Hepatocyte Growth Factor mRNA Research Articles

Hepatoprotection of a Standardized Extract of Cultured Lentinula edodes Mycelia against Liver Injury Induced by Ischemia-Reperfusion and Partial Hepatectomy.

A standardized extract of cultured Lentinula edodes mycelia (ECLM, AHCC®) has been shown to have beneficial effects on organ metabolism. ECLM has been indicated to have liver protective properties by suppressing inflammatory responses. The pathogenesis of hepatic ischemia-reperfusion injury is thought to involve the induction of inflammatory mediators. However, whether ECLM affects inflammatory mediators caused by warm hepatic ischemia-reperfusion injury and partial hepatectomy (HIRI+PH) has not been clarified. In this study, we evaluated the protective effects of ECLM against liver damage caused by HIRI+PH. Rats were fed a normal diet (HIRI+PH) or a normal diet with 2% ECLM (HIRI+PH and ECLM) for ten days, then the liver and duodenal ligament were clamped and subjected to 15 min of hepatic ischemia. After 70% hepatectomy, the inflow occlusion was released, and liver and blood samples were collected at 3, 6, and 24 h. The effect of ECLM on mortality induced by 30 min of ischemia and hepatectomy was evaluated. The results showed that ECLM attenuated pathological liver damage, including apoptosis, in the rats treated with HIRI+PH, and decreased serum aminotransferase activity; ECLM decreased mRNA levels of the inflammation-related genes inducible nitric oxide synthase and C-X-C motif chemokine ligand 1, and increased mRNA levels of interleukin 10, an anti-inflammatory cytokine; ECLM increased hepatocyte growth factor mRNA levels and Ki-67 labeled nuclei in the liver at 24 h; ECLM significantly reduced HIRI+PH-induced mortality. In conclusion, ECLM may prevent HIRI+PH-induced liver injury in part by suppressing various inflammatory responses and promoting liver regeneration.

HGF Regulation in the Tumor-Supportive Microenvironment Combined with Pan-PIM Kinases Inhibition in Malignant Cells As a Multi-Target Approach for Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), persistent clonal proliferation of malignant myeloid cells results in high severity and low cure potential. Resistance to currently available chemotherapeutics and disease relapse remains an important concern worldwide. Several molecular targets play key roles in impairing therapeutic response, including PIM-kinases, a serine/threonine kinase family associated with the progression of hepatocyte growth factor (HGF)-driven cancers through its receptor, MET. We reported that enhanced HGF secretion by bone marrow (BM) mesenchymal stromal cells (MSC) correlated with low levels of the serine protease inhibitor kunitz-type2 (SPINT2) in AML (Roversi et al., 2019), which are affected by immunosuppressive properties of the tumor microenvironment (TME). However, the interplay between SPINT2, HGF, and PIM-kinases in the TME is yet to be elucidated and could represent an important multi-target therapeutic strategy in hematological cancers. Aims: Given the importance of HGF to AML prognosis and progression, we analyzed the interplay between SPINT2, HGF, and pan-PIM kinases by evaluating the gene expression and the effects of a novel pan-PIM kinase inhibitor, PIM447 (Novartis), in leukemia cells and in the TME. Methods: SPINT2, HGF, PIM1, PIM2 and PIM3 mRNA expression data were obtained from OHSU (2018) AML study and TCGA (cBio Portal). Correlation analysis was obtained by Spearman's coefficient (r). A total of 5 healthy donors (HD) and 5 de novo AML patients BMMSC (CD90+CD105+CD73+CD45-CD34-CD31-HLA-DR-) were treated with 5-Azacitidine (Aza), and evaluated for SPINT2 gene expressions. Myeloid (OCI-AML3, MOLM13, U937) and lymphoblastic (RS4;11, Jurkat) leukemia cells were treated with increasing concentrations of PIM447, and the 101nd apoptosis rates were determined. Peripheral blood and BM mononuclear cells (PBMC/BMMC) from HD were tested for cytotoxicity. Leukemia cells were also examined for reactive oxygen species (ROS), nitric oxide (NO) production, and mitochondrial membrane potential (ΔΨm). To verify PIM447 action onto an immunosuppressive TME, cocultures of leukemia cells and monocytes or monocyte-derived macrophages (MDMs) were performed. To understand the interplay between SPINT2 and PIM-kinases, cocultures of SPINT2-overexpressed MSC and leukemia cells, were performed. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests ( P<.05). Results: The analysis of gene expression performed in MC from 188 AML patients (age median: 57.5 years [range: 18-88]) showed that levels of HGF correlated positively with PIM1, PIM2, and PIM3 ((r) =.8727;.9094;.9093, respectively, P<.0001) and negatively with SPINT2 (r=-.06402). We also verified that SPINT2 mRNA expression was upregulated in AML-BMMSC ( P<.05) after Aza treatment when compared to HD-BMMSC, suggesting that this gene is methylated in AML. Our in vitro results showed PIM447 treatment (2-20μM) yielded reduced cell viability and increased apoptosis in leukemia cells [IC50 range: 12.2-22.2μM, P<.01] without affecting HD cells. PIM447 reduced ROS, NO production, and ΔΨm (all P<.0001) in all cell lines. PIM447 treatment of macrophages differentiated from HD-PBMC revealed a shorter population of M2 macrophages (HLA-DR-CD206+) ( P<.001) and an increased population of M1 macrophages (HLA-DR+CD80+) ( P<.05). Moreover, PIM447 promoted apoptosis of leukemia cells co-cultured with MDMs ( P<.0001). Interestingly, co-culture analyses of SPINT2-overexpressed MSC enhanced PIM447 cytotoxic activity towards leukemia cells (P<.05) and reduced cell-cell adhesion by diminishing CD49b, CD49d, and CD49e (P<.05), suggesting an interplay between SPINT2 and pan-PIM kinases Conclusion: Collectively, our data identify a novel pan-PIM kinase inhibitor, PIM447, which effectively induces leukemia cell apoptosis through reducing MET receptor activation as well as acts on their tumor-supportive microenvironment by inducing a phenotypic shift from immunosuppressive M2 to anti-tumor M1 macrophages. PIM447 also interplays with SPINT2/HGF pathway in the TME, reducing AML progression. These findings suggest that a pivotal strategy to treat AML could be a multi-target therapy, acting both on leukemia cells and in the tumor microenvironment. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

