Vaccination Against Human AngiotensinⅡ Type 1 Receptor Prevents Experimental Abdominal Aortic Aneurysms And The Mechanistic Study

The effectiveness experiment of ATRQβ-001 vaccine in AngⅡ-induced abdominal aortic aneurysm.Objective: Piles of evidences demonstrated that renin-angiotensin system(RAS)intertwined with the genesis and progression of abdominal aortic aneurysm(AAA).We had developed a therapeutic anti-hypertensive vaccine named ATRQβ-001,which targeted at human AngiotensinⅡ(AngⅡ)type 1 receptor.Previous results suggested that the ATRQβ-001 vaccine ameliorated relevant pathological injuries in hypertension,diabetic nephropathy and atherosclerosis,besides anti-hypertensive effect.In this study,we employed apolipoprotein E knockout(Apo E–/–)mice subcutaneously infused with AngⅡ to explore the possibility of ATRQβ-001 vaccine in preventing AAA.Method: The ATR-001 peptide was covalently conjugated to Qβ-VLPs via the SulfoSMCC crosslinker to produce the ATRQβ-001 vaccine.Male Apo E–/– mice aged 10 weeks were randomly assigned to 6 groups as following: the control group(n=8),the AngⅡ group(n=23),the telmisartan group(n=16),the hydralazine group(n=14),the vaccine group(n=21)and the Qβ group(n=20).All the animals were subcutaneously implanted with Alzet Osmotic pumps(model 2004),receiving 4-week infusion of saline vehicle or solutions of AngⅡ(1.44mg/kg/d).Mice of the telmisartan group and the hydralazine group were administrated with telmisartan(5 mg/kg/day)or hydralazine(5 mg/kg/day)via oral gavage since AngⅡ infusion.And Mice of the vaccine group and the Qβ group were subcutaneously immunized with ATRQβ-001 vaccine or equivalent Qβ VLP at the dose of 200μg on day 0,100μg on days 14 and 28.The surgery was synchronously conducted at age 15-week(day 35).Weight of mice were measured every week.Blood pressure before and after the surgery were recorded.Serum lipid profiles and antibody titer were regularly detected.Animals were euthanized on 28 days after implantation to harvest the aorta tissues and evaluate the aneurysm occurrence.Staining slices were used for histopathology detection.Result: AngⅡ effectively elevated the blood pressure and induced abdominal aortic aneurysm in Apo E–/– mice.Compared with the AngⅡ group,systolic blood pressures(SBP)were obviously decreased in the telmisartan group,the vaccine group and the hydralazine group,except the Qβ group.Aneurysm incidence and maximal diameter were effectively alleviated by telmisartan or the vaccine,while hydralazine and Qβ VLP failed to prevent aneurysms.There’s no significant difference between groups on body weight,lipid profiles and heart rates.HE and EVG staining showed that administration of ATRQβ-001 vaccine or telmisartan blunted the aneurysmal dilation and reserved the integrality of vascular wall,compared with the AngⅡ group mice,whereas hydralazine and Qβ VLP didn’t.Conclusion: The ATRQβ-001 vaccine not only decreased the SBP elevated by AngⅡ but also blocked AngⅡ-induced abdominal aortic aneurysm formation and progression.In contrast,the anti-hypertensive drug hydralazine failed to prevent aneurysms.Part2 The effectiveness experiment of ATRQβ-001 vaccine in Ca PO4-induced abdominal aortic aneurysm.Objective: Apart from AngⅡ induced AAA model,the calcium chloride(Ca Cl2)induced AAA and calcium phosphate(Ca PO4)induced AAA model(a modified Ca Cl2 model)are also widely used in aneurysm studies.To further validate the effectiveness of ATRQβ-001 vaccine in preventing aneurysms,we introduced C57BL6/J mice perivascularly soaked with Ca Cl2 plus phosphate buffer solution(PBS)to investigate.Method: Male 10-week-old C57BL/6 mice were randomly divided into 5 groups: Control group(n=11),Ca PO4 group(n=14),Vaccine group(n=14),Qβ group(n=14)and anti-ATR-001 group(n=13).The vaccine group mice were subcutaneously vaccinated with ATRQβ-001 vaccine 200μg on days 0,100 ug on day 14 and 