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Vocal fold scar and sulcus are intractable diseases with no effective established treatments. Hepatocyte growth factor (HGF) has preclinically proven to have potent antifibrotic and regenerative effects on vocal fold scar. The current Phase I/II clinical trial aims to examine the safety and effectiveness of intracordal injection of a recombinant human HGF drug for patients with vocal fold scar or sulcus. This is an open-label, dose-escalating, first-in-human clinical trial. Eighteen patients with bilateral vocal fold scar or sulcus were enrolled and divided into three groups: Step I received 1 μg of HGF per vocal fold; Step II received 3 μg of HGF; and Step III received 10 μg of HGF. Injections were administered once weekly for 4 weeks. The protocol treatment was performed starting with Step I and escalating to Step III. Patients were followed for 6 months post-treatment. Local and systemic safety aspects were examined as primary endpoints, and therapeutic effects were assessed as secondary endpoints using voice handicap index-10; maximum phonation time; vocal fold vibratory amplitude; grade, rough, breathy, asthenic, strained scale; and jitter. The results indicated no serious drug-related adverse events in either the systemic or local examinations. In whole-subject analysis, voice handicap index-10, vocal fold vibratory amplitude, and grade, rough, breathy, asthenic, strained scale were significantly improved at 6 months, whereas maximum phonation time and jitter varied. There were no significant differences in phonatory data between the step groups. In conclusion, intracordal injection of a recombinant human HGF drug was safe, feasible, and potentially effective for human patients with vocal fold scar or sulcus.

Objectives/Hypothesis Previous animal studies demonstrated that hepatocyte growth factor (HGF) has the potential to regenerate scarred vocal folds. In addition, HGF is now produced under a good manufacturing practice (GMP) procedure. Therefore, human clinical trials of HGF are warranted in patients with vocal fold scarring. In the current study, we investigated the pharmacokinetics and the local tissue responses of HGF administered to rat vocal folds. Study Design Prospective animal experiment. Methods Five μg of recombinant human HGF was administered to the vocal folds of Sprague-Dawley rats (n = 60) using a microsyringe. The concentration of HGF in larynges and blood was investigated by enzyme-linked immunosorbent assay. To evaluate the local tissue responses caused by HGF administration, endoscopic and histological examinations were performed. Results HGF concentration in the larynges was 50.1 μg/g tissue 5 minutes after administration. The concentration decreased rapidly to 1.71 μg/g tissue at 12 hours after administration and to 0.29 ng/g tissue at 24 hours after administration. Seven days after administration, HGF concentration was minimal in one-half of the cases and was not detected in the other cases. Transmission of HGF to blood was detected in two of six cases at 5 minutes after administration, but was no longer detected 12 hours later. Endoscopic and histological examinations revealed no edema or erythema of the vocal folds in any of the cases. Conclusions The current results contribute to the safety and pharmacokinetic management of future clinical trials using HGF administered to vocal folds. Level of Evidence N/A. Laryngoscope, 124:2131–2135, 2014

Background: Extracapsular ligament injuries of the knee and ankle are common injuries. Ligaments heal slowly, usually over months or longer by scar formation rather than by tissue regeneration. This study was performed to evaluate the therapeutic effect of locally delivered recombinant hepatocyte growth factor (HGF) on the early healing of ligaments in a rabbit model. Methods: Japanese white rabbits were subjected to a standardized gap injury in the medial collateral ligaments (MCLs) of both knees. Each rabbit underwent bilateral transection of the midsubstance of the MCL, which was not repaired. During postoperative days 0-6, the rabbits were injected with 10 μg human recombinant HGF into the right MCL, while the left MCL was injected with saline alone. One, 3, 6, and 12 weeks after surgery, experimental rabbits were sacrificed. The structural properties of the femur-MCL-tibia complex were then assessed and the tissue was subjected to histological evaluation. To see the distribution of cells that express c-Met receptor, the tissue was subjected to immunohistochemistry. Results: Immunohistochemical evaluation revealed that c-Met expression was observed particularly at opposing ligament ends in the HGF-treated limbs 1 week after surgery. Histological evaluation revealed earlier neovascularization and more aligned collagen fibers in the MCLs of the HGF-treated group than the control group. In mechanical evaluations, similar ligament failure modes were noted in the two groups. After 3 weeks, HGF-treated limbs had significantly improved structural properties than the paired control limbs. Conclusions: Our findings indicate local administration of recombinant HGF promotes early steps in ligament healing and the repair of structural properties in a rabbit model. Local administration of HGF may represent a new therapeutic approach to accelerating healing and rehabilitation after ligament injury. © 2011 The Japanese Orthopaedic Association.

