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Biomedical subjects

Motokuni Aoki

Publications and source records attributed to Motokuni Aoki.

At least 19 recordsLinked to original sources
PubMed2006

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor kappaB and Ets.

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor kappaB (NFkappaB) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3 , -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NFkappaB and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NFkappaB and ets. Inhibition of NFkappaB and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.

Animals↗
PubMed2006

Inhibitory effects of NFkappaB decoy oligodeoxynucleotides on neointimal hyperplasia in a rabbit vein graft model.

Autologous vein remains the commonly used conduit for bypass grafts; however, neointimal hyperplasia is known to be one of the major disease processes in vein graft failure. In this study, we focused on the important role of NFkappaB which controls the expression of numerous genes for various cytokines and adhesion molecules in the mechanism of graft failure. Thus, we investigated the inhibitory effect of NFkappaB decoy oligodeoxynucleotides (ODN) on vein graft failure in a rabbit hypercholesterolemic model. Jugular vein to carotid artery interposition grafts in rabbits were transfected intraoperatively with NFkappaB decoy ODN (40 micromol/l) by ex-vivo pressure-mediated transfection (300 mm Hg, 10 min). Treatment with NFkappaB decoy ODN significantly suppressed intimal hyperplasia 4 weeks after vein implantation as compared to scrambled decoy ODN, and increased medial thickness, leading to a significant reduction in intima-to-media ratio. Treatment with NFkappaB decoy ODN significantly inhibited the recruitment of macrophages and the proliferation of vascular smooth muscle cells (VSMC), while apoptosis in VSMC was significantly increased by NFkappaB decoy ODN. In addition, a study of vascular reactivity demonstrated that transfection of grafts with NFkappaB decoy ODN significantly improved endothelium-mediated vasorelaxation as compared to scrambled decoy ODN. Here, we demonstrated that inhibition of NFkappaB activation using decoy ODN inhibited the development of neointimal hyperplasia, followed by suppression of inflammatory changes and accumulation of VSMC in the neointima of rabbit vein grafts. The present study raises the possibility of a novel strategy for prevention of graft failure.

Animals↗
PubMed2006

Model of vasculogenesis from embryonic stem cells for vascular research and regenerative medicine.

Embryonic stem (ES) cells are highlighted as promising cell sources for regenerative medicine. Here, we focused on providing the platform that forced ES cells to reproduce the vascular organization process, leading to efficiency and safety evaluation as preclinical testing of biological agents. Murine ES cell-derived embryoid bodies on matrigel, but not collagen or gelatin, could be differentiated into sprouting blood vessels without the addition of growth factors. The expression of endothelial cell marker CD31 and smooth muscle marker alpha-smooth muscle actin was partially colocalized and started to increase 7 days after culture on matrigel, accompanied by the induction of a number of growth factors, such as vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor, transforming growth factor-beta, and angiopoietin-1. Moreover, notch-related genes, such as Del1 or Del4 (delta-like 1/4) and hey1 or hey2 (hairy/enhancer of split related TRPW motif 1/2), were upregulated in a similar time course. The treatment of neutralizing antibodies against these growth factors failed to inhibit the differentiation into the sprouting blood vessels, whereas arginine-glycine-aspartic peptide, a selective inhibitor for the alphavbeta3-integrins, did inhibit differentiation. An anticancer drug to inhibit angiogenesis, TNP-470, also blocked the vascular formation in this model. ES cells could reproduce the vascular organization process on the biosynthetic scaffolds, such as matrigel, without the addition of growth factors. In the future, a human ES-based tissue model would be an optional tool for the screening of pharmaceutical drugs for vascular disease.

Animals↗
PubMed2006

[Therapeutic angiogenesis for ischemic diseases].

The clinical consequences of peripheral arterial disease include pain on walking, pain at rest and loss of tissue integrity in the distal ischemic limbs. Although development of beneficial drugs and intervention devices do contribute to the treatment of this disease, critical limb ischemia is estimated to develop in 500 to 1,000 individuals per million per year. As angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis" is now evaluated in the clinical fields. Recent progress in molecular biology has led to the development of gene therapy as a new strategy to treat a variety of cardiovascular diseases using angiogenic growth factors such as vascular endothelial growth factor (VEGF). Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with severe ischemic heart or peripheral arterial disease.

Angiogenesis Inducing Agents↗
PubMed2005

Does gene therapy become pharmacotherapy?

Recent progress in molecular and cellular biology has led to the development of numerous effective cardiovascular drugs. However, there are still a number of diseases for which no known effective therapy exists, such as peripheral arterial disease, ischaemic heart disease, restenosis after angioplasty, and vascular bypass graft occlusion. Currently, gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease despite its limitations. The first human trial in gene therapy for cardiovascular disease was started at 1994 to treat peripheral vascular disease using vascular endothelial growth factor (VEGF). Then, many different potent angiogenic growth factors were tested in clinical trials to treat peripheral arterial disease and ischaemic heart disease. Improvement of clinical symptoms in peripheral arterial disease and ischaemic heart disease has been reported. This review focuses on the future potential of gene therapy for the treatment of cardiovascular disease. In the future, gene therapy might become a real pharmacotherapy to treat cardiovascular disease.