28,while the Qβ group were correspondingly vaccinated with equivalent dosage of Qβ VLP.The anti-ATR-001 group mice were injected with 100μg anti-ATRQβ-001 monoclonal antibody per 7 days via caudal veins since the operation.On day 35,the latter four groups were conducted operation to expose aorta and perivascularly treated with Ca Cl2 plus PBS,while the control group treated with sodium chloride(Na Cl)plus PBS accordingly.Systolic blood pressure(SBP)was regularly recorded.4 weeks after operation,all the animals were euthanized and aorta tissues were harvested.Pathological staining sections were used to analyze elastin degradation and media calcificationResult: Ca Cl2 plus PBS accelerated aorta dilation and pathologic damages in aorta vascular wall,with the blood pressure slightly increased.The ATRQβ-001 vaccine and the anti-ATR-001 antibody effectively mitigate the dilation of abdominal aortas,although the SBP was not significantly decreased.EVG staining showed reduced fracture of the elastin layer in aortas of the vaccine group and the anti-ATR-001 antibody group,compared with the Ca PO4 group.Alizarin red staining for calcium deposition in tunica media was also alleviated by the ATRQβ-001 vaccine and the anti-ATR-001 antibody.Conclusion: ATRQβ-001 vaccine rescued Ca PO4-induced aorta dilation and media calcification beyond independent of anti-hypertensive effect.Part3 The mechanistic study in vascular-protective effect of ATRQβ-001 vaccine.Objective: Given the fact that AngⅡ-infused Apo E-/-model is more commonly used in AAA research and reproduces much more characteristics of human AAA compared with Ca PO4-induced AAA,we focused on the former to investigate the underlying mechanisms of ATRQβ-001 vaccine in preventing abdominal aortic aneurysm.Anti-ATR-001 monoclonal antibody was employed to assess the effect of ATRQβ-001 vaccine in vitro.Method: Serum concentration of IL-1β and IL-6 were detected by enzyme-linked immune sorbent assay(ELISA)kits.RNA transcriptions of inflammatory factors and relevant cytokines were detected by quantitative real-time polymerase chain reaction(q RT-PCR).Immunohistochemical staining was used to detect the expression of osteopontin(OPN),CD68 marked macrophage,matrix metalloproteinase(MMP)2 and 9,extracellular MMP inducer(EMMPRIN)in AAA sections or corresponding region in aorta.And the activities of MMP2 and 9 were estimated by gelatin zymography.We also detected contractile phenotype markers in aorta sections by immunofluorescence assay.Mouse aorta vascular smooth cell line MOVAS and mouse macrophage cell line Raw264.7 were cultured in vitro to illustrate effects of anti–ATR-001 antibody.Immunofluorescence co-localization analysis was used to examine the binding ability of anti–ATR-001 antibody to AT1 R.Western blot assay was used to assess the effectiveness of anti–ATR-001 antibody in limiting AT1 R expression and downstream signals(such as ERK and AKT)activation.Result: 1.Compared with the AngⅡgroup,ATRQβ-001 vaccine significantly mitigated circulating or local inflammation in the aortic wall;2.In vivo and in vitro experiments demonstrated that ATRQβ-001 vaccine and anti-ATR-001 antibody obviously inhibited the transformation of vascular smooth cell(VSMC)from contractile phenotype to synthetic phenotype,retarded macrophage infiltration and proteolysis of extracellular matrix(ECM);3.Anti-ATR-001 antibody effectively bound to AT1 R and suppressed AT1 R expression and downstream signals transduction.Conclusion: ATRQβ-001 vaccine prominently alleviated vascular injuries induced by AngⅡ,preventing the pathological and physiological changes of AAA.According to our results,the mechanisms beneath may be that anti-ATR-001 antibody initially suppress AngⅡ-AT1 R activation to mitigate inflammation and VSMC phenotype transition,thereby blunted the crosstalk between synthetic VSMC and macrophage,blunting ECM degradation.