In vitro differentiated human CD4+ T cells produce hepatocyte growth factor.

Differentiation of naive CD4+ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4+ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.

The Association of rs5745687 Polymorphism Located at HGF Gene with Risk of Gastric and Breast Cancer in the Helicobacter Positive Patients of Isfahan Population

Background: Hepatocyte growth factor (HGF) protein regulates cell growth, motility, and morphogenesis in a variety of cells and tissues by binding to the HGF receptor. The rs5745687 SNPs in the introns of the HGF gene could affect the splicing and expression of HGF mRNA. Objectives: In this study, the genotype frequency of rs5745687 in breast cancer (BC) and gastric cancer (GC) (positive helicobacter) patients has been investigated and compared with the healthy controls in the Isfahan population. Methods: Firstly, initial bioinformatics studies were done. Then, according to the results, bioinformatics high-resolution melt (HRM) and real-time PCR were recruited to determine genotypes rs5745678 for 432 participants in the case-control analysis (84 GC with 126 healthy control samples, as well as 111 BC cases with 111 normal controls). The conditional logistic regression model was used to measure odds ratios (OR) and 95% confidence intervals (CI) to produce these cancers based on genotype frequency. Results: The homozygote genotype of the mutant (G) allele of rs5745678 has a significant association with the lower risk of gastric cancer (P-value < 0.0001) and this allele can increase the risk of GC in a co-dominant model (OR: 5.541, P-value < 0.0001). Also, the rs5745678 SNP had a significant association with the clinicopathological features (age, smoking, Helicobacter Pylori infection) in GC patients. Conclusions: The presence of a single G allele in rs5745678 heterozygote (AG/AA) and co-dominant (AG/AA+GG) models could significantly impact GC pathogenicity in different ways. There was no significant correlation between the rs5745678 polymorphism and BC (P-value: 0.671) in the studied sample size.

Post-ischemic protection of hepatocyte growth factor requires the type II transmembrane serine protease matriptase-A reciprocal regulation of the two for neuroprotection in stroke brain.

