Upregulation Of Hepatocyte Growth Factor Research Articles

Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC.

Clusterin Promotes Corneal Epithelial Cell Growth through Upregulation of Hepatocyte Growth Factor by Mesenchymal Cells In Vitro

Although the cornea expresses high levels of clusterin (CLU), the role of CLU in the cornea is poorly understood. This study was performed to investigate the possible role of CLU in corneal epithelial homeostasis. CLU was overexpressed in 3T3 cells by transfection of a vector encoding full-length CLU (Clu-3T3). Colony-forming efficacy (CFE) was compared in mouse corneal cell line (TKE2) and human primary corneal/limbal epithelial cells that were cocultured with Clu-3T3 and mock-3T3. To determine whether feeder cells have a contact effect, cocultures without feeder-epithelium contact were also performed. Neutralizing antibody against CLU was used to assess the effects of secretory CLU in TKE2 cells cocultured with Clu-3T3 cells. The expression of growth factors associated with limbal stem/progenitor cell maintenance and growth were analyzed by RT-PCR and Western blot analysis. TKE2 cells cocultured with Clu-3T3 feeders showed higher CFE and were larger in colony size than TKE2 cells cocultured with mock-3T3 feeders. Increased CFE of TKE2 was observed without direct contact with Clu-3T3 cells, which was significantly blocked by treatment with CLU neutralizing antibody. Clu-3T3 cells expressed higher levels of HGF than mock-3T3 cells, which were significantly suppressed with anti-HGF neutralizing antibodies. Collectively, the promotion of colony-forming and cell proliferation by Clu-3T3 cells was partially mediated by the induction of HGF. Clusterin indirectly enhances the CFE of corneal/limbal epithelial cells by inducing the production of HGF by feeder cells, suggesting a role in epithelial-mesenchymal interaction.

Abstract 3383: Hepatocyte growth factor upregulation and signaling is associated with carcinogenesis and mesenchymal characteristics in hepatocellular carcinoma

Abstract Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality worldwide. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that may induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined in the cell lines and their functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics when examined following H&E staining. qPCR revealed that 1MEA cells express more HGF (200-fold) than BNL cells and confirmed by immunoblotting. There was associated decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1 and vimentin. There was also increased expression of COX-2, ERK, Akt, phosphorylated ERK and Akt and 1MEA cells formed numerous colonies in soft agar unlike BNL cells. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, increased migration and increased expression of COX-2, ERK and Akt. We conclude that development of HCC is associated with upregulation of HGF which promotes the acquisition of morphological and molecular mesenchymal characteristics, carcinogenesis and upregulation of COX-2, ERK, Akt and phosphorylated ERK and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3383. doi:10.1158/1538-7445.AM2011-3383

A synthetic prostacyclin agonist, ONO-1301, ameliorates ventricular remodeling after acute myocardial infarction via upregulation of HGF in rat

It has been shown that administration of angiogenic factors can augment collateral growth in ischemic tissues. We previously reported that ONO-1301, a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity, promotes production of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) from various cell types. We evaluated therapeutic efficacy of ONO-1301 in rat cardiac ischemia model. Ligation of left anterior descending coronary arteries was performed to 10-week-old Wistar rats, and the slow releasing form of ONO-1301 was administrated subcutaneously 3 days after the ligation (control group and ONO-1301 group). Hemodynamic parameters and plasma BNP levels were significantly improved by ONO-1301. Histological analysis revealed that ONO-1301 suppressed fibrotic changes in the myocardium (27.7±3.1% vs 23.3±3.6%, p<0.01). HGF and c-Met expression in the myocardium was significantly up-regulated by ONO-1301. Beneficial effects of ONO-1301 were abrogated by administration of a neutralizing anti-HGF antibody. These findings indicate that ONO-1301 has cardioprotective effects in the rat ischemic heart model via augmentation of endogenous HGF production.

Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines essential for cell proliferation. Overexpression of S-adenosylmethionine decarboxylase in rodent fibroblasts led to aggressive transformants (Amdc-s cells) that had unforeseen high invasive capacity in nude mice, invading rapidly from the subcutaneous injection site into the peritoneal cavity and its organs. In vitro, these cells were much more invasive than Ras-oncogene-transformed fibroblasts, or human HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells. In immunohistological characterization, Amdc-s-induced tumors showed chaotic neovascularization, with abundant pleomorphic vessel-like structures that had noncontiguous or totally missing laminin (basement membrane) and CD31 (endothelial cell) immunoreactivity. Gene expression and protein analyses of Amdc-s cells showed them to overexpress several pro-angiogenic molecules, including vascular endothelial growth factor (VEGF-A), and to exhibit profound down-regulation of the anti-angiogenic thrombospondin-1 (TSP-1). By reintroduction of TSP-1 into Amdc-s cells, the high invasiveness was efficiently inhibited in vitro. Interestingly, Amdc-s cells showed up-regulation of hepatocyte growth factor (HGF) and also expressed the MET receptor, creating thus an autocrine loop able to regulate VEGF-A and TSP-1 levels. Further, we found Amdc-s cells to express increased amounts of matrix metalloproteinase-2 (MMP-2) and the large isoform of tenascin-C (TN-C), which may also contribute to the angiogenic switch and invasiveness. Consequently, Amdc-s cells offer an excellent model to sort out the key molecules of aggressive tumor growth, and thereby help in designing rational, novel anti-vascular and other cancer therapies.

Up-Regulation of Anti-Inflammatory, STAT3-Activating Hepatocyte Growth Factor and Interleukin-11 In Polycythemia Vera Is Independent of JAK2V617F and Contributes to the Growth of Clonal Erythroblasts

AbstractAbstract 796 Context:The V617F activating mutation of JAK2, JAK2V617F, characterizes Polycythemia vera (PV). However, expression of JAK2V617F does not always result in clone expansion, implying that additional events are required for the growth of JAK2-mutated progenitors and development of the PV phenotype. In the present study, we provide evidence that anti-inflammatory cytokines are required for the growth of JAK2V617F-mutated erythroid progenitors. Methods:We searched for cytokines over-expressed in PV using cytokine antibody arrays and ELISAs for analyses of serum and bone marrow (BM) plasma, and quantitative RT-PCRs for analyses of cells purified from PV patients and controls. Transient transfection of JAK2V617F and invalidation of JAK2V617F with siRNA were used to study the effect of JAK2V617F on cytokine expression. Results:We found that PV patients over-expressed anti-inflammatory hepatocyte growth factor (HGF) and interleukin-11 (IL-11), BM stromal cells (BMSC) and erythroblasts being the main producers. HGF induced the production of IL-11 and both cytokines activate the STAT3 pathway. We used neutralizing antibodies specific for IL-11 or c-MET, the HGF receptor, to block autocrine/paracrine cytokine stimulation of erythroblasts in in vitro cultures. The growth of JAK2V617F-mutated PV erythroblasts and HEL cells was inhibited by at least 40%, indicating that JAK2V617F-mutated cells depend on HGF and IL-11 for their growth. Consistent with activation of HGF/IL-11 pathways, mRNA levels of gp130 (the receptor chain responsible for signalling common to cytokines of the IL-11/IL-6 family) and STAT3 were significantly elevated in PV erythroblasts. Moreover, mRNA levels of c-MET, HGF and IL-11 were correlated, a logical finding as c-MET, HGF and IL-11 act in cascade. No correlation was found with JAK2V617F mRNA levels. To the opposite, we provide evidence that the up-regulation of HGF and IL-11 in PV is not a consequence of JAK2V617F: transient expression of JAK2V617F in BaF-3/Epo-R cells and invalidation of JAK2V617F in HEL cells using anti-JAK2 siRNA had no effect on HGF and IL-11 expression. Conclusion:Anti-inflammatory, STAT3-activating HGF and IL-11 are up-regulated in PV independently of JAK2V617F. Both cytokines contribute to the proliferation of JAK2V617F-mutated erythroblasts, suggesting that drugs blocking the HGF/IL-11 pathways, such as c-MET antagonists, could be of interest as an additional therapeutic option in PV.Marjorie Boissinot and Cedric Cleyrat contibuted equally. Disclosures:No relevant conflicts of interest to declare.

