| Background:Preeclampsia, a serious pregnancy-specific disorder characterized by hypertension, proteinuria, abnormalities in coagulation and vascular tone, remains a leading cause of maternal and neonatal morbidity and mortality. Recent study have demonstrated that the possibility of events in fetal of preeclamptic patients, such as premature delivery, low or high birth weight and intrauterine growth retardation, significantly increased than that in decender of normal pregnant, and severely affect the early survival quality. However, except for the pathological changes of precursor, disorder of placental function and premature birth, the precise mechanism which is responsible for impaired health the preeclamptic offsprings remains unclear.In 1999, Wallukat et al for the first time reported that the autoantibody direct against the second extracellular loop (165–191) of angiotensin II receptor type 1 (AT1-AA) existed in preeclamptic patients. This autoantibody could specifically recognize the functional epitope of the second extracellular loop of AT1 receptor, and possess AT1 receptor agonist-like effect, such as increased the tissue factor level of smooth muscle cell, enhanced NADPH oxidase in trophoblast, constricted the vascular and so on. Recent studies suggested that AT1-AA belongs to Immunoglobulin G, several of which subclasses can passively transport. This report makes us proposed that AT1-AA in the matern may transfer to the fetus, and then influence the offsprings development. This study, we will use active immunized rat model to solve the following problems: (1) to determine whether AT1-AA in mother rats can transfer to the fetus; (2) there are two pathways of passive transportation--placenta and milk; however, it remains unknown that by which pathway AT1-AA in fetus was transported from pregnant mater; (3) different isoforms of the same kind of antibody may exhibit different immunological characteristics. Thus, what isoforms AT1-AA belongs to? (4) to observe whether the transported antibody remains show its activity, if so, how about its activity? The answers to these serial problems will be valuable premise for exploring the specific effect of AT1-AA on the preeclampsia fetus and guiding the health care of mater and fetus correctly in clinic.Objective:1. To determine the passive transfer capacity and pathwaies of AT1-AA in the mother rats;2. To identify the isoforms of AT1-AA in the mother rats;3. To observe the activity of AT-AA in the baby rats. Methods:Healthy female Wistar rats (who have never become pregnant, 8-week-old, 180-200g body weight) and male rats (220-250g body weight) were selected. And the female rats were divided into immunized group and control group. Peptides corresponding to the sequences of the second extracellular loop of AT1 receptor (165-191, I-H-R-N-V-F-F-I-E-N-T-N-I-T-V-C-A -F-H-Y-E-S-Q-N-S-T) were synthesed as antigen to immunize rats fortnightly. Mated the female immunized rats with normal male rats when AT1-AA reached a peak. It was considered the beginning of conception when vaginal plug was found. The control group was given antigen adjuvant only, and the rest operations were as same as the immunized group. (1) Detected AT1-AA in baby rats at 1 week old in the two groups by ELISA. (2) Used the same animal model to detected AT1-AA in fetal rats in labor (conceived 20 days) in the two groups by ELISA. Then the antibody in placental was stained by immunohistochemisty. (3) During lactation, determined the distribution of AT1-AA in milk obtained from baby rats stomach by ELISA. Then the exchange-feed experiment was carried out. (4) The IgG subclasses of AT1-AA in immunized rats were identified by ELISA. (5) The total IgGs in immunized rats, which containing large of AT1-AA, were purified with affinity column. To observe the effect of purified antibody on isolated rats'heart function and vascular activity. (6) The sera ET-1 contents in baby rats at 1 week old in the two groups were examined according to the instruction of ELISA kits.Results:1. The AT1-AA positive female rats model by active immunization was successfully establishedTo explore the active immunization characteristics of AT1-AA, AT1-AA positive female rats'model was established by active immunization. The results from ELISA showed that AT1-AA could be detected in sera from female rat at the 