The Vasoconstriction Effects of Autoantibody Against AT1 Receptor on Human Placental Blood Vessels
|Keywords||preeclampsia angiotensinⅡ1 receptor antibody placental blood vessels|
Background:Preeclampsia is one of the most significant health problems in human pregnancy. Its pathological basis is placental abnormalities associated with shallow trophoblast invasion and impairs spiral artery remodeling. However, the etiology of this syndrome is poor understanded.Recent studies indicated that the angiotensin III receptor autoantibodies (AT1-AA), the potential risk factors of preeclampsia, negatively correlates with the placental blood flow.But there was no direct evidence for the casusal relationship.Our studies showed that AT1-AA could induce a marked dose-dependent vasoconstriction in rat aortic vascular rings though AT1 receptor. Previous studies confirmed the existence of human placental chorionic AT1R. What about human placental blood vessels? If there were, the contraction of human placental blood vessels through it with the combination of AT1-AA will provide the directly evidence of AT1-AA affect the placental blood flow.In order to study the role of AT1-AA on the placenta vessles,we used Commercial synthetic ATlR-ECⅡpeptides, as antigen active immunization rats to obtain a large number of ATIR-Ab, and further determination the types and subtypes of this antibody. The nature and effects of ATIR-Ab are same with AT1-AA,will provide the theoretical basis for AT1R-Ab instead AT1-AA as an experimental tool.In this study, we will solve the following problems:(1) To detect the expression of AT1 receptor on the placental blood vessels with immunohistochemistry experiments;(2) the role of AT1-AA extracted from preeclampsia patients on human placental blood vessels; (3)To prepare and identify the chass and subclass of AT1R-Ab,observe and compare the function with AT1-AA. (4) To observe the mechanism of ATIR-Ab on human placental blood vessel contraction. Answers to these questions are to further explore the impact of AT1-AA through the placental blood flow and take part in the pathogenesis of preeclampsia, as well as tips during her pregnancy, AT1-AA increased regularly check-ups provided some experimental basis.Objective:1..To observe the expression of AT1 receptor on the human placental vessesl;2. To observe the role of AT1-AA in normal human placental blood vessels; 3. To identify the types and subtypes of ATIR-Ab, and its role in human placental blood vessels,and compared it with the role of AT1-AA.4.To observe the mechanisms of ATIR-Ab on the placenta vessels.Methods:The first step is immunohistochemistry experiments, targeting the expression of AT1 receptors on human placental blood vessels. Second, to detect the tider of AT1-AA in serum of patients with pre-eclampsia by ELISA, then observe the role of AT1-AA on normal human placental blood vessels. For the further study, a large number of ATIR-Ab was obstained for active immunization rats, and the antigen is the commercial synthetic ATlR-ECⅡpeptides.Then determinated the types and subtypes of this antibody,and observated the comparison with AT1-AA in the blood vessel contraction, the receptor mediated type in contraction, the comparison with angiotensin II and the role of AT1R-Ab in placental vascular of preeclampsia.Results:1. The expression of AT1 receptor in human placental blood vessels:In this study, we detected the expression of these receptors on the placental artery, vein and vascular endothelial cells by immunohistochemical techniques.There were strong staining in this area, while the negative control group placenta. There was no specific AT1 receptor. (Figure 1)2. AT1-AA caused the contraction of normal human placental blood vessels:The 1mmol/LIgGs from preeclampsia patients could cause significant contraction in placental vein rings (contraction amplitude of its percentage of KCl contraction amplitude of 13.33±0.9%vs. control