Suppression of lipopolysaccharide-induced corneal opacity by hepatocyte growth factor.

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.

A Nanoparticle Delivery of Plasmid Encoding Hepatocyte Growth Factor for Gene Therapy of Silicosis in Mice.

PURPOSE: Silicosis is a serious occupational disease that is characterized by pulmonary infiltrates and fibrosis and is often refractory to current treatments. New therapeutic strategies for silicosis are needed. Hepatocyte growth factor (HGF) is a latent anti-inflammatory and anti-fibrotic growth factor. METHODS: We prepared a polyethyleneimine-polyethylene glycol/pHGF/hyaluronic acid (PEG-PEI/pHGF/HA) nanomaterials loaded with plasmid DNA encoding HGF gene to increase its transfection efficiency. The characterization, including DNA entrapment efficiency, morphology, particle size, and zeta-potential of PEG-PEI/pHGF/HA was studied. And a PEG-PEI/pHGF/HA (N/P=30:1) nanoparticle with low toxicity and high transfection efficiency was used in treatment for silicosis in mice. RESULTS: The results showed that the human HGF expression in the lungs of the mice was increased, and the inflammatory cell infiltration and fibrous collagen deposition was significantly reduced. CONCLUSION: Therefore, PEG-PEI/pHGF/HA nanoparticle warrant further investigation and may be a potential therapeutic strategy for silicosis.

Effect of Hepatocyte Growth Factor-Transfected Human Umbilical Cord Mesenchymal Stem Cells on Hepatic Stellate Cells by Regulating Transforming Growth Factor-ß1/Smads Signaling Pathway.

Studies have shown that human umbilical cord mesenchymal stem cells (hUCMSCs) could ameliorate liver fibrosis (LF) through inhibiting the activation of hepatic stellate cells (HSCs). However, the specific mechanisms have not been studied clearly. The purpose of this study was to explore the possible mechanism of hepatocyte growth factor (HGF)-transfected hUCMSCs in inhibiting the proliferation and activation of HSCs-T6. The upper and lower double-cell coculture system was established among HGF-hUCMSCs, LV5-NC-hUCMSCs, hUCMSCs, and HSCs-T6 in experimental groups; HSCs-T6 were cultured alone as control group. After coculturing for 1, 2, and 3 days, results showed that HGF-transfected hUCMSCs could decrease cell viability of HSCs-T6 and promote apoptosis; inhibit their activation and reduce the expression of Collagen I, Collagen III, TGF-ß1, Smad2 and Smad3, which may be related to inhibiting the activation of TGF-ß1/Smads signaling pathway. These findings suggested that HGF-transfected hUCMSCs may be used as an alternative and novel therapeutic approach for the treatment of LF.

Serum APOA4 Pharmacodynamically Represents Administered Recombinant Human Hepatocyte Growth Factor (E3112).

BACKGROUND: Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112. METHODS: rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured. RESULTS: Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, Apoa4, was found to be induced by rh-HGF in the murine liver. By using primary cultured human hepatocytes, the significant induction of human APOA4 was observed at the mRNA and protein levels, and it was inhibited in the presence of a c-Met inhibitor. Although mice constitutively expressed Apoa4 mRNA in the small intestine and the liver, the liver was the primary organ affected by administered rh-HGF to strongly induce APOA4 in a dose- and c-Met-dependent manner. Serum APOA4 levels were increased after rh-HGF administration, not only in normal mice but also in anti-Fas-induced murine acute liver failure (ALF), which confirmed the pharmacodynamic nature of APOA4. CONCLUSIONS: APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients.

Safe and low-dose but therapeutically effective adenovirus-mediated hepatocyte growth factor gene therapy for type 1 diabetes in mice.

