Hepatocyte Growth Factor Staining Research Articles

Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors

Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation.

A phase II study of erlotinib monotherapy in pre-treated non-small cell lung cancer without EGFR gene mutation who have never/light smoking history: Re-evaluation of EGFR gene status (NEJ006/TCOG0903)

ObjectivesEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are particularly effective in non-small cell lung cancer (NSCLC) patients harboring active EGFR mutations. However, some studies have reported survival benefits in NSCLC patients with wild-type EGFR upon erlotinib treatment. This trial was conducted to evaluate the efficacy of erlotinib monotherapy and investigate the predictive values of several biomarkers. Patients and methodsPatients with previously treated NSCLC but without EGFR gene mutations that had never or light smoked were eligible for this study. Gene status screening was performed using the PNA-LNA PCR clamp method. Erlotinib was administered until disease progression or unacceptable toxicities occurred. EGFR gene status was re-evaluated using the fragment method to detect exon 19 deletions and the Cycleave-PCR method to detect point mutations. Expression of hepatocyte growth factor (HGF), Met, and thymidylate synthase (TS) were evaluated using immunohistochemistry. ResultsForty-seven patients were enrolled in the study between March 2010 and November 2011. Objective response rate (ORR) and disease control rate (DCR) were 15.2% and 41.3%. Re-evaluations for EGFR gene were performed in 32 tumor samples. EGFR gene mutations were found in eight samples (5:exon 19 deletion, 2:G719X, 1:L858R). Six patients had PR and two had SD among these eight patients. A total of 24 patients were confirmed as wild-type EGFR using different methods. ORR and DCR were 4.2% and 41.7%. The median progression free survival (PFS) and median survival times were 2.0 and 6.0 months, respectively. Patients with tumors expressing HGF showed shorter PFS but not MET or TS. ConclusionsRe-examination of EGFR gene status using different detecting method or different sample should be considered to grasp a chance of erlotinib treatment after first line treatment. In confirmed EGFR wild NSCLC, negative HGF staining could be a biomarker for longer PFS by erlotonib treatment.

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.

Hepatocyte growth factor in the neighborhood reverses resistance to BRAF inhibitor in melanoma

Hepatocyte growth factor in the neighborhood reverses resistance to <scp>BRAF</scp> inhibitor in melanoma

Immunohistochemical analysis of hepatocyte growth factor and c‐Met in plasma cell disease

Interaction with the bone marrow microenvironment is important for homing and survival of myeloma cells. One cytokine involved in this process is hepatocyte growth factor (HGF). HGF, by binding to the receptor tyrosine kinase c-Met, mediates a broad range of tumour progression activities. Our aims were to investigate whether HGF and c-Met are present in bone marrow and extramedullary tumours from patients with monoclonal plasma cell disease, and whether c-Met is activated. Expression of HGF, c-Met and phospho-c-Met was studied by immunohistochemistry in biopsies from 80 patients with monoclonal plasma cell disease. Cytoplasmic staining for HGF in plasma cells was demonstrated in 58 of 68 biopsies from multiple myeloma patients (85%), but also in biopsies from nine of 10 healthy individuals. Membranous staining for c-Met was found in 25 of 63 multiple myeloma patients (40%) and in none of 10 healthy individuals. Membranous staining for phospho-c-Met was found in biopsies from 15 of 21 c-Met-positive myeloma patients (71%). Our data point to c-Met expression as one of the factors that distinguishes normal from malignant plasma cells, and indicate that the HGF/c-Met system is activated in multiple myeloma patients.

The expression of hepatocyte growth factor (HGF) and c-Met in uterine serous carcinoma

