[Show abstract][Hide abstract] ABSTRACT: Vaccines are commonly used as a preventive medicine for infectious diseases worldwide; however, the trial for an amyloid beta vaccine against Alzheimer's disease will open a new concept in vaccination. In case of therapeutic vaccines for cancer, their targets are usually specific antigens in cancer cells, allowing activated cytotoxic T cells (CTLs) to attach and remove the antigen-presenting cancer cells. In our therapeutic vaccines against hypertension, the target is angiotensin II (Ang II) and induced anti-Ang II antibodies could efficiently ameliorate high blood pressure. Similarly, we developed the therapeutic vaccine against DPP4 for diabetes mellitus. However, because Ang II or DPP4 is an endogenous hormone, we must avoid autoimmune disease induced by these vaccines. Therefore, our system was used to design a therapeutic vaccine that elicits anti-Ang II or DPP4 antibodies without CTL activation against Ang II or DPP4. In this review, we will describe our concept of therapeutic vaccines for hypertension and diabetes mellitus.
[Show abstract][Hide abstract] ABSTRACT: Magnetic drug delivery system (MDDS) is a technique to effectively accumulate drugs, which are combined with ferromagnetic particles, into the affected area using magnetic force control. This study intends to apply MDDS for immunotherapy by enhancing immune responses by a surface treatment of a ferromagnetic particle. The objective of this study is to give the adjuvant effect to a ferromagnetic particle by the surface treatment with alum, which is known as one of the common adjuvants that activates inflammasome pathway. First, magnetite was prepared as a ferromagnetic particle and coated with alum. Alum-coated magnetite increased the expression of caspase-1, which is an activated indicator of inflammasome, in the culture of human monocyte cell (THP-1 cell). To evaluate the potential of the surface coated particles, the particles were subcutaneously injected to mice with a peptide vaccine. As a result, the antibody titer was increased by the surface coated particles as assessed by ELISA. Although a magnetic force has not yet applied in this study, the administration experiment to mice using magnetic force control is our next step. In conclusion, we modified the immune response to magnetite by coating the surface with alum. This can lead to a clinical application for vaccine therapy in future.
No preview · Article · May 2015 · Journal of Applied Physics
[Show abstract][Hide abstract] ABSTRACT: Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG(-/-) mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG(-/-) mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl2, or H2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.
Preview · Article · May 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Periostin is an extracellular matrix glycoprotein and has various cellular effects. Previously, we demonstrated the neuroprotective effects of periostin during the acute stage of cerebral ischemia. However, its expression during the chronic stage remains unknown. Herein, we examined the expression of full-length periostin (periostin 1; Pn1) and its splicing variant lacking exon 17 (periostin 2; Pn2) during the 28 days following transient middle cerebral artery occlusion in mice. Real-time reverse transcription-PCR showed that the expression of Pn2 was dramatically upregulated between days 3 and 28, and the highest expression was observed on day 7. The expression of Pn1 was also increased, but delayed compared with Pn2. Immunohistochemistry showed that periostin was weakly expressed in reactive astrocytes in the peri-infarct region and in microglia/macrophages in infarct regions, on days 3 and 7. Periostin was also expressed around CD31-positive cells in both the peri-infarct and the sub-ventricular zone (SVZ) on days 3 and 7. SOX-2 positive cells, which are neural stem cells, also expressed periostin on day 7. The highest periostin immunoreactivity that occurred co-localized with collagen I and fibronectin in the peri-infarct region between days 7 and 28. Thus, the expression pattern of periostin mRNA was dependent on the splicing variant, and it continued to be expressed up to 28 days after cerebral ischemia. As periostin was expressed in various cells, such as reactive astrocytes/microglia, fibroblasts and neuronal progenitor cells, periostin might be associated with pathophysiology in post-ischemic inflammation and neurogenesis.Hypertension Research advance online publication, 20 March 2014; doi:10.1038/hr.2014.36.