AIMS: Hepatocyte growth factor (HGF) is a multifunctional cytokine that plays important roles in pancreatic physiology. Approvals of gene therapy drugs have highlighted gene therapy as an innovative new drug modality, but the very recent reports of deaths in clinical trials have provided a warning that high-dose gene therapy can cause dangerous liver toxicity. The present study aimed to develop a safe and low-dose but therapeutically effective adenovirus-mediated HGF gene therapy for streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. MAIN METHODS: A single intravenous injection of a low dose (3 × 108 plaque forming units) of adenoviral vector expressing the HGF gene under the transcriptional control of a strong promoter, i.e., the cytomegalovirus immediate-early enhancer and a modified chicken ß-actin promoter (Ad.CA-HGF), was given to T1D mice. KEY FINDINGS: Low-dose HGF gene therapy significantly attenuated the elevation of blood glucose concentrations at the acute phase of T1D, and this effect persisted for several weeks. Temporal upregulation of plasma insulin at the acute phase was maintained at a normal level in Ad.CA-HGF-treated mice, suggesting that the therapeutic mechanism may involve protection of the remaining ß-cells by HGF. Liver enzymes in plasma were not elevated in any of the mice, including the Ad.CA-HGF-treated animals, all of which looked healthy, suggesting the absence of lethal adverse effects observed in patients receiving high intravenous doses of viral vectors. SIGNIFICANCE: A low dose of intravenous Ad-mediated HGF gene therapy is clinically feasible and safe, and thus represents a new therapeutic strategy for treating T1D.

HGF alleviates septic endothelial injury by inhibiting pyroptosis via the mTOR signalling pathway.

BACKGROUND: Endothelial injury is one of the predominant pathophysiological characteristics of sepsis and is the major cause of sepsis-induced multiple organ failure. Endothelial pyroptosis is a fatal mechanism of endothelial injury in sepsis, and specific, effective therapies are lacking. Although hepatocyte growth factor (HGF) has been shown to have anti-apoptotic and anti-necrotic effects, whether it prevents pyroptosis to improve endothelial injury in sepsis remains unclear. METHODS: Recombinant HGF was intravenously injected into mice with sepsis caused by caecal ligation puncture (CLP). Histopathological examination and transmission electron microscopy (TEM) were used to measure lung vascular endothelial injury. Lipopolysaccharide (LPS) was transfected into EA.hy926 cells to induce endothelial pyroptosis, and the cells were treated with HGF in the presence of inhibitors of c-Met and mTOR, namely, PHA-665752 and rapamycin, respectively. The mTOR signalling pathway and mitochondrial physiology were assessed using Western blot and flow cytometry. RESULTS: Intravenous HGF effectively alleviated pulmonary vascular endothelial injury and acute lung injury in the septic mice. The TEM results of lung tissue revealed that HGF attenuated pulmonary vascular endothelial pyroptosis, which was confirmed in vitro. Transfected LPS induced the pyroptosis of EA.hy926 cells and damaged their paracellular permeability, and these effects were ameliorated by treating the cells with recombinant HGF. The protective effect of HGF against pyroptosis was dependent on c-Met/mTOR signalling. mTOR activation effectively protected mitochondrial physiology and decreased reactive oxygen species (ROS) production in EA.hy926 cells in vitro. CONCLUSIONS: These results demonstrated that HGF protected mitochondrial physiology by activating mTOR signalling to partially ameliorate endothelial pyroptosis and attenuate vascular endothelial injury and acute lung injury in sepsis animal model.

Phase I/II Study of Intrathecal Administration of Recombinant Human Hepatocyte Growth Factor in Patients with Acute Spinal Cord Injury: A Double-Blind, Randomized Clinical Trial of Safety and Efficacy.