ObjectiveThe hepatocyte growth factor (HGF) receptor c-Met plays an important role in tumor dissemination by activating mitogenic signaling pathways. The goal of this study was to investigate immunohistochemical (IHC) staining patterns of HGF and c-Met in endometrioid endometrial cancer (EC) and uterine serous cancer (USC) and to correlate staining with patient outcomes. MethodsA tissue microarray was created using tissue from patients with atrophic endometrium (AE), grade 1 EC, grade 3 EC, and USC. Immunohistochemistry was used to detect c-Met, phosphorylated c-Met (p-c-Met), and HGF expression. Differences between IHC staining intensity were calculated using t-tests. Correlations between staining and clinicopathologic variables were determined by Chi-square testing for categorical variables and t-tests for continuous variables. Kaplan–Meier curves were constructed to analyze survival in USC. ResultsPatients with cancer had more total c-Met and HGF expression than those with AE (p=0.037, p<0.001 respectively), but no difference in p-c-Met staining. Total c-Met and HGF staining was significantly different between groups (p=0.042, p<0.001 respectively). This difference was accounted for by greater c-MET expression in USC compared to AE (p=0.027). Depth of invasion, stage, and lymph node status were not significantly related to staining intensity. Patients with strong c-Met and HGF staining had decreased overall survival compared to patients with weaker staining. ConclusionsHGF and c-Met may play a role in the progression of endometrial cancer and should be studied further as prognostic and therapeutic tools.

Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo

Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. To analyse the involvement of the fibroblast-derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.

MCP-1 Antibody Treatment Enhances Damage and Impedes Repair of the Alveolar Epithelium in Influenza Pneumonitis

Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti-MCP-1-treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus-infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.

Evaluation of hepatocyte growth factor in patients with biliary atresia

Background Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes in primary culture and seems to be a trigger factor for liver regeneration and proliferation after liver injury. The aim of this study was to evaluate the expression of HGF in patients with biliary atresia (BA). Methods Plasma levels of HGF were determined using an enzyme-linked immunosorbent assay from 19 patients with BA, 10 at the time of Kasai procedure (KP) and 9 at liver transplantation (LT), and 10 patients with nonicteric choledochal cyst served as control. Hepatic HGF level was quantitatively estimated using enzyme-linked immunosorbent assay and Western blotting. Hepatocyte growth factor messenger RNA (mRNA) expression of liver was measured with quantitative reverse transcriptase polymerase chain reaction. Immunohistochemical study of liver sections was evaluated by HGF staining. Results Plasma HGF levels in LT patients were significantly higher than in KP patients (2977.30 ± 1251.42 vs 960.17 ± 559.82 pg/mL, P < .01). Western blots showed increased hepatic HGF levels in LT group than in KP group (2.02 ± 0.86 vs 1.44 ± 0.72 pg/ μg total protein, P < .05). However, there were no differences in HGF mRNA expression. Immunohistochemical study showed significantly increased HGF staining in hepatocytes of LT livers than of KP livers. Conclusions Elevation of plasma and liver HGF levels could be related to the progression of liver cirrhosis in patients with BA. Hepatocyte growth factor might have a protective role, probably through the induction of hepatocyte proliferation, during the development of BA-associated liver cirrhosis.

Hepatocyte Growth Factor Expression in Bone Marrow Microenvironment Is Critical for Progression of MGUS to Myeloma.

Hepatocyte growth factor (HGF) augments proliferation, survival and increases migration of plasma cells in vitro and in addition it induces haemangiogeneis and lymphangiogenesis. C-met (HGF receptor) is expressed on plasma cells and HGF is constitutively elevated in serum of patients with myeloma (MM) and MGUS patients. We investigated the distribution of HGF in bone marrow biopsies to better understand its role in progression of monoclonal gammopathy.Materials and methods: Bone marrow trephines were analysed by immunohistochemistry of paraffin embedded bone marrow (BM) trephine specimens. Trephines were dewaxed without antigen retrieval. Plasma cell enumeration was done using CD38 staining (Novocastra, UK). HGF (Goat antihuman IgG, R&D systems) staining was performed using Vectastain® ABC kit with DAB reactivity. Slides were analysed using an Optical microscope (Zeiss Standard 20, Germany).Results: Archived BM specimens of MM (n= 7), MGUS (n= 5) and normal controls (n=6) were stained in duplicate. Trephines were graded from Grade 1–3 (weak, moderate, strong) based on the intensity of staining. The localisation of staining to paratrabecular, perivascular and intertrabecular areas was analysed. Interstitial and cytoplasmic staining patterns were analysed. HGF staining localised to paratrabecular regions was observed in all normal individuals with staining intensity of Grade 1–2 in 3/6(50%) and Grade 3 in 3/6(50%). In 4/5(80%) patients with MGUS a distinct intertrabecular pattern of staining was observed in discrete regions, where plasma cells were localised. Intensity of staining was Grade 2 in 3/5(60%) and Grade 3 in 2/5(40%) MGUS patients. Plasma cell percentages were between 5–20% in MGUS patients. In MM patients we found a correlation between the plasma cell counts and the degree of HGF staining. In 2/7 (28.5%) patients plasma cell percentages ranged between 5% and 15% and Grade 1–2 discrete paratrabecular staining was observed with none or low perivascular staining. In 5/7 (71.5%) MM patients plasma cell percentages were >20% and Grade 3 confluent staining with interstitial and cytoplasmic location of HGF was observed. In addition, patients with Grade 3 staining presented a strong perivascular localisation of HGF.Conclusion: HGF distribution in bone marrow microenvironment of MGUS and MM patients is non random. We observed that MGUS patients have a discrete intertrabecular pattern and MM patients have a confluent interstitial pattern of staining with strong perivascular localisation. Other groups have shown that high HGF levels increase angiogenesis. We hypothesise that HGF localisation and pattern of distribution in BM microenvironment leads to neo angiogenesis and progression from MGUS to MM. Ongoing studies are evaluating this hypothesis.

