Hepatocyte Growth Factor Gene Therapy Research Articles

Recent advances in the therapeutic efficacy of hepatocyte growth factor gene-modified mesenchymal stem cells in multiple disease settings.

Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co‐transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune‐related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.

Clinical study of hepatocyte growth factor in the treatment of diabetic lower limb arteriosclerosis obliterans

OBJECTIVE: The objective of this study was to investigate the safety and efficacy of hepatocyte growth factor (HGF) gene therapy in the patients with diabetic lower limb arteriosclerosis obliterans, provide theoretical support for better coordination of clinical operation and anesthesia, ensure medical safety, and improve the quality of nursing.MATERIALS AND METHODS: Twenty-one patients with Grade 4–6 (Rutherford classification) and limb ischemia who met the inclusion criteria were divided into four dose groups, including three cases in the 4 mg group, six cases in the 8 mg group, five cases in the 12 mg group, and seven cases in the 16 mg group. The dosage of each group was divided into equal doses, which were injected intramuscularly on the 1st and 15th days of treatment, respectively. The dosage of each injection point was 0.25 mg/0.5 ml. Adverse reactions, visual analog scale (VAS) scores for pain and ulcer healing were observed on the 1st, 8th, 15th, 21st, 28th, 59th, and 91st days after the treatment. The ankle-brachial index (ABI) was measured on the 1st, 28th, 59th, and 91st days after the treatment.RESULTS: Complications, serious adverse reactions, and abnormal test results did not occur in the patients during the operation. After the operation, the pain had significantly reduced in 17 cases, the average VAS score of all cases decreased from 5.95 to 1.64 (P < 0.05), and the pain disappeared completely in four cases. The average ABI of all cases increased from 0.49 to 0.63 (P < 0.05), five cases had ulcer, and four cases had gangrene. After treatment, one case of ulcer and one case of gangrene healed completely, and three cases of ulcer and one case of gangrene improved significantly, and one case of ulcer and two cases of gangrene had no significant change.CONCLUSION: HGF gene therapy can effectively treat diabetic lower limb arteriosclerosis obliterans, relieve the clinical symptoms and improve the quality of life. The smooth operation is inseparable from the cooperation of nurses, including careful preoperative visit, strict aseptic operation during the operation, follow-up visit regularly after the operation, and mastering the changes in patients' condition in the whole process to provide high-quality nursing for patients.

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

AimsHepatocyte 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 methodsA 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 β-actin promoter (Ad.CA-HGF), was given to T1D mice. Key findingsLow-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 β-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. SignificanceA low dose of intravenous Ad-mediated HGF gene therapy is clinically feasible and safe, and thus represents a new therapeutic strategy for treating T1D.

Novel Drug Treatments for Diabetes Complications

Diabetes mellitus is a metabolic disease associated with elevated levels of blood glucose. It has become the most prevalent disease in the Saudi population according to the last report from the national prevention program. Diabetes cases can be divided into three types: type I, which requires insulin therapy; type II, which requires the use of hypoglycemic drugs, with or without insulin, and is the most common type in Saudi Arabia; and gestational diabetes, which occurs during pregnancy. We must remember that diabetes can be very dangerous, causing complications in many parts of the body, like the nerves, heart, eyes, and kidneys. According to previous studies, these complications vary depending on the severity of diabetes. They may also affect the blood vessels of the retina, which can develop into diabetic retinopathy. Complications may also be fatal, in the cases of heart attacks, angina, and atherosclerosis. Additionally, they may cause poor circulation that leads to infection and gangrene, or cause neuropathy with numbness and a loss of pain sensations. Diabetic nephropathy is another harmful effect of diabetes that attacks the renal blood vessels. In this article, my aim was to review newly discovered drugs that target and decrease the complications produced by diabetes. My focus here was on certain drugs, including those using nanotechnology, cell-based therapy, angiopoietin like 4, baricitinib, and hepatocyte growth factor (HGF) gene therapy.

Combined Analysis of Clinical Data on HGF Gene Therapy to Treat Critical Limb Ischemia in Japan.