Spinal cord injury (SCI) is an abrupt traumatic injury that leads to permanent functional loss, and no practical treatment is available. We have developed pharmaceutical recombinant human hepatocyte growth factor (KP-100), and its efficacy for SCI has been verified using animal models. The purpose of this study was to evaluate the safety and efficacy of intrathecal KP-100 administration for SCI patients in the acute phase. This investigation was a multi-center, randomized, double-blind study. Subjects with modified Frankel grade A/B1/B2 at 72 h after SCI were included. KP-100 was administered intrathecally. Subjects were followed up for 168 days after the first administration. Outcomes were evaluated using American Spinal Injury Association (ASIA) scores and subjected to analysis of covariance. Our results demonstrated that the subjects did not show any serious adverse events caused by KP-100. Forty-three subjects underwent neurological function testing (26 in KP-100 group; 17 in placebo group), which revealed that KP-100 contributed to motor improvement at Days 140 (p = 0.050) and 168 (p = 0.079). In the subset of subjects with Frankel grade A, the proportions of subjects who gained at least 1 point on their lower-extremity motor scores were 33.3% (5/15) and 6.3% (1/16) in the KP-100 and placebo groups, respectively (p = 0.083). Therefore, KP-100 has the potential to be useful and beneficial for SCI patients during the acute phase. However, this was a phase I/II trial and did not definitely address the question of efficacy; a larger phase III trial would be required to assess the efficacy.

Traction and attraction: haptotaxis substrates collagen and fibronectin interact with chemotaxis by HGF to regulate myoblast migration in a microfluidic device.

Cell migration is central to development, wound healing, tissue regeneration, and immunity. Despite extensive knowledge of muscle regeneration, myoblast migration during regeneration is not well understood. C2C12 mouse myoblast migration and morphology were investigated using a triple-docking polydimethylsiloxane-based microfluidic device in which cells moved under gravity-driven laminar flow on uniform (=) collagen (CN=), fibronectin (FN=), or opposing gradients (CN-FN or FN-CN). In haptotaxis experiments, migration was faster on FN= than on CN=. At 10 h, cells were more elongated on FN-CN and migration was faster than on the CN-FN substrate. Net migration distance on FN-CN at 10 h was greater than on CN-FN, as cells rapidly entered the channel as a larger population (bulk-cell movement, wave 1). Hepatocyte growth factor (HGF) stimulated rapid chemotaxis on FN= but not CN=, increasing migration speed at 10 h early in the channel at low HGF in a steep HGF gradient. HGF accelerated migration on FN= and bulk-cell movement on both uniform substrates. An HGF gradient also slowed cells in wave 2 moving on FN-CN, not CN-FN. Both opposing-gradient substrates affected the shape, speed, and net distance of migrating cells. Gradient and uniform configurations of HGF and substrate differentially influenced migration behavior. Therefore, haptotaxis substrate configuration potently modifies myoblast chemotaxis by HGF. Innovative microfluidic experiments advance our understanding of intricate complexities of myoblast migration. Findings can be leveraged to engineer muscle-tissue volumes for transplantation after serious injury. New analytical approaches may generate broader insights into cell migration.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Hepatocyte Growth Factor in the Treatment of Critical Limb Ischemia.

NL003 is a plasmid engineered to simultaneously express two isoforms of hepatocyte growth factor. This phase II study was performed to assess the clinical safety and efficacy of intramuscular injection of NL003 in critical limb ischemia (CLI) patients for 6 months. Two hundred patients (Rutherford scale 4-5) were randomly assigned: placebo (n = 50), low-dose NL003 (n = 50), middle-dose NL003 (n = 50), or high-dose NL003 (n = 50). The drug was administered in the affected limb of 197 patients on days 0, 14, and 28. No significant differences in the incidence of adverse events (AEs) or serious AEs were found among the groups. At 6 months, pain severity was significantly reduced in all NL003 groups, but not in the placebo group (p < 0.05). The proportion of patients with complete ulcer healing in the high-dose group was significantly higher than that of the placebo group (p = 0.0095). There were no statistically significant differences in transcutaneous oxygen pressure (TcPO2), ankle-brachial index (ABI), or toe-brachial index (TBI) value among the four groups throughout the study period. These results provide the first effective evidence of significant improvements in total healing of ulcers in treated legs, complete pain relief without analgesics, and safety for NL003 in patients with Rutherford stage 4-5.

Peglated-H1/pHGFK1 nanoparticles enhance anti-tumor effects of sorafenib by inhibition of drug-induced autophagy and stemness in renal cell carcinoma.