Background: Although the liver has a large regenerative capacity, in many hepatopathies, these repair mechanisms fail. The therapeutic potential of hepatocyte growth factor (HGF) has been proven in numerous toxin-induced liver failure models in rodents, but never in spontaneously occurring liver diseases in larger animal models. Aim: The aim of this study was to induce liver growth in a hypoplastic liver by the administration of exogenous recombinant HGF. The natural hypoplastic liver model used is the canine congenital portosystemic shunt (CPSS) characterized by strongly reduced liver growth and function. Methods: Recombinant HGF (rHGF), 200 μg/kg, was given twice daily during 3 weeks by an intravenous injection in six dogs with CPSS. Liver volumes were determined by computed tomography before and at 1, 2, 3 and 7 weeks after the initiation of treatment. Portosystemic shunting was evaluated with an ammonia tolerance test and liver portal perfusion was quantified with scintigraphy. Simultaneously, blood parameters for liver function were assayed and liver biopsies were taken for histology, immunohistochemistry and gene-expression measurements. Results: During 3 weeks of HGF treatment, hepatocyte proliferation increased and an increase in liver volume up to 44% was seen, persisting in two dogs up to 4 weeks after the termination of treatment. Ki-67 expression, gene expression of E2F1 and CDC6, phosphorylated-c-MET and phosphorylated-ERK1/2 protein levels confirmed increased hepatocyte proliferation and HGF signalling. The aberrant portal perfusion did not change during treatment. Conclusions: Transient in vivo liver growth is shown using CPPS as a naturally occurring large animal model, indicating the therapeutic potential of HGF in liver disease.

Aim: Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis. In the present study, we examined whether and how nephrin expression is sustained by podocytes during the HGF-mediated attenuation of albuminuria. Methods: Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction. Results: Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration. Conclusion: HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation.

Purpose This study was performed to evaluate the therapeutic effect of hepatocyte growth factor (HGF) on tendon-bone healing in a rabbit model. Methods In adult rabbits the long digital extensor tendon was detached from the lateral femoral condyle, and the free end of the tendon was inserted into a tunnel drilled into the proximal tibial metaphysis. Cancellous bone obtained during drilling of the tibial hole was soaked in saline solution or solution containing 100-μg/mL human recombinant HGF and then transplanted into the bone tunnel. Junctional healing between the tendon and the bone was evaluated by histologic analysis and uniaxial load-to-failure testing at 2, 4, 6, 8, and 12 weeks after surgery. Results In the saline solution–treated control group, Sharpey-like fibers, which connected the tendon graft and the bone tissue, appeared 6 weeks after treatment. At 8 weeks after treatment, maturation of lamellar bone was seen, and at 12 weeks, the adhesion between tendon and bone appeared to be supported by indirect insertion of fibrocartilaginous tissue, wherein the border between the fibrocartilaginous tissue and tendon or bone was significant. In the HGF-treated group, the fibrous tissues were parallel to the load axis, and lamellar bone and Sharpey-like fibers appeared as early as 4 weeks after treatment. At 12 weeks, junctional tissue, characterized by a continuous 4-layer structure of bone, calcified cartilage, fibrocartilage, and tendon, was regenerated by a direct insertion. On biomechanical testing, the HGF-treated group had significantly better biomechanical properties than the control group at 2 and 4 weeks. The histologic improvement caused by HGF treatment was associated with the biomechanical improvement. Conclusions Local administration of recombinant HGF promotes the adhesive healing process at the tendon-bone junction, both histologically and mechanically, after ligament reconstruction in a rabbit model. Clinical Relevance Application of HGF may be considered as a new therapeutic approach to accelerate healing and rehabilitation after ligament reconstruction.

