Hepatocyte growth factor promotes retinal pigment epithelium cell activity through MET/AKT signaling pathway

AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepithelial transition factor(MET)inhibitor SU11274 in vitro.Cell viability was detected by a Cell Counting Kit-8 assay.Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay,respectively.The expression levels of MET,phosphorylated MET,protein kinase B(AKT),and phosphorylated AKT proteins were determined by Western blot assay.The MET and phosphorylated MET proteins were also determined by immunofluorescence assay.RESULTS:HGF increased ARPE-19 cells’viability,proliferation and migration,and induced an increase of phosphorylated MET and phosphorylated AKT proteins.SU11274 significantly reduced cell viability,proliferation,and migration and decreased the expression of MET and AKT proteins.SU11274 suppressed HGF-induced increase of viability,proliferation,and migration in ARPE-19 cells.Additionally,SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins.CONCLUSION:HGF enhances cellular viability,proliferation,and migration in RPE cells through the MET/AKT signaling pathway,whereas this enhancement is suppressed by the MET inhibitor SU11274.HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.

Research progress on the development of hepatocyte growth factor/c-Met signaling pathway in gastric cancer:A review

Hepatocyte growth factor(HGF)and its receptor,c-Met,play important roles in the occurrence,development,and treatment of gastric cancer(GC).This review explored the function of the HGF/c-Met signaling pathway in GC and its potential targeted therapeutic mechanisms.As one of the most common malignant tumors worldwide,GC has a complex pathogenesis and limited therapeutic options.Therefore,a thorough understanding of the molecular mechanism of GC is very important for the development of new therapeutic methods.The HGF/c-Met signaling pathway plays an important role in the proliferation,migration,and invasion of GC cells and has become a new therapeutic target.This review summarizes the current research progress on the role of HGF/c-Met in GC and discusses targeted therapeutic strategies targeting this signaling pathway,providing new ideas and directions for the treatment of GC.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Microvesicles derived from mesenchymal stem cells inhibit acute respiratory distress syndrome-related pulmonary fibrosis in mouse partly through hepatocyte growth factor

BACKGROUND Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome(ARDS)patients.Mesenchymal stromal cell-derived microvesicles(MSC-MVs)have been shown to exert antifibrotic effects in lung diseases.AIM To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models.METHODS MSC-MVs with low hepatocyte growth factor(HGF)expression(siHGF-MSC-MVs)were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model.Following intubation,respiratory mechanics-related indicators were measured via an experimental small animal lung function tester.Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging.Immunohistochemical,western blotting,ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators.RESULTS The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice.Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores.However,low expression of HGF(siHGF-MSC-MVs)significantly inhibited the effects of MSC-MVs(P<0.05).Compared with the ARDS pulmonary fibrosis group,the MSC-MVs group exhibited suppressed expression of type I collagen antigen,type III collagen antigen,and the proteins transforming growth factor-βandα-smooth muscle actin,whereas the siHGF-MVs group exhibited significantly increased expression of these proteins.In addition,pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group,and the effects of the MSC-MVs were significantly inhibited by low HGF expression(all P<0.05).CONCLUSION MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.

Effect of hepatocyte growth factor on inflammatory factors associated with CCL_(4)-induced hepatocyte injury

Objective:The aim of this study is to investigate the effects of Hepatocyte Growth Factor(HGF)on the expression levels of IL-8,TNF-α,IL-4,and IL-21 in mice with liver injury induced by CCL_(4).Methods:An acute liver injury mouse model was established using CCL_(4),and hepatocytes and white blood cells were separated by gradient density centrifugation.Different concentrations of HGF were added in vitro,and the expression levels of cytokines were detected using ELISA.Results:In the in vivo injury model,the hepatocyte experiment results showed that the expression level of IL-8 was reduced in the 10 ng/mL HGF group compared to the injured hepatocyte group(P<0.05),and increased in the 50 ng/mL HGF group compared to the 10 ng/mL HGF group(P<0.05).For IL-4,the expression levels were reduced in both the 25 ng/mL HGF group(P<0.05)and the 50 ng/mL HGF group(P<0.05)compared to the injured hepatocyte group.The white blood cell experiment results showed that the expression levels of TNF-αwere reduced in both the 10ng/ml HGF group(P<0.05)and the 25 ng/mL HGF group(P<0.05)compared to the injured white blood cell group.In the in vitro injury model,hepatocyte experiment results showed that the expression levels of TNF-αwere reduced in both the 25 ng/mL HGF group(P<0.05)and the 50 ng/mL HGF group(P<0.05)compared to the normal control group.For IL-4,the expression level was reduced in the 25 ng/mL HGF group compared to the normal control group(P<0.05).The white blood cell experiment results showed that the expression level of TNF-αwas increased in the 50 ng/mL HGF group compared to the 10 ng/mL HGF group(P<0.001);for IL-21,the expression levels were reduced in the CCL_(4) model group(P<0.05),10 ng/mL HGF group(P<0.05),25 ng/mL HGF group(P<0.05),and 50 ng/mL HGF group(P<0.05)compared to the normal control group.Conclusion:when the liver of mice is acutely damaged by CCL_(4),HGF can reduce the expression levels of inflammatory cytokines IL-8,TNF-α,IL-4 in hepatocytes,and TNF-αin liver white blood cells.

Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.

Effect of IL-10 on the expression of HSC growth factors in hepatic fibrosis rat

AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor （PDGF） of hepatic stellate cells （HSCs） of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by CCI4 administration intra-peritoneally. Sixty clean male Sprague-Dawley （SD） rats were randomly divided into three groups： normal control group （GN, 8 rats）, hepatic fibrosis model group （GC, 28 rats） and IL-10 treated group （GI, 24 rats）. At the beginning of the 7^th and 11^th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS： Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7^th and 11^th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN （P = 0.001/0.042, 0.001/0.001, 0.001/0.001） and GI （P= 0.001/0.007, 0.002/0.001, 0.001/0.001）. For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7^th wk （P= 0.005） and 11^th wk （P= 0.049）. For HGF, mRNA level in GI decreased compared with GN at the 7^th wk （P = 0.001） and 11^th wk （P= 0.021）. Between these two time points, TGF-β1 expression at the 7^th wk was higher than that of the 11^th wk （P = 0.049）, but for EGF, the former was lower than the latter （P = 0.022）. As for PDGF mRNA, there was no significant difference between thesegroups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION： The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

Expression of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 in normal and malignant tissues of gastrointestinal tract

AIM： To provide the expression profile of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 （HAI-1） in normal and malignant tissues of gastrointestinal tract at mRNA level for further study on their correlations with tumor progression and metastasis. METHODS： Total RNAs were prepared from 37 samples of colorectal cancer tissues, 40 samples of gastric cancer tissues, and their adjacent normal tissues. The expression of SNC19/matriptase and HAI-1 in these samples was detected by real-time fluorescent quantitative PCR using glyceraldehyde-3-phosphate dehydrogenase as internal standard, and the clinical significance for the correlation with clinicopathological parameters was evaluated. RESULTS： In gastric cancer tissues the expression of HAI-1 and SNC19/matriptase was significantly lower than that in the corresponding adjacent normal tissues （Z = -3.280, P= 0.006; Z= -4.651, P= 0.000）. HAI-1：SNC19/matriptase ratio showed no difference between normal and malignant tissues （P〉0.05）. Analysis of clinicopathological parameters showed decreased expression of HAI-1 and HAI-1：SNC19/ matriptase ratio associated with stage Ⅲ/Ⅳ gastric tumors as compared to stage Ⅰ/Ⅱ ones （Z= -2.140, P= 0.031; Z = -2.155, P = 0.031）, and with lymph node-positive gastric cancer tissues as compared to lymph node-negative ones （Z = -2.081, P = 0.036; Z= -2.686, P = 0.006）. The expression of SNC19/matriptase had no relationship with stages and lymph node metastasis （P〉0.05）. The expression of HAI-1 and HAI-1：SNC19/matriptase ratio increased in well-differentiated gastric cancer tissues, but there was no statistical significance （P〉0.05）. The difference of SNC19/matriptase expression was not significant in gastric cancer tissues of different histological differentiation status （P〉0.05）. In colorectal cancer tissues, the expression of HAI-1 and SNC19/matriptase was also markedly lower than that in their adjacent normal tissues （Z= -3.100, P = 0.002; Z= -2.731, P = 0.006）, whereas HAI-1：SNC19/matriptase ratio showed no difference. Decreased expression of HAI-1 was associated with increased invasive depth and lymph node metastasis, but there was no statistical significance （P〉0.05）. The difference of SNC19/matriptase expression and HAI-1： SNC19/matriptase ratio was not significant in different stages and different lymph node metastasis status （P〉0.05）. The expression of SNC19/matriptase, HAI-1 or HAI-1： SNC19/matriptase ratio showed no difference in colorectal cancer tissues of different histological differentiation status （P〉0.05）. CONCLUSION： The expressions of SNC19/matriptase and its inhibitor HAI-1 are decreased in gastrointestinal cancer tissues compared to their normal counterparts, and the decreased expression of HAI-1 may correlate with invasion and lymph node metastasis. The possible mechanisms involved need to be further investigated.

