Combination Of Hepatocyte Growth Factor Research Articles

Chronic treatment with a combination of hepatocyte growth factor and JNK inhibitor ameliorates cavernosal veno-occlusive dysfunction: a rat model of cavernous nerve injury.

Structural alterations of the penis, including cavernosal apoptosis and fibrosis, induce venous leakage into the corpus cavernosum or cavernosal veno-occlusive dysfunction, a key pathophysiology associated with erectile dysfunction after radical prostatectomy. We hypothesized that the effect of JNK inhibitors on reducing apoptosis and hepatocyte growth factor (HGF) on inducing tissue regeneration could be another treatment mechanism of erectile dysfunction after radical prostatectomy. To investigate whether JNK inhibition combined with intracavernosal administration of HGF can completely preserve cavernosal veno-occlusive function (CVOF) in a rat model of erectile dysfunction induced via bilateral cavernosal nerve crush injury (CNCI). A total of 42 male Sprague-Dawley rats were randomly assigned to sham control (group S), CNCI (group I), and CNCI treated with a combination of JNK inhibitor and HGF (group J + H) for 5weeks after surgery. Rats in each group were evaluated via dynamic infusion cavernosometry (DIC), caspase-3 activity assay, Masson trichrome staining, immunohistochemical staining of α-smooth muscle actin, and immunoblotting at 5weeks after surgery. Regarding CVOF, group I showed decreased papaverine response, increased maintenance, and drop rates of DIC when compared with group S. Group J + H showed significant improvement in the 3 DIC parameters vs group I. No differences in the 3 DIC parameters were found between group J + H and group S. Regarding the structural integrity of the corpus cavernosum, group I showed increased caspase-3 activity, decreased smooth muscle (SM):collagen ratio, decreased SM content, decreased protein expression of PECAM-1, and decreased phosphorylation of c-Jun and c-Met. Group J + H showed significant attenuation in histologic and molecular derangement as compared with group I. There were no differences in caspase-3 activity, SM content, SM:collagen ratio, PECAM-1 protein expression, c-Jun phosphorylation, and c-Met phosphorylation between groups J + H and S. Our results suggest that antiapoptotic and regenerative therapy for the corpus cavernosum is a potential mechanism of penile rehabilitation after radical prostatectomy. This study provides evidence that combination treatment of JNK inhibitor and HGF preserves erectile function by restoring corporal SM and endothelium. However, additional human studies are needed to confirm the clinical effect. Chronic treatment with JNK inhibitor and HGF may preserve CVOF to levels comparable to sham control by preserving the structural integrity of the corpus cavernosum and so represents a potential therapeutic option for preventing the development of cavernosal veno-occlusive dysfunction.

Hepatocyte Growth Factor Promotes Differentiation Potential and Stress Response of Human Stem Cells from Apical Papilla

Harsh local microenvironment, such as hypoxia and lack of instructive clues for transplanted stem cells, presents the serious obstacle for stem cell therapies’ efficacy. Therefore, continued efforts have been taken to improve stem cells’ viability and plasticity. Hepatocyte growth factor (HGF) has previously been reported to mitigate the complications of various human diseases in animal model studies and in some clinical trials. Besides, human stem cells from the root apical papilla (SCAP) are deemed a better resource of mesenchymal stem cells due to derived stem cells holding greater amplification ability in vitro compared with those from other dental resources. To move forward, evaluating effects and understanding underlying molecular mechanisms of HGF on SCAP for periodontal regeneration are needed. In this study, HGF was transgenically expressed in SCAP, and it was found that HGF enhanced osteo/dentinogenic differentiation capacity of SCAP compared with those of non-treated control in an ectopic mineralization model. Moreover, HGF reduced the apoptosis of SCAP under both normoxic and hypoxic conditions, whereas the combination of HGF and hypoxia exposure had inhibitory effects on cell proliferation during an 8-day in vitro culture period. Transcriptome analysis further revealed that suppressed cell cycle progression and activated BMP/TGFβ, Hedgehog, WNT, FGF, HOX, and other morphogen family members result upon HGF overexpression, which may render SCAP recapitulate part of neural crest stem cell characteristics. Moreover, strengthened stress response modulation such as unfolded protein response, macroautophagy, and anti-apoptotic molecules might explain the increased viability of SCAP. In all, our results imply that these potential mechanisms underlying HGF-promoting SCAP differentiation could be further elucidated and harnessed to improve periodontal tissue regeneration.

