Antibodies Against The Ectodomains Of AT₁ Receptor And AT₁ Receptor Vaccine Against Hypertension

Part IAutoantibodies against the AT₁ receptor induce Ca²⁺ increase in cultured rat vascular smooth muscle cellsObjective: AT₁ receptor autoantibodies (AT₁-AAs) were described in the patients with malignant hypertension or preeclampia. We also found the autoantibodies were highly associated with refractory hypertension. Function of vascular smooth muscle cells (VSMC) is important in the regulation of blood pressure. We investigated and compared the ability of angiotensin II (Ang II) and AT₁-AA to stimulate the intracellular calcium mobilization and cellular proliferation of rat vascular smooth muscle cells (VSMC).Methods: 22 patients with refractory hypertension, 24 patients with non-refractory hypertension and 37 normotensives were recruited randomly. The serum of each patient was detected for the presence of AT₁-AAs by ELISA. Ang II and AT1-AA from the sera of patients were used to stimulate rat VSMC in vitro, and celluar proliferation was evaluated by a Brdu ELISA kit.Results: AT1-AAs were detected in 45.5, 12.5 and 8.1% of patients with refractory hypertension, non-refractory hypertension normotensives, respectively. AT1-AAs led the increase intracellular Ca2+ mobilization in a dose-dependent manner and cellular proliferation of VSMC just as Ang II. Both of these effects caused by AT1-AAs were blocked by losartan and by a peptide corresponding to a part of the second extracellular loop of the AT1 receptor. In contrast, control IgG had no effects. Conclusion: Since AT1-AAs exhibited pharmacological activity in VSMC just as Ang II, they may play a role in the elevation of peripheral vascular resistance and in vascular remodeling. AT1-AAs were suggested to involve in resistance to antihypertensive therapy.Part II Influence of active immunizations against AT₁ receptor on blood pressure in spontaneously hypertensive rats.Objective: We designed four different peptides according to the extracellular parts of the rat AT₁A-receptor and used as antigens. We observed the influence of four different antibodies on blood pressure of SHRs to select peptides which can reduce the blood pressure.Methods: the four kinds of peptides (coded ATR11188, ATR12181, ATR13263 and ATR13275) were designed based on the amino acid sequences of rat AT_1A-receptor extracellular parts. The peptides were synthesized, purified, conjugated to tetanus toxoid (TT) and used as antigens. SHRs were divided into 5 groups: (1) Control group (n=7) were 0.9% sodium chloride hypodermically. (2) ATR11188 group (n=7). (3) ATR12181 group (n=8). (4) ATR13263 group(n=6). (4)ATR13275 group (n=6). All peptide groups were repeatedly immunized with peptide-TT conplex hypodermically. The systolic blood pressure (SBP) was recorded and the titres of specific antibodies were measured by EL1SA. And the immunogobulin (Ig)G was obtained from the SHRs' serum, and used as antibodies to prove the affinity with AT₁-receptor in the cultured vascular smooth muscles (VSMC) by western blotting and immunofluorescence. IgG was used to stimulate VSMC, and celluar prolifreation was evaluated by a Brdu ELISA kit.Results: the SHRs in all peptide groups developed positive antibodies after repeated immunizations. Western blotting with IgG from ATR12181 produced bands of about 41KD molecular weight. Furthermore, fluorescence microscopy of vascular smooth muscle cells showed colocalization of IgG against ATR12181. In ATR12181 group, the SBP was found to be significantly decreased (SBP of ATR12181, 167.3 ± 10.4 mmHg, SBP of control group 180.6 ± 8.0 mmHg, n=7 per group, P < 0.05) in 8^th week. Other peptides did not reduce the SBP. In addition, proliferation of vascular smooth muscle cells, in response to angiotensin II, could be inhibited by antibodies against peptide ATR12181 in vitro.Conclusion: The results indicated that long-term immunized with ATR12181 effectively reduced blood pressure of SHRs. And active immunization with different peptides from the AT₁-receptor extracellular parts may have different effects on AT₁ -receptor.Part III Target organ protection from an angiotensin II receptor (AT₁) vaccine ATR12181 in spontaneously hypertensive ratsObjective: We have previously established that an angiotensin II receptor (AT₁) vaccine-ATR12181 attenuated the development of high blood pressure (BP) inspontaneously hypertensive rat (SHR) model of human essential hypertension. Ourobjective was to determine whether this attenuation of high BP is associated withprevention of target organ damages induced by hypertensive state.Methods: SHRs were immunized against a peptide (coded ATR12181) from theextracelluar portion of the AT_1A receptor by repeated subcutaneous injections ofpeptide- tetanus-toxoid complex in combination with Freund's adjuvant. A 24 weekshort-term and a 64 weeks long-term observation were performed. At the end ofobsevations, all SHRs were sacrificed for morphological and ultrastructureexamination of the heart and kidneys. At last, c-fos and c-jun mRNA level of leftventricular muscles and renal cortex were detected by RT-PCR.Results: At the end of observations, vaccinated SHRs expressed lower BP, decreasedcardiac hypertrophy and attenuation of kidney injures. mRNA levels of c-fos andc-jun in heart and kidneys had decrease in vaccinated SHRs.Conclusion: These data demonstrate that repeated immunization against a domain ofthe extracellular portion of the AT₁ receptor is able to cause a target organ protectionagainst hypertension. Active immunization against the AT₁ receptor may beconsidered a promising new strategy in the treatment of hypertension.Part IV Safty of vaccine-ATR12181Objective: since a self-antigen (peptide ATR12181 from the rat AT_1A receptor) wasused, safety of vaccine was concerned. Our objective was to evalaute the safty ofATR12181-vaccine.Methods: SHRs were immunized against a peptide (coded ATR12181) from theextracelluar portion of the AT_1A receptor by repeated subcutaneous injections of peptide- tetanus-toxoid complex in combination with Freund's adjuvant. A 64 weeks long-term observation was performed. In 64^th weeks, all SHRs were weighed and sacrificed for morphological and ultrastructure examination of important organs including heart, kidneys, liver, spleen, aorta and artery.Results: Body weight (BW) of SHR in ATR12181 group was significant heavier than control group(BW of control group 383±18g, BW of ATR12181 group 538±36g), and behaviour of ATR12181 group was normal. Morphological examination of important organs did not find any signs of autoimmune damage. Kidney was has abundant arterioles and capillary, where the AT_1A receptors are abundantly expressed. There were no signs of perivascular cellular inflammation. Although the thickening basement membrane of glomcruluses was found under TEM, control group also developed similar ultrastructure changes that were more serious. In addition, there were no signs of depositions in basement membrane or other places of glomcruluses in vaccinated group.Conclusion: Preliminary evaluation found that the vaccine-ATR12181 was safe. Since a self-antigen was used, the more experiment needed be done to evaluate the safty of vaccine.