HGF Gene Expression Research Articles

Methionine can subside hair follicle development prejudice of heat-stressed rex rabbits.

In the present experiment, we study the function of methionine on hair follicle development in heat-stressed Rex Rabbits and its potential molecular mechanism. Rex rabbits were randomly divided into 5 groups (30 replicates per group): control group (20-25°C, fed basic diet), heat stress group (30-34°C, fed basic diet), heat stress + methionine group (30-34°C, fed 0.15% methionine in addition to the basic diet). fed basic diet (control), heat stress + methionine group (30-34°C, fed 0.3% methionine in addition to the basic diet), heat stress + methionine group (30-34°C, fed 0.45% methionine in addition to the basic diet). The results show that heat stress decreases the hair follicle density of Rex rabbits, and the diet methionine addition significantly increases the hair follicle density of heat-stressed Rabbits (p < .05). Heat stress increased serum HSP70 concentration and skin HSP70 gene expression, 0.15%-0.3% methionine but not 0.45% addition alleviated the effect of heat stress. Dietary 0.15% methionine addition significantly increases the gene expression of Wnt10b, β-catenin, LEF, FZD4, LRP6, Shh, HGF, EGF, and Noggin in heat-stressed Rex rabbits and observably decreases the gene expression of BMP2/4 and TGFb. There was no significant effect of methionine on the expression of IGF1 and FGF5/7 gene expression. In conclusion, methionine maybe promotes hair follicle development via TGFβ-BMP/Shh-Noggin, Wnt10b/β-catenin, EGF, and HGF signaling pathways in heat-stressed rabbits.

Hgf gene expression in rat hepatocytes under conditions of glutamate-induced steatohepatosis and when using drugs with pro- and prebiotic action

Hgf gene expression in rat hepatocytes under conditions of glutamate-induced steatohepatosis and when using drugs with pro- and prebiotic action

A drug screen with approved compounds identifies amlexanox as a novel Wnt/β-catenin activator inducing lung epithelial organoid formation.

Background and Purpose:Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signalling and assess its potential to activate lung epithelial cells and repair.Experimental Approach:We screened 1216 human-approved compounds for Wnt/β-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model.Key Results:The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt/β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces proregenerative signalling in emphysema.Conclusion and Implications:Using a drug screen based on Wnt/β-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

DEVELOPMENT OF MULTIPLE MYELOMA CELL LINES WITH HIGH EXPRESSION OF HEPATOCYTE GROWTH FACTOR

Objective:&#x0D; To determine the overexpress HGF gene in non HGF producing humane myeloma cell lines (INA-6).&#x0D; Materials and Methods:&#x0D; In this study, retroviral- expression constructs were designed and constructed. The sequence of the cloned fragments in the vector constructs was determined and the effect of construct was examined using qRT-PCR and Western blot analysis. At department of cancer research &amp; molecular medicine, norwegien university of science &amp; technology, Trondheim Norway, in June 2016 to April 2018.&#x0D; Results:&#x0D; We established expression constructs based on HGF mRNA sequence. The HGF non producing human myeloma cell line INA-6 transduced with lentiviral HGF-overexpression transduction particles, were analyzed for their effect on the HGF gene expression. HGF overexpression was analyzed both at mRNA level and protein levels. Two myeloma cell line INA-6 HGF-1 and INA-6HGF-2 showed significant over expression of HGF mRNA and protein.&#x0D; Conclusion:&#x0D; In conclusion, the HGF overexpression cell lines can be used as tissue culture models to investigate the role of HGF protein in pathology of multiple myeloma.

