Naotaka Hashiya

Safety Evaluation of Clinical Gene Therapy Using Hepatocyte Growth Factor to Treat Peripheral Arterial Disease

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with critical limb ischemia (CLI). Because hepatocyte growth factor (HGF) has potent angiogenic activity, we investigated the safety and efficiency of HGF plasmid DNA in patients with CLI as a prospective open-labeled clinical trial. Intramuscular injection of naked HGF plasmid DNA was performed in ischemic limbs of 6 CLI patients with arteriosclerosis obliterans (n= 3) or Buerger disease (n= 3) graded as Fontaine III or IV. The primary end points were safety and improvement of ischemic symptoms at 12 weeks after transfection. Severe complications and adverse effects caused by gene transfer were not detected in any patients. Of particular importance, no apparent edema was observed in any patient throughout the trial. In addition, serum HGF concentration was not changed throughout the therapy period in all patients. In contrast, a reduction of pain scale of more than 1 cm in visual analog pain scale was observed in 5 of 6 patients. Increase in ankle pressure index more than 0.1 was observed in 5 of 5 patients. The long diameter of 8 of 11 ischemic ulcers in 4 patients was reduced > 25%. Intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs. Although the present data are conducted to demonstrate the safety as phase I/early phase IIa, the initial clinical outcome with HGF gene transfer seems to indicate usefulness as sole therapy for CLI.

Angiogenesis and Antifibrotic Action by Hepatocyte Growth Factor in Cardiomyopathy

Impairment of cardiac function in cardiomyopathy has been postulated to be related to decreased blood blow and increased collagen synthesis. Therefore, a therapeutic approach to alter the blood flow or fibrosis directly by means of growth factors may open a new therapeutic concept in dilated cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with antifibrosis and angiogenesis effects. Using the hereditary cardiomyopathic Syrian hamster as a model of genetically determined cardiomyopathy and heart failure, the effects of overexpression of HGF on fibrosis and microvascular dysfunction were examined. HGF gene or control vector was injected by the Hemagglutinating Virus of Japan–liposome method into the anterior heart of cardiomyopathic hamsters (Bio 14.6) under echocardiography once a week, from 12 to 20 weeks of age (total, 8 times). Blood flow, as assessed by a laser Doppler imager score, and the capillary density in hearts, as assessed by alkaline phosphatase staining, were significantly increased in hamsters transfected with HGF gene compared with control-vector-transfected hamsters (P <0.01). In contrast, the fibrotic area was significantly decreased in hamsters transfected with HGF gene compared with control (P <0.01). Overall, in vivo experiments demonstrated that transfection of HGF gene into the myocardium of cardiomyopathic hamsters stimulated blood flow through the induction of angiogenesis and reduction of fibrosis. These results suggest that HGF gene transfer may be useful to protect against myocardial injury in cardiomyopathy through its cardioprotective effects such as antifibrosis and angiogenesis actions.

Angiogenesis Induced by Endothelial Nitric Oxide Synthase Gene Through Vascular Endothelial Growth Factor Expression in a Rat Hindlimb Ischemia Model

Background—Because the mechanism of the angiogenic property of nitric oxide (NO) was not fully understood in vivo, we focused on the role of vascular endothelial growth factor (VEGF) in angiogenesis induced by endothelial NO synthase (eNOS) gene transfer. Methods and Results—After intramuscular injection of eNOS DNA into a rat ischemic hindlimb, transfection of eNOS vector resulted in a significant increase in eNOS protein 1 week after transfection. In addition, tissue concentrations of nitrite and nitrate were significantly increased in rats transfected with the eNOS gene up to 2 weeks after transfection. The increase in tissue nitrite and nitrate concentrations was completely inhibited by NG-nitro-l-arginine methyl ester (L-NAME). In contrast, serum concentrations of nitrite and nitrate and blood pressure were not changed by eNOS gene transfer. Importantly, overexpression of the eNOS gene resulted in a significant increase in peripheral blood flow, whereas L-NAME inhibited the increase in blood flow. Interestingly, basal blood flow was significantly lower in rats treated with L-NAME than in control rats. A significant increase in capillary number was consistently detected in rats transfected with the eNOS gene at 4 weeks after transfection, accompanied by a significant increase in VEGF. Moreover, administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS plasmid injection. Conclusions—Overall, intramuscular injection of bovine eNOS plasmid induced therapeutic angiogenesis in a rat ischemic hindlimb model, a potential therapy for peripheral arterial disease. The stimulation of angiogenesis by NO might be due to upregulation of local VEGF expression.

Therapeutic angiogenesis using hepatocyte growth factor (HGF).

