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We have developed a partial peptide of RANKL (receptor activator of NF-κB ligand) that suppresses TLR (toll-like receptor)-related inflammation via RANKL/RANK (receptor activator of nuclear factor-κB) signals in the acute phase of ischemic stroke. This peptide has been found to be a therapeutic agent for ischemic stroke that can be used in combination with tPA in a mouse model. Based on the findings, we are working on translational research to aim for clinical application of this peptide through collaboration with pharmacy companies. However, the problem is that the need for development of medication in the acute stage of ischemic stroke is currently low in pharmaceutical companies due to the failure of many investigational drugs in the past. To overcome the problem, we are examining the effects of this peptide in other diseases included in the company's priority areas and explaining the environmental changes in the clinical trials due to the development of endovascular treatment in the acute stage of ischemic stroke.

Hypertension, osteoporosis and vascular calcification are major diseases in the recent aging society and may share the same backgrounds genetically and environmentally. As treatments to prevent aging-related diseases simultaneously are desirable, we investigate common backgrounds underling these diseases. Renin- angiotensin system, which causes high blood pressure, is found to be involved in bone metabolism. Angiotensin II has been shown to accelerate osteoporosis through RANKL up-regulation in osteoblast. RANKL, in turn, contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins. Angiotensin type 1 receptor blockers (ARBs) ameliorate osteoporosis and vascular calcification beyond their blood pressure lowering effects. These pleiotropic effects of antihypertensive drugs such as ARBs might benefit especially hypertensive postmenopausal women.

The number of patients with high blood pressure and osteoporosis are increased year by year in our society. In hypertension patients, excess urinary calcium secretion induces secondary parathyroidism to increase serum calcium level by calcium release from bone, which may accelerate osteoporosis. In this aspect, there are several reports that anti-hypertensive drugs, especially thiazides, increase bone mineral density and decrease the incidence of bone fracture. In addition, we demonstrated that renin-angiotensin system can be involved in the process of osteoporosis. Angiotensin II significantly induced the expression of RANKL (receptor activator of NF-κB ligand) in osteoblasts, leading to the activation of osteoclasts, while these effects were completely blocked by an Ang II type 1 receptor blockade. Recently, it has been reported that angiotensin receptor blockade clinically decreased the incidence of bone fracture. Renin-angiotensin system might be common molecule to regulate both hypertension and osteoporosis.

Angiotensin II is a potent stimulator of vascular smooth mustle cells that increases blood pressure, and angiotensin type 1 receptors have been identified on osteoblasts; thus, the renin-angiotensin system has been suggested to be involved in bone metabolism. The authors reported that angiotensin II significantly increased TRAP-positive multinuclear osteoclasts with the up-regulation of RANKL expression through extracellular kinase of osteoblast. These effects were abolished with co-treatment of angiotensin converting enzyme (ACE) inhibitors or angiotensin type 1 receptor blockers (ARB). Recently, RANK-RANKL-OPG system is proved to be involved in vascular calcification and RAAS also might be involved in the development of the disease. Thus, target therapy for RAAS might bring additional beneficial effects on bone metabolic diseases such as osteoporosis, rheumatoid arthritis and vascular calcification other than blood pressure lowering effect.

The patients with "Hypertension" and "Chronic Kidney Disease (CKD) " are accompanied with an osteoporosis. In hypertension patients, excess urinary calcium secretion induces secondary parathyroidsim to increase serum calcium (Ca) level, which may lead to Ca release from bone. In this aspect, there are several reports that anti-hypertensive drugs, especially thiazides, increase bone mineral density and decrease the incidence of bone fracture. In addition, we demonstrated that renin-angiotensin system can be involved in the process of osteoporosis. Angiotensin II significantly induced the expression of RANKL (receptor activator of NF-κB ligand) in osteoblasts, leading to the activation of osteoclasts, while these effects were completely blocked by an Ang II type 1 receptor blockade. As for CKD, excess phosphorus (P) due to renal dysfunction induces secondary parathyroidism to decrease serum P level, which similarly leads to osteoporosis. Moreover, excess P can increase FGF23 expression and decrease activated vitamin D, which also resulted in progression of osteoporosis. Both "Hypertension" and "Chronic Kidney Disease (CKD) " are inducible factor to osteoporosis.

The complications of diabetes mellitus have a significant impact on patient survival and quality of life with respect to ischemic disease. Therapeutic gene therapy was performed for peripheral artery disease (PAD) using vascular endothelial growth factor (VEGF), but VEGF gene therapy in phase III clinical trial for PAD did not succeed. Although hepatocyte growth factor (HGF) is also a potent angiogenic growth factor in animal models of ischemia, their characteristics are not the same in clinical trials. In this background, we demonstrated that HGF, but not VEGF, attenuated angiotensin II-induced senescence of endothelial progenitor cells through a reduction of oxidative stress by inhibition of the phosphatidylinositol-3,4,5-triphosphate/Akt pathway. Therapeutic gene therapy still may improve patients with the complications of diabetes mellitus.