The initial delay to mitotic activity in primary cultures of equine satellite cells is reduced by combinations of growth factors.

Satellite cell (SC) activation is defined as the time frame during which the stem cell becomes poised to reenter G1 of the cell cycle. The growth factors and events leading to full mitotic activation in equine SCs remain largely unknown. Insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), and fibroblast growth factor 2 (FGF2) are sequentially transcribed during the muscle repair and recovery period following strenuous exercise in adult horses. Expression of IGF-I occurs within 24 h of the postexercise recovery period suggesting it may affect early SC actions. As a first step, gluteus medius muscle cryosections from adult horses (n = 9) were examined for the presence of central nuclei (CN), a marker of SC addition to the fiber. Results demonstrate few CN fibers prior to exercise with a 3-fold increase (P = 0.05) 24 h postexercise. Cultures of SC (n = 4 isolates) were treated with 100 ng/mL IGF-I for varying times prior to measurement of myogenic events. Results demonstrate that IGF-I does not affect the initial lag period, proliferation, or subsequent differentiation of equine SC in vitro (P > 0.05). However, media containing a combination of IGF-I and 10 ng/mL FGF2 and 25 ng/mL HGF hastens (P < 0.05) the time to S-phase entry in fresh isolates of SCs. Media supplementation with optimal concentrations of FGF2, HGF, or a combination of HGF and FGF2 suppresses (P < 0.05) the percentage of myogenin immunopositive SCs to levels below that found in control- or IGF-I-treated SCs. These results provide new insight into the combinatorial roles growth factors play during equine SC myogenesis.

The cytokines HGF and CXCL13 predict the severity and the mortality in COVID-19 patients

The objective of the present study was to identify biological signatures of severe coronavirus disease 2019 (COVID-19) predictive of admission in the intensive care unit (ICU). Over 170 immunological markers were investigated in a ‘discovery’ cohort (n = 98 patients) of the Lausanne University Hospital (LUH-1). Here we report that 13 out of 49 cytokines were significantly associated with ICU admission in the three cohorts (P < 0.05 to P < 0.001), while cellular immunological markers lacked power in discriminating between ICU and non-ICU patients. The cytokine results were confirmed in two ‘validation’ cohorts, i.e. the French COVID-19 Study (FCS; n = 62) and a second LUH-2 cohort (n = 47). The combination of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 13 (CXCL13) was the best predictor of ICU admission (positive and negative predictive values ranging from 81.8% to 93.1% and 85.2% to 94.4% in the 3 cohorts) and occurrence of death during patient follow-up (8.8 fold higher likelihood of death when both cytokines were increased). Of note, HGF is a pleiotropic cytokine with anti-inflammatory properties playing a fundamental role in lung tissue repair, and CXCL13, a pro-inflammatory chemokine associated with pulmonary fibrosis and regulating the maturation of B cell response. Up-regulation of HGF reflects the most powerful counter-regulatory mechanism of the host immune response to antagonize the pro-inflammatory cytokines including CXCL13 and to prevent lung fibrosis in COVID-19 patients.