The objective of this combined analysis of data from clinical trials in Japan, using naked plasmid DNA encoding hepatocyte growth factor (HGF), was to document the safety and efficacy of intramuscular HGF gene therapy in patients with critical limb ischemia (CLI). HGF gene transfer was performed in 22 patients with CLI in a single-center open trial at Osaka University; 39 patients in a randomized, placebo-controlled, multi-center phase III trial, 10 patients with Buerger's disease in a multi-center open trial; and 6 patients with CLI in a multi-center open trial using 2 or 3 intramuscular injections of naked HGF plasmid at 2 or 4 mg. Resting pain on a visual analogue scale (VAS) and wound healing as primary endpoints were evaluated at 12 weeks after the initial injection. Serious adverse events caused by gene transfer were detected in 7 out of 77 patients (9.09%). Only one patient experienced peripheral edema (1.30%), in contrast to those who had undergone treatment with VEGF. At 12 weeks after gene transfer, combined evaluation of VAS and ischemic ulcer size demonstrated a significant improvement in HGF gene therapy group as compared to the placebo group (P=0.020). The long-term analysis revealed a sustained decrease in the size of ischemic ulcer in HGF gene therapy group. In addition, VAS score over 50 mm at baseline (total 27 patients) demonstrated a tendency (P=0.059), but not significant enough, to improve VAS score in HGF gene therapy as compared to the placebo group. The findings indicated that intramuscular injection of naked HGF plasmid tended to improve the resting pain and significantly decreased the size of the ischemic ulcer in the patients with CLI who did not have any alternative therapy, such as endovascular treatment (EVT) or bypass graft surgery. An HGF gene therapy product, CollategeneTM, was recently launched with conditional and time-limited approval in Japan to treat ischemic ulcer in patients with CLI. Further clinical trials would provide new therapeutic options for patients with CLI.

Anti-inflammatory activities of hepatocyte growth factor in post-ischemic heart failure.

Hepatocyte growth factor (HGF) alleviates acute and chronic inflammation in experimental inflammatory bowel disease, glomerulonephritis, and airway inflammation. However, the anti-inflammatory effects of HGF on myocardial infarction are not defined. The current study assessed the anti-inflammatory effects of HGF in post-ischemic heart failure. The left anterior descending coronary artery was ligated in rats, and adenovirus containing human HGF (Ad-HGF) or control virus (Ad-GFP) was administered intramyocardially. The quantity of proinflammatory cytokines secreted by cardiomyocytes, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, was evaluated. Cardiac function and LV remodeling were assessed using echocardiography and collagen deposition, respectively. Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) four weeks after injection were significantly increased in Ad-HGF-treated animals compared to the Ad-GFP group. HGF gene therapy improved ventricular geometry with a significantly decreased left ventricular end-diastolic diameter (LVEDD) and markedly reduced myocardial collagen deposition. Treatment with Ad-HGF significantly decreased the mRNA levels of TNF-α, IL-6, and IL-1β in the non-infarcted region four weeks after injection. Changes of the TNF-α, IL-6, and IL-1β levels in the non-infarcted region positively correlated with the LVEDD 4 weeks after infarction. Treatment of acute myocardial infarction (AMI) with Ad-HGF in the early stage of MI reduced the pro-inflammatory cytokine levels and preserved cardiac function. These findings indicated that Ad-HGF gene therapy alleviated ventricular remodeling after infarction by reducing inflammation.

Hepatocyte Growth Factor Gene Therapy for Ischemic Diseases.

Stem cells and gene therapy have become promising strategies for treating ischemic diseases and regenerating tissue. Hepatocyte growth factor (HGF) is an angiogenic growth factor with multiple functions, including promoting angiogenesis, regulating inflammation, inhibiting fibrosis, and activating tissue regeneration. Numerous preclinical experiments and clinical trials have demonstrated the feasibility and efficacy of HGF gene therapy in the treatment of ischemic diseases and tissue regeneration. This review summarizes the current advances of therapeutic angiogenesis using HGF gene transfer and modified stem cells. The physiological roles of HGF in angiogenesis and tissue regeneration are revisited. The current advances of clinical trials of plasmid and adenovirus HGF in the treatment of critical limb ischemia and coronary heart disease in China are introduced. Furthermore, valuable insight is provided into the prospective future of novel regenerative strategies using HGF-modified mesenchymal stem cells. HGF gene therapy is presented as a promising therapeutic approach in the treatment of ischemic diseases and regenerative medicine.

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.

Novel Combination BMP7 and HGF Gene Therapy Instigates Selective Myofibroblast Apoptosis and Reduces Corneal Haze In Vivo.