BACKGROUND: Tumor targeting small molecular inhibitors are the most popular treatments for many malignant diseases, including cancer. However, the lower clinical response and drug resistance still limit their clinical efficacies. HGFK1, the first kringle domain of hepatocyte growth factor, has been defined as a potent anti-angiogenic factor. Here, we aimed to develop and identify novel nanoparticles-PH1/pHGFK1 as potential therapeutic agents for the treatment of renal cell carcinoma (RCC). METHODS: We produced a novel cationic polymer-PH1 and investigated the anti-tumor activity of PH1/pHGFK1 nanoparticle alone and its combination therapy with sorafenib in RCC cell line xenografted mice model. Then, we figured out its molecular mechanisms in human RCC cell lines in vitro. RESULTS: We firstly demonstrated that intravenous injection of PH1/pHGFK1 nanoparticles significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice, as well as synergistically enhanced anti-tumor activities of sorafenib. Furthermore, we elucidated that recombinant HGFK1 improved sorafenib-induced cell apoptosis and arrested cell cycle. In addition, HGFK1 could also decrease sorafenib-induced autophagy and stemness via blockading NF-κB signaling pathway in RCC both in vitro and in vivo. CONCLUSIONS: HGFK1 could inhibit tumor growth, synergistically enhance anti-tumor activities of sorafenib and reverse its drug resistance evolution in RCC. Our results provide rational basis for clinical application of sorafenib and HGFK1 combination therapy in RCC patients.

Intrathecal delivery of recombinant AAV1 encoding hepatocyte growth factor improves motor functions and protects neuromuscular system in the nerve crush and SOD1-G93A transgenic mouse models.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease resulting from motor neuron degeneration that causes muscle weakness, paralysis, and eventually respiratory failure. We investigated whether recombinant adeno-associated virus encoding human hepatocyte growth factor (rAAV-HGF) could generate beneficial effects in two mouse models with neuromuscular problems when intrathecally delivered to the subarachnoid space. We chose AAV serotype 1 (rAAV1) based on the expression levels and distribution of HGF protein in the lumbar spinal cord (LSC). After a single intrathecal (IT) injection of rAAV1-HGF, the protein level of HGF in the LSC peaked on day 14 and thereafter gradually decreased over the next 14 weeks. rAAV1-HGF was initially tested in the mouse nerve crush model. IT injection of rAAV1-HGF improved mouse hindlimb strength and rotarod performance, while histological analyses showed that the length of regenerated axons was increased and the structure of the neuromuscular junction (NMJ) was restored. rAAV1-HGF was also evaluated in the SOD1-G93A transgenic (TG) mouse model. Again, rAAV1-HGF not only improved motor performance but also increased the survival rate. Moreover, the number and diameter of spinal motor neurons (SMNs) were increased, and the shape of the NMJs restored. Data from in vitro motor cortical culture experiments indicated that treatment with recombinant HGF protein (rHGF) increased the axon length of corticospinal motor neurons (CSMNs). When cultures were treated with an ERK inhibitor, the effects of HGF on axon elongation, protein aggregation, and oxidative stress were suppressed, indicating that ERK phosphorylation played an important role(s). Taken together, our results suggested that HGF might play an important role(s) in delaying disease progression in the SOD1-G93A TG mouse model by reducing oxidative stress through the control of ERK phosphorylation.

Application of Hepatocyte Growth Factor for Acute Spinal Cord Injury: The Road from Basic Studies to Human Treatment.

Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes, and has also gained attention as a strong neurotrophic factor in the central nervous system. We found that during the acute phase of spinal cord injury (SCI) in rats, c-Met, the specific receptor for HGF, increases sharply, while the endogenous HGF up-regulation is relatively weak. Introducing exogenous HGF into the spinal cord by injecting an HGF-expressing viral vector significantly increased the neuron and oligodendrocyte survival, angiogenesis, and axonal regeneration, to reduce the area of damage and to promote functional recovery in rats after SCI. Other recent studies in rodents have shown that exogenously administered HGF during the acute phase of SCI reduces astrocyte activation to decrease glial scar formation, and exerts anti-inflammatory effects to reduce leukocyte infiltration. We also reported that the intrathecal infusion of recombinant human HGF (intrathecal rhHGF) improves neurological hand function after cervical contusive SCI in the common marmoset, a non-human primate. Based on these collective results, we conducted a phase I/II clinical trial of intrathecal rhHGF for patients with acute cervical SCI who showed a modified Frankel grade of A/B1/B2 72 h after injury onset, from June 2014 to May 2018.