Hepatocyte growth factor regulation: An integral part of why wounds become chronic

Hepatocyte growth factor (HGF) is a cytokine known to play multiple roles during the various stages of wound healing. This study addresses the ongoing key questions regarding the role of HGF in wound healing, namely: are HGF and its regulators expressed differently in chronic and acute wounds? Biopsies from normal skin (n=10), acute (n=10), and chronic wounds (n=17) were analyzed by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR for the presence of HGF, its receptor cMet, its activators and its inhibitors. Immunohistochemical staining for HGF, HGF activators, and HGF inhibitors was similar, with expression being greater in chronic wound dermis compared with acute wound dermis. While expression of cMet in chronic wound dermis was less than in the acute wound dermis. PCR quantification of these proteins showed similar trends although the differences did not reach statistical significance. The results of this study provide important data confirming the role of HGF in wound healing. In addition, we have demonstrated the aberrant expression of the HGF receptor cMet and activation inhibitors (HAI-1 and HAI-2) in chronic wounds. This aberrant expression may have a value in predicting the process of healing and nonhealing wounds and constitute important targets of future therapies.

Expression of hepatocyte growth factor in Hashimoto’s thyroiditis with nodular lesions

Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease frequently associated with hyperplastic nodules (HN)s. Hepatocyte growth factor (HGF) is expressed in benign thyroid nodules and over-expressed in malignant thyroid nodules, particularly in papillary thyroid carcinomas. To elucidate the role of HGF in the development of HNs in association with HT we evaluated, by immunohistochemistry, the expression of HGF in both nodular and extranodular tissues, obtained from 30 HTs and 15 goiter samples. Six normal thyroid glands were used as controls. All normal control tissue samples exhibited no evidence of HGF immunoreaction. HNs showed weak to moderate HGF immunoreaction, which was located exclusively in the cytoplasm of stromal cells (fibroblasts and endothelial cells). However, the percentage of positive cases was higher in HNs arisen in the context of HT, compared to HNs not associated with HT (30/30 or 100% vs 4/15 or 40%; p<0.001). HGF immunoreactivity was also detected in all extranodular tissues from HT specimens (30/30 or 100%), but we found some significant differences. In fact, while in HNs observed in the context of HT lesions HGF was expressed only in stromal cells, in the extranodular tissues from the same thyroid gland affected by HT it was also detected in the cytoplasm of the epithelial follicular cells. Furthermore, HTs showed a much higher HGF staining grade in the extranodular tissue compared to HNs. Finally, a clear positive correlation was observed in HT between the proportion of HGF expressing follicular cells and the grade of lymphoid aggregates of the thyroid gland. In conclusion, HGF is much more frequently and highly expressed in thyroid tissue with HT, compared to goiter. In HT glands HGF can be detected in both follicular thyroid cells and stromal cells, while in HNs, either from goiters or associated with HT, its expression is restricted only to the stromal cells. These data indicate that HGF may play a role in cell proliferation processes occurring in thyroid glands affected by HT, probably under the regulation of the lymphoid infiltrate.