PurposeWe tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models.MethodsEighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests.ResultsPEI2-GNP–mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in −therapy group; P < 0.001). Corneas that received BMP7+HGF demonstrated significantly reduced mRNA levels of profibrotic genes: α-SMA (3.2-fold; P < 0.01), fibronectin (2.3-fold, P < 0.01), collagen I (2.1-fold, P < 0.01), collagen III (1.6-fold, P < 0.01), and collagen IV (1.9-fold, P < 0.01) compared to the −therapy corneas. Furthermore, BMP7+HGF-treated corneas showed significantly fewer myofibroblasts compared to the −therapy controls (83%; P < 0.001). The PEI2-GNP introduced >104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity.ConclusionsLocalized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts.

Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by AttenuatedSalmonellain Rats

The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 109 cfu), vehicle (TP, 1 × 109 cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm2, which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm2, respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm2, which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site.

El factor de crecimiento del hepatocito disminuye la expresión vascular de mediadores inflamatorios y la hipertensión en ratas espontáneamente hipertensas

ObjectiveThis study was designed to examine the effects of hepatocyte growth factor (HGF) gene delivery on vascular inflammation and hypertension in spontaneously hypertensive rats (SHR). We speculated that HGF deficiency could play a key role in the pathogenesis of hypertension in SHR, and that increasing HGF levels will produce prolonged decreases in blood pressure due to reduced vascular inflammation. Materials and methodsFifteen-week old male SHRs received weekly hydrodynamic injections of a naked plasmid containing human HGF (pCMV-HGF) (1mg/kg) or empty vector (pcDNA3.1) for 6 weeks. Two groups of Wistar-Kyoto (WKY) rats were used as controls (n=6) and treated in the same manner. The activation of NF-κB was assessed by Western blot and mRNA expression of pro-inflammatory cytokines by real-time PCR and Western blot. ResultsBlood pressure, NF-κB activation and expression of IL-6, MCP-1 and RANTES were significantly higher in SHR than in the control WKY. The HGF gene therapy normalized NF-κB activity, pro-inflammatory cytokines expression, and decreased the hypertension in SHR. ConclusionThese observations suggest that decreased aorta HGF concentration may have a role in the vascular inflammation observed in SHR, and demonstrate that increasing HGF is a potential therapeutic target in the treatment of hypertension.

Stimulatory effect of HGF-overexpressing adipose tissue-derived mesenchymal stem cells on thymus regeneration in a rat thymus involution model.

The thymus is the central lymphoid organ providing a unique and essential microenvironment for T-cell precursor development into mature functionally competent T-lymphocytes. Thus, it is important to develop the strategies for enhancing thymic regeneration from involution induced by a variety of clinical treatments and conditions. Hepatocyte growth factor (HGF) promotes proliferation in a variety of cell types. We have used stem cell-based HGF gene therapy to enhance regeneration from acute thymic involution. HGF-overexpressing human adipose tissue-derived mesenchymal stem cells (HGF-hATMSCs) were generated by liposomal transfection with the pMEX expression vector, constructed by inserting the HGF gene. Significantly increased HGF expression in these cells was confirmed by reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay. HGF produced by HGF-hATMSCs enhanced the proliferation of a mouse thymic epithelial cell line and the expression of interleukin-7 in vitro. We also examined the effect of HGF-hATMSCs on thymic regeneration in rats with acute thymic involution. Significant increases in thymus size and weight, as well as the number of thymocytes (especially, early thymocyte progenitors), were seen in the HGF-hATMSCs-treated rats compared to saline-treated control animals. A stimulatory effect of HGF-hATMSCs on thymic regeneration has therefore been shown, highlighting the clinical value of HGF-hATMSCs for treating thymic involution.

Therapeutic Angiogenesis by Gene Therapy for Critical Limb Ischemia: Choice of Biological Agent.

Peripheral artery disease (PAD) is caused by atherosclerosis, hardening and narrowing arteries over time due to buildup of fatty deposit in vascular bed called plaque. Severe blockage of an artery of the lower extremity markedly reduce blood flow, resulting in critical limb ischemia (CLI) manifested by a variety of clinical syndromes including rest pain in the feet or toes, ulcer and gangrene with infection. Despite significant advances in clinical care and interventions for revascularization, patients with CLI remain at high risk for amputation and cardiovascular death. To overcome this unmet need, therapeutic angiogenesis using angiogenic growth factors has evolved in an attempt to increase blood flow in ischemic limb. Initial animal studies and phase I clinical trials with vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF) demonstrated promising results, inspiring scientists to progress forward. However, more rigorous phase II and III clinical trials have failed to demonstrate beneficial effects of these angiogenic growth factors to date. Recently, two multicenter, double-blind, placebo-controlled clinical trials in Japan (phase III) and US (phase II) demonstrated that hepatocyte growth factor (HGF) gene therapy for CLI significant improved primary end points and tissue oxygenation up to two years in comparison to placebo. These clinical results implicate a distinct action of HGF on cellular processes involved in vascular remodeling under pathological condition. This review presents data from phase I-III clinical trials of therapeutic angiogenesis by gene therapy in patients with PAD. Further, we discuss the potential explanation for the success or failure of clinical trials in the context of the biological mechanisms underlying angiogenesis and vascular remodeling, including cellular senescence, inflammation, and tissue fibrosis.