Safety, Tolerability, and Pharmacodynamics of Intrathecal Injection of Recombinant Human HGF (KP-100) in Subjects With Amyotrophic Lateral Sclerosis: A Phase I Trial.

Hepatocyte growth factor is an endogenous pleiotropic factor shown to act as a potent neuroprotectant against disease progression in animal models of amyotrophic lateral sclerosis, which is a devastating, adult-onset motor neuron disease. To evaluate the safety, tolerability, and pharmacokinetics of recombinant 5-residue-deleted human hepatocyte growth factor (KP-100) injected intrathecally through an implantable catheter connected to a subcutaneous port, we conducted a first-in-human phase I trial of intrathecal KP-100 in 15 Japanese patients with amyotrophic lateral sclerosis. The regimen was a single injection of 3 escalating doses (0.2, 0.6, and 2.0 mg/body) in 9 subjects followed by 2 doses (0.6 and 2.0 mg/body) repeated 5 times at 1-week intervals in 6 subjects (3 subjects/group). With single-dose administration, the mean half-life of KP-100 in the cerebrospinal fluid was 1.2 to 1.4 days, with its maximum concentration increasing in a dose-dependent manner. With multiple-dose administration, the trough KP-100 concentrations in the cerebrospinal fluid generally remained constant for any dose, despite multiple dosing. There were no deaths, serious adverse events, or device malfunctions leading to discontinuation. In all subjects, plasma KP-100 concentrations were <1 ng/mL, or below the lower limit of detection at all time points of measurement. Anti-KP-100 antibody was not detected in the cerebrospinal fluid or plasma specimens from any of the subjects throughout the KP-100 dosing period. These results suggest that KP-100, as well as the device used to administer it, is safe and tolerable. A phase II trial is warranted in patients with various central nervous system diseases such as amyotrophic lateral sclerosis.

Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTR.

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in the CFTR chloride channel, the most frequent of which is Phe508del. Phe508del causes not only intracellular retention and premature degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life when experimentally rescued to the plasma membrane (PM). Despite recent successes in the functional rescue of several CFTR mutations with small-molecule drugs, the folding-corrector/gating-potentiator drug combinations approved for Phe508del-CFTR homozygous patients have shown only modest benefit. Several factors have been shown to contribute to this outcome, including an unexpected intensification of corrector-rescued Phe508del-CFTR PM instability after persistent co-treatment with potentiator drugs. We have previously shown that acute co-treatment with hepatocyte growth factor (HGF) can significantly enhance the chemical correction of Phe508del-CFTR. HGF coaxes the anchoring of rescued channels to the actin cytoskeleton via induction of RAC1 GTPase signalling. Here, we demonstrate that a prolonged, 15-day HGF treatment also significantly improves the functional rescue of Phe508del-CFTR by the VX-809 corrector/VX-770 potentiator combination, in polarized bronchial epithelial monolayers. Importantly, we found that HGF treatment also prevented VX-770-mediated destabilization of rescued Phe508del-CFTR and enabled further potentiation of the rescued channels. Most strikingly, prolonged HGF treatment prevented previously unrecognized epithelial dedifferentiation effects of sustained exposure to VX-809. This was observed in epithelium-like monolayers from both lung and intestinal origin, representing the two systems most affected by adverse symptoms in patients treated with VX-809 or the VX-809/VX-770 combination. Taken together, our findings strongly suggest that co-administration of HGF with corrector/potentiator drugs could be beneficial for CF patients.

Plasmid-based gene therapy with hepatocyte growth factor stimulates peripheral nerve regeneration after traumatic injury.