The present study was aimed at determining whether human recombinant hepatocyte growth factor (HGF) ameliorates cerebral edema induced by microsphere embolism (ME). Rats were injected with 700 microspheres (48 microm in diameter). Continuous administration of HGF at 13 microg/3 days/animal into the right ventricle was started from 10 min after embolism to the end of the experiment by using an osmotic pump. On day 3 after the ME, the rats were anesthetized, and their brains were perfused with an isotonic mannitol solution to eliminate constituents in the vascular and extracellular spaces. Thereafter, tissue water and cation contents were determined. A significant increase in tissue water content of the right hemisphere by ME was seen. This ME-induced increase in water content was associated with increases in tissue sodium and calcium ion contents and decreases in tissue potassium and magnesium ion contents of the right hemisphere. The treatment of the animal with HGF suppressed the increases in water and sodium and calcium ion contents, but not the decreases in potassium and magnesium ion contents. These results suggest that HGF suppresses the formation of ischemic cerebral edema provoked intracellularly in rats with ME.

Background Ischemia–reperfusion (IR) injury after lung transplantation leads to significant morbidity and mortality in recipients, which remains the major obstacle in clinical lung transplantation. To reduce pulmonary graft dysfunction and improve prognosis after lung transplantation, prevention of IR-induced lung injury in the peri-operative period is required. In the present study, we investigated the effects of recombinant hepatocyte growth factor (HGF) on pulmonary IR injury using a murine model system. Methods To assess the protective effect of HGF against lung injury, mice with pulmonary IR were divided into two groups and injected with 500 μg/kg of human recombinant HGF or the same dose of saline alone as a control. Results After pulmonary IR injury, the lung injury score increased in a time-dependent manner up to 24 hours. A significant reduction of lung injury score was observed with the administration of exogenous HGF. Moreover, the ratio of apoptotic cells was significantly reduced in mice treated with HGF. Significantly increased expression of Bcl-xL was observed after IR in mice administered HGF as compared with saline-treated controls. In contrast, expression of Bax was reduced significantly in HGF-treated mice. Serum levels of endogenous murine HGF were increased significantly in HGF-treated mice. Conclusions Our findings indicate that administration of exogenous HGF ameliorates the pulmonary tissue injury induced by IR, which may provide an alternative for prevention of IR-induced lung injury in the peri-operative period in lung transplantation.

BACKGROUND In heart transplantation, myocardial apoptosis during hypothermic storage contributes to graft dysfunction. On the other hand, hepatocyte growth factor (HGF) has been reported to be an antiapoptotic factor in the heart. Therefore, we assessed whether the administration of recombinant human HGF (rh-HGF) prevents apoptosis in the prolonged preserved myocardium, resulting in an improvement in the cardiac function of the graft. METHODS Isolated rat hearts were subjected to 4 hr (group A), 6 hr (group B), and 8 hr (group C: without rh-HGF vs. group D: with 100 microg of rh-HGF) of hypothermic storage followed by 60 min of normothermic reperfusion (n=5 in each group). RESULTS Compared with non-HGF-treated hearts (group C), HGF-treated hearts (group D) showed a significantly higher recovery rate of left ventricular developed pressure (38+/-5% vs. 58+/-6%, P

Excessive activity of the Fas system in the liver is an essential event and contributor to fulminant hepatic failure, whose prognosis is extremely poor with high mortality due to lack of effective therapy. Administration of agonistic anti-Fas antibody to mice rapidly led to massive liver apoptosis and fulminant hepatic failure. In contrast, administration of human recombinant hepatocyte growth factor (HGF) abrogated Fas-induced massive liver apoptosis and the lethal hepatic failure. Addition of anti-Fas antibody to hepatocytes in primary culture induced cell death, but Fas-mediated cell death was potently suppressed by HGF. HGF strongly induced Bcl-xL expression and subsequently blocked Fas-mediated signaling pathway upstream of CPP32 in the liver. These results implicate a potential therapeutic usage of HGF for treatment of fulminant hepatic failure.

Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

Liver fibrosis/cirrhosis is characterized by hyper-accumulation of fibrous tissue components and is commonly observed in later or terminal states of chronic hepatic diseases. In ongoing work, we found that the administration of human recombinant hepatocyte growth factor (hrHGF) suppressed the onset of liver fibrosis/cirrhosis in several distinct models and accelerated the recovery from liver fibrosis/cirrhosis in rats. Repeated administration of porcine serum for 10 weeks to rats induced liver fibrosis without any accompanying hepatocellular injuries; in addition, the intravenous (i.v.) administration of hepatocyte growth factor (HGF) to these rats suppressed increases in fibrous components and hydroxyproline contents in the liver, thus preventing the onset of liver fibrosis. Repeated administration of dimethylnitrosamine (DMN) for four weeks induced liver cirrhosis, as characterized by the hyper-accumulation of fibrous components, infiltration of mononuclear leukocytes, and hepatic dysfunction. When HGF was injected daily for four weeks along with DMN-treatment, the onset of DMN-induced hepatic fibrosis/cirrhosis was suppressed; the numbers of infiltrating mononuclear cells, fibrous tissue components, and hydroxyproline content in the liver were decreased. When HGF was injected for two weeks following four weeks of DMN-treatment, HGF accelerated the recovery from liver cirrhosis and prevented death due to hepatic dysfunction. Likewise, HGF-injection suppressed the onset of liver fibrosis, when liver fibrosis had been induced by long-term treatment with carbon tetrachloride (CCl4). Thus, the administration of HGF holds great promise for treating subjects with liver fibrosis/cirrhosis as a result of chronic hepatic injury.

Portal branch ligation (PBL) or embolization prior to extensive hepatectomy has been employed to increase the functional reserve of the remaining liver. This study investigated the effects of human recombinant hepatocyte growth factor (rh-HGF) on liver regeneration following PBL in dogs. Beagle dogs were subjected to PBL and were divided into two groups, a control group (n = 11) without rh-HGF and a treated group (n = 12) receiving postoperative rh-HGF at 250 ng/kg via the portal vein. Dogs were killed 72 h or 14 days following PBL. We studied the changes in serum HGF level, DNA synthesis of the liver, hepatocyte size, liver weight, and liver function tests. In the HGF group, the ratio of whole liver weight to body weight increased significantly, and both ligated and nonligated lobes showed marked increases in weight. The nonligated lobes in the HGF group showed significant increases in both DNA synthesis and hepatocyte size. Moreover, ligated lobes in the HGF group showed an increase in DNA synthesis without hypertrophy compared with the control group. Administration of rh-HGF did not significantly affect liver function tests. Ligation of the portal branch supplying the portion of liver to be resected, coupled with the administration of rh-HGF, is a useful strategy to increase hepatic reserve in advance of major hepatectomy.

Hepatocyte growth factor is a mesenchyme-derived pleiotropic factor that regulates the growth, motility and morphogenesis of various types of cells, and is also a member of the angiogenic growth factors. Hepatocyte growth factor is secreted by vascular endothelial cells and smooth muscle cells, and the hepatocyte growth factor receptor, c-met, was also observed in these vascular cells. Treatment of human aortic endothelial cells with recombinant hepatocyte growth factor resulted in a significant increase in cell proliferation, accompanied by mitogen-activated protein kinase and Akt/protein kinase B phosphorylation. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As preclinical study of gene therapy using hepatocyte growth factor to treat peripheral arterial disease, naked hepatocyte growth factor plasmid was intramuscularly injected into the ischemic hind limb of rabbits in order to evaluate its angiogenic activity. Intramuscular injection of hepatocyte growth factor plasmid once on day 10 following surgery, produced significant augmentation of collateral vessel development in the ischemic limb on day 30. In the clinical setting, the authors further investigated the safety and efficacy of hepatocyte growth factor plasmid DNA in patients with critical limb ischemia, in a prospective open-labeled trial. Intramuscular injection of naked plasmid DNA was performed in the ischemic limbs of six patients with critical limb ischemia with arteriosclerosis obliterans (n = 3) or Buerger disease (n = 3) graded as Fontaine III or IV. In the efficacy evaluation, a reduction of pain scale of more than 1 cm on a visual analog pain scale was observed in five out of six patients. An increase in ankle pressure index of more than 0.1 was observed in five out of five patients. The long diameter of eight out of 11 ischemic ulcers in four patients was reduced by more than 25%. Intramuscular injection of naked hepatocyte growth factor plasmid is safe, feasible and can achieve successful improvement of ischemic limbs. Although the present data were obtained to demonstrate safety in a Phase I/early Phase II trial, the initial clinical outcome with hepatocyte growth factor gene transfer seems to indicate its usefulness as sole therapy for critical limb ischemia. Randomized placebo-controlled clinical trials of alternative dosing regimens of gene therapy will be required to define the efficiency of this therapy.

