonstrate that neutralizing TAF-derived HGF with ficlatuzumab effectively mitigates c-Met signaling and decreases HNSCC proliferation, migration, and invasion. Thus, ficlatuzumab effectively mitigates stromal influences on HNSCC progression.

Estradiol-mediated hepatocyte growth factor is involved in the implantation of endometriotic cells via the mesothelial-to-mesenchymal transition in the peritoneum.

The pathogenesis of endometriosis, a chronic painful gynecological disease characterized by the presence of endometrial tissue located outside of the uterus and often adhering to the peritoneum, is known to be estrogen dependent. However, the precise pathophysiology of endometriosis remains elusive. Recent studies indicate that the epithelial-to-mesenchymal transition (EMT) of human endometrial cells is important for the progression of endometriosis, and another previous study has implicated hepatocyte growth factor (HGF) in endometriosis progression. The aim of the present study was to examine the role of estradiol in the regulation of HGF production and progression of peritoneal endometriosis, focusing on the interactions between the peritoneum and endometriotic cells. Consequently, estradiol was found to promote the proliferation, invasion, and migration of immortalized human endometrial epithelial cells (hEECs) via HGF upregulation, and the estradiol-induced direct binding of estrogen receptor-α to the HGF promoter was confirmed on a chromatin immunoprecipitation (ChIP) assay. Estradiol also induced the EMT in hEECs by promoting HGF production. Furthermore, human mesothelial cells underwent the mesothelial-to-mesenchymal transition (MMT) during culture with estradiol-stimulated hEEC conditioned medium. Importantly, estradiol itself did not induce the MMT, and the estradiol-stimulated hEEC-conditioned medium in the presence of HGF antibodies reversed the MMT process. These results, which were obtained using immortalized hEECs, indicate that estradiol-induced HGF production may play a crucial role in the peritoneal implantation of human endometriotic cells by exerting proliferative and invasive effects via the EMT in hEECs and promoting the MMT in mesothelial cells.

Expression of HGF and c-Met Proteins in Human Keratoconus Corneas.

Keratoconus (KC) is a progressive degenerative inflammatory-related disease of the human cornea leading to decreased visual function. The pathogenesis of KC remains to be understood. Recent genetic studies indicate that gene variants of an inflammation-related molecule, hepatocyte growth factor (HGF), are associated with an increased susceptibility for developing KC. However HGF protein expression in KC has not been explored. In this initial study, we investigated late-stage KC and control corneas for the expression of HGF and its receptor mesenchymal-epithelial transition factor (c-Met/Met). KC buttons (~8 mm diameter) (n = 10) and whole control corneas (n = 6) were fixed in 10% formalin or 2% paraformaldehyde, paraffin embedded and sectioned. Sections were immunolabelled with HGF and c-Met antibodies, visualised using immunofluorescence, and examined with scanning laser confocal microscopy. Semiquantitative grading was used to compare HGF and c-Met immunostaining in KC and control corneas. Overall, KC corneas showed increased HGF and c-Met immunostaining compared to controls. KC corneal epithelium displayed heterogeneous moderate-to-strong immunoreactivity for HGF and c-Met, particularly in the basal epithelium adjacent to the cone area. Taken together with the recent genetic studies, our results further support a possible role for HGF/c-Met in the pathogenesis of KC.

Abstract A42: Evaluation of HGF and MET protein expression in NSCLC tumor specimens from patients previously treated with targeted or chemotherapies.

Abstract Background: Although epidermal growth factor receptor (EGFR) T790M mutation accounts for majority of the resistance to the first generation EGFR tyrosine kinase inhibitors (TKIs), activation of the MET pathway through increased expression of hepatocyte growth factor (HGF) or amplification of MET (the HGF receptor) has also been identified as a resistance mechanism in patients. Effective treatment of patients that have progressed on first-generation TKIs requires diagnostic assays that detect the major resistance mechanisms and direct therapy selection. We have developed immunohistochemistry (IHC) assays to detect both HGF and MET protein expression in formalin-fixed paraffin-embedded (FFPE) tissues, and evaluated expression levels in patients previously treated with EGFR TKIs or chemotherapy. Methods: The IHC assays were developed using the Ventana Benchmark automated staining system. The MET antibody (SP44) was obtained from Ventana. Five different HGF antibodies from several vendors were evaluated. The assay was established using cell pellets generated from cell lines overexpressing HGF as well as a FFPE metastatic cancer tissue array. In addition, tissue specimens obtained from remnant material from surgery or autopsy were used for the assay optimization. Results: The MET (SP44) assay was validated using the recommended protocol supplied by Ventana. Robust staining was observed in multiple FFPE samples, and both cytoplasmic and membranous staining were defined as positive. The expression was scored on the basis of intensity and fraction of positive cells. In contrast, among the 5 HGF antibodies tested for specificity, only the IBL antibody demonstrated HGF-specific staining in cell lines and the metastatic tissue array. The selected MET and HGF antibodies were used to evaluate a set of 25 NSCLC patient FFPE tissues. 19/25 specimens tested showed positive MET expression. Of these, 13 were 3+ whereas 6 were 2+ in terms of staining intensity. Additionally, 2 of the specimens with 2+ score exhibited predominantly cytoplasmic staining. In contrast, HGF expression was more variable with 5/24 with strong, 9/24 with weak and 10/24 with negative staining (one specimen was not evaluable for HGF). There was no clear correlation between MET and HGF staining patterns. In this sample set, there was not a clear correlation between MET or HGF expression and patient status as relapsed versus newly diagnosed. Conclusions: We have developed robust assays for evaluation of MET and HGF expression in FFPE sections and applied these assays in analyzing 25 NSCLC patient specimens, documenting a higher than expected proportion of the samples as MET or HGF positive. A more expansive dataset, potentially evaluating factors beyond protein expression levels, will be needed to elucidate a relationship between MET pathway activation and response to EGFR TKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A42. Citation Format: Sabita Sankar, Jennifer Wright, Chad Galderisi. Evaluation of HGF and MET protein expression in NSCLC tumor specimens from patients previously treated with targeted or chemotherapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A42.