Long-term Production of Glycogen and Hepatic-Derived, Cell-Invasion-Promoting Chemokines by Ultrasound-Driven Hepatic-Differentiated Human Bone Marrow Mesenchymal Stem Cells

The current treatment for liver failure is restricted to surgical liver transplantation, which is technically complicated, limited by the shortage of available organs and presents major risks to the patient. Bone marrow mesenchymal stem cells (BMSCs) represent promising sources of hepatocyte-like cells for cell transplantation treatment. However, a safe and efficient induction method for their differentiation remains to be defined. Here we further optimized an effective technique by combining high-dose treatment with hepatocyte growth factor (HGF) and ultrasound stimulation. The optimized ultrasound parameter (1.0 W/cm2 intensity, 1 MHz frequency, 20% duty cycle, 100 Hz pulse repetition frequency, 60-s irradiation duration, triple times in three days) combined with different HGF doses (10, 20 and 50 ng/ml) was used to treat BMSCs. The results showed that the specific hepatic markers, including α-fetoprotein (αFP/AFP), cytokeratin 18 (CK18), albumin (ALB) and glycogen, were increased in a dose-dependent manner. Their concentration was then further increased when ultrasound irradiation was administered (P < 0.05), as indicated by PCR, Western blot and immunofluorescence staining as well as a glycogen synthesis test. Furthermore, analysis of the hepatocyte-derived chemokines showed elevated stromal cell-derived factor 1alpha (SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) after HGF treatment. Again, concentrations of those chemokines were further increased by ultrasound radiation (P < 0.05). The observed increased effect was sustained for 21 days. To summarize, we further defined the optimal combination of HGF and ultrasound treatment to increase the differentiation and chemotaxis of BMSCs in a safe, sustained and efficient manner. These findings provide a new perspective for stem cell orientation in the field of tissue engineering.

Co-transfection of hepatocyte growth factor and truncated TGF-β type II receptor inhibit scar formation.

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-β plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-β type II receptor (t-TGF-βRII) is unable to continue signal transduction but is still capable of binding to TGF-β, thereby blocking the TGF-β signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-βRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-βRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-βRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-βRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.

Combination of HGF and IGF-1 promotes connexin 43 expression and improves ventricular arrhythmia after myocardial infarction through activating the MAPK/ERK and MAPK/p38 signaling pathways in a rat model.

In this study, we hypothesized that the combination of hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) alters the expression of connexin 43 (Cx43) and results in a reduced frequency of induced ventricular arrhythmia in rats after myocardial infarction (MI) and explored the preliminary mechanisms involved. Cardiomyocytes were cultured in vitro in medium with PBS, HGF, IGF-1, GFs (HGF + IGF-1), HGF + p38 inhibitor, HGF + ERK inhibitor, IGF-1 + p38 inhibitor or IGF-1 + ERK inhibitor. The expression of Cx43 was tested by real-time PCR and Western blotting after 48 hours. MI was induced in 48 male Sprague-Dawley rats. The rats were randomly divided into four groups and received an injection of PBS, HGF, IGF-1 or GFs into the infarct border zone two weeks after MI. Six weeks after injection, the expression levels of Cx43 and programmed stimulation-induced ventricular arrhythmias were examined. In vitro, the expression of Cx43 mRNA and the Cx43 protein in cardiomyocytes was higher in the HGF, IGF-1, and GFs groups than in the PBS group. GFs had a combinatorial effect on the Cx43 mRNA level but not on the Cx43 protein level. There was a significant reduction in Cx43 mRNA and Cx43 protein levels in the IGF-1 + p38 inhibitor group and IGF-1 + ERK inhibitor group compared to the IGF-1 group. In vivo, programmed stimulation significantly decreased the frequency of ventricular arrhythmia in the GFs, HGF and IGF-1 groups, and this effect was accompanied by increased immunohistochemical staining for Cx43, myocardial Cx43 protein levels and Cx43 mRNA levels in the infarct border zone of the left ventricle compared with those in the PBS group. The combinatorial effect of GFs on Cx43 expression was only observed at the mRNA level. Both HGF and IGF-1 enhanced the expression of Cx43 and improved induced ventricular arrhythmia in rats with MI. Both synergistic and antagonistic effects of HGF and IGF-1 were not observed. In addition, IGF-1 may function through the MAPK/p38 and ERK1/2 signaling pathways to regulate Cx43 expression.