Bone metabolism is closely regulated by hormones and cytokines, which have effects on both bone resorption and deposition. Since renin-angiotensin system (RAS) has been known to play an important role to regulate remodeling in several tissues, we focused on the potential role of RAS in bone metabolism. It is known that the receptors of angiotensin II are expressed in culture osteoclasts and osteoblasts, and angiotensin II induced the differentiation and activation of osteoclasts responsible for bone resorption. Of importance, angiotensin II significantly induced the expression of RANKL (receptor activator of NF-kappaB ligand) in osteoblasts, leading to the activation of osteoclasts, while these effects were completely blocked by an angiotensin II type 1 receptor blockade. In a rat ovariectomy model of estrogen deficiency, administration of angiotensin II accelerated the increase in TRAP activity, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. As it has been known that osteoclast differentiation is regulated by a variety of hormones, local factors and inflammatory cytokines, such as IL-1 and TNF-alpha, RAS might also be involved in this autocrine system. Of importance, sub-analysis of recent clinical study demonstrated that the usage of angiotensin-converting enzyme inhibitors significantly reduced the fracture risk. RAS might be a novel target to treat the subgroups of hypertensive patients with osteoporosis.

Better understanding of the mechanism of aging, particularly in vivo mice or C. elegans, will have an important impact on drug discovery for several kinds of human diseases. In the history of gene therapy, the initial target diseases were cancer or congenital diseases. However, since the concept of therapeutic angiogenesis had been proposed, the cardiovascular diseases were currently new targets of gene therapy. We used the naked plasmid of hepatocyte growth factor (HGF) as an angiogenic factor for peripheral arterial diseases, and showed the efficacy and safety in clinical trial. Recent reports suggest that HGF has an anti-inflammatory action and attenuated cellular senescence. Thus, HGF gene therapy might be an anti-aging therapy in cardiovascular diseases.

Werner's syndrome is a typical progeroid syndrome with many specific features of aging early in life. Clinical features of Werner's syndrome closely resemble accelerated aging, such as cataract, scleroderma skin, diabetes and tumorigenesis. The causative gene of this syndrome is denoted as WRN, which encodes a homolog of the E. coli RecQ DNA helicase and is located on chromosome 8p2-p11.2. WRN is not only a helicase but also an exonuclease and ATPase. WRN protein plays a key role in genome stability, particularly during DNA replication and telomere metabolism. In this review, we introduce the clinical characteristics of Werner's syndrome and recent topics concerning WRN in comparison with other progeroid syndromes.

Gene therapy based on ultrasound with microbubbles offers a novel approach for the prevention and treatment of variety of diseases. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cancer or cardiovascular medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc.) have been used in the clinical trials already. But some severe side effects were reported in clinical gene therapy using such viral, so people desire safe and efficient clinical gene therapy. Recently, ultrasound-mediated gene transfer has been reported to augment the transfection efficiency and facilitate local gene expression. Interestingly, gene transfer into the fetal central nervous system was successfully achieved by intrauterine injection with microbubble-enhanced ultrasound. Compared to other viral vectors, there are some theoretical advantages including safety, simplicity of preparation, and local gene transfer. Thus, we focused on the development of gene transfer using naked plasmid DNA with an ultrasound or microbubble-enhanced ultrasound method.

Arteriosclerosis of the extremities is a disease of the blood vessels characterized by hardening and/or narrowing of the arteries that supply the legs and feet. This causes a decrease in blood flow that can injure nerves and other tissues. Therapeutic angiogenesis using angiogenic growth factor is expected to be a new treatment for patients with critical limb ischemia. The first human clinical trial treating peripheral vascular disease was started in 1994 using vascular endothelial growth factor. To date, other potent angiogenic growth factors, such as hepatocyte growth factor(HGF), have been also estimated in clinical trials for peripheral arterial disease. Several results from phase 1 or 2 trials using HGF gene were encouraging. Phase 3 trials are now ongoing and their results are expected.

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.

Useful drug therapy for inhibiting the extension of an aortic aneurysm or promoting its involution has not yet been established. Hypertension is a known risk factor for extension of an aortic aneurysm. However, on a cellular level it is believed that activation of nuclear factor (NF)-kappaB and matrix metalloproteinase (MMP) is involved in the extension of an aortic aneurysm. Nifedipine has been evaluated in a rat model of aortic aneurysm and it was found to inhibit activation of MMP and extension of the aortic aneurysm. Nifedipine also inhibited activation of NF-kappaB and these effects were independent of the antihypertensive effect of the drug. With antihypertensive and vasculoprotective effects and inhibitory actions on activation of NF-kappaB and MMP, nifedipine has potential to be a useful option in the drug treatment of patients with aortic aneurysm.