Updates on the hepatocyte growth factor/c-Met axis in hepatocellular carcinoma and its therapeutic implications

Hepatocellular carcinoma(HCC) is the fifth most common cancer and is the second leading cause of cancer death. Since the diagnosis of HCC is difficult, in many cases patients with HCC are diagnosed advanced stage of development. Hepatocyte growth factor(HGF)/c-mesenchymal-epithelial transition receptor(c-Met) axis is a key signaling pathway in HCC, either via canonical or non-canonical pathways. Available treatments against HCC based upon HGF/c-Met inhibition can increase patient lifespan, but do not reach the expected therapeutic benefits. In HCC, c-Met monomers can bind other receptor monomers, activating several noncanonical signaling pathways, leading to increased cell proliferation, invasion, motility, and drug resistance. All of these processes are enhanced by the tumor microenvironment, with stromal cells contributing to boost tumor progression through oxidative stress, angiogenesis, lymphangiogenesis, inflammation, and fibrosis. Novel treatments against HCC are being explored to modulate other targets such as microR NAs, methyltransferases, and acetyltransferases, which are all involved in the regulation of gene expression in cancer. This review compiles basic knowledge regarding signaling pathways in HCC, and compounds already used or showing potential to be used in clinical trials.

Hepatocyte growth factor gene therapy prevents radiation-induced liver damage

AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8rats received HGF-EGT 100 μg 48 h before WLIR.Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.RESULTS: Marked decrease of apoptotic cells and downregulation of transforming growth factor-beta 1 (TGF-β1)mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.

Effects of hepatocyte growth factor on MMP-2 expression in scleral fibroblasts from a guinea pig myopia model

AIMTo investigate the effects of hepatocyte growth factor (HGF) on MMP-2 expression in scleral fibroblasts from guinea pig with LIM.

Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6 (Hyper-IL-6, HIL-6) and hepatocyte growth factor (HGF) (Ad-HGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF (Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure (ACLF).METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or Ad-HGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes.RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1 (HMGB1), endotoxin, tumour necrosis factor (TNF)-&#x003b1; and interferon-&#x003b3; were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. Likewise, reduced hepatic damage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGF-HIL-6 treatment groups, more predominantly in the latter group.CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects.

Hepatocyte Growth Factor Gene-modified Bone Marrow-derived Mesenchymal Stem Cells Transplantation Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia

Summary： Angiogenic gene therapy and cell-based therapy for peripheral arterial disease （PAD） have been studied intensively currently. This study aimed to investigate whether combining mesenchymal stem cells （MSCs）transplantation with ex vivo human hepatocyte growth factor （HGF） gene transfer was more therapeutically efficient than the MSCs therapy alone in a rat model of hindlimb ischemia. One week after establishing hindlimb ischemia models, Sprague-Dawley （SD） rats were randomized to receive HGF gene-modified MSCs transplantation （HGF-MSC group）, untreated MSCs transplantation （MSC group）, or PBS injection （PBS group）, respectively. Three weeks after injection, angiogenesis was significantly induced by both MSCs and HGF-MSCs transplantation, and capillary density was the highest in the HGF-MSC group. The number of transplanted cell-derived endothelial cells was greater in HGF-MSC group than in MSC group after one week treatment. The expression of angiogenic cytokines such as HGF and VEGF in local ischemic muscles was more abundant in HGF-MSC group than in the other two groups. In vitro, the conditioned media obtained from HGF-MSCs cultures exerted proproliferative and promigratory effects on endothelial cells. It is concluded that HGF gene-modified MSCs transplantation therapy may induce more potent angiogenesis than the MSCs therapy alone. Engraftment of MSCs combined with angiogenic gene delivery may be a promising therapeutic strategy for the treatment of severe PAD.

