BACKGROUND: The early stages of tumor bone metastasis are closely associated with changes in the vascular niche of the bone microenvironment, and abnormal angiogenesis accelerates tumor metastasis and progression. However, the effects of lung adenocarcinoma (LUAD) cells reprogrammed by the bone microenvironment on the vascular niche within the bone microenvironment and the underlying mechanisms remain unclear. This study investigates the effects and mechanisms of LUAD cells reprogrammed by the bone microenvironment on endothelial cells and angiogenesis, providing insights into the influence of tumor cells on the vascular niche within the bone microenvironment. METHODS: The culture media from bone-metastatic LUAD cell A549-GFP-LUC-BM3 (BM3-CM) and A549-GFP-LUC (A549-CM) were separately applied to human umbilical vein endothelial cell (HUVEC). A colony formation assay, scratch assay, and tube formation assay were conducted to evaluate the proliferation, migration, and angiogenesis of HUVEC. Gene set enrichment analysis (GSEA) was conducted to identify enriched pathways, while reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA) were performed to quantify hepatocyte growth factor (HGF), a protein that plays a crucial role in angiogenesis. Furthermore, the pivotal function of HGF and its underlying molecular mechanisms have been substantiated through the utilisation of recombinant proteins, neutralising antibodies, pathway inhibitors, immunofluorescence staining, and Western blot. RESULTS: BM3-CM demonstrated a more pronounced impact on the proliferation, migration, and angiogenesis of HUVEC compared to A549-CM. Bioinformatics analysis, combined with in vitro experiment, demonstrated that the secretory protein HGF was significantly elevated in BM3 cells and BM3-CM (P<0.05). The addition of HGF neutralizing antibodies to BM3-CM inhibited the promoting effect of BM3-CM on HUVEC (P<0.05), while the addition of recombinant HGF to A549-CM reproduced that promoting effect of BM3-CM on HUVEC (P<0.05). HGF can enhance the activation of YAP (Yes-associated protein) in HUVEC, and this promotion effect may be achieved by activating Src and activating YAP into the nucleus (P<0.05), but this effect can be inhibited by HGF neutralizing antibodies (P<0.05). Furthermore, the addition of recombinant HGF to A549-CM can recapitulate the YAP activation effect of BM3-CM in HUVEC (P<0.05). CONCLUSIONS Bone microenvironment reprogrammed bone-metastatic LUAD cells BM3 promote the proliferation, migration, and angiogenesis of HUVEC through the HGF/YAP axis, potentially playing a significant role in the modifications of the vascular niche.
Humans ; Hepatocyte Growth Factor/genetics* ; Signal Transduction ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells ; Bone Neoplasms/blood supply* ; Adenocarcinoma of Lung/genetics* ; Adaptor Proteins, Signal Transducing/genetics* ; Lung Neoplasms/genetics* ; Cell Movement ; Cell Proliferation ; YAP-Signaling Proteins ; Transcription Factors/genetics* ; Cell Line, Tumor ; Tumor Microenvironment ; Angiogenesis

Targeted therapy is an important therapeutic method for advanced non-small cell lung cancer with driver gene alteration.However,resistance to targeted therapy will inevitably happen in clinical practice,which has become a major issue demanding prompt solution.Studies have demonstrated that bypass resistance mediated by the activation of hepatocyte growth factor(HGF)/mesenchymal-epithelial transition factor(MET)signaling pathway is a common cause of resistance to targeted therapy.Presently,relevant studies have accumulated rich experience in the specific mechanisms.To be brief,HGF/MET is an important target for overcoming the resistance to targeted therapy and promises to be a leading biomarker for judging and observing the occurrence of resistance.This paper introduces the recent studies concerning the effects and mechanisms of HGF/MET signaling pathway on resistance to targeted therapy.
Carcinoma, Non-Small-Cell Lung/genetics* ; Epithelial-Mesenchymal Transition ; Hepatocyte Growth Factor ; Humans ; Lung Neoplasms/genetics* ; Proto-Oncogene Proteins c-met/metabolism* ; Signal Transduction

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

BACKGROUND: Hemoglobin concentration reportedly is positively associated with muscle strength, for example, handgrip strength. However, hemoglobin cannot repair muscle directly, but is beneficial only in a supportive role. Since hepatocyte growth factor (HGF) regulates muscle satellite cell production and differentiation, which is stimulated by organ injury, the supportive effect of hemoglobin should thus be stronger for participants with high HGF than for those with low HGF. However, the association between hemoglobin concentration and handgrip strength in relation to HGF levels remains unknown. METHODS: We conducted a cross-sectional study of 255 Japanese elderly men aged 60-69 years who participated in annual health check-ups in 2014-2015. The study population was categorized on the basis of a median value of HGF of 300.6 pg/mL. RESULTS: Among present study population, 128 participants showed low HGF. For participants with low HGF, hemoglobin concentration showed no significant association with handgrip strength (standardized parameter estimate (β) = 0.03, p = 0.767), but for those with high HGF, hemoglobin concentration was significantly positively associated with handgrip strength (β = 0.23, p = 0.014). CONCLUSIONS A significant positive association between hemoglobin level and handgrip strength was established for elderly Japanese men aged 60-69 years with high HGF but not for participants with low HGF. Our finding indicates that HGF levels could determine the relationship of hemoglobin concentration with handgrip strength in elderly Japanese men aged 60-69 years. This result can be expected to serve as an effective tool for the clarification of the roles played by HGF and hemoglobin concentration in maintenance of muscle strength.
Aged ; Cross-Sectional Studies ; Hand Strength ; physiology ; Hemoglobins ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Japan ; Male ; Middle Aged

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

OBJECTIVETo observe the inhibition of NK4 protein in the proliferation of human Raji lymphoma xenografts in nude mice, and to explore its molecular mechanism.