Effects of Basic Fibroblast Growth Factor on Rat Vocal Fold Fibroblasts

The overarching goal of this line of research is to translate basic fibroblast growth factor (bFGF) treatment for vocal fold scarring into practical clinical use. In a previous canine investigation, we demonstrated that bFGF improves phonation threshold pressure, mucosal wave amplitude, and histologic measures in vocal folds treated after injury. In the present study, we studied the effects of bFGF on gene expression of the extracellular matrix and growth factors in rat vocal fold fibroblasts. Fibroblasts harvested from the vocal folds of 5 rats were treated with 3 concentrations of bFGF (0, 10, and 100 ng/mL). The fibroblasts were collected at 24 hours and 72 hours after bFGF administration. Quantitative polymerase chain reaction was then used to investigate the gene expression of the investigated growth factors and extracellular matrices. The results revealed significantly down-regulated expression of procollagen I and significantly up-regulated expression of hyaluronic acid synthase (HAS) 2 and fibronectin in fibroblasts treated with bFGF. The administration of bFGF also resulted in the up-regulation of bFGF and hepatocyte growth factor (HGF). No changes in the expression of HAS-1, tropoelastin, or procollagen III were observed between the treatment and control conditions. Treatment with bFGF induces the down-regulation of procollagen I and the up-regulation of HAS-2 in vocal fold fibroblast cell cultures. These gene expression alterations to key mediators of the wound healing process may translate into potential benefits in the remediation of vocal fold injury. The up-regulation of HGF, an antifibrotic effector molecule, may demonstrate additional benefits by optimizing the wound healing environment and by accelerating the wound repair cascade. These findings may provide fuel for additional discoveries into the development of growth factor therapy for the treatment of vocal fold scar.

A Comparative Study of Carotid Atherosclerotic Plaque Microvessel Density and Angiogenic Growth Factor Expression in Symptomatic Versus Asymptomatic Patients

Objective A challenge facing clinicians is identifying patients with asymptomatic carotid disease at risk of plaque instability. We hypothesise that locally released angiogenic growth factors contribute to plaque instability. Methods Carotid endarterectomy specimens from eight symptomatic and eight asymptomatic patients were interrogated for microvessel density and angiogenic growth factor expression histologically using immunofluorescence, and biochemically using quantitative real-time polymerase chain reaction (q-RT-PCR). Bio-Plex™ suspension array was used to assess circulating biomarkers in venous blood from the same patients and six healthy age-matched controls. Results Immunofluorescence demonstrated significantly greater neovessel density in symptomatic plaques ( P = 0.010) with elevated expression of hepatocyte growth factor ( HGF) ( P = 0.001) and its receptor MET ( P = 0.011) than in asymptomatic plaques. The q-RT-PCR demonstrated up-regulation of Endoglin (CD105), HGF ( P = 0.001) and MET ( P = 0.011) in the plaques of symptomatic versus asymptomatic patients. Bio-Plex™ suspension array demonstrated elevated HGF ( P = 0.002) serum levels in symptomatic versus asymptomatic patients and healthy controls, and decreased platelet-derived growth factor (PDGF) ( P = 0.036) serum levels in symptomatic versus asymptomatic patients. Conclusion Plaque instability may be mediated by HGF-induced formation of new microvessels, and decreased vessel stability resulting from decreased PDGF. Suspension array technology has the potential to identify circulating biomarkers that correlate with plaque rupture risk.

Effect of Exogenous Hepatocyte Growth Factor on Vocal Fold Fibroblasts

We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the treatment of vocal fold scarring, although how exogenous HGF affects gene expression of endogenous HGF or extracellular matrix components in the vocal fold fibroblasts remains unclear. In this in vitro study, we aimed to clarify this aspect in order to better understand the effects of HGF on the vocal folds. Fibroblasts were obtained from the lamina propria of the vocal folds of 5 Sprague-Dawley rats and were cultured with HGF at concentrations of 100, 10, 1, and 0 ng/mL. The cells were collected on days 1, 3, and 7, and the expression of endogenous HGF, its receptor c-Met, transforming growth factor-beta1 (TGF-beta1), procollagen types I and III, and hyaluronic acid synthase (HAS)-1 and HAS-2 messenger RNAs (mRNAs) was examined by real-time reverse transcription-polymerase chain reaction. The expression of endogenous HGF and HAS-1 mRNAs increased significantly when exogenous HGF was administered at a concentration of 1 ng/mL. On day 1, the expression of TGF-beta1 and HAS-2 mRNAs increased significantly in response to 1 ng/mL HGF. Exogenous HGF triggered the up-regulation of endogenous HGF, TGF-beta1, HAS-1, and HAS-2 mRNAs in vocal fold fibroblasts.