Animals↗
PubMed2005

Development of novel method of non-viral efficient gene transfer into neonatal cardiac myocytes.

To establish new treatment for cardiovascular disease, the development of safe and highly efficient vectors is necessary. Especially, non-viral vectors are considered to be ideal for human gene therapy, since recent adverse events with retroviral or adenoviral vectors have highlighted the issue of safety. Although we previously reported safety and high efficiency of HVJ-liposome method, we have modified the envelope of HVJ (Sendai virus). In this novel non-viral vector, the envelope of HVJ alone was utilized as a carrier to deliver proteins, genes and oligodeoxynucleotides (ODN). Thus, we optimized the transfection efficiency of HVJ-envelope vector into neonatal cardiac myocytes in this study, since cardiac myocytes is one of the most difficult cells to be transfected. HVJ-envelope, obtained after complete destruction of HVJ genome, containing FITC-labeled ODN or luciferase plasmid was incubated with cardiac myocytes. In addition, the concentration of protamine sulfate was modified (0-700 microg/ml) to increase transfection efficacy. Without HVJ-envelope vector, few cells showed fluorescence, whereas most cells demonstrated fluorescence with HVJ-envelope vector. Consistent with the high transfection efficiency of ODN, high luciferase activity was also detected using HVJ-envelope vector. Moreover, the transfection efficiency varied according to the concentration of protamine sulfate. No obvious cytotoxicity was observed in cells transfected with HVJ-envelope vector. The present study demonstrated the development of a highly efficient novel non-viral vector for cardiac myocytes, suggesting that further development may provide a new useful tool for research and clinical gene therapy in the field of cardiovascular disease.

Animals↗
PubMed2005

A calcium-channel blocker, benidipine, improves forearm reactive hyperemia in patients with essential hypertension.

The pathophysiological role of endothelial cells is important in the mechanism of progression of atherosclerosis and improvement of endothelial function may be important for cardiovascular morbidity. Calcium antagonists are reported to have protective effects on the endothelium in vitro and in vivo. In this clinical study, we investigated the effect of calcium antagonist, benidipine, on endothelial function in the patients with essential hypertension, which causes endothelial dysfunction. Twenty-five patients with hypertension without other risk factors for atherosclerosis were treated with monotherapy (8 mg benidipine, n=25) for 8 weeks. Blood pressure was reduced significantly. Endothelial function was evaluated using forearm blood flow by strain-gauge plethysmography after 8 weeks of treatment. Changes in vasodilator response to reactive hyperemia were significantly improved (p<0.01), while the response to nitroglycerin was not changed, suggesting the improvement of endothelial function. Moreover, we focused on hepatocyte growth factor (HGF), which is a novel angiogenic growth factor with an anti-apoptotic action on endothelial cells, and evaluated involvement of HGF in improvement of endothelial function. Serum HGF concentration in subjects treated with benidipine was significantly elevated at 8 weeks (p<0.05). Overall, these results demonstrated that benidipine improved endothelial dysfunction in patients with hypertension. Interestingly, an increase in serum HGF concentration by benidipine might contribute to the improvement of endothelial dysfunction.

Aged↗
PubMed2005

Hepatocyte growth factor gene therapy reduces ventricular arrhythmia in animal models of myocardial ischemia.

It was recently reported that gene therapy using hepatocyte growth factor (HGF) has the potential to preserve cardiac function after myocardial ischemia. We speculated that this HGF gene therapy could also prevent ventricular arrhythmia. To investigate this possibility, we examined the antiarrhythmic effect of HGF gene therapy in rat acute and old myocardial infarction models. Myocardial ischemia was induced by ligation of the left descending coronary artery. Hemagglutinating virus of Japan (HVJ)-coated liposome containing HGF genes were injected directly into the myocardium fourteen days before programmed pacing. Ventricular fibrillation (VF)was induced by programmed pacing. The VF duration was reduced and the VF threshold increased after HGF gene therapy ( p< 0.01). Histological analyses revealed that the number of vessels in the ischemic border zone was greatly increased after HGF gene injection. These findings revealed that HGF gene therapy has an anti-arrhythmic effect after myocardial ischemia.

Acute Disease↗
PubMed2004

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.

Adult↗
PubMed2004

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.

Animals↗
PubMed2004

Local delivery of E2F decoy oligodeoxynucleotides using ultrasound with microbubble agent (Optison) inhibits intimal hyperplasia after balloon injury in rat carotid artery model.