Hepatocyte growth factor induction of macrophage chemoattractant protein‐1 and osteophyte‐inducing factors in osteoarthritis

In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors.

Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro

Hepatocyte growth factor (HGF) regulates granulosa cell (GC) steroidogenesis and suppresses apoptosis in non-ovarian cells. The hypothesis was thus developed that intraovarian HGF supports folliculogenesis by mediating steroidogenesis and suppressing apoptosis. To investigate the latter, the anti-apoptotic actions of HGF were tested in GCs and follicles isolated from immature rats. Results showed that HGF suppressed apoptosis in GC and follicle cultures as visualized using apoptosis indicator dye, YO-PRO-1. Immunohistochemistry was used to investigate the distribution of HGF, c-met, and HGF activator (HGFA) protein during folliculogenesis in equine chorionic gonadotropin (eCG)-primed rats. Immunoreactive HGF content was the greatest in GCs within preantral follicles. Following eCG, large antral follicles showed elevated HGF staining in theca and interstitial cells when compared with GCs. Intense c-met staining was observed in GCs within non-primed small preantral follicles; following eCG, the level of c-met was diminished in GCs, but increased within theca and interstitial cells. Theca, interstitium, and GCs in non-primed and primed ovaries contained HGFA. Following eCG, HGFA was more apparent in theca cells and the interstitium when compared to that in GCs within large antral follicles. The presence of HGF, c-met, and HGFA in preantral follicles would potentially enable the anti-apoptotic effects of HGF that were observed in vitro to occur in vivo. Advanced folliculogenesis led to a change in the cellular distribution of the HGF, c-met, and HGFA, suggesting that the ovarian HGF system is hormonally regulated in vivo.

Expression of hepatocyte growth factor and c-Met in hypopharyngeal squamous cell carcinoma

Conclusion. Our results suggest that hepatocyte growth factor (HGF) may play an important role in the progression of hypopharyngeal cancer. Objective. HGF, a potent stimulator of hepatocyte growth, stimulates the motility, invasiveness, proliferation and morphogenesis of epithelium and may be involved in physiologic and pathologic processes such as embryogenesis, wound healing, organ regeneration, inflammation and tumor invasion. We therefore examined the role of HGF and c-Met in the invasion and metastasis of hypopharyngeal squamous cell carcinoma (SCC). Material and methods. We performed immunohistochemical staining of 40 specimens each of normal mucosa and SCC of the hypopharynx with HGF and c-Met antibodies. For reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting, fresh normal and cancer tissue from the hypopharynx obtained from five patients were used. Results. Positive rates of HGF and c-Met expression in hypopharyngeal SCC were 77.5% and 70%, respectively. HGF staining was significantly correlated with lymph node metastasis and pathologic stage (p < 0.05). c-Met staining was only significantly correlated with lymph node metastasis (p < 0.05). Increased expression of c-Met mRNA (RT-PCR) and protein (Western blotting) was detected in hypopharyngeal cancer tissue.

Clinical Significance of Hepatocyte Growth Factor and Its Specific Receptor c-Met Expression in Colorectal Cancer Progression

To clarify the clinical significance of hepatocyte growth factor (HGF) and its specific receptor c-Met in colorectal cancer, we performed serological analysis of HGF in 108 patients who underwent colon cancer surgery and 200 age-matched control subjects, and immunohistochemical analysis of HGF and c-Met in surgically resected samples in 50 of the 108 patients. Serological analysis demonstrated that preoperative serum HGF level was significantly higher in the cancer group than in the control group (p<0.0001), and increased in patients with advance of depth of invasion (p=0.0173), liver metastasis (p=0.0024), and tumor stage (p=0.0069). Immunohistochemical analysis demonstrated the strong expression and colocalization of HGF and c-Met in the cancer cells. Tissue HGF staining score was increased in patients with advance of venous invasion (p=0.0092), whereas tissue c-Met staining score was decreased in patients with advance of lymphatic invasion (p=0.0234), liver metastasis (p=0.0176), and serum HGF level over 300 pg/mL (p=0.018). The present results suggest that the verification of both preoperative serum HGF level and tissue HGF and c-Met levels contributes to the prognostic evaluation of tumor progression in the preoperative period. Furthermore, it is likely that HGF oversupply-associated c-Met downregulation occurs in colorectal cancer via autocrine, paracrine and endocrine mechanisms.