HGF mediates the anti-inflammatory effects of PRP on injured tendons.

Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2.

Attenuation of hepatic fibrosis through ultrasound-microbubble-mediated HGF gene transfer in rats

ObjectiveThe objective was to explore the feasibility of ultrasound-microbubble-mediated hepatocyte growth factor (HGF) gene transfer for treating rat hepatic fibrosis induced by CCl4. MethodsForty-eight male SD rats were divided into ultrasound-microbubble-HGF group (U-M-HGF group), ultrasound-HGF group (U-HGF group), microbubble-HGF group (M-HGF group), HGF group (HGF group), CCl4 group (control group), and normal group. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total protein, albumin (ALB), and globulin (GLB) and the ratio of ALB/GLB were determined after treatment. The degree of hepatic fibrosis was evaluated by histopathological numerical scores. The protein expressions of HGF, collagen I, collagen III, and α-smooth muscle antibody (α-SMA) were detected by immunohistochemistry. ResultsUltrasound-microbubble-mediated HGF therapy significantly reduced the serum level of ALT and AST to 59.88% and 49.18% of the control group, respectively. Ultrasound-microbubble-mediated HGF therapy prevented liver fibrosis, with an obvious decrease in fibrosis areas and extracellular matrix production of collagen I, collagen III, and α-SMA. The gene therapy could induce HGF delivery into the fibrotic liver effectively. ConclusionsUltrasound-microbubble-mediated HGF gene therapy can reduce liver fibrosis, which provides a novel strategy for gene therapy of chronic liver disease.

Contrary Regulation of TIMP-1 and MMP-9 by Hepatocyte Growth Factor Antibody after Lung Injury

To study the influence of hepatocyte growth factor (HGF) antibody on the lung expression level of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Thirty male Wistar rats were randomly divided into 3 groups: control group, model group, and intervention group. Endotoxin was intratracheally infused in the model and intervention groups. HGF antibody was injected in the rats of the intervention group from day 1 to day 14, while the same volume of saline was injected in the control group. The rats were sacrificed on day 28 after endotoxin treatment. The amounts of MMP-9 mRNA and TIMP-1 mRNA were measured by reverse transcription-polymerase chain reaction, and protein expression levels of MMP-9 and TIMP-1 were measured by immunohistochemistry. In the model group, both mRNA and protein expression levels of TIMP-1 were significantly increased, the same as MMP-9. In the intervention group, the increase of TIMP-1 was remarkably reduced compared with the model group, while the mRNA and protein expression levels of MMP-9 were still increased. HGF activity may accelerate the repair of lung injury through contrary regulating the expression levels of TIMP-1 and MMP-9.

Abstract 893: Associations between HGF, MET, and phospho-MET expression, overall survival, and HGF genotypes in epithelial ovarian cancer