In a rat middle cerebral artery occlusion (MACo) model of ischemic stroke, intracerebroventricular administration of human recombinant hepatocyte growth factor (HGF) mitigated motor impairment and cortical infarction. Recombinant HGF reduced MCAo-induced TNFα and IL1β expression, and alleviated perilesional reactivation of microglia and astrocyte. All of the aforementioned beneficial effects of HGF were antagonized by an inhibitor to the type II transmembrane serine protease matriptase (MTP). MCAo upregulated MTP mRNA and protein in the lesioned cortex. MTP protein, not the mRNA, was increased further by recombinant HGF but reduced when MTP inhibitor (MTPi) was added to the treatment. Changes of the endogenous active HGF by MCAo, HGF or MTPi paralleled with the changes of MTP protein under the same conditions whilst neither HGF mRNA nor the total endogenous HGF protein were altered. These data showed that the therapeutic effects of HGF in stroke brain is attributed to its proteolytic activation and that MTP is a main protease of the event. MCAo enhanced MTP mRNA and thus protein expression; the initial use of the recombinant active HGF stabilized MCAo-induced MTP protein and subsequent activation of endogenous latent HGF which in turn stabilized further MTP protein. A reciprocal regulation between MTP and HGF appears to be present where MTP promotes HGF activation and the active HGF prevents MTP protein turnover. This study, for the first time, shows that MTP can participate in neural protection in stroke brain through activation of HGF. The cycles of HGF-MTP regulation achieved preservation of the neurological activity.

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells.

Background: CRISPR/Cas9 system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Hepatocyte growth factor (HGF) is a well-known growth factor that plays a crucial role in cell growth and organ development. According to recent studies, it has been reported that HGF promoted growth of hepatocellular carcinoma (HCC) cells. Here, we investigated the apoptotic effects in HCC cells. Methods: Crispr-HGF plasmid was constructed using GeneArt CRISPR Nuclease Vector. pMex-HGF plasmid that targets HGF overexpressing gene were designed with pMex-neo plasmid. We performed real time-polymerase chain reaction to measure the expression of HGF mRNA. We performed cell counting assay and colony formation assay to evaluate cell proliferation. We also carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. Furthermore, we performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. Collectively, we performed annexin V/PI staining assay and Western blot assay. Results: In Crispr-HGF-transfected group, the mRNA expression levels of HGF were markedly downregulated compared to pMex-HGF-transfected group. Moreover, Crispr-HGF inhibited cell viability in HCC cells. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Expression of the phosphorylation of mitogen activated protein kinases and c-Met protein was regulated in Crispr-HGF-transfected group. Interestingly, we found that the expression of HGF protein in conditioned media significantly decreased in Crispr-HGF-transfected group. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh7 and Hep3B cells.

Abstract LB215: Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is one of the most common cancers. Ultraviolet radiation exposure leads to DNA damage in keratinocyte stem cells leading to transformation. Epidermal cancer stem cells (ECS cells) display aggressive spheroid formation, invasion, and migration, and form rapidly growing and highly vascularized tumors. The transglutaminase 2 (TG2) cancer stem cell maintenance proteins is present at high levels in ECS cells and is required for maintenance of the ECS cell phenotype. Identifying how TG2 maintains the ECS cell phenotype is an important goal. RNA transcriptomic analysis, comparing gene expression in wild-type and TG2 knockout cells, reveals that hepatocyte growth factor (HGF) signaling is markedly reduced in TG2 knockout cells, a finding that was confirmed by showing that TG2 knockdown/knockout reduces HGF and MET mRNA and protein. HGF/MET signaling appears to play an important role in maintaining the cancer phenotype, as inhibition of HGF/MET reduces ECS cell spheroid formation, invasion and migration. Stem cell and EMT marker expression and MEK1/2 and ERK1/2 activity are also reduced. These changes phenocopy the response to TG2 knockdown. Treatment with MEK1/2 and ERK1/2 inhibitors suppresses the ECS cell phenotype indicating that MAPK signaling is a major HGF/MET target, and MET knockout reduces ECS cell tumor formation which is associated with reduced MEK1/2 and ERK1/2 activity. These findings suggest that TG2 maintains HGF/MET signaling which stimulates MEK1.2 and ERK1/2 activity to enhance the aggressive ECS cell cancer phenotype and drive tumor formation. Citation Format: Xi Chen, Gautam Adhikary, Suruchi Shrestha, Wen Xu, Richard Eckert. Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB215.

