Shijun Yang

Effectiveness and Safety of a Therapeutic Vaccine Against Angiotensin II Receptor Type 1 in Hypertensive Animals

Primary hypertension is a chronic disease with high morbidity, and the rate of controlled blood pressure is far from satisfactory, worldwide. Vaccination provides a promising approach for treatment of hypertension and improvement in compliance. Here, the ATRQ&bgr;-001 vaccine, a peptide (ATR-001) derived from human angiotensin II (Ang II) receptor type 1 conjugated with Q&bgr; bacteriophage virus-like particles, was developed and evaluated in animal models of hypertension. The ATRQ&bgr;-001 vaccine significantly decreased the blood pressure of Ang II–induced hypertensive mice up to 35 mm Hg (143±4 versus 178±6 mm Hg; P=0.005) and that of spontaneously hypertensive rats up to 19 mm Hg (173±2 versus 192±3 mm Hg; P=0.003) and prevented remodeling of vulnerable hypertensive target organs. No obvious feedback activation of circulating or local renin-angiotensin system was observed. Additionally, no significant immune-mediated damage was detected in vaccinated hypertensive and nonhypertensive animals. The half-life of the anti-ATR-001 antibody was 14.4 days, surpassing that of existing chemical drugs. In vitro, the anti–ATR-001 antibody specifically bound to Ang II receptor type 1 and inhibited Ca2+-dependent signal transduction events, including protein kinase C-&agr; translocation, extracellular signal-regulated kinase 1/2 phosphorylation (72% decrease; P=0.013), and elevation of intracellular Ca2+ (68% decrease; P=0.017) induced by Ang II, but without inhibiting Ang II binding to the receptor. In conclusion, the ATRQ&bgr;-001 vaccine decreased the blood pressure of Ang II–induced hypertensive mice and spontaneously hypertensive rats effectively through diminishing the pressure response and inhibiting signal transduction initiated by Ang II. Thus, the ATRQ&bgr;-001 vaccine may provide a novel and promising method for the treatment of primary hypertension.

Therapeutic Vaccines against Human and Rat Renin in Spontaneously Hypertensive Rats

Vaccination provides a promising approach for treatment of hypertension and improvement in compliance. As the initiation factor of renin-angiotensin system, renin plays a critical role in hypertension. In this study, we selected six peptides (rR32, rR72, rR215, hR32, hR72, and hR215) belonging to potential epitopes of rat and human renin. The main criteria were as follows: (1) include one of renin catalytic sites or the flap sequence; (2) low/no-similarity when matched with the host proteome; (3) ideal antigenicity and hydrophilicity. The peptides were coupled to keyhole limpet hemocyanin and injected into SpragueDawley (SD) rats, spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats. The antisera titers and the binding capacity with renin were detected. The effects of the anti-peptides antibodies on plasma renin activity (PRA) and blood pressure were also determined. All peptides elicited strong antibody responses. The antisera titers ranged from 1:32,000 to 1:80,000 in SD rats on day 63. All antisera could bind to renin in vitro. Compared with the control antibody, the antibodies against the rR32, hR32, rR72 and hR72 peptides inhibited PRA level by up to about 50%. Complete cross-reactivity of the anti-rR32 antibody and the anti-hR32 antibody was confirmed. The epitopes rR32 and hR32 vaccines significantly decreased systolic blood pressure (SBP) of SHRs up to 15mmHg (175±2 vesus 190±3 mmHg, P = 0.035; 180±2 vesus 195±3 mmHg, P = 0.039), while no obvious effect on SD rats. Additionally, no significant immune-mediated damage was detected in the vaccinated animals. In conclusion, the antigenic peptide hR32 vaccine mimicking the 32Asp catalytic site of human renin may constitute a novel tool for the development of a renin vaccine.

ATRQβ-001 vaccine prevents atherosclerosis in apolipoprotein E-null mice.