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.

Background —Previous studies have shown that repeated systemic administration of human recombinant hepatocyte growth factor (hrHGF) in mg/kg levels modulates the wound-healing process in various diseases. Recently, HGF has been characterized as one of the most potent endothelial-cell–specific growth factors. We tested our hypothesis that local delivery of hrHGF, even at low μg/kg levels (≥2 orders of magnitude lower than systemically administered doses), might attenuate neointimal hyperplasia in response to vascular injury via accelerated reendothelialization. Methods and Results —The iliac artery was denuded in 16 New Zealand White rabbits (3 kg), followed by administration, via a drug delivery catheter, of either hrHGF (10 μg; n=11) or control vehicle (n=5) over 20 minutes. In pilot studies using this device, the drug permeated into the medial tissues, where it persisted for ≥24 hours. Four weeks after the local delivery of hrHGF, computer-assisted morphometric analysis revealed significant reduction in the intimal area (hrHGF, 0.37±0.21 versus control, 0.68±0.16 mm 2 , mean±SD; P 2 ). Scanning electron microscopy revealed extensive endothelialization with regular and confluent endothelial cell layer regeneration in the hrHGF-treated vessels. Conclusions —Accelerated endothelialization after local delivery of hrHGF, a novel and potent endothelial cell mitogen, effectively attenuates neointimal proliferation even after single low-dose administration. This observation could have potential therapeutic implications in the prevention of restenosis after angioplasty.

A porcine hepatocyte based bioartificial liver (BAL) is still insufficient to replace liver transplantation. In this experiment, to strengthen the performance of a BAL, the effect of human recombinant hepatocyte growth factor (rhHGF) on the proliferation and function of xenogeneic porcine hepatocytes was studied. Isolated porcine hepatocytes were seeded at various densities (5 × 10 3 to 8 × 10 4 cells/well) on a collagen coated 96 well plate in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. After 4 hours, the medium was changed to DMEM with added insulin and dexamethasone. Subsequently, rhHGF was added at various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 ng/ml) and cultured for an additional 24, 48, and 72 hours, respectively. The proliferation of porcine hepatocytes in response to rhHGF reached a plateau at 2.5 ng/ml at 24 hours and subsequently decreased. The levels of porcine albumin vs protein present in the supernatant increased when cultured at high cell density. In conclusion, rhHGF was found to stimulate proliferation of porcine hepatocytes at low cell density and low concentration. rhHGF can also increase albumin synthesis at higher cell density, thus indicating its potential use in a more satisfactory porcine hepatocyte based BAL.

Abstract —Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients ( P P P

Background.Acute endotoxemia is known to cause activation of Kupffer cells as well as serious injury in parenchymal and nonparenchymal cells in the liver. We have recently shown that a continuous recombinant hepatocyte growth factor (rHGF) supply prevents lipopolysaccharide (LPS)-induced liver injury in rats. As an attempt to elucidate the mechanism, here we investigate the cytoprotective effect of rHGF on sinusoidal endothelial cells (SECs) in LPS-induced liver injury in rats. Materials and methods.In order to supply rHGF continuously to the liver, syngenic rat fibroblasts genetically modified to secret rat rHGF were implanted in the spleen. Fourteen days after cell implantation, we injected LPS intravenously and evaluated SEC damage histologically and blood chemically. Results.Phosphotungstic acid–hematoxylin staining revealed that rHGF treatment greatly attenuated intrasinusoidal LPS-induced fibrin deposition. The ultrastructural changes in SECs caused by LPS administration in control rats were barely detectable in rHGF-treated rats. Blood chemical analyses showed that rHGF potently suppressed the LPS-induced increase in serum hyaluronic acid and transaminase levels. Conclusions.Our results indicate an important role for HGF in SEC protectionin vivoand would suggest a novel therapeutic strategy for liver diseases with SEC injury.