Peripheral nerve injury remains a common clinical problem with no satisfactory treatment options. Numerous studies have shown that hepatocyte growth factor (HGF) exerts neurotrophic effect in motor, sensory, and parasympathetic neurons in addition to mitogenic, morphogenic, angiogenic, antiapoptotic, antifibrotic, and anti-inflammatory effect on various tissues and cells. In our study we examined efficacy of gene therapy with HGF-bearing plasmid (pC4W-hHGF) to improve consequences of traumatic nerve injury in mice. Treatment by pC4W-hHGF led to restoration of nerve structure and functional recovery compared to similar parameters in control animals. Compound action potentials (CAP) in experimental groups treated with 100 or 200 µg of pC4W-hHGF demonstrated increased amplitude and latency decrease compared to spontaneous recovery control group. In HGF-treated mice histological analysis showed a three-fold increase in axon number in nerve portion located distal to the lesion site compared to control. Moreover, significant functional recovery of n. peroneus communis triggered by pC4W-hHGF gene therapy was observed using the footprints analysis. Obtained results provide evidence for plasmid-based HGF gene therapy as a potential treatment for traumatic injury of peripheral nerve.

[Construction and bioactivity evaluation of hepatocyte growth factor-loaded poly (lactic-co-glycolic acid) nanoparticles].

OBJECTIVE: To explore the optimum conditions for preparing poly(lactic-co-glycolic) acid (PLGA) nanoparticles and evaluate the bioactivity of hepatocyte growth factor (HGF)-loaded PLGA nanoparticles. METHODS: Bovine serum albumin (BSA)-loaded PLGA nanoparticles were prepared using a double emulsion-solvent evaporation method. The preparation process of nanoparticles was optimized by orthogonal test with the particle size, encapsulation efficiency (EE), drug loading (DD), and recovery as the indexes. HGF-loaded nanoparticles were then prepared under the optimized conditions. The EE, DD and release characteristics of BSA?loaded nanoparticles and HGF-loaded nanoparticles were evaluated using a BCA kit and HGF ELISA kit. The bioactivity of HGF-loaded nanoparticles was evaluated using CCK8 proliferation assay. RESULTS: The HGF-loaded nanoparticles prepared under the optimized conditions had a uniform size with a mean diameter of 234.4∓4.8 nm, an EE of (77.75∓3.04)% and a recovery rate of (49.33∓9.34)%. The in vitro release curve highlighted an initial burst drug release followed by sustained release from the nanoparticles. HGF-loaded nanoparticles obviously promoted the proliferation of Hacat keratinocytes in vitro. CONCLUSION: HGF-loaded nanoparticles prepared using double emulsion?solvent evaporation method under optimized conditions possesses a high EE with a good sustained drug release profile and a good bioactivity.

Overexpression of Hepatocyte Growth Factor mRNA Induced by Gene Transfer Attenuates Neointimal Hyperplasia After Balloon Injury.

Hepatic growth factor (HGF) has been widely used in studies on arterial remodeling after injury, and results turn out to be inconsistent. The changes of endogenous HGF expression after injury also remain controversial. This study clarified the role of exogenous human HGF (hHGF) gene transfer in neointimal hyperplasia and investigated the associated alterations of endogenous HGF and c-Met expressions under endothelial denudation with or without hHGF gene transfer using a balloon-injured rabbit aorta model. Sixty-one rabbits were randomly divided into normal controls, endothelial injury, endothelial injury with hHGF, or the control vector gene transfer groups. On weeks 1, 2, 4, and 8 after injury, neointimal hyperplasia and endothelialization were evaluated by the ratio of neointimal area to medial area (N/M ratio), CD31-positive staining, α-smooth muscle actin, and endothelial nitric oxide synthase expressions using histological analysis, immunohistochemistry staining, or real-time quantitative reverse transcriptase polymerase chain reaction. Endogenous rabbit HGF (rHGF) and c-Met expressions were detected with immunohistochemistry staining and quantitative reverse transcriptase polymerase chain reaction. It was found that expressions of endogeneous rHGF and c-Met in endothelial injury upregulated with peak levels on week 2 or week 4 after injury (p < 0.01). On week 1 after hHGF transfer, neointimal hyperplasia was significantly inhibited (p < 0.001), with decreased α-smooth muscle actin expression (p < 0.05) and improved endothelial cells regeneration and function (p < 0.01). More remarkable overexpression of endogenous rHGF and c-Met mRNAs were detected, and lowered positive staining of rHGF and c-Met was shown in the neointima (p < 0.05). These results demonstrated hHGF gene transfer induced further overexpression of endogenous rHGF and c-Met mRNAs but lowered immunoreactivities of rHGF and c-Met in the neointima, thus leading to significant attenuation of neointimal hyperplasia.