IFATS Collection: Fibroblast Growth Factor-2-Induced Hepatocyte Growth Factor Secretion by Adipose-Derived Stromal Cells Inhibits Postinjury Fibrogenesis Through a c-Jun N-Terminal Kinase-Dependent Mechanism

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs.

Estrogen Up-regulation of Hepatocyte Growth Factor (HGF) Activator in the Mouse Uterus Is Associated with HGF Activation.

Estrogen Up-regulation of Hepatocyte Growth Factor (HGF) Activator in the Mouse Uterus Is Associated with HGF Activation.

90 EVIDENCE OF A VASOPRESSIN-INDEPENDENT AQP2 EXPRESSION AND WATER RETENTION IN REFRACTORY ASCITES

Liver transplantation is the only curative treatment for advanced liver cirrhosis. Therapies aimed at halting the progression of the disease are urgently needed. Previous studies have shown that the administration of recombinant insulin like growth factor-I (IGF-I) induces hepatoprotective effects in experimental cirrhosis. However, the necessity of using high daily doses of the protein for a long period of time makes this therapy very costly hampering their clinical application. As an alternative therapeutic approach, we have evaluated whether sustained IGF-I expression within the liver from viral vectors based on the Simian Virus 40 (rSVIGF-I) could exert therapeutic effects in liver cirrhosis. We have previously shown that intraportal injection of rSVIGF-I in rats prior to the development of liver cirrhosis induces the expression of hepatocyte growth factor (HGF) in the liver and delays the progression of the disease. In the present study we have tested the effect of the vector in rats that had already developed advanced liver cirrhosis. Liver cirrhosis was induced in Sprague-Dawley male rats by intragastric administration of CCl4 for 12 weeks and then a single dose of rSVIGF-I (1011 vp/rat) was administered by different routes. The following administration routes were tested: intraportal (IP), intrahepatic (IH), intraarterial (IA) and intrabiliar (IB). At the end of the study, cirrhotic rats treated with rSVIGF-I showed reduced serum bilirubin, transaminases and liver fibrosis scores as well as increased expression of serum albumin as compared to mock-infected cirrhotic animals. The effects were more prominent in rats treated by IP or IA injection, suggesting that these routes could allow better access of the vector to hepatocytes in fibrotic livers. IGF-I expression correlated with the increase of mRNA levels of IGF-I binding protein 3 (whose synthesis is induced by IGF-I) and with upregulation of HGF, a factor that seems to mediate hepatoprotective effects of IGF-I. These results indicate that established cirrhosis can be reverted by IGF-I and also, that rSVIGF-I are promising vectors for the treatment of this disease.

Effects of adenoviral-mediated hepatocyte growth factor on liver regeneration after massive hepatectomy in rats.

Resection is the only curative treatment for liver metastasis of colorectal cancers. Despite the supreme regenerative potential of the liver, major hepatectomy sometimes leads to liver failure, and the limitation of resectable liver volumes makes advanced tumors inoperable. This study was attempted to promote liver regeneration using hepatocyte growth factor (HGF) gene transfection by venous-administered adenovirus and to improve the survival of rats after massive hepatectomy. The adenovirus that encodes HGF was administered to rats before 85%-hepatectomy. The administration of HGF gene improved the survival of rats after massive hepatectomy, while the administration of control adenovirus deteriorated their survival. Gene transfection of HGF showed up-regulation of serum HGF, stimulation of hepatocellular proliferation and rapid liver regeneration. Moreover, HGF administration reduced apoptosis of hepatocytes. The administration of HGF gene prevented liver dysfunction after major hepatectomy and may be a new assist for surgery.