group, P<0.01). Anti-human IgG antibody(lmmol/L) and AT1R-ECⅡ(1mmol/L,AT1 receptor second extracellular loop epitope peptides), respectively, after pre-incubation could effectively abolish the role of IgGs in preeclampsia serum. (Figure 2,3).3. The class,subclass and the role of AT1R-Ab on the human placental blood vessels are consistent with the AT1-AA:3.1 The preparation of Anti-ATIR antibody (ATIR-Ab) and the identification of its types and subtypes, and the results is similar with AT 1-AA:By ELISA method, the AT1R-Ab belongs to IgG class immunoglobulin (OD value, of 2.77±0.03 vs.0.40±0.04, P<0.01 in the control group), but no IgM and IgA types (P> 0.05, vs. the same period the control group, Figure 5). This antibody mainly blongs to IgG2b subtype (OD value of 0.32±0.04 vs. OD value of 0.08±0.01, P<0.01 in the control group). There are very few parts of belonging to IgG2a subtype (OD value of 0.10±0.01 vs.0.07±0.01, P＜0.05, in the control group Figure 6).3.2 The comparison of the AT1R-Ab’contraction with AT1-AA’:Both could cause the contraction of placental veins. The same concentration of 1mmo/L AT1-AA and AT1R-Ab may show the contraction rate of the placental veins and had no statistics difference. (13.33±0.9% vs.14.00±3.2% P>0.05) (Figure 7)4.The mechanism of AT1R-Ab on the human placenta blood vessels:4.1 The contraction of AT1R-Ab on the placental vein and artery had no significant difference:AT1R-Ab (10mmol/L) could enhance the contraction rate of the placental veins which was29.21±3.7%. The rate of placental arteries was 21.35±2.8%, and there was no significant difference between them. (Figure 8).4.2 AT1R-Ab contracted the human placental blood vessels in a concentration-dependent manner, and the AT1R-ECⅡcould effectively abolish the role of this antibody:The rate of 0.1,1,10mmol/L of the AT1R-Ab contracted the human placental vascular ring was 2.73±1.1%、14.00±3.2%and 33.30±5.6%, respectively.There were significant difference between them(P＜0.01). (Figure 9)AT1R-ECⅡ(lmmol/L,AT1 receptor second extracellular loop epitope peptides) could effectively abolish the role of this antibody. (Figure 10)4.3 The contraction of AT1R-Ab was mediated by the AT1 receptor, but not by AT2 receptor:Losartan(lmmol/L) could effectively neutralize the role of ATIR-Ab in vascular rings (14.00±3.2% vs.-6.33±0.9%, P<0.05). But the PD123319 had no this role (14.00±3.2% vs.13.50±3.3%,P>0.05). (Figure 11)4.4 The contraction of AT1R-Ab was similar with angiotensin II, but had the more time.The contraction of ATIR-Ab was similar with angiotensin II. Losartan(lmmol/L) could effectively neutralize the role of AngⅡ(-2.60±0.2% vs.8.11±1.6%, P<0.05). But the PD123319 had no this role (6.98±1.7%vs.8.11±1.6%, P>0.05). (Figure 12)In the experiment, we observed an interesting phenomenon:AT1R-Ab caused the continued contraction’time in placental blood vessels significantly which was longer than AngⅡ’s. The contraction time of AT1R-Ab was 33.60±3.1 minutes, while Ang II’was 2.93±0.3 minutes. Obviously, the time of ATIR-Ab caused the continued contraction in placental vessels significantly longer than angiotensinⅡ’, and the trend has no desensitization. (Figure 13) 4.5 ATIR-Ab could contract the preeclamptic placental blood vessels:The rate of 0.1,1,10mmol/L of the AT1R-Ab contracted the human placental vascular ring were 1.74±0.3%、5.58±1.4%and 13.73±2.5%, respectively. It had significant difference between them(P<0.01). (Figure 14)Conclusion:1. There was expression of AT1R in human placental blood vessel;2. The AT1-AA could cause the contraction of normal human placental blood vessels;3. AT1R-Ab could replace AT1-AA as an experimental tool;4. AT1R-Ab could contract the blood vessels not only in normal placental blood vessels, but also in the placental vessels with preeclampsia though the AT1 receptors. The role of ATIR-Ab was similar with angiotensin II, but had the longer time. The trend has no desensitization.