ESTABLISHMENT OF MULTIPLE MYELOMA CELL LINES WITH REDUCED EXPRESSION OF HEPATOCYTE GROWTH FACTOR USING RNAI APPROACH

Objective: To establish the gene knockdown system (RNAi) using different Multiple Myeloma Cell lines (MMCL). Material and Methods: RNAi is generally used in biomedical research to study the outcome of knockdown of gene expression. In this research, retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) were designed and constructed. The sequence of the cloned fragments in the vector constructs was determined, and the effect of constructs in MMCLs was examined using qRT-PCR and Western blot analysis. Results: In this work, we established a method for making retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) design. The HGF producing human myeloma cell line JJN-3 transduced with lentiviral HGF-shRNA transduction particles were analyzed for their effect on the HGF gene expression. HGF knockdown MMCL was analyzed both at mRNA level and protein levels. Two out of five clones of myeloma cell line JJN-3 (JJN3-sh3308 and JJN3-sh47137) were transduced with lentiviral HGF–shRNA transduction particles were showing 50-60 % knockdown in their HGF mRNA and protein expression. Conclusion: HGF knockdown cell lines can be used as tissue culture models to investigate the role of HGF protein in the pathology of multiple myeloma. Further, the retroviral-HGF-shRNA expression constructs can study HGF response in various multiple myeloma via gene knockdown studies.

Comparative study on effect of mesenchymal stem cells and endothelial progenitor cells on treatment of experimental CCL4-induced liver fibrosis

Background We speculated impacts of BM-MSCs and UC-EPCs on reversal of hepatic injury induced by carbon tetrachloride (CCl4). Fifty adult rats were divided into five groups: control group, CCl4A group, CCl4B group, CCl4/BM-MSCs group and CCl4/UC-EPCs group. Blood samples were driven to measure concentration of albumin and ALT. Quantitative expression of HGF, TGF-β, MMP-2, and VEGF were assessed by PCR. Histological and immunohistochemistry examination of the liver tissue were performed. Results There was elevating albumin (p < .05) and reducing ALT (p < .05) concentrations in groups treated with BM-MSCs and UC-EPCs compared to untreated CCL4A&B groups. UC-EPCs treated group have significantly higher MMP-2 and VEGF (p < .01) genes expression than BM-MSCs treated group. Furthermore, UC-EPCs were more valuable than BMMSCs in increasing gene expression of HGF (p < .05) and immunohistochemistry of α-SMA and Ki-67 (p < .01). BM-MSCs have significantly lower TGF-β (p < .00) compared to UC-EPCs. Conclusion This study highlighted on liver regeneration role of both UC-EPCs and BM-MSCs in liver fibrosis.

Comparative Study on Effect of Mesenchymal Stem Cells and Endothelial Progenitor Cells on Treatment of Experimental CCL4 Induced Liver Fibrosis

Abstract Background Bone marrow mesenchymal stem cells (BM-MSCs) and human umbilical cord endothelial progenitor cells (UC-EPCs) have several benefits for liver regeneration. We speculate huge impacts of rat BM-MSCs and UC-EPCs on reversal of hepatic injury and improvement of liver function in liver fibrosis induced by carbon tetrachloride (CCl4). Methods Forty adult rats were divided into 4 groups; control group, CCl4 group, CCl4/BM-MSCs group and CCl4/UCEPCs group. Blood samples were driven from rats at 1, 2 and 3months to measure serum concentration of albumin and alanine aminotransferase (ALT). Quantitative expression of HGF,TGF-β, MMP-2, and VEGF were assessed by polymerase chain reaction. Histological examination of the liver tissue was performed. α-SMA immunohistochemistry to identify the myoepithelial cells (MECs) and Ki-67 to identify cell prolifration immunohistochemistry are detected in groups injected with cells to confirm cells regeneration. Results Regarding liver function, there was elevating albumin (P &amp;lt; 0.05) and reducing ALT (P &amp;lt; 0.05) concentrations in groups treated with BM-MSCs and UC-EPCs effect compared to untreated CCL4 group. Concerning gene expression, UC-EPCs treated group have significantly higher MMP-2 and VEGF (P &amp;lt; 0.01) genes expression than BM-MSCs treated group. Furthermore, UC-EPCs were more valuable than BM-MSCs in increasing gene expression of HGF (P &amp;lt; 0.05) and immunohistochemistry of α-SMA and Ki-67 (P &amp;lt; 0.01). BM-MSCs have significantly lower TGF- β (P &amp;lt; 0.00) compared to UC-EPCs. Conclusion This study highlighted on liver regeneration role of both human UC-EPCs and BM-MSCs in liver fibrosis by different signaling mechanistic.