Abstract Background. The hepatocyte growth factor (HGF) signaling pathway regulates key cellular functions related to tumor invasion and metastasis including cell motility, cell proliferation, and morphogenesis. We previously found that minor alleles at a single-nucleotide polymorphism (SNP; rs5745709) in the gene encoding the cytokine HGF was associated with poorer overall survival in women with invasive epithelial ovarian cancer (EOC) enrolled at Mayo Clinic (MAY) and The Cancer Genome Atlas (TCGA) (HR 1.6, 95% CI 1.3-1.9, p=8.9×10−5). In addition, we found that that genotype correlated with HGF expression in ovarian cancer tissue from TCGA women, suggesting that genetically-regulated expression of HGF may play a role in post-diagnosis survival. To further examine this possibility, we evaluated intensity of antibody immunostaining for HGF, MET (HGF's transmembrane tyrosine kinase receptor), and phospho-MET (pMET; MET's activated form) in tumor cells using tissue microarrays (TMAs) created from genotyped primary peritoneal and EOC cases, and correlated these expression levels with overall survival and HGF SNP genotypes. Methods. Participants were 326 white non-Hispanic women over 20 years of age with histologically-confirmed invasive EOC diagnosed between 1998 and 2009. All cases were enrolled at MAY within one year of diagnosis. Five μm sections of TMAs including three 0.6 mm cores per case were immunostained with HGF, MET, and pMET primary antibodies. Staining levels were classified according to combinations of staining extent and intensity as negative (&amp;lt; 10% cells stained), weak, moderate, or strong. The maximal expression value recorded over the multiple cores for each case was used. Cox proportional hazards regression was used to examine associations of overall survival with HGF, MET and pMET expression levels, adjusted for relevant clinical variables. Associations between expression levels and HGF SNP rs5745709 genotypes were examined using linear regression models. Results. HGF and MET expression were not associated with overall survival in our series of EOC cases (p=0.48 and 0.90, respectively). pMET expression was associated with survival, but in the opposite direction of that hypothesized (HR 0.77, 95% CI 0.39-1.52 for moderate expression; HR 0.55, 95% CI 0.27-1.10 for strong expression; each referent to negative/weak, p=0.01). Expression levels were not significantly associated with HGF rs5745709 genotypes (p=0.80 for HGF expression, p=0.89 for MET, p=0.41 for pMET). Conclusion. HGF expression levels were not associated with overall survival or with HGF SNP genotypes that correlated with survival in a previous study. Functional studies such as this one are crucial as a follow-up to discovery-based studies in order to assess biologic plausibility of observed results. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2011-893

Abstract LB-395: Hepatocyte growth factor: a potential therapeutic target in pancreatic cancer

Abstract Pancreatic stellate cells (PSCs, which produce the desmoplastic reaction of pancreatic cancer) interact with pancreatic cancer (PC) cells to potentiate PC progression. A candidate factor that may mediate this interaction is the hepatocyte growth factor (HGF). High serum HGF levels in PC patients correlate with poor outcome. However, there is limited knowledge about the role of HGF in PC. Aims: To i) determine whether human PSCs and PC cell lines express HGF; and ii) assess the effects of HGF inhibition on PC progression in an orthotopic mouse model. Methods: i) HGF expression in PSCs and PC cells assessed by RT-PCR, immunoblotting/immunocytochemistry. ii) Orthotopic model: AsPC-1 (a human PC cell line) ± human PSCs implanted into the pancreas mice. One week later, mice divided into groups (n=8 mice/group) and treated with: HGF antibody AMG102 [(Amgen Inc.), 300 or 600μg IP biweekly] or isotype IgG (600 μg IP biweekly). Seven weeks later, tumour size and metastasis assessed. Results: i) PSCs express HGF at both mRNA and protein levels. In contrast, PC cells (MiaPaCa2, Panc-1, AsPC-1) express negligible HGF mRNA. ii) Orthotopic model: a) IgG treated AsPC-1+PSC mice showed larger tumours than mice injected with AsPC-1 alone (Table); b) 300 and 600μg AMG102 inhibited tumour growth in AsPC-1+PSC mice compared to the IgG treated group. However, AMG102 did not reduce tumour size in mice injected with AsPC-1 alone (Table). c) 600μg AMG102 inhibited metastasis (liver, diaphragm, mediastinum) in AsPC-1+PSC mice (p&amp;lt;0.05) compared to IgG treated mice. Conclusions: We have shown for the first time that i)human PSCs synthesise HGF; ii)HGF inhibition reduces tumour growth and metastasis of tumours representative of human PC i.e., exhibiting both tumour elements and a stromal reaction, but not in the clinically non-representative cancers formed by PC cells alone. Implication: Targeting the stromal reaction with relevant specific inhibitors may represent a novel therapeutic approach in PC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-395. doi:10.1158/1538-7445.AM2011-LB-395

Hepatocyte growth factor and antibodies to HLA and MICA antigens in heart transplant recipients