MiR-93-5p promotes insulin resistance to regulate type2 diabetes progression in HepG2 cells by targeting HGF.

Insulin resistance is a common feature of type 2 diabetes mellitus (T2DM). However, the mechanisms underlying insulin resistance are not completely understood. The present study aimed to investigate the effect of microRNA (miR)-93-5p on insulin resistance in T2DM cells. Human hepatocellular carcinoma (HCC; HepG2) cells were cultured in medium with high glucose content (30 mM glucose) to establish an in vitro insulin-resistant cell model (IR group). Glucose consumption and glycogen synthesis assays were performed to assess glucose consumption and glycogen synthesis, respectively. By performing immunoprecipitation assays, the abundance of the Met-insulin receptor complex was detected in HepG2 cells. miR-93-5p and hepatocyte growth factor (HGF) mRNA expression levels were measured via reverse transcription-quantitative PCR, and HGF protein expression levels were measured via western blotting. A dual-luciferase reporter assay was conducted to investigate the interaction between miR-93-5p and HGF. Cell Counting Kit-8, BrdU and caspase-3 activity assays were performed to evaluate cell viability, proliferation and apoptosis, respectively, in insulin-resistant HepG2 cells following transfection with small interfering RNA-HGF, HGF overexpression vector, miR-93-5p mimic or miR-93-5p inhibitor. The results demonstrated that miR-93-5p expression was significantly increased and HGF expression was significantly decreased in HCC tissues isolated from patients with or without T2DM compared with adjacent healthy tissues isolated from patients without T2DM. Compared with the IR group, miR-93-5p overexpression significantly increased cell proliferation, glucose consumption and glycogen synthesis, but significantly inhibited apoptosis in insulin-resistant HepG2 cells. By contrast, compared with the IR group, HGF overexpression significantly inhibited cell proliferation, glucose consumption and glycogen synthesis, but significantly enhanced cell apoptosis in insulin-resistant HepG2 cells. Following co-transfection with HGF overexpression vector and miR-93-5p mimic, miR-93-5p mimic-mediated induction of HepG2 cell proliferation, glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells was inhibited. Collectively, the results of the present study indicated that miR-93-5p enhanced insulin resistance to regulate T2DM progression in HepG2 cells by targeting HGF.

Abnormalities in reparative function of lung-derived mesenchymal stromal cells in emphysema.

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in chronic obstructive pulmonary disease (COPD). We hypothesized that lung-derived MSCs (LMSCs) from patients with emphysema are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged microenvironment. LMSCs were isolated from the lung tissue of controls and patients with severe emphysema and characterized at baseline. In addition, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential, and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of FGF10 and HGF messenger RNA (mRNA) and hepatocyte growth factor (HGF) and decorin protein. When seeded on control decellularized lung tissue scaffolds, control- and COPD-derived LMSCs showed no differences in engraftment, proliferation, or survival within 2 wk, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and the ability to deposit new matrix were not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from patients with COPD compared with controls show less expression of FGF10 mRNA, HGF mRNA and protein, and decorin protein, whereas other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation, and functioning on native lung tissue scaffolds. The damaged, emphysematous microenvironment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema.

Chronic Fetal Leucine Infusion Does Not Potentiate Glucose-Stimulated Insulin Secretion or Affect Pancreatic Islet Development in Late-Gestation Growth-Restricted Fetal Sheep

Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic β-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising β-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, β-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50-100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising β-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and β-cell consequences in IUGR fetuses.

Alleviation of remote lung injury following liver ischemia/reperfusion: Possible protective role of vildagliptin