Human hepatocytes stimulated with human recombinant hepatocyte growth factor (h-rHGF) (10 ng/mL) displayed a characteristic lag period before entering into the S phase. The duration of this delay was dependent on the timing of h-rHGF addition to cultures. The highest peak of DNA synthesis was observed at 120 hours of culture when hepatocytes were stimulated with h-rHGF at 72 hours of culture. This was accompanied by an early peak of c-jun and c-fos synthesis (3 hours after addition of h- rHGF) followed by c-myc (6 hours) and increased expression of cyclins A, B, D, and E (12 hours after h-rHGF). A significant dose-dependent increase in inositol 1,4,5-P 3 was observed within 45 seconds after stimulation with the factor. This was followed by an immediate increase in the cytosolic-free calcium. Cyclic adenosine monophosphate (cAMP) levels did not change after stimulation with the factor. Tyrosine phosphorylation seems to be an early event in the course of the stimulatory effect of h-rHGF on DNA synthesis of hepatocytes; genistein, a tyrosine kinase inhibitor, impaired the stimulatory effect of h-rHGF on DNA synthesis dose dependently. On the other hand, the action of the factor was negatively regulated by protein kinase C activation, as shown by the increased stimulatory effect of h-rHGF on DNA synthesis upon inhibition of protein kinase C by H7. (Hepatology 1996 May;23(5):1012-9)

Introduction There has been increasing interest in the possibility of using spontaneous canine hepatic disease as a model for those in human beings. 1 Dogs with congenital portosystemic shunts (CPSS) have hypoplasia of the liver and intrahepatic portal veins. While the condition is extremely rare in people, it is more common in dogs. Surgical CPSS attenuation results in liver growth and development of the intrahepatic portal vasculature, associated with clinical improvement. 2 The precise mechanism of this hepatic response is unknown; we hypothesised that it is due to hepatic regeneration and angiogenesis. The study aimed to measure the mRNA expression of growth factors and receptors involved in liver regeneration and angiogenesis in liver biopsies from dogs with CPSS before and after partial attenuation. Methods Dogs treated for CPSS were prospectively recruited to the study and liver biopsy samples were collected and placed in RNAlater . The expression of nine genes related to liver regeneration and angiogenesis were evaluated using quantitative polymerase chain reaction (qPCR). Differences in gene expression were assessed using independent or paired T tests. Significance was set at the 5% level (p=0.05). Results Liver biopsies were collected from 49 CPSS dogs to seven control dogs. 24 dogs tolerated complete attenuation of their CPSS and 25 tolerated partial attenuation. A second surgery was performed in all partial attenuation dogs to achieve complete attenuation and a follow-up biopsy was taken. HGF mRNA expression was significantly decreased in CPSS dogs compared with controls. There were significant increases in mRNA expression of HGF, MAT2α andVEGFR2 following partial CPSS attenuation. In addition, dogs that could tolerate complete attenuation had significantly greater MAT2α, VEGFR2 and TGFβR2 mRNA expression. Conclusion The results of this study indicate that the liver regeneration and angiogenesis are involved in the hepatic response to surgery in dogs with CPSS. This suggests that canine CPSS could be a useful, naturally occurring model of liver regeneration. Competing interests None declared. References 1. Kruitwagen HS , Arends B, Spee B, et al . Recombinant hepatocyte growth factor treatment in a canine model of congenital liver hypoplasia. Liver Int 2011; 31 :940–9. 2. Lee KC , Lipscomb VJ, Lamb CR, et al . Association of portovenographic findings with outcome in dogs receiving surgical treatment for single congenital portosystemic shunts: 45 cases (2000-2004). J Am Vet Med Assoc 2006; 229 :1122–9.

When rat serum was subjected to gel filtration on a Sephadex G-200 column, a factor, "hepatotropin", that promoted hepatocyte growth in primary culture was separated. Its Mr was about 150 KD and it was an anionic protein that was unstable on acid- and heat-treatments. Hepatotropin was purified 20-fold further by affinity chromatography on heparin-Sepharose CL-6B. The purified hepatotropin was effective at 20 micrograms/ml and maximally effective at 120 micrograms/ml, and its effect was additive with that of insulin plus epidermal growth factor. In rats after partial hepatectomy, the hepatotropin activity in the serum increased time-dependently reaching a maximum of about 5-fold the initial level 24 h after the operation. Various known growth factors, such as fibroblast growth factor, platelet derived growth factor, somatomedin, thrombin and transferrin, did not stimulate DNA synthesis in cultured hepatocytes. These results suggest that hepatotropin is a new growth factor.