Endothelial progenitor cells in clinical settings.

Senescence of cells is associated with shortened or damaged telomeres and is characterized by permanent exit from the cell cycle and altered function. Cellular senescence is caused by repeated cell division, and also conditions of stress including inflammation and reactive oxygen species can lead to the development of premature senescence. At the cellular level, proliferative and oxidative-stress induced cell senescence related to a pro-inflammatory state might strongly contribute to age-associated impaired tissue and organ functions. Vascular cells (endothelial cells, vascular smooth muscle cells) and bone marrow-derived endothelial progenitor cells have been repeatedly shown to have pivotal role in the maintenance and regeneration of cardiovascular tissue. Therefore, the molecular mechanisms of vascular cell senescence have been extensively studied. However, therapeutic approaches to prevent cellular senescence in cardiovascular disease (CVD) are still limited. Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) are all potent angiogenic growth factors in animal models of ischemia, but their therapeutic effects are not the same in animal experiments and clinical trials. A multicenter, double-blind, placebo-controlled phase III clinical trial in Japan and a US phase II clinical trial of HGF gene therapy for critical limb ischemia (CLI) demonstrated a significant improvement in primary end points and an increase in transcutaneous partial pressure of oxygen even after one year compared with placebo, whereas effectiveness of VEGF and FGF treatment for CLI has not yet been shown. Moreover, our recent publication and another researcher demonstrated that HGF acts as an anti-inflammatory cytokine, while VEGF and FGF act as pro-inflammatory cytokine. This review overviews the outcomes of clinical trials using angiogenic growth factors, which have shown a dramatic effect in several animal studies. Additionally, interventions with HGF aimed at improving the regenerative capacity of stem/progenitor cells and vascular cells by preventing cellular senescence are discussed.

Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Reduce Radiation-Induced Lung Injury

Abstract Effective therapeutic strategies for radiation-induced lung injury (RILI) are lacking. Mesenchymal stem cells (MSCs), as gene therapy delivery vehicles, possess the ability to repair injured lung. In this study, we conducted MSC-based hepatocyte growth factor (HGF) gene therapy for RILI. Mice received single-dose radiation with 20 Gy of γ rays locally to the lung, and then were administered normal sodium, Ad-HGF-modified MSCs, or Ad-Null-modified MSCs. Ad-HGF-modified MSCs (MSCs-HGF) improved histopathological and biochemical markers of lung injury. MSCs-HGF could reduce secretion and expression of proinflammatory cytokines, including tumor necrosis factor-α, interferon-γ, interleukin (IL)-6, and intercellular adhesion molecule-1, and increase the expression of antiinflammatory cytokine IL-10. It could also decrease expression levels of profibrosis factors transforming growth factor-β, Col1a1 (collagen type 1, α1), and Col3a1, and inhibit fibrosis progress. MSCs-HGF could promote proliferation of lung epithelial cells and protect them from apoptosis, and improve the expression of endogenous HGF and its receptor c-Met significantly. We also found that sphingosine-1-phosphate receptor-1 expression was increased in injured lung. These results suggest MSC-based HGF gene therapy not only reduces inflammation but also inhibits lung fibrosis.

Long-Term Follow-Up Evaluation of Results From Clinical Trial Using Hepatocyte Growth Factor Gene to Treat Severe Peripheral Arterial Disease

As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.

Novel therapy for idiopathic pulmonary arterial hypertension: Can hepatocyte growth factor be beneficial?