HGF is a mesenchyme-derived pleiotropic factor, which regulates cell growth, cell motility, and morphogenesis of various types of cells and is thus considered a humoral mediator of epithelial-mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis. Although HGF was originally identified as a potent mitogen for hepatocytes, it has also been identified as a member of angiogenic growth factors. Interestingly, the presence of its specific receptor, c-met, is observed in vascular cells and cardiac myocytes. In addition, among growth factors, the mitogenic action of HGF on human endothelial cells was most potent. Recent studies have demonstrated the potential application of HGF to treat cardiovascular diseases such as peripheral vascular disease, myocardial infarction and cerebrovascular disease. In this review, we will discuss a potential therapeutic strategy using HGF in cardiovascular disease.

In Vivo Evidence of Angiogenesis Induced by Transcription Factor Ets-1 Ets-1 Is Located Upstream of Angiogenesis Cascade

Background—A transcription factor, ets-1, regulates the transcription of metalloproteinase genes, the activity of which is necessary for matrix degradation and the migration of endothelial cells. However, no study has demonstrated that ets-1 itself has an angiogenic action in vivo. Thus, we examined (1) the effects of overexpression of the ets-1 gene on angiogenesis in a rat hindlimb ischemia model, and (2) how ets-1 induced angiogenesis. Methods and Results—In this study, we used the HVJ-liposome method, which is highly effective for transfection, to transfect the human ets-1 gene. At 4 weeks after transfection, the capillary density and blood flow were significantly increased in a hindlimb transfected with the human ets-1 gene compared with control. These data clearly demonstrated that ets-1 has the ability to stimulate angiogenesis in vivo. To elucidate the molecular mechanisms by which ets-1 induced angiogenesis, we focused especially on the expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), potent angiogenic growth factors, because the promoter regions of both genes contain ets binding sites. Interestingly, overexpression of ets-1 upregulated both tissue HGF and VEGF concentrations in rat hindlimb. More importantly, administration of neutralizing antibody against HGF and VEGF attenuated the increase in blood flow and BrdU-positive cells induced by ets-1. Upregulation of HGF and VEGF by ets-1 was also confirmed by in vitro experiments using human vascular smooth muscle cells. Conclusions—The present study demonstrated that ets-1 regulated angiogenesis through the induction of angiogenic growth factors (VEGF and HGF). Overexpression of ets may provide a new therapeutic strategy to treat peripheral arterial disease.

Phase I/IIa clinical trial of therapeutic angiogenesis using hepatocyte growth factor gene transfer to treat critical limb ischemia.

Objective—To evaluate the safety and feasibility of intramuscular gene transfer using naked plasmid DNA-encoding hepatocyte growth factor (HGF) and to assess its potential therapeutic benefit in patients with critical limb ischemia. Methods and Results—Gene transfer was performed in 22 patients with critical limb ischemia by intramuscular injection of HGF plasmid, either 2 or 4 mg, 2 times. Safety, ankle-brachial index, resting pain on a 10-cm visual analog scale, wound healing, and walking distance were evaluated before treatment and at 2 months after injection. No serious adverse event caused by gene transfer was detected over a follow-up of 6 months. Of particular importance, no peripheral edema, in contrast to that seen after treatment with vascular endothelial growth factor, was observed. In addition, the systemic HGF protein level did not increase during the study. At 2 months after gene transfer, the mean±SD ankle-brachial index increased from 0.46±0.08 to 0.59±0.13 (P<0.001), the mean±SD size of the largest ischemic ulcers decreased from 3.08±1.54 to 2.32±1.88 cm (P=0.007), and the mean±SD visual analog scale score decreased from 5.92±1.67 to 3.04±2.50 cm (P=0.001). An increase in ankle-brachial index by >0.1, a reduction in ulcer size by >25%, and a reduction in visual analog scale score by >2 cm at 2 months after gene transfer were observed in 11 (64.7%) of 17 limbs, 18 (72%) of 25 ulcers, and 8 (61.5%) of 13 limbs, respectively. Conclusion—Intramuscular injection of naked HGF plasmid is safe and feasible and can achieve successful improvement of ischemic limbs as sole therapy.

Inhibition of NFκB activation using cis-element ‘decoy’ of NFκB binding site reduces neointimal formation in porcine balloon-injured coronary artery model