Moderate hyperthermic heating encountered during thermal ablation increases tumor cell activity

Purpose The aim of this study was to determine whether moderate hyperthermic doses, routinely encountered in the periablational zone during thermal ablation, activate tumor cells sufficiently to secrete pro-tumorigenic factors that can induce increased proliferation. Material and methods R3230 rat mammary tumor cells and human cancer cell lines, MCF7 breast adenocarcinoma, HepG2 and Huh7 HCC, and HT-29 and SW480 colon adenocarcinoma, were heated in to 45 ± 1 °C or 43 ± 1 °C in vitro for 5-10 min and incubated thereafter at 37 °C for 1.5, 3 or 8 hr (n = 3 trials each; total N = 135). mRNA expression profiles of cytokines implicated in RF-induced tumorigenesis including IL-6, TNFα, STAT3, HGF, and VEGF, were evaluated by relative quantitative real-time PCR. HSP70 was used as control. c-Met and STAT3 levels were assessed by Western blot. Finally, naïve cancer cells were incubated with medium from R3230 and human cancer cells that were subjected to 43–45 °C for 5 or 10 min and incubated for 3 or 8 h at 37 °C in an xCELLigence or incuCyte detection system. Results Cell-line-specific dose and time-dependent elevations of at least a doubling in HSP70, IL-6, TNFα, STAT3, and HGF gene expression were observed in R3230 and human cancer cells subjected to moderate hyperthermia. R3230 and several human cell lines showed increased phosphorylation of STAT3 3 h post-heating and increased c-Met following heating. Medium of cancer cells subject to moderate hyperthermia induced statistically significant accelerated cell growth of all cell lines compared to non-heated media (p < 0.01, all comparisons). Conclusion Heat-damaged human tumor cells by themselves can induce proliferation of tumor by releasing pro-tumorigenic factors.

Stepwise participation of HGF/MET signaling in the development of migratory muscle precursors during vertebrate evolution

BackgroundThe skeletal musculature of gnathostomes, which is derived from embryonic somites, consists of epaxial and hypaxial portions. Some hypaxial muscles, such as tongue and limb muscles, undergo de-epithelialization and migration during development. Delamination and migration of these myoblasts, or migratory muscle precursors (MMPs), is generally thought to be regulated by hepatocyte growth factor (HGF) and receptor tyrosine kinase (MET) signaling. However, the prevalence of this mechanism and the expression patterns of the genes involved in MMP development across different vertebrate species remain elusive.ResultsWe performed a comparative analysis of Hgf and Met gene expression in several vertebrates, including mouse, chicken, dogfish (Scyliorhinus torazame), and lamprey (Lethenteron camtschaticum). While both Hgf and Met were expressed during development in the mouse tongue muscle, and in limb muscle formation in the mouse and chicken, we found no clear evidence for the involvement of HGF/MET signaling in MMP development in shark or lamprey embryos.ConclusionsOur results indicate that the expressions and functions of both Hgf and Met genes do not represent shared features of vertebrate MMPs, suggesting a stepwise participation of HGF/MET signaling in MMP development during vertebrate evolution.

Erythropoietin induces production of hepatocyte growth factor from bone marrow mesenchymal stem cells in vitro

BackgroundHepatocyte Growth Factor (HGF) plays a pivotal role in hematopoiesis, motility, growth and mobilization of hematopoietic stem/progenitor cells (HSPCs). HGF mainly is produced by bone marrow mesenchymal stem cells (BM-MSCs). MSCs express erythropoietin (EPO) receptor. In this study, we aimed to assess the effect of EPO on HGF secretion in BM-MSCs. MethodsThe BM-MSCs treated with EPO (4 IU/ml) for 6, 24 and 48 h. HGF gene expression and protein level were assessed using quantitative real time PCR (qRT-PCR) and Enzyme-linked immunosorbant Assay. In order to show the effect of secreted HGF on migration of HSPCs, hematopoietic stem cells (HSCs) were isolated from cord blood and evaluated using transwell migration assay. ResultsWe observed a significant increase in level of HGF in cell supernatant after 48 h compared to control group (P < 0.05). Also, qRT-PCR results demonstrated a significant elevation in HGF expression level after 24 and 48 h treatment with EPO compared to control group (P < 0.05). Finally, migration assay results showed a significant increase in migration of HSCs in treated group after 48 h. ConclusionOur data indicated that EPO may play an important role in stem cell mobilization through up regulating HGF in MSCs and inducing migration of HSCs.