Full-text · Article · Mar 2014 · Hypertension Research
[Show abstract][Hide abstract] ABSTRACT: The increasing prevalence of type 2 diabetes mellitus is associated with a significant economic burden. We developed a dipeptidyl peptidase 4 (DPP4)-targeted immune therapy to increase glucagon-like peptide 1 hormone levels and improve insulin sensitivity for the prevention and treatment of type 2 diabetes mellitus. Immunization with the DPP4 vaccine in C57BL/6J mice successfully increased DPP4 titer, inhibited plasma DPP4 activity, and induced an increase in the plasma glucagon-like peptide 1 level. Moreover, this elevated titer was sustained for 3 mo. In mice fed a high-fat diet, DPP4 vaccination resulted in improved postprandial glucose excursions and insulin sensitivity and, in the diabetic KK-A(y) and db/db mice strains, DPP4 vaccination significantly reduced glucose excursions and increased both plasma insulin and pancreatic insulin content. Importantly, T cells were not activated following challenge with DPP4 itself, which suggests that this vaccine does not induce cell-mediated autoimmunity. Additionally, no significant immune-mediated damage was detected in cells and tissues where DPP4 is expressed. Thus, this DPP4 vaccine may provide a therapeutic alternative for patients with diabetes.
Preview · Article · Mar 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Vaccines are commonly used as a preventive medicine for infectious diseases worldwide, however, clinical trials on an amyloid beta vaccine for Alzheimer's disease represents a new concept in the field of vaccinations. Several recent studies indicate the potential of therapeutic vaccines as well as classical vaccines as preventive medicines. A number of therapeutic vaccines for cancer have been developed as novel immunotherapies. Their targets are usually specifi c antigens in cancer cells, allowing activated cytotoxic T cells (CTLs) to attach and remove the antigen-presenting cancer cells. Recently, we and others have attempted to develop a therapeutic vaccine against hypertension. The vaccine target is angiotensin II (AngII), and induced anti-AngII antibodies could efficiently ameliorate high blood pressure. However, because AngII is an endogenous hormone, we must avoid the induction of autoimmune diseases by administration of an AngII vaccine. Therefore, our system was used to design a therapeutic vaccine that elicits anti-AngII antibodies without CTL activation against AngII. Because the target antigen itself does not include T cell epitopes, the immunogenic molecule (ie, KLH) provides antigen that supports the activation of T cells. In particular, helper T cells may activate B cells that produce antibodies against our specific antigen. In this review, we will explain our concept of therapeutic vaccines based on our recent data.
No preview · Article · Mar 2014 · International Heart Journal
[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus, hypertension and metabolic syndrome are major risk factors for the occurrence of cardiovascular events. In this study, we used spontaneous hypertensive rat (SHR)/NDmcr-cp (cp/cp) (SHRcp) rats as a model for metabolic syndrome to examine the effects of dipeptidyl peptidase (DPP)-4 inhibition on hypertension, glucose metabolism and endothelial dysfunction. First, we confirmed that SHRcp rats showed very severe obesity, hypertension and endothelial dysfunction phenotypes from 14 to 54 weeks of age. Next, we examined whether the DPP-4 inhibitor teneligliptin (10 mg kg(-1) per day per os for 12 weeks) could modify any of these phenotypes. Treatment with teneligliptin significantly improved hyperglycemia and insulin resistance, as evidenced by an oral glucose tolerance test and homeostasis model assessment for insulin resistance, respectively. Teneligliptin showed no effects on systolic blood pressure or heart rate. In regard to endothelial function, the vasodilator response to acetylcholine was significantly impaired in SHRcp rats when compared with WKY rats. Long-term treatment with teneligliptin significantly attenuated endothelial dysfunction through the upregulation of endothelium-derived nitric oxide synthase mRNA. These results demonstrate that long-term treatment with teneligliptin significantly improved endothelial dysfunction and glucose metabolism in a rat model of metabolic syndrome, suggesting that teneligliptin treatment might be beneficial for patients with hypertension and/or diabetes.Hypertension Research advance online publication, 13 March 2014; doi:10.1038/hr.2014.53.
No preview · Article · Mar 2014 · Hypertension Research
[Show abstract][Hide abstract] ABSTRACT: We developed DNA vaccine for vascular endothelial growth factor (VEGF), which may provide the therapeutic option instead of anti-VEGF antibody, bevacizumab. Plasmid containing VEGF mini-gene was constructed in the insertion of B-cell epitope of Hepatitis B core protein [HBc-VEGF], which was an epitope carrier. High titer of anti-VEGF antibody was observed in BALB/c mice which were intramuscularly immunized with HBc-VEGF by electropolator. In mice inoculated with colon 26 cells, tumor volume and microvessel density was decreased in HBc-VEGF with a significant prolonged survival. Co-treatment of purified IgG from immunized mice with HBc-VEGF showed in vitro neutralizing activity for VEGF-induced ERK phosphorylation and tube formation in cultured endothelial cells. Furthermore, intravitreally injection of this purified IgG reduced the neovessel formation in the mouse oxygen-induced retinopathy and laser-induced choroidal neovascularization models. These results first provided that DNA vaccine against VEGF possessed the anti-angiogenic effect, leading to prolonged survival in mouse cancer model.