Lung injury is a serious condition encountered following hepatic ischemia/reperfusion (IR). This study aimed to explore whether a dipeptidyl peptidase-4 inhibitor agent vildagliptin (V) could alleviate the lung injury caused by hepatic IR in a rat model and if so elucidate its molecular protective mechanism. Three groups of rats were used. Sham group: received normal saline and exposed to a sham operation, IR group: received normal saline and subjected to the operation of hepatic I (45 min)/ R (180 min), V+IR group: received for 10 days intraperitoneal injection of V (10 mg/kg/day). After reperfusion, liver and lung were collected for biochemical and histological evaluation. Hepatic IR exhibited significant elevation in serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) enzyme levels, serum and lung malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) in addition to lung nitric oxide (NO) levels, hypoxia-inducible factor 1-alpha (HIF-1α) mRNA and protein levels, hepatocyte growth factor (HGF) mRNA expression, and inducible nitric oxide synthase (iNOS) mRNA and protein expressions in lung tissue along with a marked reduction in the serum and lung content of catalase in comparison to the sham group. Moreover, liver and lung injury in the IR group was detected by histopathological examination. Vildagliptin ameliorated markedly the biochemical changes as well as liver and lung architecture in comparison to the IR group. Vildagliptin mitigated the induced lung injury by hepatic IR via suppression of oxidative stress markers, pro-inflammatory cytokine TNF-α as well as the HIF1-α/iNOS/HGF expressions in lung tissue.

A Chronic Fetal Leucine Infusion Potentiates Fetal Insulin Secretion and Increases Pancreatic Islet Size, Vascularity, and β Cells in Late-Gestation Sheep

Background:Infusion of a complete amino acid mixture into normal late-gestation fetal sheep potentiates glucose-stimulated insulin secretion (GSIS). Leucine acutely stimulates insulin secretion in late-gestation fetal sheep and isolated fetal sheep islets in vitro. Objectives:We hypothesized that a 9-d leucine infusion would potentiate GSIS in fetal sheep. Methods:Columbia-Rambouillet fetal sheep at 126 days of gestation received a 9-d leucine infusion to achieve a 50%–100% increase in leucine concentrations or a control infusion. At the end of the infusion we measured GSIS, pancreatic morphology, and expression of pancreatic mRNAs. Pancreatic islet endothelial cells (ECs) were isolated from fetal sheep and incubated with supplemental leucine or vascular endothelial growth factor A (VEGFA) followed by collection of mRNA. Data measured at multiple time points were compared with a repeated-measures 2-factor ANOVA. Data measured at 1 time point were compared using Student's t test or the Mann–Whitney test. Results:Glucose-stimulated insulin concentrations were 80% higher in leucine-infused (LEU) fetuses than in controls (P < 0.05). In the pancreas, LEU fetuses had a higher proportion of islets >5000 μm2 than controls (75% more islets >5000 μm2; P < 0.05) and a larger proportion of the pancreas that stained for β cells (12% greater; P < 0.05). Pancreatic and pancreatic islet vascularity were both 25% greater in LEU fetuses (P < 0.05). PancreaticVEGFAand hepatocyte growth factor (HGF) mRNA expressions were 38% and 200% greater in LEU fetuses than in controls (P < 0.05), respectively. In isolated islet ECs,HGFmRNA was 20% and 50% higher after incubation in ce:supplemental leucine (P < 0.05) or VEGFA (P < 0.01), respectively. Conclusions:A 9-d leucine infusion potentiates fetal GSIS, demonstrating that glucose and leucine act synergistically to stimulate insulin secretion in fetal sheep. A greater proportion of the pancreas being comprised of β cells and higher pancreatic vascularity contributed to the higher GSIS.