Hepatocyte Growth Factor Establishes Autocrine and Paracrine Feedback Loops for the Protection of Skin Cells after UV Irradiation

Hepatocyte growth factor (HGF) is a multifunctional cytokine, which, among various other activities, acts as a growth factor for melanocytes and has recently been implicated in the pathogenesis of malignant melanoma. In the skin, the main source for HGF is dermal fibroblasts (FB). Here, we have investigated the regulation of HGF production and secretion by cytokines derived from UV-irradiated keratinocytes (KC) and by direct UV irradiation. We demonstrate that supernatants of ultraviolet (UV)B-irradiated KC strongly induce HGF production in FB, and that this effect was mediated primarily by IL-1alpha. Direct irradiation of FB with UVB had no effect on HGF expression. In contrast, irradiation with UVA1 strongly upregulated HGF mRNA production and secretion of the functional protein. Addition of neutralizing anti-HGF antibodies after UVA1 irradiation, as well as transfection of FB with HGF small-interfering RNA (siRNA); which completely abrogated HGF secretion led to a dramatic rise of FB apoptosis demonstrating that autocrine HGF efficiently protected FB from UVA1-induced apoptosis. Our data suggest that upregulation of HGF plays a role in skin homeostasis after UV irradiation. However, a negative side effect of UV-induced HGF secretion by dermal FB might represent a decisive factor for induction and/or progression of melanoma.

4028 POSTER Current outcomes of clinical trials in carbon ion radiotherapy for prostate cancer

S-adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines essential for cell proliferation. Overexpression of S-adenosylmethionine decarboxylase in rodent fibroblasts led to aggressive transformants (Amdc-s cells) that had unforeseen high invasive capacity in nude mice, invading rapidly from the subcutaneous injection site into the peritoneal cavity and its organs. In vitro, these cells were much more invasive than Ras-oncogene-transformed fibroblasts, or human HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells. In immunohistological characterization, Amdc-s-induced tumors showed chaotic neovascularization, with abundant pleomorphic vessel-like structures that had noncontiguous or totally missing laminin (basement membrane) and CD31 (endothelial cell) immunoreactivity. Gene expression and protein analyses of Amdc-s cells showed them to overexpress several pro-angiogenic molecules, including vascular endothelial growth factor (VEGF-A), and to exhibit profound down-regulation of the anti-angiogenic thrombospondin-1 (TSP-1). By reintroduction of TSP-1 into Amdc-s cells, the high invasiveness was efficiently inhibited in vitro. Interestingly, Amdc-s cells showed up-regulation of hepatocyte growth factor (HGF) and also expressed the MET receptor, creating thus an autocrine loop able to regulate VEGF-A and TSP-1 levels. Further, we found Amdc-s cells to express increased amounts of matrix metalloproteinase-2 (MMP-2) and the large isoform of tenascin-C (TN-C), which may also contribute to the angiogenic switch and invasiveness. Consequently, Amdc-s cells offer an excellent model to sort out the key molecules of aggressive tumor growth, and thereby help in designing rational, novel anti-vascular and other cancer therapies.

Hepatocyte growth factor promotes endogenous repair and functional recovery after spinal cord injury

Many therapeutic interventions using neurotrophic factors or pharmacological agents have focused on secondary degeneration after spinal cord injury (SCI) to reduce damaged areas and promote axonal regeneration and functional recovery. Hepatocyte growth factor (HGF), which was identified as a potent mitogen for mature hepatocytes and a mediator of inflammatory responses to tissue injury, has recently been highlighted as a potent neurotrophic and angiogenic factor in the central nervous system (CNS). In the present study, we revealed that the extent of endogenous HGF up-regulation was less than that of c-Met, an HGF receptor, during the acute phase of SCI and administered exogenous HGF into injured spinal cord using a replication-incompetent herpes simplex virous-1 (HSV-1) vector to determine whether HGF exerts beneficial effects and promotes functional recovery after SCI. This treatment resulted in the significant promotion of neuron and oligodendrocyte survival, angiogenesis, axonal regrowth, and functional recovery after SCI. These results suggest that HGF gene delivery to the injured spinal cord exerts multiple beneficial effects and enhances endogenous repair after SCI. This is the first study to demonstrate the efficacy of HGF for SCI.