Improvement of endothelial dysfunction by angiotensin II blockade accompanied by induction of vascular hepatocyte growth factor system in diabetic spontaneously hypertensive rats.

Hepatocyte growth factor (HGF) is a unique growth factor with many protective functions. Previously, we demonstrated that HGF stimulated growth of endothelial cells without replication of vascular smooth muscle cells (VSMC) and that angiotensin (Ang) II significantly decreased local HGF production in VSMC. Moreover, we also reported that high glucose significantly decreased local vascular HGF production. Therefore, we examined effects of Ang II blockade on vascular HGF expression and endothelial injury in diabetic hypertensive rats. An angiotensin-converting enzyme inhibitor (quinapril) and an Ang II type 1 receptor antagonist (GA-0113) or vehicle was administrated to diabetic spontaneously hypertensive rats (SHR-DM), in whom diabetes was induced by streptozotocin. Endothelial function was evaluated by the vasodilator response to acetylcholine, and the expression of vascular HGF and its receptor, c-met, was examined by immunohistochemistry. Both quinapril and GA-0113 significantly improved the vasodilator response to acetylcholine ( P < 0.01), while vehicle did not as compared to untreated normotensive Wistar-Kyoto rats (WKY). We next examined the effects of Ang II blockade on vascular HGF expression in SHR-DM. Importantly, the vascular HGF level was markedly decreased in SHR-DM as compared to WKY, while Ang II blockade by quinapril or GA-0113 significantly increased positive staining for HGF in SHR-DM. Similarly, staining of its specific receptor, c-met, was less in the blood vessels of SHR-DM as compared to WKY. In contrast, Ang II blockade also significantly increased c-met production in SHR-DM. The present data demonstrated the improvement of endothelial dysfunction by Ang II blockade in SHR-SM, accompanied by an increase in vascular HGF and c-met.

Hepatocyte growth factor and macrophage-stimulating protein are upregulated during excisional wound repair in rats.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.

Papillary Carcinoma of the Thyroid: Hepatocyte Growth Factor (HGF) Stimulates Tumor Cells to Release Chemokines Active in Recruiting Dendritic Cells

Tissue distribution of dendritic cells was investigated in eight cases of papillary carcinoma of the thyroid using immunohistochemistry. Most dendritic cells had an immature phenotype (CD1a++, CD11c+, CD40+, CD86-, HLA-DR-) and were located at the invasion edge of the tumor. This pattern of distribution was profoundly different from that of CD68+ macrophages, which were evenly distributed throughout the tumor. The ability of tumor cells to release chemotactic factors active on dendritic cells was investigated in primary cultures of the same cases of papillary carcinoma, and was compared to that of the corresponding normal thyroid cells obtained from the tumor-free contralateral lobe. Chemotactic activity of culture supernatants was tested against dendritic cells in a chemotaxis chamber. It was found that papillary carcinoma cells were active in releasing chemotactic activity, that hepatocyte growth factor (HGF; 100 ng/ml) or interleukin (IL)-1beta (10(3) U/ml) induced a fourfold increase in the amount of chemotactic activity released, and that normal thyroid cells obtained from the same patients were as effective as tumor cells. Characterization of chemokines at RNA level revealed that unstimulated cells contain large amounts of IL-8 and monocyte chemotactic protein (MCP)-1 RNAs, and that stimulation with HGF or IL-1beta induced RNAs for regulated upon activation normal T expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-3alpha, interferon-gamma-inducible protein 10 (IP-10), and, to a lesser extent, MIP-1alpha and MIP-1beta. The possibility that HGF/Met interaction has a biological role in vivo was investigated in serial sections of six tumors immunostained for CD1a+, Met protein, and HGF. It was found that all six tumors were intensely and diffusely positive for Met protein, that HGF staining was present in tumor cells of the advancing edge, and that HGF+/Met+ tumor cell nests were infiltrated by CD1a+ dendritic cells. The foregoing observations are consistent with the possibility that HGF stimulation of Met+ tumor cells is one of the molecular mechanisms involved in the recruitment of dendritic cells.