Combined Action of GDNF and HGF Up-Regulates Axonal Growth by Increasing ERK1/2 Phosphorylation.

A stimulating effect of a combination of hepatocyte growth factor (HGF) and glial neurotrophic factor (GDNF) on the growth of neurites in the spinal ganglion model was demonstrated. The mechanism of neurite growth in the spinal ganglion model is associated with transactivation of HGF c-met receptor in the presence of both HGF and GDNF. The combination of HGF and GDNF significantly activated mitogenic signaling cascade mediated by protein kinases ERK1/2, which can be a mechanism for increasing the number of neurites. Our findings can be used for developing effective methods for restoring impaired peripheral nerve function after traumatic and ischemic injury using a combination of GDNF and HGF.

Adrenergic receptor agonists induce the differentiation of pluripotent stem cell-derived hepatoblasts into hepatocyte-like cells

Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.

Abstract 4293: Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study

Abstract Backgrounds: Hepatocyte growth factor (HGF) induces ERK pathway which is associated with carcinogenesis by promoting cell proliferation. Inhibition of ERK signal transduction pathways by genistein, one of the isoflavones, has been reported by several in vitro studies. Therefore, gastric cancer risk by HGF and isoflavone concentrations was evaluated in this study. Methods: Nested case-control study with 93 gastric cancer cases and 1:1 matched controls was conducted within the Korean Multi-center Cancer Cohort (KMCC). HGF and ISF (genistein, daidzein, equol) concentrations in plasma were measured with enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, respectively. Odds ratios (ORs) and 95% confidence intervals (95% CIs) on gastric cancer risk according to HGF and ISF level was calculated using conditional logistic regression models. Results: Compared to the group with low level of HGF (&amp;lt;315pg/mL) and high level of genistein (≥212.5nmol/L), the group having high level of HGF and lower level of genistein was associated with increased gastric cancer risk (OR = 5.08, 95% CI = 1.39-18.57). Elevated gastric cancer risk in the subjects having high HGF level and low daidzein level than subjects with low HGF and high daidzein levels was also shown (OR = 3.67, 95% CI = 1.18-11.45). There was significant interaction between HGF and genistein levels on gastric cancer was found (p for interaction&amp;lt;0.01). Continual increase in gastric cancer risk according to the counts of risky status by HGF, genistein, and daidzein levels was found (p for trend = 0.03). Conclusion: Our study suggests that combination of HGF, genistein and daidzein concentrations has potential as a biomarker for gastric cancer risk. Citation Format: Jieun Jang, Yunji Hwang, Choonghyun Ahn, Kwang-Pil Ko, Keun-Young Yoo, Sue K. Park. Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4293.

HGF and IGF-1 promote protective effects of allogeneic BMSC transplantation in rabbit model of acute myocardial infarction.

To explore effects of hepatocyte growth factor (HGF) combined with insulin-like growth factor 1 (IGF-1) on transplanted bone marrow mesenchymal stem cells (BMSCs), for treatment of acute myocardial ischaemia. After ligation of the left anterior descending artery, rabbits were divided into a Control group, a Factors group (HGF+IGF-1), a BMSC group and a Factors+BMSCs group. Allogenous BMSCs (1 × 10(7)) and/or control-released microspheres of 2 μg HGF+2 μg IGF-1 were intramyocardially injected into infarcted regions. Apoptosis and differentiation of implanted BMSCs, histological and morphological results, and cardiac remodelling and function were evaluated at different time points. In vitro, BMSCs were exposed to HGF, IGF-1 and both (50 ng/ml) and subsequently proliferation, migration, myocardial differentiation and apoptosis induced by hypoxia, were analysed. Four weeks post-operatively, the above indices were significantly improved in Factors+BMSCs group compared to the others (P < 0.01), although Factors and BMSCs group also showed better results than Control group (P < 0.05). In vitro, HGF promoted BMSC migration and differentiation into cardiomyocytes, but inhibited proliferation (P < 0.05), while IGF-1 increased proliferation and migration, and inhibited apoptosis induced by hypoxia (P < 0.05), but did not induce myocardial differentiation. Combination of HGF and IGF-1 significantly promoted BMSCs capacity for migration, differentiation and lack of apoptosis (P < 0.05). Combination of HGF and IGF-1 activated BMSCs complementarily, and controlled release of the two factors promoted protective potential of transplanted BMSCs to repair infarcted myocardium. This suggests a new strategy for cell therapies to overcome acute ischemic myocardial injury.