Application of DNA technology to regulate the transcription of disease-related genes has important therapeutic potential. The transcription factor NFκB plays a pivotal role in the transactivation of inflammatory and adhesion molecule genes, leading to vascular lesion formation. Double-stranded DNA with high affinity for NFκB may be introduced as ‘decoy’ cis elements to bind NFκB and block the activation of genes mediating inflammation, resulting in effective drugs for treating intimal hyperplasia. In this study, we tested the feasibility of NFκB decoy therapy to treat neointimal formation in a porcine coronary artery balloon injury model as a pre-clinical study.An angioplasty catheter was introduced into the left anterior descending coronary artery of the pig to cause vascular injury. First, we tested the feasibility of transfection of FITC-labeled NFκB decoy ODN using a hydrogel balloon catheter. Fluorescence due to NFκB decoy ODN could be detected throughout the medial layer. Therefore, we transfected NFκB decoy ODN into the balloon-injured LAD using a hydrogel catheter. Histological evaluation demonstrated that the neointimal area in the balloon-injured artery was significantly reduced by NFκB decoy ODN as compared to scrambled decoy ODN at 1 week after single transfection, accompanied by a significant reduction in PCNA-positive stained cells (P<0.01). Interestingly, the reduction of ICAM-positive staining was observed, accompanied by the inhibition of migration of macrophages. Of importance, intravascular ultrasound (IVUS) confirmed that neointimal area in the balloon-injured artery was significantly reduced by NFκB decoy ODN at 4 weeks after transfection (P<0.01). Interestingly, the inhibition of neointimal area was only limited to the lesion transfected with NFκB decoy ODN, while other lesions without NFκB decoy ODN demonstrated a marked increase in neointimal formation.Here, we report the successful in vivo transfer of NFκB decoy ODN using a hydrogel catheter to inhibit vascular lesion formation in balloon-injured porcine coronary artery.

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

Objective—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. Methods and Results—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. Conclusion—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.

Molecular strategy using cis-element 'decoy' of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model.

Transcription factor E2F plays a pivotal role in the transactivation of cell cycle regulatory genes, leading to vascular lesion formation. Double-stranded DNA with high affinity for E2F as ‘decoy’ cis elements may block the activation of genes mediating the cell cycle, resulting in an effective therapeutic agent for treating intimal hyperplasia. In this study, we tested the feasibility of E2F decoy therapy to treat neointimal formation in a porcine coronary artery balloon injury model. An angioplasty catheter was inserted in the left anterior descending coronary artery of pigs to cause vascular injury. Initially, we tested the feasibility of transfection of FITC-labeled E2F decoy ODN using a hydrogel balloon catheter. Fluorescence due to E2F decoy ODN could be detected throughout the medial layer. Therefore, we transfected E2F decoy ODN into the balloon-injured artery using hydrogel catheter. Of importance, intravascular ultrasound (IVUS) and histological evaluation demonstrated that plaque area in the balloon-injured artery was significantly reduced by E2F decoy ODN compared with mismatched decoy ODN at 1 month after a single transfection (P < 0.01). In contrast, luminal and total vessel areas were significantly increased in vessels treated with E2F decoy ODN as compared with mismatched decoy. Endothelial function after angioplasty was not affected by E2F decoy transfection. Finally, we tested the acute toxicity of E2F decoy ODN in monkeys, and no apparent side-effects were detected. Here, we report the successful in vivo transfer of E2F decoy ODN using a hydrogel catheter to inhibit vascular lesion formation in balloon-injured porcine coronary artery without any apparent side-effects.

Effect of nifedipine on endothelial function in normotensive smokers: potential contribution of increase in circulating hepatocyte growth factor

Calcium antagonists are reported to have protective effects on the endothelium in vitro and in vivo. Especially, nifedipine, among many calcium antagonists, was shown to improve endothelial dysfunction in patients with hypertension. However, no report has determined whether the improvement of endothelial dysfunction by nifedipine is due to direct effects or indirect effects such as its hypotensive effect. Thus, in this study, we evaluated the direct effects of nifedipine on smoking-induced endothelial dysfunction, since cigarette smoking itself is a major factor in damage of endothelial cells, as well as hypertension. We examined whether nifedipine improves endothelial function in 10 normotensive smokers without any risk factors for atherosclerosis. The subjects were treated with 20u2009mg nifedipine monotherapy (n=10) or placebo (n=10) for 4 weeks. Nifedipine did not affect blood pressure and heart rate of normotensive smokers. We measured forearm blood flow (FBF) by strain-gauge plethysmography after 2 and 4 weeks of treatment. Changes in vasodilator response to reactive hyperaemia were significantly improved in nifedipine-treated subjects (P<0.05), while there was no significant change in FBP response in control subjects. Response to nitroglycerin was not changed in either group. Moreover, to evaluate the mechanisms of the direct effects of nifedipine on the endothelium, we focused on hepatocyte growth factor (HGF), which is a novel angiogenic growth factor with an antiapoptotic action on endothelial cells. Interestingly, serum HGF concentration in smokers treated with nifedipine was significantly elevated both at 2 and 4 weeks (P<0.05). Overall, these results demonstrated direct effects of nifedipine in the improvement of endothelial dysfunction in normotensive smokers. The increase in serum HGF concentration by nifedipine might contribute to the improvement of endothelial dysfunction.