Expression of hepatocyte growth factor and c-Met after spinal cord injury in rats

Since hepatocyte growth factor (HGF) plays a pivotal role in the development of the central nervous system and pathological conditions, we examined the long-term changes in the mRNA and protein expression of HGF and its receptor c-Met after spinal cord injury (SCI) in rats. HGF mRNA was significantly increased from 7 days after SCI in the injured segment, and the peak was at 7 days after SCI as assessed by real-time RT-PCR. Importantly, c-met mRNA expression was up-regulated from 1 day after SCI, and reached a peak at 14 days after SCI. Although up-regulation of HGF and c-met mRNA expression in the injured segment gradually decreased, the increased expression level persisted until 56 days after SCI. Consistent with HGF mRNA expression, HGF protein level was significantly increased mainly in the injured region, which persisted until 56 days after SCI. Immunohistochemistry showed that most of GFAP-positive reactive astrocytes expressed HGF and c-Met both on 14 days and 56 days after SCI. Staining with the mitotic indicator, bromodeoxyuridine (BrdU), revealed that a small number of BrdU-incorporated cells were co-localized with HGF/GFAP-positive or c-Met/GFAP-positive cells both on 14 and 56 days. These data suggest that HGF and c-Met were up-regulated mainly in the reactive astrocytes around the injured region in the subacute to chronic stage of spinal cord injury. Since HGF plays a critical role in neurotrophic activity, activation of the HGF/c-Met signaling system might be involved in the process of post-traumatic regeneration.

Bone Marrow Transplantation Attenuates the Myopathic Phenotype of a Muscular Mouse Model of Spinal Muscular Atrophy

Bone marrow (BM) transplantation was performed on a muscular mouse model of spinal muscular atrophy that had been created by mutating the survival of motor neuron gene (Smn) in myofibers only. This model is characterized by a severe myopathy and progressive loss of muscle fibers leading to paralysis. Transplantation of wild-type BM cells following irradiation at a low dose (6 Gy) improved motor capacity (+85%). This correlated with a normalization of myofiber number associated with a higher number of regenerating myofibers (1.6-fold increase) and an activation of CD34 and Pax7 satellite cells. However, BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%). These data suggest that BM transplantation was able to attenuate the myopathic phenotype through an improvement of skeletal muscle regeneration of recipient mutant mice, a process likely mediated by a biological activity of BM-derived cells. This hypothesis was further supported by the capacity of muscle protein extracts from transplanted mutant mice to promote myoblast proliferation in vitro (1.6-fold increase). In addition, a tremendous upregulation of hepatocyte growth factor (HGF), which activates quiescent satellite cells, was found in skeletal muscle of transplanted mutants compared with nontransplanted mutants. Eventually, thanks to the Cre-loxP system, we show that BM-derived muscle cells were strong candidates harboring this biological activity. Taken together, our data suggest that a biological activity is likely involved in muscle regeneration improvement mediated by BM transplantation. HGF may represent an attractive paracrine mechanism to support this activity.

Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat

In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl4) administration in the rat. Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl4 (1ml/kg, twice per week) and dalteparin (50IU/kg, daily) for 7 weeks. Dalteparin did not prevent acute CCl4-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24h after simultaneous administration of CCl4 and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12h after injection of CCl4 were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl4 treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl4-induction of smooth muscle alpha-actin expression, alpha1(I)procollagen and transforming growth factor (TGF)-beta1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2'deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner. Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl4 treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.

A scientific rationale for the CREST trial results: Evidence for the mechanism of action of cilostazol in restenosis

The Cilostazol for RESTenosis (CREST) clinical trial was initiated to evaluate the efficacy of cilostazol, an antiplatelet drug, in inhibiting restenosis after stent implantation in a native coronary artery as evaluated by quantitative coronary angiography. Preliminary results suggest that cilostazol reduces restenosis by 36% over standard therapy alone. Restenosis after coronary stenting is primarily attributed to neointimal formation. Cilostazol decreases the activity of phosphodiesterase type 3, leading to the accumulation of cyclic adenosine monophosphate, which initiates a cascade of events including upregulation of anti-oncogenes p53 and p21 and upregulation of hepatocyte growth factor (HGF). The increase in p53 protein blocks cell cycle progression and induces apoptosis in vascular smooth muscle cells (VSMCs), leading to an antiproliferative effect. Upregulation of local HGF stimulates rapid regeneration of endothelial cells, which inhibits neointimal formation via two mechanisms: inhibition of abnormal VSMC growth and improvement of endothelial function. These mechanisms may be responsible for the improvement in restenosis shown in the CREST trial and a number of other trials in patients who underwent percutaneous transluminal coronary angioplasty. These effects, in addition to antithrombotic and vasodilatory attributes of cilostazol, make it a potentially viable treatment option for preventing restenosis following coronary stenting.