Effect of FK506 on Expression of Hepatocyte Growth Factor in Murine Spinal Cord Following Peripheral Nerve Injury

This study is to investigate the effect of FK506 on expression of hepatocyte growth factor （HGF） in rats＇ spinal cord following peripheral nerve injury and to elucidate the mechanisms for neuroprotective property of FK506. Fifty male rats were randomly divided into normal group, injury group and treatment group. Models of peripheral nerve injury were established by bilateral transection of sciatic nerve 0.5 cm distal to piriform muscle. Then the treatment group received subcutaneous injection of FK506 （1 mg/kg） at the back of neck, while the injury group was given 0.9% saline. The L4-6 spinal cords were harvested at various time points after the surgery. Western blotting and immunofluorescent staining were used to detect the level and position of HGF in spinal cord. Immunofluorescent staining showed that HGF-positive neurons were located in anterior horn, intermediate zone and posterior horn of gray matter in normal spinal cord. Western blotting revealed that there was no significant difference in the expressions of HGF between the injury group and the normal group, while the expression of HGF was significantly higher in the treatment group than in the injury group 7 and 14 days after surgery. It is suggested that peripheral nerve injury does not result in up-regulation of the expression of HGF in spinal cord, while FK506 may induce high expression of endogenous HGF after injury thereby protecting neurons and promoting axonal outgrowth.

Osteoprotegerin,interleukin and hepatocyte growth factor for prediction of diabetes and hypertension in the third trimester of pregnancy

BACKGROUND Gestational diabetes mellitus(GDM)raises the risk of high blood pressure and may cause a series of life-threatening complications in pregnant women.Screening and management of GDM and gestational hypertension(GH)in pregnancy helps to control and reduce these risks and prevent adverse effects on mothers and their fetuses.Currently,the majority criteria used for screening of diabetes mellitus is oral glucose tolerance tests,and blood pressure test is usually used for the screening and diagnosis of hypertension.However,these criteria might not anticipate or detect all GDM or GH cases.Therefore,new specific predictive and diagnostic tools should be evaluated for this population.This study selected three biomarkers of osteoprotegerin(OPG),interleukin(IL)and hepatocyte growth factor(HGF)for GDM and GH predication and diagnosis.AIM To explore the feasibility of changes in placental and serum OPG,IL and HGF as tools for prediction and diagnosis of diabetes and hypertension in pregnant women.METHODS From January 2018 to January 2019,44 pregnant women with GDM and GH were selected as an observation group,and 44 healthy pregnant women were selected as a control group in the same period.Serum OPG,IL and HGF were compared between the two groups.RESULTS The levels of OPG and HGF in the observation group were lower than in the control group,and the level of IL-1βwas higher in the observation group than in the control group(all P<0.05).Furthermore,OPG and HGF were negatively associated with gestational diabetes and gestational hypertension,while IL-1βwas positively associated with GDM complicated with GH(all P<0.05).CONCLUSION The evaluation of serum OPG,HGF and IL-1βlevels in patients with coexistent gestational diabetes complicated with hypertension can predict the degree of disease and play an important role in the follow-up treatment and prognosis prediction.

HEPATOCYTE GROWTH FACTOR PROTECTS AGAINST APOPTOSIS INDUCED BY ADVANCED GLYCATION END PRODUCTS IN ENDOTHELIAL CELLS

Objective To investigate the effects of hepatocyte growth factor(HGF)on vascular endothelial cells apoptosis induced by advanced glycation end products(AGEs)and its possible mechanism. Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and intervened by different concentrations of AGEs and HGF.The cell inhibitory rates of each group with different culture time(12, 24, 48, and 72 hours)were measured by methyl thiazolyl tetrazolium(MTT)assay. The early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining, morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the expression of apoptosis-associated genes Bax and Bcl-2 were determined by Western blotting.The activity of caspase-3 was detected by enzyme-linked immunosorbent assay (ELISA).Results Morphological observation indicated that high concentration of AGEs induced characteristic apoptotic changes in HUVECs.Within a certain concentration range, HUVECs apoptosis inducing rates by AGEs were in both dose- and time-dependent manners.HGF significantly inhibited the apoptosis of HUVECs induced by AGEs (P< 0.05).AGEs significantly promoted expression of Bax protein, but not Bcl-2.Whereas HGF significantly promoted the expression of Bcl-2(P<0.01)and decreased the activity of caspase-3(P<0.05)without affecting Bax level.Conclusions AGEs can induce the apoptosis of endothelial cells in vitro.HGF may effectively attenuate AGEs-induced endothelial cells apoptosis through upregulating Bcl-2 gene expression and inhibiting caspase-3 activation.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Hepatocyte growth factor gene transfer effects on the femoral and intramuscular nerve in a canine model of lower limb ischemia