Full-text · Article · Nov 2013 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Gene therapy and cell-based therapy have emerged as novel therapies to promote therapeutic angiogenesis in critical limb ischemia (CLI) caused by peripheral artery disease (PAD). Although researchers initially focused on gene therapy using proangiogenic factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factors (HGF), cell therapy using bone marrow mononuclear cells (BMMNCs), mesenchymal stem cells (BMMSCs), G-CSF-mobilized peripheral blood mononuclear cells (M-PBMNCs), and endothelial progenitor cells (EPCs) have also been extensively studied. Based on the elaborate studies and favorable results of basic research, some clinical phase I/II trials have been performed, and the results demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI. However, the phase 3 clinical trials have thus far been limited to gene therapy using the HGF gene. Further studies using well-designed larger placebo-controlled and long-term randomized control trials (RCTs) will clarify the effectiveness of gene therapy and cell-based therapy for the treatment of CLI. Furthermore, the development of efficient gene transfer systems and effective methods for keeping transplanted cells healthy will make these novel therapies more effective and ease the symptoms of CLI.
[Show abstract][Hide abstract] ABSTRACT: Objective:
Vascular calcification is accelerated by hypertension and also contributes to hypertension; however, it is an enigma why hypertension and vascular calcification are a vicious spiral. The present study elucidates the cross-talk between renin-angiotensin II system and receptor activator of nuclear factor-κB ligand (RANKL) system in vascular calcification.
Approach and results:
Angiotensin (Ang) II (10(-7) mol/L) significantly increased calcium deposition as assessed by Alizarin Red staining, associated with a significant increase in the expression of RANKL, RANK, and bone-related genes, such as cbfa1 and msx2, in human aortic vascular smooth muscle cells. Infusion of Ang II (100 ng/kg per minute) in ovariectomized ApoE(-/-) mice under high-fat diet significantly increased the expression of RANKL system and calcification in vivo, whereas administration of Ang II receptor blocker (olmesartan, 3 mg/kg per day) decreased the calcification and bone markers' expression. In addition, male OPG(-/-) mice showed a significant increase in vascular calcification followed by Ang II infusion as compared with wild type. Conversely, RANKL significantly increased Ang II type 1 receptor and angiotensin II-converting enzyme expression in vascular smooth muscle cells via extracellular signal-regulated protein kinase phosphorylation.
The present study demonstrated that Ang II significantly induced vascular calcification in vitro and in vivo through RANKL activation. In addition, RANKL activated renin-angiotensin II system, especially angiotensin II-converting enzyme and Ang II type 1 receptor. Cross-talk between renin-angiotensin II system and RANKL system might work as a vicious cycle to promote vascular calcification in atherosclerosis. Further studies to inhibit renin-angiotensin II system and RANKL may provide new therapeutic options to prevent and regress vascular calcification.
[Show abstract][Hide abstract] ABSTRACT: The effectiveness of angioplasty and stenting in intracranial atherosclerotic diseases is controversial due to high rates of delayed restenosis and hemorrhage compared with extracranial arteries. However, the mechanisms underlying these differences are still unclear, because their pathophysiology is yet to be examined. To address this issue, we established a novel vascular injury model in the intracranial internal carotid arteries (IICAs) in mice, and analyzed the remodeling process in comparison to that of the femoral arteries (FAs). In IICAs, neointimal hyperplasia was observed from day 14 and grew until day 56. Although smooth muscle cells (SMCs) emerged in the neointima from day 28, SMCs in the injured media were continuously lost with eventual extinction of the media. Re-endothelialization was started from day 7 and completed on day 28. Accumulation of macrophages was continued in the adventitia until day 56. Compared with FAs, the following points are unique in IICAs: (1) delayed continuous formation of neointima; (2) accumulation of macrophages in the media on day 14; (3) continuous loss of SMCs in the media followed by extinction of the media itself; and (4) continuously growing adventitia. These pathophysiologic differences might be associated with unfavorable outcomes in percutaneous transluminal angioplasty and stenting in intracranial arteries.Journal of Cerebral Blood Flow & Metabolism advance online publication, 10 April 2013; doi:10.1038/jcbfm.2013.62.