Combined effect of bone marrow derived mesenchymal stem cells and nitric oxide inducer on injured gastric mucosa in a rat model.

To study the effect of intravenous injection of bone marrow mesenchymal stem cells (BMMSCs), alone and combined with NO inducer in gastric ulcer healing in a rat model. Rats were divided into controls, gastric ulcer, gastric ulcer receiving mesenchymal stem cells (MSCs), gastric ulcer receiving NO inducer (l-Arginine), gastric ulcer receiving MSCs plus NO inducer (l-Arginine) groups. MSCs were given in a dose of (106cells) by intravenous injection. l-Arginine was given 300mg/kg body weight intraperitoneally. 24h and 7days after BMMSCs and NO inducer injection, VEGF, PGE, TNF-α were assessed by ELISA. Gene expression of HGF, caspase-3, eNOS and BAX/Bcl-2 in gastric tissues were studied by real time PCR. Histopathology staining of gastric tissues was performed. Injection of MSCs or NO inducer or both to the gastric ulcer group significantly decreased caspase-3 and BAX genes expression (apoptotic factors) and increased Bcl-2 gene expression (anti-apoptotic factor) compared to that of the gastric ulcer group after both 24h and 7days with more significant results in the gastric group received both MSCs and NO inducer. HGF gene expression was significantly increased in the groups injected with MSCs or NO inducer or both compared with the corresponding gastric ulcer group (p<0.05, p<0.05 & p<0.001 respectively). There was a significant decrease in the mean PGE2 and TNF-α levels in the gastric ulcer group receiving MSCs, the gastric ulcer group receiving NO and the gastric ulcer group receiving both MSCs andNO compared to the gastric ulcer group after both 24h and 7days. Histopathological examination of gastric tissue of groups that received stem cells or NO alone, showed mucosal regenerative changes with increased thickness together with reduced inflammatory cellular infiltrate in the submucosa and decreased congestion. There was complete restoration in gastric mucosa in the group that received both stem cells and NO. Administration of MSCs, NO, or MSCs plus NO may exert a therapeutic effect on the mucosal lesion in gastric ulcer through their anti-inflammatory, angiogenic and antiapoptotic actions.

Decreased expression of hepatocyte growth factor in the nitrofen model of congenital diaphragmatic hernia.

Pleuroperitoneal folds (PPFs) are essential for normal diaphragmatic development, representing the only source of the diaphragm's muscle connective tissue. Hepatocyte growth factor (Hgf), which is secreted in PPFs, plays a crucial role in the formation of the muscular diaphragmatic components by regulating the migration of myogenic progenitor cells into the primordial diaphragm. Hgf is also a known downstream target of Gata4 and it has been demonstrated that the expression of Hgf was significantly downregulated in PPF cells of Gata4 knockouts with congenital diaphragmatic hernia (CDH). Furthermore, mutations in PPF-derived cells have been shown to result in CDH. We hypothesized that Hgf expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH. Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms (n=72) were divided into control and nitrofen-exposed specimens (n=12 per time-point and experimental group, respectively). Diaphragmatic gene expression of Hgf was analyzed by qRT-PCR. Immunofluorescence double staining for Hgf and the mesenchymal marker Gata4 or muscular progenitor marker Myogenin was performed to evaluate protein expression and localization in fetal diaphragms. Relative mRNA expression of Hgf was significantly downregulated in PPFs of nitrofen-exposed fetuses on D13 (3.08±1.46 vs. 5.24±1.93; p<0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (2.01±0.79 vs. 4.10±1.50; p<0.05) and fully muscularized diaphragms of nitrofen-exposed fetuses on D18 (1.60±0.78 vs. 3.21±1.89; p<0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic immunofluorescence of Hgf in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls, which was associated with disruptions in muscle connective tissue formation and reduced myogenic progenitor cell invasion. Decreased diaphragmatic expression of Hgf may disturb the formation of muscle connective tissue in PPFs and thus prevent essential migration of muscle progenitor cells into the developing diaphragm, leading to diaphragmatic defects in the nitrofen CDH model.