Hepatocyte growth factor-modified adipose tissue-derived stem cells improve erectile function in streptozotocin-induced diabetic rats

TGFβ1-Smad signaling pathway is closely related to various tissues fibrosis. Hepatocyte growth factor (HGF) has been shown to antagonize TGFβ1-Smad signaling and may improve kidney tissue fibrosis in diabetic models. Penile fibrosis is a pathological condition which occurs during diabetic erectile dysfunction (ED). The aim of this study was to examine the effect of the treatment of ED in diabetic rats with a combination of HGF and adipose tissue-derived stem cells (ADSC). In this diabetes model, rats were injected intraperitoneally with 60 mg streptozotocin (STZ) to induce diabetes. Three months later, the diabetic rats were divided into a negative control(NC) group, an ADSC-treated group and an ADSC + HGF-treated group while normal rats were assigned into a sham group. Rats in the sham and NC groups were injected in the corpus cavernosum with phosphate-buffered saline, while rats in the other groups were injected with either ADSC or ADSC + HGF. One month later, erectile function was examined in each group and penile tissues were collected for experiments. The expression of smooth muscle actin (SMA) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) was analyzed by Western blotting. The smooth muscle and collagen deposition in corpus cavernosum was evaluated by Masson staining, while endothelial changes were assessed immunohistochemically. Cell apoptosis was detected by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. The results revealed that ADSC alone can significantly improve erectile function in diabetic rats, but in combination with HGF the improvement was more prominent, showing higher content of smooth muscle and endothelial cells and lower cell apoptotic index in corpus cavernosum. Treatment with HGF can significantly enhance the beneficial effect of ADSC on erectile function in diabetic rats, and this effect might be closely related to the down-regulation of TGFβ1-Smad signaling.

Differentiation and Migration of Bone Marrow Mesenchymal Stem Cells Transplanted through the Spleen in Rats with Portal Hypertension

AimsThe goals of this paper were to evaluate the differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro, and to determine whether stem cells can migrate and plant into the liver with portal hypertension accompanied by the end-stage of liver disease. MethodsBMSCs were isolated from rats and amplified with hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4). The expression of alpha-fetoprotein (AFP), cytokeratin 18 (CK-18), and albumin (ALB) was detected by immunofluorescence in induced cells. Rats were randomly divided into experimental (with common bile duct ligation) and control groups. After injection of fluorescence labeled cells, cell distribution was observed under a fluorescence microscope. The integrated optical density (IOD) and cell distribution scores were evaluated using Image-Pro Plus 6.0 software. The portal pressure was measured before the rats were killed. ResultsAfter being induced with HGF and FGF-4, the Golgi apparatus, endoplasmic reticulum, ribosomes, and mitochondria all significantly increased in the fifth generation cells. Immunofluorescent analysis showed that the induced cells expressed AFP, CK-18, and ALB. BMSCs were stained by CM-Dil, and the labeling rate was as high as 95.5%. The portal pressure in experimental group was much higher than that of the control group (18.04±2.35 vs. 9.75±1.40cm H2O p<0.01). The IOD of transplanted cells in the experimental group was also significantly higher than that of the control group (11.30±2.09×105 vs. 2.93±0.88×105, p<0.01). In addition, the cell distribution score in the experimental group was lower than that of the control group (1.99±0.36 vs. 2.36±0.27, P<0.05). ConclusionsThe combination of HGF and FGF-4 induces the differentiation of BMSCs into hepatocyte-like cells, which express AFP, CK-18, and ALB. In addition, the recruitment of BMSCs (after transplantation in the spleen) was improved in rats with portal hypertension.