SO023HEPATOCYTE GROWTH FACTOR (HGF) PRECLUDE HIGH-FAT DIET-INDUCED OBESITY AND IMPROVED INSULIN RESISTANCE IN MICE

Abstract Background and Aims Obesity and its associated chronic inflammation in adipose tissue initiate insulin resistance, which is related to several pathologies including hypertension and atherosclerosis. Previous reports demonstrated that circulating hepatocyte growth factor (HGF) level was associated with obesity and type 2 diabetes. However, its precise role in obesity and related-pathology is unclear. Method In this experiment, cardiac-specific over-expression of human HGF in mice (HGF-Tg mice) which showed 4-5 times higher serum HGF levels than wild-type mice were used. We chose cardiac specific HGF overexpression, as other strain of HGF transgenic mice such as liver and kidney specific HGF overexpression mice develop cancer and cystic diseases, which are rare in the heart. In the present study, using HGF-Tg mice and anti-HGF neutralizing antibody (HGF-Ab), we explored the role of HGF in obese and insulin resistance induced by high fat diet (HFD) for 14 weeks (200 or 400ug/week). Results With normal chow diet (ND), there were no significant changes in body weight between WT and HGF-Tg mice. While body weight in wild-type mice fed with HFD for 14 weeks was significantly increased accompanied with insulin resistance, HGF-Tg mice prevented body weight gain and insulin resistance. Insulin resistance in obesity arises from the combination of altered functions of insulin target cells (e.g., liver, skeletal muscle, and adipose tissue) and the accumulation of macrophages that secrete pro-inflammatory mediators in adipose tissue. The accumulation of macrophages and elevated levels of inflammatory mediators in adipose tissue were significantly inhibited in HGF-Tg mice as compared to wild-type mice. In the gWAT, the mRNA levels of the mature macrophage marker F4/80, the chemoattractants, MCP-1 and CXCL2, and the inflammatory cytokines, such as TNF-α and iNOS, were significantly increased in WT mice fed with HFD. However, these levels were markedly reduced in HGF-Tg mice fed with HFD. Additionally, activation of Akt by insulin administration was significantly reduced in the gWAT SM, and liver by HFD; however, this activation was restored in HGF-Tg mice. Moreover, insulin-induced Akt signaling was decreased in HGF-Ab groups as compared to saline group under HFD condition. Importantly, HFD significantly increased the level of HGF mRNA by approximately 2 fold in gWAT, SM, and liver without changing cMet expression. All together, these data indicate that the HGF as one of the systemic gWAT, SM, and liver-derived growth factor plays a role in compensatory mechanism against insulin-resistance through the at least anti-inflammatory effect in adipose tissue. The HFD-induced obesity in wild-type mice treated with HGF-neutralizing antibody showed an exacerbated response to the glucose tolerance test. Conclusion HGF suppresses inflammation in adipose tissue induced by a high-fat diet, and as a result improves systemic insulin resistance. These gain-of-function and loss-of-function studies demonstrated that the elevated HGF level induced by HFD have protective role against obesity and insulin resistance.

Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease.

Background: Chemical modification of mRNA (mmRNA) substantially improves their stability and translational efficiency within cells. Nanofibrillar collagen scaffolds were previously shown to enable the spatially localized delivery and temporally controlled release of mmRNA encoding HGF both in vitro and in vivo. Materials &methods: Herein we developed an improved slow-releasing HGF mmRNA scaffold and tested its therapeutic efficacy in a porcine model of peripheral arterial disease. Results & conclusion: The HGF mmRNA wasreleased from scaffolds in a temporally controlled fashion in vitro with preserved transfection activity. The mmRNA scaffolds improved vascular regeneration when sutured to the ligated porcine femoral artery. These studies validate the therapeutic potential of HGF mmRNA delivery from nanofibrillar scaffolds for treatment of peripheral arterial disease.

Efficacy of autologous bone marrow mononuclear cell transplantation in dogs with chronic spinal cord injury.

Background:Spinal cord injury (SCI) is relatively common in dogs and is a devastating condition involving loss of sensory neurons and motor neurons. However, the main clinical protocol for the management of SCI is surgery to decompress and stabilize the vertebra. Cell transplantation therapy is a very promising strategy for the treatment of chronic SCI, but extensive preclinical and clinical research work remains.Aim:The aim of this study is to confirm the effect of bone marrow-derived mononuclear cell (BM-MNC) transplantation for chronic SCI in dogs.Methods:We tested the treatment efficiency of chronic SCI in 12 dogs using BM-MNC transplantation. Neurological evaluation used the Texas Spinal Cord Injury Scale (TSCIS). Concurrently, we characterized the transplanted cells by evaluation using quantitative real-time polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay.Result:All dogs had a pre-transplantation TSCIS score of 0. Two animals did not show any improvement in their final TSCIS scores. The remaining 10 dogs (83.4%) achieved improvement in the final TSCIS scores. Five of them (41.7%) regained ambulatory function with a TSCIS score greater than 10. We determined that canine BM-MNCs expressed hepatocyte growth factor (HGF) mRNA at higher levels than other cytokines, with significant increases in HGF levels in cerebrospinal fluid within 48 hours after autologous BM-MNC transplantation into the subarachnoid space of the spinal dura matter in dogs.Conclusions:BM-MNC transplantation may be effective for at least some cases of chronic SCI.