Study on inflammation-related genes and microRNAs, with special emphasis on the vascular repair factor HGF and miR-574-3p, in monocytes and serum of patients with T2D.

BackgroundRecently, we reported signs of inflammation (raised IL-8, reduced miR-146a) and signs of vascular repair (raised HGF) in the serum of Ecuadorian patients with type 2 diabetes (T2D). In contrast, we found that the circulating monocytes lacked up-regulation of classical inflammatory genes (IL-1B, IL-6, and TNF) and there was even significant down-regulation of PTGS2. Notably, genes and a microRNA involved in adhesion, cell differentiation and morphology (CD9, DHRS3, PTPN7 and miR-34c-5p) were up-regulated in the T2D monocytes, suggesting a role of the anti-inflammatory cells in adhesion, vascular repair and invasion.AimTo determine the gene expression of the vascular repair factor HGF in the circulating monocytes of patients with T2D and to investigate the relationship between HGF and the expression of the other previously tested monocyte genes and the contribution to the raised serum level of HGF. In addition, we tested the level of 6 microRNAs, which were previously found abnormal in the circulating monocytes, in the serum of the patients.MethodsA gene and microRNA expression study in monocytes and serum of 64 Ecuadorian patients with T2D (37–85 years) and 44 non-diabetic controls (32–87 years).ResultsThe gene expression of HGF was significantly raised in the monocytes of the patients with T2D and associated with the expression of genes involved in adhesion, cell differentiation and morphology. HGF gene expression did not correlate with the serum level of HGF. The monocyte expression of pro-inflammatory cytokine genes was also not associated with the serum levels of these cytokines. The level of miR-574-3p was significantly decreased in the serum of the patients with T2D, and correlated in expression with the decreased well-established inflammation-regulating miR-146a. The level of the microRNAs in serum did not correlate with their expression level in monocytes.ConclusionIn circulating monocytes of Ecuadorian T2D patients, the microRNA and gene expression of important inflammatory/chemotactic/motility/vascular repair factors differs from the expression in serum. While monocytes show a gene expression profile compatible with an anti-inflammatory state, serum shows a molecular profile compatible with an inflammatory state. Both compartments show molecular signs of vascular repair support, i.e. up-regulated HGF levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0113-5) contains supplementary material, which is available to authorized users.

PDGFRα plays a crucial role in connective tissue remodeling.

Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling.

Structural characterisation of immunoglobulin G glycosylation in multiple myeloma

smoldering MM (SMM). Samples from patients with MGUS and healthy donors were also included in the study. LEN treatment was performed at concentration ranging from 0,1 to 2 M during all the differentiation culture period. Moreover moDC differentiation was performed in the presence of CM of human TERT transfected-hMSCs (hTERT-hMSCs) treated for 5 days with LEN. DC maturation markers (CD83, HLA-DR, CD80, CD86 and CD209) were evaluated by flow cytometry. The levels of soluble factors produced by mo-DCs were measured in the conditioned media (CM) of mo-DC by a Bio-Plex Multiplex System. The expression levels of Ikaros and Aiolos were also evaluated by western blot. LEN treatment induced a reduction of both number and % of mature mo-DC as compared to untreated controls. On the other hand, LEN treatment significantly increased the median intensity expression of CD86 HLA-DR and CD209 but not of CD80 by moDCs derived from BM. Similarly increased CD209 expression has been also seen in mo-DCs derived from PB in both MM and SMM patients. LEN treatment enhanced the production of IL-8 and MCP1 and decreased TNFlevels. Ikaros mediated the effect of LEN on DCs as we observed a significant down-regulation of Ikaros levels in THP1-DCs after LEN treatment with a dose dependent effect. On the other hand, we found that LEN blunted the inhibitory effect of hTERT-hMSCs on mo-DC differentiation in three independent experiments and slightly down-regulated IL-6 and IDO but not TGFB1, IL-8, TNF and HGF gene expression. The effect of LEN on the immunomodulatory properties of hMSC was likely to be not mediated by Ikaros or Aiolos because both transcription factors were not expressed by hMSCs. These evidences underline a possible new effect of IMiDs on the alloreactivity against MM cells.