A Potential Role of Progestin-Induced Laminin-5/α6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland

Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and α6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-κB ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

Hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D together stimulate human bone marrow-derived stem cells toward the osteogenic phenotype by HGF-induced up-regulation of VDR

Bone formation and remodeling require generation of osteoprogenitors from bone marrow stem cells (MSC), which are regulated by growth factors and hormones, with putative roles in mesenchymal cell differentiation. Hepatocyte growth factor (HGF) is a pleiotropic growth factor, and together with its high affinity receptor cMet are widely expressed in normal tissues. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D; produced mainly in the kidney, but also by osteoblasts. We previously reported that HGF and 1,25OHD act together to increase osteogenic differentiation of human MSC (hMSC) potentially through increasing p53. Although p53 does not induce the vitamin D receptor (VDR), p63, a member of the p53 family of transcription factors has been reported to up-regulate VDR expression in some tumor cell lines, and thus might play a part in HGF-regulated VDR expression. Our hypothesis is that the combination of HGF and 1,25OHD can induce hMSC differentiation by up-regulation of 1,25OHD and/or VDR expression to increase cell response(s) to 1,25OHD. Using real-time RT-qPCR, Western blots, luciferase reporter assays, and siRNAs, as well as antibodies to specific signaling molecules we showed that HGF induced VDR gene expression, as well as up-regulated p63 gene expression. p63 gene knockdown by siRNA eliminated the effects of HGF on VDR gene expression as measured by RT-qPCR, Western blots and luciferase reporter assay, and downstream on osteogenic differentiation markers, including alkaline phosphatase staining. Differentiation is a coordinated process of cell cycle exit and tissue-specific gene expression. These results suggest HGF might be a good candidate to coordinate the regulation of these two processes during hMSC osteogenic differentiation. p63 could be a key connecting molecule on the pathway of HGF-induced VDR expression. Understanding the role of these factors and their actions could have important clinical implications for the use of hMSC in the development of novel stem cell therapies.

In Vitro Differentiation of Rat Bone Marrow Mesenchymal Stem Cells into Hepatocytes

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

English

Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells were induced by sodium butyrate as inducer for 7 days in the first phase, followed by the combination of hepatocyte growth factor and dexamethasone as inducers for 12 days in the second phase based on an adherent culture system with a two-step induction. The results from the morphology, gene expression, protein molecular markers and cell function of ES-D3 cells derived hepatocyte-like cells demonstrated that mES cells can be differentiated efficiently in vitro to functional hepatocytes under chemically defined conditions, which might be useful as an in vitro system for hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words : Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, hepatocyte growth factor, dexamethason.

Generation of insulin-secreting cells from adult rat pancreatic ductal epithelial cells induced by hepatocyte growth factor and betacellulin-δ4

The present study was conducted to identify effective factors that could induce differentiation of pancreatic ductal epithelial cells (PDECs) into insulin-producing cells. We examined the effect of hepatocyte growth factor (HGF) and betacellulin-δ4 (BTC-δ4) on PDECs. We isolated PDECs from adult rats and stimulated them with HGF and BTC-δ4 for 28days. We observed several characteristics of PDECs after induction, including functional changes in PDECs, differentiation rate, and expression levels of critical transcription factors. We observed that insulin content and glucose-induced insulin secretion were significantly higher in induced PDECs compared to control PDECs. The expression of markers specific to mature β-cells, i.e., insulin and glucose transporter-2 (GLUT2) was also observed. An analysis of the expression of different transcription factors (PDX-1, Ngn3, NeuroD, and Pax4) revealed that cell-specific gene expression patterns varied from 7 to 28days after treatment. Hence, we conclude that treatment with a combination of HGF and BTC-δ4 is effective in inducing the differentiation of PDECs into insulin-secreting cells.