Transplantation of Hepatocyte Growth Factor-Modified Dental Pulp Stem Cells Prevents Bone Loss in the Early Phase of Ovariectomy-Induced Osteoporosis.

Investigations based on mesenchymal stem cells (MSCs) for osteoporosis have attracted attention recently. MSCs can be derived from various tissues, such as bone marrow, adipose, umbilical cord, placenta, and dental pulp. Among these, dental pulp-derived MSCs (DPSCs) and hepatocyte growth factor (HGF)-modified DPSCs (DPSCs-HGF) highly express osteogenic-related genes and have stronger osteogenic differentiation capacities. DPSCs have more benefits in treating osteoporosis. The purpose of this study was to investigate the roles of HGF gene-modified DPSCs in bone regeneration using a mouse model of ovariectomy (OVX)-induced bone loss. The HGF and luciferase genes were transferred into human DPSCs using recombinant adenovirus. These transduced cells were assayed for distribution or bone regeneration assay by transplantation into an OVX-induced osteoporosis model. By using bioluminogenic imaging, it was determined that some DPSCs could survive for >1 month in vivo. The DPSCs were mainly distributed to the lung in the early stage and to the liver in the late stage of OVX osteoporosis after administration, but they were scarcely distributed to the bone. The homing efficiency of DPSCs is higher when administrated in the early stage of a mouse OVX model. Micro-computed tomography indicated that DPSCs-Null or DPSCs-HGF transplantation significantly reduces OVX-induced bone loss in the trabecular bone of the distal femur metaphysis, and DPSCs-HGF show a stronger capacity to reduce bone loss. The data suggest that systemic infusion of DPSCs-HGF is a potential therapeutic approach for OVX-induced bone loss, which might be mediated by paracrine mechanisms.

Efficacy of the dual controlled release of HGF and bFGF impregnated with a collagen/gelatin scaffold.

BACKGROUND: We previously developed collagen/gelatin sponges (CGS) able to sustain and release basic fibroblast growth factor (bFGF) and reported that this CGS impregnated with bFGF promoted dermis-like tissue formation. We herein confirmed the single-sustained release of hepatocyte growth factor (HGF) and the dual sustained release of HGF and bFGF from CGSs, and explored its efficacy using a murine model of skin defects. MATERIALS AND METHODS: The sustained release of HGF alone and both HGF and bFGF from CGSs were evaluated in vitro. CGSs (8 mm in diameter) impregnated with normal saline solution (NSS) (NSS group), HGF solution (10 or 50 µg/cm2) (HGF-L or HGF-H group), bFGF solution (7 µg/cm2) (bFGF group), or HGF (10 µg/cm2) and bFGF (7 µg/cm2) solution (HGF + bFGF group) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, dermis-like tissue formation and newly formed capillaries were evaluated. RESULTS: The single release of HGF and the dual release of HGF and bFGF from CGSs were confirmed. At week 1, the wound closure and neoepithelium length were promoted in the HGF-L group compared with the NSS group. At week 2, the wound closure, neoepithelium length, dermis-like tissue formation and newly formed capillary formation were promoted in the bFGF and HGF + bFGF groups compared with the NSS and HGF-H groups. Newly formed capillary formation was superior in the HGF + bFGF group compared with the bFGF group. CONCLUSIONS: The dual release of HGF and bFGF from CGS was a promising treatment for full-thickness skin defects.

A phase I/II exploratory clinical trial for intracordal injection of recombinant hepatocyte growth factor for vocal fold scar and sulcus.

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.