Since restenosis after angioplasty still remains a major clinical problems, inhibition of neointimal formation is an important subject. In this study, we focused on the transcription factor, E2F, that plays a pivotal role in the transactivation of cell-cycle regulatory genes, and also we developed a newly delivery system of decoy oligodeoxynucleotides (ODN). We transfected E2F decoy ODN mixed with an echo-contrast microbubble agent (Optison) into rat carotid artery balloon-injured model by using therapeutic ultrasound (US) to inhibit neointimal formation. Two weeks after transfection, the intimal to medial area ratio in E2F decoy+Optison+US group was significantly decreased (P < 0.01). Inhibition of cell growth was also confirmed by PCNA staining. No apparent toxicity such as inflammation could be detected in blood vessels transfected with E2F decoy ODN with Optison and ultrasound. Overall, the present studies demonstrated a novel non-viral ODN transfer method into blood vessels. A novel therapeutic strategy using E2F decoy ODN with Optison using ultrasound may be useful to inhibit restenosis in clinical practice without a viral vector.

Angioplasty, Balloon, Coronary↗
PubMed2004

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice.

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.

Animals↗
PubMed2004

Novel therapeutic strategy to treat brain ischemia: overexpression of hepatocyte growth factor gene reduced ischemic injury without cerebral edema in rat model.

BACKGROUND: Although cerebral occlusive disease leads to cerebral ischemic events, an effective treatment has not yet been established. An ideal therapeutic approach to treat ischemia might have both aspects of enhancement of collateral formation and prevention of neuronal death. Hepatocyte growth factor (HGF) is a potent angiogenic factor that also acts as a neurotrophic factor. Thus, in this study, we examined the therapeutic effects of HGF on brain injury in a rat permanent middle cerebral artery occlusion model. METHODS AND RESULTS: Gene transfer into the brain was performed by injection of human HGF gene with hemagglutinating virus of Japan-envelope vector into the cerebrospinal fluid via the cisterna magna. Overexpression of the HGF gene resulted in a significant decrease in the infarcted brain area as assessed by triphenyltetrazolium chloride staining, whereas rats transfected with control vector exhibited a wide area of brain death after 24 hours of ischemia. Consistently, the decrease in neurological deficit was significantly attenuated in rats transfected with the HGF gene at 24 hours after the ischemic event. Stimulation of angiogenesis was also detected in rats transfected with the HGF gene compared with controls. Of importance, no cerebral edema or destruction of the blood-brain barrier was observed in rats transfected with the HGF gene. CONCLUSIONS: Overall, the present study demonstrated that overexpression of the HGF gene attenuated brain ischemic injury in a rat model, without cerebral edema, through angiogenic and neuroprotective actions. In particular, the reduction of brain injury by HGF may provide a new therapeutic option to treat cerebrovascular disease.

Animals↗
PubMed2004

Increase in peripheral blood flow by intravenous administration of prostaglandin E1 in patients with peripheral arterial disease, accompanied by up-regulation of hepatocyte growth factor.

Since endothelial damage is a trigger for the progression of atherosclerosis, we evaluated the clinical utility of prostaglandin E1 (PGE1) in relation to peripheral blood flow and regulation of hepatocyte growth factor (HGF), an angiogenic growth factor, in patients with peripheral arterial disease (PAD). Fourteen male patients with PAD who showed the characteristic symptoms of arteriosclerosis obliterans (Fontaine I: n=2; Fontaine II: n=4; Fontaine III: n=2; Fontaine IV: n=6), confirmed by angiography, were enrolled in this study. Patients were administrated synthetic PGE1 at a dose of 120 microg per day for 14 consecutive days. Measurement of peripheral blood flow and serum HGF concentration was performed before PGE1 treatment and after 14 days of administration. Interestingly, intravenous administration of PGE1 for 2 weeks significantly increased the blood flow as assessed by a laser Doppler imager (p<0.01). In patients with Fontaine III and IV, serum HGF concentration was significantly higher than that in patients with Fontaine I or II and normal subjects. Of importance, administration of PGE1 further increased serum HGF concentration as compared to that before treatment (p<0.01). The increase in circulating HGF might work as a compensatory mechanism to decrease local HGF expression in patients with PAD, since HGF acts as an angiogenic growth factor with anti-apoptotic actions on endothelial cells. Moreover, to confirm the stimulatory effect of PGE1 on HGF in vessels, we employed an in vitro culture system. PGE1 increased HGF production and the growth of human cultured vascular endothelial cells. The stimulatory effect of PGE1 on HGF production might be due to an increase in cAMP, since forskolin and 8-bromo-cAMP induced HGF production. In conclusion, we demonstrated that administration of PGE1 stimulated peripheral blood flow, accompanied by an increase in systemic HGF concentration. Also, our in vitro data suggested that PGE1 augmented not only the systemic HGF level, but also local HGF production, probably through cAMP accumulation, resulting in improvement of endothelial function and blood flow.

Aged↗
PubMed2004

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.

Animals↗
PubMed2003

Inhibition of experimental abdominal aortic aneurysm in the rat by use of decoy oligodeoxynucleotides suppressing activity of nuclear factor kappaB and ets transcription factors.

BACKGROUND: Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)-kappaB, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-kappaB and ets by using chimeric decoy oligodeoxynucleotides (ODN). METHODS AND RESULTS: AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-kappaB and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-kappaB decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. CONCLUSIONS: We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-kappaB and ets in rat model. NF-kappaB and ets are considered to play an important role in the pathogenesis of AAA.

Animals↗
PubMed2003

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.

Animals↗