D012 Development of novel therapeutic strategies to stimulate revascularization in heart failure: biopolymeric delivery of a synergistic combination of angiogenic growth factors

Therapeutic angiogenesis is a promising approach to re-establish cardiac perfusion following ischemia or in heart failure. Recently, it has been proposed that a combination of growth factors may be key to the generation of stable vascular networks. However, it is currently unknown what growth factor combinations are optimal for therapeutic angiogenesis and what mode of administration is most adapted to achieve functional revascularization of the heart. The aim of this study was to investigate the effects of novel growth factor combinations and to evaluate their angiogenic effects in vivo following delivery with a new biodegradable polymer system. First, we found that FGF-2 (Fibroblast Growth Factor-2) and HGF (Hepatocyte Growth Factor) display synergistic stimulatory effects on the migration and proliferation of murine endothelial cells in vitro. Furthermore, we show that whereas HGF potentiates the proliferative effect of FGF-2 in smooth muscle cells, the HGFinduced mobility response in these cells is enhanced by FGF-2. The molecular mechanisms of the reciprocal stimulatory effects involve upregulation in endothelial cells of the HGF receptor, c-Met, by FGF-2, and vice versa upregulation of the FGFR-1 by HGF stimulation. Next, we determined the release potential of a new polymeric delivery system based on biodegradable alginate microparticules adapted for cardiac injection. We found that FGF-2 is liberated during less than 1 month with a strong initial level of release during the first ten days. In contrast, HGF is continuously liberated during 40 days with a maximal release around day eighth. Our in vivo data, from a Matrigel plug assay in mice, indicate a significantly increased efficacy of angiogenic therapy using growth factors delivered by alginate polymers. Moreover, we found that a combination of HGF and FGF-2 displays a potent synergistic angiogenic effect in vivo. In conclusion, we demonstrate the potential of the specific combination of FGF-2 and HGF in obtaining a synergistic stimulatory effect in vitro and in vivo. Further, our new alginate-based injectable polymeric system seems well adapted for delivery of growth factors for therapeutic angiogenic approaches to ameliorate cardiac perfusion.

Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells

Hepatocyte growth factor (HGF) is a pleiotropic protein with renoprotective functions, which have been attributed at least in part to its ability to counteract the profibrotic effects of transforming growth factor beta (TGF-beta). A major downstream mediator of TGF-beta is connective tissue growth factor (CTGF). However, the molecular mechanisms of CTGF regulation by HGF have not yet been investigated. CTGF expression was analysed in human primary tubular epithelial cells (hPTECs) and the cell line HKC-8 by western blotting. Morphological alterations were analysed by immunocytochemistry. HGF induced a transient expression of CTGF, which was maximal after 6 h and returned to baseline after 24 h. Coincubation with TGF-beta increased CTGF protein at 6 h, whereas HGF significantly decreased CTGF induction by TGF-beta after 24 h. Furthermore, HGF induced cell scattering associated with reorganization of focal adhesions and formation of lamellipodia and filopodia. The early induction of CTGF was linked to the HGF-mediated alterations of cell morphology. The PP2 inhibitor of Src-family kinases, which regulate focal adhesion turnover, reduced HGF-mediated upregulation of CTGF. In addition, inhibition of the Rho-kinase, which modulates the actin cytoskeleton, impaired CTGF expression. Combination of both inhibitors further decreased CTGF expression. Comparable inhibitory effects were obtained, when CTGF was induced by the combination of HGF and TGF-beta. We provide evidence for a dual effect of HGF on CTGF regulation in human tubular epithelial cells: transient upregulation of CTGF in the absence of TGF-beta, which was related to alterations of cell morphology, and interference with TGF-beta-mediated CTGF induction after prolonged incubation.