Topical Application of Oleuropein Induces Anagen Hair Growth in Telogen Mouse Skin.

We observed that oleuropein, the main constituent of the leaves and unprocessed olive drupes of Olea europaea, protected mice from high-fat diet-induced adiposity by up-regulation of genes involved in Wnt10b-mediated signaling in adipose tissue. The activation of Wnt/β-catenin pathway is also well established to positively regulate the anagen phase of hair growth cycle in mice skin.Methodology and Principal FindingsOleuropein promoted cultured human follicle dermal papilla cell proliferation and induced LEF1 and Cyc-D1 mRNA expression and β-catenin protein expression in dermal papilla cells. Nuclear accumulation of β-catenin in dermal papilla cells was observed after oleuropein treatment. Topical application of oleuropein (0.4 mg/mouse/day) to C57BL/6N mice accelerated the hair-growth induction and increased the size of hair follicles in telogenic mouse skin. The oleuropein-treated mouse skin showed substantial upregulation of Wnt10b, FZDR1, LRP5, LEF1, Cyc-D1, IGF-1, KGF, HGF, and VEGF mRNA expression and β-catenin protein expression.Conclusions and SignificanceThese results demonstrate that topical oleuroepin administration induced anagenic hair growth in telogenic C57BL/6N mouse skin. The hair-growth promoting effect of oleuropein in mice appeared to be associated with the stimulation of the Wnt10b/β-catenin signaling pathway and the upregulation of IGF-1, KGF, HGF, and VEGF gene expression in mouse skin tissue.

Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

BackgroundHepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma.MethodsHGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA.ResultsHGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF.ConclusionsWe here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of particular importance for patients harbouring a myeloma clone which produces large amounts of HGF.

Does platelet-rich plasma freeze-thawing influence growth factor release and their effects on chondrocytes and synoviocytes?

PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1β and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

IntroductionBased on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl4-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl4 injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl4 treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl4-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI−/− mice with a lethal CCl4-induced acute liver failure.ConclusionsOur study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.

Experimental Research on Treatment of Injured Facial Nerves Induced by Hepatocyte Growth Factor Mediated by Ultrasound-Targeted Microbubble Destruction

The purpose of our study was to explore the regeneration effect of the ultrasound (US)-targeted microbubble destruction-mediated eukaryotic coexpression vector (pIRES2-enhanced green fluorescent protein [EGFP]/hepatocyte growth factor [HGF]) with EGFP and HGF gene system on facial nerve injury in rats. Forty rats were randomly divided into 4 groups after the models of facial nerve injury were established: A, phosphate-buffered saline (PBS) group; B, HGF and microbubble (HGF + MB) group; C, HGF and US (HGF + US) group; and D, HGF + US + microbubble (HGF + MB/US) group. Gene and protein levels of HGF were detected by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot, respectively. The expression of pEGFP in facial nerve trunks was examined by laser scanning confocal microscope; HGF gene and protein expression were significantly higher in D group compared with those of the other groups (P < 0.05). The expression of pEGFP was the strongest in D group (P < 0.05). These data indicate that US-targeted microbubble destruction effectively transfects the HGF gene into target tissues and has a significant effect on an injured facial nerve, thus providing a new strategy for gene therapy in facial nerve injury.