Recent unconfirmed literature data suggest that elevated concentrations of the multifunctional cytokine hepatocyte growth factor (HGF) might be a marker of increased incidence of acute rejection after organ transplantation. The aim of this study was to test the hypothesis that HGF levels may correlate with the rejection and/or with the production of HLA and MHC Class I chain-related antigens A (MICA) specific antibodies. Sixty-three heart transplant recipients were included into the study. Hundred and eighty-five endomyocardial biopsies (EMB) obtained up to 6 months after transplantation were retrospectively analyzed for signs of cellular (CR) and antibody-mediated rejection (AMR). Pre- and post-transplant sera were tested for HGF concentrations and antibodies to HLA class I, class II and MICA antigens by xMap technology (Luminex). Pre-transplant HGF did not correlate with the incidence of CR or AMR. However, higher HGF concentrations correlated significantly with HLA antibody production before and after transplantation (P = 0.006 and P < 0.0001 respectively). Patients with both HLA class I and class II antibodies before transplantation had significantly lower AMR-free survival. Furthermore, recipients with pre-transplant donor-specific antibodies (DSA) had significantly lower AMR-free survival (50%) than recipients without pre-transplant HLA antibodies (90%) and patients with antibodies not specific to donor antigens (92%) (P = 0.005). Post-transplant MICA antibodies tended to be more frequent in patients with AMR (P = 0.063). In conclusion, elevated HGF concentrations in our study were not associated with the incidence of CR and/or AMR but with the presence of HLA-specific antibodies. Testing for DSA before heart transplantation by Luminex may be helpful for the identification of patients with increased risk of AMR.

Factor VII Transcription Correlates with Hepatocyte Proliferation and Hepatocyte Growth Factor Expression in a Rodent Extrahepatic Portal Vein Obstruction Model

Extrahepatic portal vein obstruction (EHPVO) is clinically associated with decreased liver-dependent coagulation factors and smaller than normal liver volumes. We developed a rodent EHPVO model to describe changes in coagulation factor regulation and liver homeostasis. Fifteen rats underwent controlled narrowing of the portal vein (PV) at the hilum, and 15 underwent sham operations. Three animals from each group were sacrificed at postoperative days (PODs) 1, 2, 4, 7, and 21. Their livers were studied for apoptosis, proliferation, and hepatocyte growth factor (HGF) expression using terminal transferase dUTP nick end labeling (TUNEL) assays, Ki-67, and hepatocyte growth factor antibody. Liver total RNA was harvested for reverse transcriptase-polymerase chain reaction (RT-PCR), using primers for factors V, VII, VIII, X, protein C, and antithrombin. Appropriate statistical analysis was applied. Ligation of the portal vein in the experimental group resulted in a 59+/-17% (mean +/- standard deviation) decrease in distal portal vein diameter. Proportional body weight was equivalent in both groups, but spleen weight was higher in the experimental group at PODs 4, 7, 21, and liver weight was higher at POD 7 (p < 0.05). The percentage of apoptotic cells in the experimental group increased from that of the control group at POD 4 (p < 0.05). The percentages of proliferating cells, HGF expression, and factor VII transcription in the experimental group were lower than those of controls at POD 2 (p < 0.05) but higher at POD 7 (p < 0.05). Ki-67/TUNEL double staining showed no grouping of apoptotic and proliferating cells. Factor V transcription in the experimental group was increased compared with that in controls at POD 2 (p < 0.05). Transcription of factors VIII, X, protein C, and antithrombin was unchanged. Our model demonstrated an early increase in hepatocyte apoptosis, impairment of factor VII transcription, and decrease in proliferative indices. These data support the hypothesis that EHPVO disrupts hepatic homeostasis and leads to dysregulation of coagulation factor synthesis.

Pharmacokinetics and Safety of a Fully Human Hepatocyte Growth Factor Antibody, AMG 102, in Cynomolgus Monkeys

AMG 102, a fully human monoclonal antibody that binds to hepatocyte growth factor (HGF), is a potential cancer therapeutic agent because of its ability to disrupt HGF/c-Met signaling pathways which have been implicated in most tumor types. To support a phase 1 study, the pharmacokinetic and safety profile of AMG 102 was assessed in cynomolgus monkeys. Serum concentration-time data from single- (i.v. and s.c.) and repeated-dose (i.v.) studies of up to 13 weeks were used for pharmacokinetic analysis. Safety was assessed in a single-dose safety pharmacology study with i.v. doses of 0 (vehicle), 25, 100, or 300 mg/kg and a 4-week toxicity study with once weekly i.v. doses of 0 (vehicle), 5, 25, or 100 mg/kg. AMG 102 exhibited linear pharmacokinetics over a 600-fold dose range (0.5 to 300 mg/kg) with a mean terminal half-life of 5.6 days after i.v. dosing. Clearance and volume of distribution at steady state were 1.22 ml/h and 198.3 ml, respectively. Estimated bioavailability was 72% for s.c. administration. Antibody response to AMG 102 was observed in a small percentage of monkeys. No treatment-related cardiovascular, respiratory, or CNS changes were observed. Administration of AMG 102 for 4 weeks was well tolerated at doses up to 100 mg/kg. Potential treatment-related effects were limited to minimal/moderate gastric mucosa hemorrhage in the mid- and high-dose groups. The nonclinical pharmacokinetic and safety profile of AMG 102 effectively supports clinical investigation.