No preview · Article · Apr 2013 · Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein(a) [Lp(a)] is an unique lipoprotein consisting of the glycoprotein apolipoprotein(a) [apo(a)] in low-density lipoprotein. Although Lp(a) is a well-known independent risk factor for cardiovascular disease; however, there is no drugs to decrease plasma Lp(a) level. Thus, to inhibit the biological activity of Lp(a), we developed DNA vaccine for apo(a) by the targeting to the selected 12 hydrophilic amino acids in the kringle-4 type 2 domain of apo(a). Hepatitis B virus core protein was used as an epitope carrier to enhance the immunogenicity. Intramuscular immunization with apo(a) vaccine resulted in the significant inhibition of neointima formation in carotid artery ligation model using Lp(a) transgenic mice, associated with anti-apo(a) antibody and decrease in vascular Lp(a) deposition. Overall, this study provided the first evidence that the pro-atherosclerotic actions of Lp(a) could be prevented by DNA vaccine directed against apo(a), suggesting a novel therapeutic strategy to treat cardiovascular diseases related to high Lp(a).
[Show abstract][Hide abstract] ABSTRACT: Vaccines have been recently developed to treat various diseases such as cancer, rheumatoid arthritis and Alzheimer's disease in addition to infectious diseases. However, before use in the clinical setting, vaccines targeting self-antigens must be demonstrated to be effective and safe, evoking an adequate humoral immune response from B cells while avoiding T cell activation in response to self. Although the vaccine targeting angiotensin II (Ang II) is efficient in rodents and humans, little is known regarding the immunological activation and safety of the vaccine. In this study, we evaluated the efficiency and safety of an Ang II peptide vaccine in mice. Immunization with Ang II conjugated to keyhole limpet hemocyanin (KLH) successfully induced the production of anti-Ang II antibody, which blocked Ang II signaling in human aortic smooth muscle cells. However, Ang II itself did not activate T cells, as assessed by the proliferation and lymphokine production of T cells in immunized mice, whereas KLH activated T cells. In an Ang II-infused model, the non-immunized mice showed high blood pressure (BP), whereas the immunized mice (Ang II-KLH) showed a significant decrease in systolic BP, accompanied by significant reductions in cardiac hypertrophy and fibrosis. Importantly, anti-Ang II antibody titer was not elevated even after the administration of large amounts of Ang II, indicating that Ang II itself boosted antibody production, most likely due to less activation of T cells. In addition, no accumulation of inflammatory cells was observed in immunized mice, because endogenous Ang II would not activate T cells after immunization with Ang II-KLH. Taken together, these data indicate that vaccines targeting Ang II might be effective to decrease high BP and prevent cardiovascular complications without severe side effects.
[Show abstract][Hide abstract] ABSTRACT: Objectives: The aim of this study is to design DNA vaccines for high blood pressure (BP) toward to human vaccine therapy to treat hypertension. Design and methods: We selected angiotensin II (Ang II) as a target antigen. Plasmid vector encoding Hepatitis B core (HBc)-Ang II fusion protein was constructed, as HBc forms sphere-like structure by self-aggregation and the presented fusion peptide in the position of B cell epitope of HBc protein could induce high titer antibody to the target peptide. Plasmid DNA (HBc-Ang II or HBc) or saline were injected to the back skin of spontaneously hypertensive rats (SHR) three times (at 0, 2, 4 weeks) by needle less injection system. Results: Anti-Ang II antibody was successfully produced, and sustained at least up to 6 months. Consistently, systolic BP was lower in HBc-Ang II group after the immunization (at 12 weeks: 188.1 +/- 15.8 mmHg in HBc-Ang II group vs. 221.5 +/- 14.6 mmHg in HBc group, P < 0.01), and BP reduction was continued at least up to 6 months. Of importance, perivascular fibrosis in heart tissue was significantly reduced in HBc-Ang II group. T cell activity of cytokine production responded to Ang II or angiotensinogen was not detected without any apparent pathologically toxic observation. Conclusions: The present study demonstrated that Ang II DNA vaccine to SHR significantly lowered high BP at least up to 6 months. Future development of DNA vaccine to hypertension might provide new therapeutic option to treat hypertensive population.