Therapeutic angiogenesis using angiogenic growth factor is expected to be a new treatment for with patients with critical limb ischemia. The first human clinical trial treating peripheral vascular disease was started in 1994 using vascular endothelial growth factor (VEGF). To date, other potent angiogenic growth factors, such as fibroblast growth factor(FGF) or hepatocyte growth factor(HGF), have been also estimated in clinical trials for peripheral arterial disease. Several results from phase 1 or 2 trials using VEGF, FGF and HGF gene were encouraging. Phase 3 trials are now ongoing and their results are expected.

Cardiovascular diseases are the leading causes of mortality and morbidity in developed countries. Most conventional therapy is inefficient and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy based on ultrasound with microbubbles offers a novel approach for the prevention and treatment of cardiovascular diseases. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cardiovascular medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc) have been used in clinical trials already. However, some severe side effects were reported in clinical gene therapy using such viral vector, so people wish for safe and efficient clinical gene therapy. Transfection with ultrasound and microbubbles has been reported as a new powerful tool in a new class for gene therapy.

Most of cardiac myocytes have lost their ability to proliferate and differentiate into new myocytes. Therefore, myocyte regeneration and replacement in adult myocardium was thought to be impossible by medical community. However, recent findings in cardiovascular researches provide the possibility that gene therapy and stem cell therapy could have supportive effects on myocyte regeneration and myocardial revascularization in damaged heart. Here, we presented recent progress, especially, in gene therapy for myocardial regeneration under ischemic heart disease and heart failure.

Gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease, for which no known effective therapy exists. The first human trial in cardiovascular disease was started in 1994 to treat peripheral vascular disease using VEGF. Since then, at least 5 different potent angiogenic growth factors has been tested in clinical trials to treat peripheral arterial disease. In addition, therapeutic angiogenesis using VEGF gene was applied to treat ischemic heart disease. Results from these clinical trials seems to be more than expected. Improvement of clinical symptoms in peripheral arterial disease or ischemic heart disease has been reported. In this review, we have focused on the future potential of gene therapy for the treatment of cardiovascular disease as a new pharmacological therapy.

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment of patients with severe ischemic diseases. Indeed, human gene therapy for peripheral arterial disease(PAD) using VEGF gene demonstrated the beneficial effects. In contrast, we have reported the potent angiogenic activity of hepatocyte growth factor (HGF) in animal study and we planned gene therapy for ASO and Buerger disease using HGF gene (TREAT-HGF). In a prospective, open-labeled clinical trial, we investigated the safety and biological efficiency of this gene therapy in patients with peripheral arterial disease(PAD) who had failed conventional therapy.

The HMG-CoA reductase inhibitors reduce levels of low-density lipoproteins, raise high-density lipoproteins, and lower triglycerides. Treatment with HMG-CoA reductase inhibitors reduces the risk of cardiovascular events across a broad spectrum of patient profiles, as evidenced by both primary and secondary prevention trials. Improved survival by way of reduced deaths from coronary heart disease was also reported with these agents, which are primarily indicated for substantial reduction in LDL-cholesterol levels. However, HMG-CoA reductase inhibitors are extremely complex drugs and exhibit a wide variety of vascular effects that may or may not be dependent on their lipid-modifying properties. These so-called pleiotropic effects include alternations of endothelial function, inflammation, coagulation and plaque stability. The relative contribution of the nonlipid effects of HMG-CoA reductase inhibitor therapy to the well-documented clinical benefits is currently under intense investigation.

The treatment strategies of cerebral infarction have been studied in order to prevent neuronal cell death. Gene therapy is one of the most promising therapy and has several advantage over classical drug therapies. There has been a problem that drug proteins are unable or difficult to pass through blood brain barrier. In gene therapy, however, drug proteins are expressed in the brain with transgene transfer technique. Ischemic neural death proceeds with a complex series of pathophysiological events in the neurons. But molecular mechanism of ischemic neuronal cell death gradually understood. It has been known that a number of genes can be potent candidates for treatment factors of cerebral infarction. Actually, many investigators have been studied treatment strategies of cerebral infarction using a variety of neurotrophic factors such as bcl-2, heat shock protein 72, glial cell line-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). Moreover, the development of new vectors and gene delivery systems have been studied. Gene therapy would be a strong strategy for treatment of cerebral infarction in the future.