METHODSModels of human Raji lymphoma xenograft transfected with HGF gene were established by subcutaneous inoculation in nude mice. After establishment of the models, the mice received continuous NK4 protein via tail vein for 4 weeks, and the weight and tumor growth were monitored every week. After 8 weeks, the expression of HGF mRNA and c-Met mRNA of tumor tissues was measured by real-time fluorescent quantitation PCR. The apoptotic index (AI) and microvessel density (MVD) were evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry, respectively.

RESULTSThe models of human Raji lymphoma xenograft were successfully established. Although the animal weights of all groups declined, especially in the groups with NK4 protein injection, there was no statistical significance (P > 0.05). The tumor volume in HGF gene transfected group was larger than those of the control groups (P < 0.01), and there was no statistical significance among the control groups (P > 0.05). However, the tumor volume of the NK4 protein injection group decreased significantly (P < 0.01). Expression of HGF mRNA and c-Met mRNA in HGF gene transfected group increased significantly after injection of NK4 protein (P < 0.01). AI in HGF gene transfected group (33.5% ± 12.3%) was significantly lower than that of control groups (89.1% ± 22.3% vs. 81.9% ± 27.0%, P < 0.05), but became significantly higher (119.1% ± 18.9%) after NK4 protein injection (P < 0.01). MVD in HGF gene transfected group (28.5 ± 2.0) was higher than that of control groups (12.2 ± 1.4, 13.8 ± 1.3, P < 0.01), although declined (15.5 ± 2.5) after NK4 protein injection (P < 0.01).

CONCLUSIONSNK4 protein suppresses significantly the growth of human Raji lymphoma xenografts transfected with HGF gene. The pathogenesis may be involved in promoting tumor cell apoptosis and restraining tumor angiogenesis through competitive interrupting HGF/Met signal pathway.

Animals ; Apoptosis ; Hepatocyte Growth Factor ; genetics ; metabolism ; Heterografts ; Humans ; Lymphoma ; genetics ; metabolism ; therapy ; Mice ; Mice, Nude ; Microvessels ; pathology ; Neovascularization, Pathologic ; Proto-Oncogene Proteins c-met ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; T-Box Domain Proteins ; administration & dosage ; Transfection ; Transplantation, Heterologous

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-beta1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.
Animals ; Cell Engineering ; Cells, Cultured ; *Genetic Engineering ; Hepatocyte Growth Factor/analysis/*genetics ; Humans ; Liver/metabolism/pathology ; Liver Cirrhosis/pathology/*therapy ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*metabolism ; Rats ; Rats, Sprague-Dawley ; *Up-Regulation

Hepatocyte growth factor (HGF) is a potent angiogenic factor that can stimulate the production of blood vessels in ischemic tissue. We investigated whether gene therapy using HGF-expressing adenovirus could enhance skin flap survival. Sprague-Dawley rats were randomly divided into three groups. Rats were subdermally injected with HGF-expressing adenovirus (HGF virus group), recombinant HGF (rhHGF group), or phosphate buffered saline (PBS group) 2 days before and immediately after 3 x 9 cm caudal flap elevation. The survival area of the skin flap, the ratio of blood flow, CD31-positive vessels and, VEGF expression were examined. Skin flap viability was significantly increased in the HGF virus group compared to the rhHGF and PBS groups (71.4% +/- 5.9%, 63.8%+/- 6.4%, and 39.2% +/- 13.0%, respectively) (P = 0.025). Furthermore, the blood flow ratio was significantly increased in the HGF virus group. In the HGF virus group, the number of CD31-positive vessels and vascular endothelial growth factor (VEGF) expression were significantly increased. Gene therapy using HGF-expressing adenovirus increase VEGF expression, the number of viable capillaries, and blood flow to the flap, thereby improving skin flap survival.
Adenoviridae/genetics ; Animals ; Genetic Therapy/*methods ; Graft Survival/genetics ; Hepatocyte Growth Factor/biosynthesis/*genetics ; Male ; Models, Animal ; Neovascularization, Physiologic/*genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reconstructive Surgical Procedures ; Skin Transplantation/*methods ; Surgical Flaps/*surgery

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.
Animals ; Antigens, CD34 ; genetics ; metabolism ; Antigens, Ly ; genetics ; metabolism ; Cell Differentiation ; drug effects ; genetics ; physiology ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; ultrastructure ; Epidermal Growth Factor ; pharmacology ; Flow Cytometry ; Gene Expression Regulation, Developmental ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Liver ; cytology ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microfilament Proteins ; metabolism ; Microscopy, Atomic Force ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Time Factors