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive, nearly fatal condition that until recently has had very few treatment options. Median survival time for untreated IPAH was 2.8 years without effective drug intervention. IPAH is characterized by deregulated proliferation of pulmonary arterial endothelial and intimal smooth muscle cells resulting in progressive pulmonary vascular remodeling and an increase in pulmonary arterial pressure. In order to alleviate their symptoms, anticoagulants, diuretics, calcium channel blockers and inotropic agents have been used to treat patients with PAH. Moreover, specific targeted therapies using prostacyclins, endothelin-receptor antagonists and phosphodiesterase type-5 inhibitors have been developed recently. However, because of the insufficient proof of efficacy and poor tolerability of these agents, new therapeutic modalities continue to be explored. Hepatocyte growth factor (HGF), which was first purified as a mitogen for hepatocytes from the plasma of patients with fulminant hepatic failure, has mitogenic, motogenic, morphogenic, and antiapoptotic activities in various cell types. The pluripotent activities of HGF are mediated by a membrane- spanning tyrosine kinase receptor encoded by the c-Met proto-oncogene. HGF acts as a safe and effective organotrophic factor for protection from injury and ischemia of various organs. Phase-II and Phase-III clinical trials of HGF gene therapy for the treatment of peripheral arterial disease have been completed in both the USA and Japan. In the lung, biological and pulmotrophic roles for HGF have been well documented. In response to acute lung injury, HGF plays a role in lung regeneration and protection. Furthermore, research elucidating the pulmotrophic role of HGF has led to the development of therapeutic approaches for the treatment of chronic lung diseases.

Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle.

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.

Attenuation of hepatic fibrosis through ultrasound-microbubble-mediated HGF gene transfer in rats

ObjectiveThe objective was to explore the feasibility of ultrasound-microbubble-mediated hepatocyte growth factor (HGF) gene transfer for treating rat hepatic fibrosis induced by CCl4. MethodsForty-eight male SD rats were divided into ultrasound-microbubble-HGF group (U-M-HGF group), ultrasound-HGF group (U-HGF group), microbubble-HGF group (M-HGF group), HGF group (HGF group), CCl4 group (control group), and normal group. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total protein, albumin (ALB), and globulin (GLB) and the ratio of ALB/GLB were determined after treatment. The degree of hepatic fibrosis was evaluated by histopathological numerical scores. The protein expressions of HGF, collagen I, collagen III, and α-smooth muscle antibody (α-SMA) were detected by immunohistochemistry. ResultsUltrasound-microbubble-mediated HGF therapy significantly reduced the serum level of ALT and AST to 59.88% and 49.18% of the control group, respectively. Ultrasound-microbubble-mediated HGF therapy prevented liver fibrosis, with an obvious decrease in fibrosis areas and extracellular matrix production of collagen I, collagen III, and α-SMA. The gene therapy could induce HGF delivery into the fibrotic liver effectively. ConclusionsUltrasound-microbubble-mediated HGF gene therapy can reduce liver fibrosis, which provides a novel strategy for gene therapy of chronic liver disease.

Inhibition of Lp(a)-induced functional impairment of endothelial cells and endothelial progenitor cells by hepatocyte growth factor

BackgroundLipoprotein (a) (Lp(a)) is one of the risk factors for peripheral artery disease (PAD). Our previous report demonstrated that hepatocyte growth factor (HGF) gene therapy attenuated the impairment of collateral formation in Lp(a) transgenic mice. Since risk factors for atherosclerosis accelerate endothelial senescence and impair angiogenesis, we examined the role of Lp(a) in dysfunction and senescence of endothelial progenitor cells (EPC) and endothelial cells. MethodsIn vitro and in vivo incorporation assays were performed using ex-vivo expanded DiI-labeled human EPC. Senescence of cultured endothelial cells, production of oxidative stress and angiogenesis function were evaluated by SA-β-galactosidase staining, dihydroethidium (DHE) staining and Matrigel assay, respectively. ResultsEPC transplantation significantly stimulated recovery of ischemic limb perfusion, while EPC pre-treated with Lp(a) did not increase ischemic limb perfusion. Impairment of angiogenesis by EPC with Lp(a) was associated with a significant decrease in CD31-positive capillaries and DiI-labeled EPC. Importantly, Lp(a) significantly accelerated the onset of senescence and production of reactive oxygen species (ROS) in human aortic endothelial cells, accompanied by a significant increase in the protein expression of p53 and p21. On the other hand, HGF significantly attenuated EPC dysfunction, senescence, ROS production, and p53 and p21 expression induced by Lp(a). ConclusionLp(a) might affect atherosclerosis via acceleration of senescence, ROS production, and functional impairment of the endothelial cell lineage. HGF might have inhibitory effects on these atherogenic actions of Lp(a).