Objective: Angiotensin II (AngII) type 1 receptor (AT1R) blockers have been proved to reduce atherosclerosis. Previously, we have invented ATRQ&bgr;-001 vaccine which showed a desirable blocking effect for AT1R. The purpose of this study was to investigate whether ATRQ&bgr;-001 vaccine would prevent atherosclerosis in apolipoprotein E-null (ApoE–/–) mice. Methods: Male ApoE–/– mice were administered with ATRQ&bgr;-001 vaccine, Q&bgr; virus-like particles, valsartan or vehicle over a period of 24 weeks. In vitro, human coronary artery endothelial cells preincubated with the anti-ATR-001 antibody, the neutralization antibody or valsartan for 2 h, were treated with AngII for 24 h. Histological stain and molecule biology methods were used to assess the atheroprotective effect of the vaccine. Results: ATRQ&bgr;-001 vaccine significantly reduced the lesion area and promoted the stability of atherosclerotic plaque. Meanwhile, macrophage infiltration as well as the expressions of adhesion molecules and monocyte chemoattractant protein-1 was obviously decreased in the ATRQ&bgr;-001 vaccine group. Additionally, the vaccine markedly reduced the apoptosis in the lesions of the ApoE–/– mice. In vitro, the anti-ATR-001 antibody inhibited endothelial apoptosis induced by AngII. Furthermore, ATRQ&bgr;-001 vaccine exhibited a dramatical attenuation in the expressions of lectin-like oxidized low-density lipoprotein receptor-1 and AT1R in the aortic. More importantly, compared with the valsartan group, no obvious feedback of the plasma renin–angiotensin system was elicited in the vaccine group. Conclusion: The results demonstrated that ATRQ&bgr;-001 vaccine reduced the progression of atherosclerosis in ApoE–/– mice without obvious feedback of renin–angiotensin system.

Functional analysis of a novel antagonistic antibody against the short epitope of the α1A-adrenergic receptor

AIMS The alpha1A-adrenergic receptor (α1A-AR) regulates various vascular functions and participates in the pathogenesis of primary hypertension. However, highly specific and subtype-selective antagonists of α1A-AR have not been developed. METHODS AND RESULTS Two novel antibodies against the short peptides CP-7, which is located in the second extracellular loop of α1A-AR, and CPE-8, which is located in the third extracellular loop of α1A-AR, were prepared. The two antibodies specifically bound to α1A-AR. However, neither antibody prevented [(3)H]-epinephrine or [(3)H]-prazosin from binding to the receptor. In vitro, the anti-CP-7 antibody inhibited Ca(2+)-dependent signal transduction processes including protein kinase C translocation and extracellular signal-regulated kinase (ERK1/2) phosphorylation induced by phenylephrine (PHE). The anti-CP-7 antibody decreased the beating rates of neonatal rat cardiomyocytes with or without PHE stimulation and reduced the blood pressure of spontaneously hypertensive rats that were immunized with CP-7-keyhole limpet haemocyanin. CONCLUSION The anti-CP-7 antibody specifically inhibited the activation of α1A-AR both in vitro and in vivo. No competition binding was found between anti-CP-7 and [(3)H]-epinephrine or [(3)H]-prazosin. An antibody that specifically inhibits a receptor could be useful in research on G-protein-coupled receptors that lack specific antagonists. The antibody against the epitope CP-7 might have potential in a therapeutic application for treating primary hypertension.

Immune Response of A Novel ATR-AP205-001 Conjugate Anti-hypertensive Vaccine

We developed a virus-like particle (VLP)-based therapeutic vaccine against angiotensin II receptor type 1, ATR-AP205-001, which could significantly reduce the blood pressure and protect target organs of hypertensive animals. In this study, we focused on the immunological effect and safety of the VLP-based vaccine. By comparing to the depolymerized dimeric vaccine ATR-Dimer-001, we found that ATR-AP205-001 reached subcapsular sinus of lymph node shortly after administration, followed by accumulation on follicle dendritic cells via follicle B cell transportation, while ATR-Dimer-001 vaccine showed no association with FDCs. ATR-AP205-001 vaccine strongly activated dendritic cells, which promoted T cells differentiation to follicular helper T cells. ATR-AP205-001 vaccine induced powerful germinal center reaction, which was translated to a boost of specific antibody production and long-lasting B cell memory, far superior to ATR-Dimer-001 vaccine. Moreover, neither cytotoxic T cells, nor Th1/Th17 cell-mediated inflammation was observed in ATR-AP205-001 vaccine, similar to ATR-Dimer-001 vaccine. We concluded that ATR-AP205-001 vaccine quickly induced potent humoral immunity through collaboration of B cells, follicular dendritic cells and follicular helper T cells, providing an effective and safe intervention for hypertension in the future clinical application.