No preview · Article · Sep 2012 · Journal of Hypertension
[Show abstract][Hide abstract] ABSTRACT: Recent studies have demonstrated that some antihypertensive drugs reduced the risk of bone fracture. Although calcium channel blockers (CCB) are used as first-line agents, there is no evidence that they prevent osteoporosis. In this study, we investigated the effects of 2 types of CCB, benidipine (L-/T-type CCB) and amlodipine (L-type CCB), on bone metabolism. In ovariectomized SHR, administration of benidipine resulted in a significant increase in the ratio of ALP to TRAP and a decrease in the number of osteoclasts in the tibia. Moreover, bone mineral density was significantly higher in the benidipine group compared with the amlodipine group, associated with a significant decrease in urinary deoxypyridinoline. In an in vitro study, benidipine promoted ALP expression and decreased receptor activator of NF-kB (RANK) ligand expression of human osteoblasts, indicating suppression of osteoclast differentiation. These pleiotropic effects of antihypertensive drugs such as benidipine might provide additional benefits in treating hypertensive postmenopausal women.
Full-text · Article · Aug 2012 · Current Cardiovascular Risk Reports
[Show abstract][Hide abstract] ABSTRACT: Both osteoporosis and high blood pressure are major diseases in the recent aging society and may share the same background genetically and environmentally. Although several antihypertensive drugs, thiazides, beta (β)-blockers and angiotensin converting enzyme (ACE) inhibitors, are reported to decrease the risk of bone fractures, calcium channel blockers (CCBs), which are widely used as first line drugs, have not shown significant effects. As treatments to prevent aging-related diseases simultaneously are desirable, a simple approach to identify drugs effective for both diseases is essential. In this study, we screened various antihypertensive drugs with a focus on osteoclast function of acid secretion, bone resorption and reactive oxygen species (ROS) activity. Extracellular acidification in the resorption lacunae of osteoclasts was identified by acridine orange staining under a fluorescence microscope. Low pH of activated osteoclasts was identified as red to orange waves, while neutral pH of resting osteoclasts was identified as yellow to green waves. These phenomena were well correlated with the findings of excavated pit areas formed on the synthetic bone surface by active or resting stage osteoclasts, respectively. It was shown that benidipine, cilnidipine, olmesartan and calvedilol strongly attenuated acid secretion of osteoclasts, azelnidipine moderately, and hydrochlorothiazide mildly reduced the secretion, while amlodipine and telmisartan did not. Overall, screening drugs by osteoclast acidification by fluoresence microscopy might be a useful approach for evaluating the action of drugs on bone metabolism.
[Show abstract][Hide abstract] ABSTRACT: Estrogen deficiency increases the cardiovascular risks and metabolic dysfunction in postmenopausal women. The potential effects of estrogen are partially related to its direct effects on the vascular wall, stimulating endotheliumderived NO synthesis (eNOS). HMG-CoA reductase inhibitors (statins) may also improve the endothelial function through upregulation of eNOS. In this study, we examined whether the treatment of pitavastatin for a short period (1 week) may improve the endothelial function or glucose metabolism under estrogen deficiency induced by ovariectmy. At 4 weeks after ovariectomy, hyperglycemia and hyperinsulinemia were observed in estrogen deficient mice as assessed by intraperitoneal glucose tolerance test, and endothelial dysfunction was also observed as assessed by the vasodilator response to acetylcholine. At 3 weeks after ovariectomy, we started to administer pitavastatin to ovariectomized mice for 1 week. Although the treatment of pitavastatin for a week did not affect obesity, the treatment of pitavastatin significantly attenutaed hyperglycemia and hyperinsulinemia as well as endothelial dysfunction induced by estrogen deficiency. These results demonstrated that treatment of pitavastatin rapidly improved glucose metabolism and endothelial function under estrogen deficiency.
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein(a) [Lp(a)] is known as an independent risk factor for cardiovascular diseases (CVD). Lp(a) is a unique lipoprotein consisting of the glycoprotein apolipoprotein(a) [apo(a)] covalently linked to apolipoproteinB-100 (apoB) in low-density lipoprotein (LDL) by a single disulfide bond. Although Lp(a) level is hugely variable and under strict genetic control, largely by apo(a) gene with kringle-4 type2 repeats, elevated level of plasma Lp(a) is correlated with CVD. Smaller isoforms with fewer kringle-4 repeats are associated with higher plasma Lp(a) level, leading to an increased risk for CVD. Lipid lowering agents (i.e. statins) have little or no effect on plasma Lp(a) level. Although it was reported that administration of niacin or estrogen may reduce the Lp(a) levels, there are no specific agents for the reduction of plasma Lp(a). In this review, we put together recent evidence and describe the overview of Lp(a) with the future direction.