Fibrin Deposit on the Peritoneal Surface Serves as a Niche for Cancer Expansion in Carcinomatosis Patients

Peritoneal metastasis (PM) is a very serious complication of gastrointestinal and gynecological malignancies which is poorly documented. Modified mesothelial cell layer and their microenvironments can favor fibrin deposition for cancer cell adhesion. Scanning and transmission electron microscopy of peritoneal surface and cancer cell clusters from cancer patients was done. Ascites and its impact on mesothelial cells were assessed by cytokine array. Neprilysin, matrix metalloprotease, epithelial mesenchymal transition (EMT) related molecules (E-cadherin, Snail, Slug, Twist, Vimentin and Fibronectin), tissues factor (TF), endothelial protein C receptors (EPCR) were quantified by q-PCR. Fibrin in the simples were stained using anti fibrin F1E1 antibody. Migration ability was assessed by scratch assay. Cell viability and neprilysin activity were analyzed by bioluminescence. Cancer cells-fibrin interaction was investigated by scanning electron microscopy (SEM) and microcinematography (MCG). Mesothelial cells change their morphology after incubation with carcinomatosis peritoneal fluids in vitro. EMT associated with upregulation of neprilysin, matrix metalloproteinase-2, tissue factor and cytokines secretions such as interleukin-6, and 8, hepatocyte growth factor and granulocyte chemotactic protein-2 mRNA and protein were observed. EPCR expression as a natural anticoagulant was decreased. In parallel, carcinomatosis cell clusters extracted from peritoneal fluids were found to be associated with fibrin. Kinetic analysis of cancer cell-fibrin interaction in vitro studied by MCG showed that fiber filaments generated from clots inhibited cancer cell adhesion on fibrin clots. These results indicated that fibrin deposit on the peritoneal surface serve as niches for cancer expansion in carcinomatosis patients.

Downregulation of lncRNA UCA1 facilitates apoptosis and reduces proliferation in multiple myeloma via regulation of the miR-1271-5p/HGF axis.

Long noncoding RNAs (lncRNAs) are considered to be a novel prognostic and therapeutic target in many cancers. This study identified dysregulation of lncRNA urothelial carcinoma associated 1 (UCA1) and hepatocyte growth factor (HGF) mRNA via the Gene Expression Omnibus (GEO) database, which was traced to the mutual target miRNA, miR-1271-5p, and their effects were explored in multiple myeloma (MM). RNA expression profiles of MM were downloaded from the GEO database and analyzed using R packages. The expression of RNAs in MM tissue samples and cells was evaluated through quantificational real-time polymerase chain reaction (qRT-PCR). A luciferase reporter assay was utilized to confirm the binding relationships between UCA1/HGF and miR-1271-5p. To assess cell proliferation and apoptosis, CCK-8 assays and flow cytometry were conducted. Additionally, tumor progression was demonstrated in vivo. LncRNA UCA1 and HGF expression was higher in the cells and samples of patients with MM than in normal plasma cells. miR-1271-5p was confirmed to be the target of lncRNA UCA1 and HGF and to be negatively correlated with them. Moreover, downregulation of lncRNA UCA1 and HGF inhibited cell proliferation and facilitated cell apoptosis in RPMI 8226 cells (human MM cell line). However, miR-1271-5p overexpression affected the proliferation decrease and apoptosis increase. Moreover, in vivo experiments indicated that down or upregulation of lncRNA UCA1 repressed or enhanced the tumor growth of MM, respectively, in xenograft models. LncRNA UCA1 promoted proliferation and inhibited apoptosis by regulating miR-1271-5p and HGF in the human MM cell line RPMI 8226. Our investigations might contribute to a better understanding of the lncRNA UCA1/miR-1271-5p/HGF axis as a potential therapeutic strategy in MM.