BACKGROUND： Recent advancements in gene therapy have provided new methodology for treating ischemia in lower extremities. Gene transfer of angiogenic factors to ischemic tissues may promote local proliferation of new vessels and form collateral circulation. OBJECTIVE： To observe histopathological changes in the femoral and intramuscular nerve three months after intramuscular injection of hepatocyte growth factor （HGF） into the peripheral skeletal muscle in a canine model of lower limb ischemia. DESIGN： Randomized occlusion modelled and verification animal study. SETTING： Experimental Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA. MATERIALS： This study was performed at Animal Experimental Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from September to November 2006. A total of eight male mongrel dogs, weighing 12–15 kg and 1.5–3 years of age, were selected for this study. This experimental study was in accordance with local ethics standards. Recombinant plasmid carrying HGF （pUDKH） and occlusion model plasmid （pUDK） were provided by the Third Laboratory of Radiation Medical Institute, Academy of Military Medical Sciences of PLA. METHODS： Grouping and model establishment： under anesthesia, complete vascular occlusion models were established on the left lower extremities. The experimental dogs were randomly divided into a model group and a pUDKH treatment group, with four dogs in each group. Dogs in the pUDKH group were injected with 0.15 mg/kg pUDKH. Ten minutes later, intramuscular injections were performed at three spots into the peripheral skeletal muscle of the left hind limb, as well as lateral injections at two spots. The injection volume at each spot was 0.2 mL. Dogs in the model group were injected with pUDK, and dosage and injection method were identical to the treatment group. MAIN OUTCOME MEASURES： Histopathological changes in the femoral nerve, as well as internal and external intramuscular nerve tissues in the hind limb of dogs three months after plasmid injection under optic microscope. RESULTS： （1） Histopathological changes in the femoral nerve： tiny nerves from the femoral nerve to the intramuscular nerve exhibited marked degeneration in the model group. The degenerating features included neurites, myelin sheaths, and Schwann cell nuclei. Neuropathy in the pUDKH treatment group was not detected. （2） Histopathological changes of the intramuscular nerve： large and irregular vacuoles were present on several longitudinal sections of intramuscular nerve fibers in the model group, as well as annular-shaped blank regions on transverse sections of peripheral neurites. In the pUDKH treatment group, large, blank regions were present in several segments of partial nerve fibers of the longitudinal intramuscular nerve region, but only a few nerve fibers exhibited annular-shaped blank regions on the transverse section of peripheral neurites. CONCLUSION： Local pUDKH injection may relieve or block femoral and intramuscular nerve tissue injury in a canine mocel of lower limb ischemia.

Improvement of cardiac function by hepatocyte growth factor via intracoronary transfection in the swine myocardial infarction model

Objective： To study the amelioration effect of AdenovirusS-mediated human hepatocyte growth factor （AdsHGF） on postinfarction heart failure in the swine myocardial infarction model. Methods： Twelve SuZhong young swine were randomly divided into 2 groups with 6 swine in each group： Ad5-HGF-treated group and null-Ad5 group. Four weeks after ligation at left anterior descending coronary artery in swine hearts, Ad5-HGF was transferred to the swine myocardium. Simultaneously, Gated myocardial perfusion imaging was performed to evaluate cardiac perfusion and heart function. After three weeks, Gated myocardial perfusion imaging was performed again, then the hearts were harvested and sectioned to examine the expression of HGF through ELISA. Results： High expression of human HGF was observed in the myocardium of Ad5-HGF-treated group. From 4 weeks to 7 weeks after operation, Left ventricular ejection fraction was increased in Ad5-HGF-treated group. The improvement in LVEF was greater in Ad5-HGF-treated group than that in null-Ad5 group at 7 weeks after operation. Cardiac perfusion was significantly improved in the Ad5-HGF-treated group. Conclusion： High expression of human HGF was observed in the myocardium through intracoronary transfection, which suggests that HGF can ameliorate heart function in swine with postinfarction heart failure.

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

Objective 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). Methods 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. Results 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. Conclusion HGF activity may accelerate the repair of lung injury through contrary regulating the expression levels of TIMP-1 and MMP-9.