Identification of HGF as a novel target of miR-15a/16/195 in gastric cancer

Background Gastric malignancy is the third most frequently encountered cancer globally and have been documented to confer extremely poor prognosis, given their limited treatment options. The up-regulation of hepatocyte growth factor (HGF) has been found in various tumor tissues, including GC tissue, and has been linked with tumor development. Nevertheless, the pathways leading to HGF upregulation have yet to be fully explored. Methods Immunohistochemistry (IHC) assay was used to detect HGF expression in human gastric tumor tissues, while western blotting allowed quantification of protein levels. Bioinformatics tools were used to predict potential miRNA that may target HGF mRNA. Relative levels of miR-15a/16/195 as well as the target mRNA levels were analyzed with qRT-PCR. Direct targeting between miRNA and mRNA was then validated by luciferase assay. Finally, a mouse xenograft tumor model was selected to demonstrate the in vivo effects of miR-15a/16/195. Results HGF protein expressions were markedly raised, while miR-15a/16/195 levels were dramatically down-regulated in tumor tissues of GC. miR-15a/16/195 were shown to directly bind with the 3'-UTR of HGF mRNA. This study demonstrated that HGF can be repressed by overexpressed miR-15a/16/195, which resulted in the suppression of GC cell proliferation and migration. Furthermore, in the xenograft mouse model, miR-15a/16/195 were also found to have a tumor growth suppression effect. Conclusions miR-15a/16/195 suppresses tumorigenesis by targeting HGF and may have a potential therapeutic application in the clinical treatment of GC.

Microvesicles derived from human Wharton's Jelly mesenchymal stem cells ameliorate acute lung injury partly mediated by hepatocyte growth factor.

Several studies have highlighted the underlying role of mesenchymal stem cells microvesicles (MSC-MVs) in acute lung injury (ALI). Hepatocyte growth factor (HGF) derived from MSC-MVs is partly involved in their therapeutic effects; however, the detailed mechanism remains unclear. MVs were isolated from human Wharton's Jelly MSCs. The rat model of ALI was established by intratracheal instillation of bleomycin (BLM). A co-culture model of alveolar epithelial cells or pulmonary endothelial cells and MSC-MVs was utilized. Total protein content in bronchoalveolar lavage fluid (BALF) was determined by bicinchoninic acid method. White blood cell (WBC) and neutrophil in BALF were counted. ELISA was used for the determination of cytokines and HGF in BALF. Apoptosis was determined by TUNEL assay and Annexin V-FITC/PI staining as well as caspase-3 activity detection. HE and Masson staining of lung tissues was used for histopathology analysis. The expression of HGF and proteins involved in the PI3K/AKT/mTOR pathway were measured by quantitative Real-Time PCR (qRT-PCR) and western blotting. Treatment with MSC-MVs significantly inhibited BLM-induced apoptosis and fibrosis in lung tissues and PI3K/AKT/mTOR activation, which was reversed by HGF mRNA deficient MVs. Intriguingly, these effects were completely abrogated by PI3K inhibitor. The therapeutic effect of MSC-MVs in ALI was partly mediated through HGF mRNA.

Influence of hepatocyte growth factor-transfected bone marrow-derived mesenchymal stem cells towards renal fibrosis in rats.

Background & objectives:Hepatocyte growth factor (HGF) produced by endothelial cells, fibroblasts, fat cells and other interstitial cells, can promote angiogenesis, repair damaged tissues and resist fibrosis. Mesenchymal stem cells (MSCs) are located in bone marrow and secrete a variety of cytokines and are often used in the repair and regeneration of damaged tissues. This study was aimed to investigate the influence of HGF-transfected bone marrow-derived MSCs towards renal fibrosis in rats.Methods:The HGF gene-carrying adenoviral vector (Ad-HGF) was transfected into MSCs, and the Ad-HGF-modified MSCs were transplanted into rats with unilateral ureteral obstruction (UUO). The localization of renal transplanted cells in the frozen section was observed with fluorescence microscope. The Masson's trichrome staining was performed to observe the renal collagen deposition, and the immunohistochemistry was performed to detect the expressions of α-smooth muscle actin (α-SMA) and HGF in renal tissues. Reverse transcription (RT)-PCR was used to detect the mRNA expressions of α-SMA, HGF and fibronectin (FN).Results:Ad-HGF-modified MSCs could highly express HGF in vitro. On the post-transplantation 3rd, 7th and 14th day, the 4',6-diamidino-2-phenylindole (DAP)-labelled transplanted cells were seen inside renal tissues. Compared with UUO group, the renal collagen deposition in transplantation group was significantly reduced, and the expressions of α-SMA mRNA and protein were significantly decreased, while the expressions of HGF mRNA and protein were significantly increased, and the expression of FN mRNA was significantly decreased (P<0.001).Interpretation & conclusions:Trans-renal artery injection of HGF-modified MSCs can effectively reduce the renal interstitial fibrosis in UUO rat model.