2nd weeks after initial active immunization, and the level of the autoantibody quickly increased and reached the peak at 8th week (OD value: 2.76±0.07 vs. 0.43±0.05,P < 0.01, vs. control group at the same time point) as shown in Fig. 1a. However, AT1-AA was not detected in the concurrent control, suggesting that the active immunization model was successfully established. In addition, the isotype of AT1-AA belonged to IgG, but not IgM or IgA (Fig.1b and 1c).2. AT1-AA could transfer from mother to the fetusTo detect the transfer capability of AT1-AA in immunized mother rats, the antibody was detected in baby rats at 1 week old in the two groups by ELISA. As summary in Fig. 2, AT1-AA was strong positive in offsprings (n=8) delivered by immunized mother rats who garnered the antibody. The P/N value exceed 2.1 (OD value was 1.49±0.25). But there was no AT1-AA in the control group baby rats (OD value was 0.13±0.03), and the P/N value was less than 1.5. There was significant difference.3. AT1-AA transfered across the placentaUse the same animal model to observe the pathway of AT1-AA across the placenta. The serum of fetal rats in labor (conceive 20 days) was detected by ELISA. The content of AT1-AA was higher in offsprings (n=12,OD value was 2.32±0.35 ) delivered by immunized mother rats than that in baby rats in control group (n=12,OD value was 0.13±0.04). There was significant difference (P < 0.01, Fig 3a). In order to track the line of AT1-AA in placenta, we detected the antibody by immunohistochemisty. There were strong staining in the trophoblast of villi and vascular endothelial cells in immunized group placenta, which indicated AT1-AA (Fig. 3b). There was no staining in control placenta (Fig. 3b).4. Transfer of AT1-AA via mother rats'milkBreast-feeding to 1 week, the milk obtained from neonate stomach in immunized group contained high titer of AT1-AA (OD value was 1.33±0.26 vs. 0.19±0.10,P < 0.01, vs. control group at the same period,Fig. 4a). The P/N value exceeded 2.1, which means the AT1-AA in the offspring was positive. However, there was no AT1-AA in the offspring in control group.Then the exchange-feed experiment was carried out, to exclude the interference to the milk in stomach caused by many factors. The ELISA showed that, after 7 days, AT1-AA occurred in the female newborn rats of control group fed by immunized mother (OD value was 2.60±0.12 vs. 0.16±0.07,P < 0.01,vs. male newborn rats in control group, Fig. 4b). On the contrary, the level of AT1-AA in immunized group female newborn rats decreased after fed by control mother (OD value was 1.04±0.26 vs.2.62±0.08,P < 0.15,vs. male newborn rats in immunized group,Fig. 4b).5. Identified the IgG subtybes of AT1-AA in immunized ratsThe total IgGs in immunized rats, which containing large of AT1-AA, were purified with affinity column. The purity of the IgGs was detected by SDS-PAGE. It is clear that, there were two bands at 55KDa and 25KDa, which represent IgG heavy chain and light chain (Fig. 5a). Then, the IgG subclasses of AT1-AA in immunized rats were identified by ELISA, and it is proved to belong to IgG2b mainly (Fig. 5b). 6. Positive effects of IgG fractions from immunized group offsprings on cardiac function in isolated perfusing rat heartCompared with the baseline, IgG fractions (0.1μmol/L) from AT1-AA positive newborn rats remarkably enhanced the LVDP, +dp/dtmax, -dp/dtmax of isolated perfusing rat heart. There were significant differences respectively (P < 0.05, P < 0.01, P < 0.05). The effects of AT1-AA-positive immunoglobulin were similar to that observed with angiotensin II (0.1μmol/L), the agonist of AT1 receptor. And both of their effects could be obviously blocked by losartan (1μmol/L). However, IgG fractions from newborn rats in control group had no effect on cardiac function, even its concentration reached to 1μmol/L (Table 1).7. IgG fractions from AT1-AA positive newborn rats constricted rat thoracic aorta ringsConsistent to the results in Table 1, 0.01μmol/L of IgG fraction from AT1-AA positive sera newborn rats caused significant vasoconstriction that is similar to that observed with the same concentration of Ang II (the contraction value were 0.44±0.05g, 0.52±0.07g, respectively). Moreover, vasoconstrictive response to 0.1μmol/L of immunoglobulin was completely blocked by 1μmol/L losartan (0.05±0.02g, P < 0.01, vs. IgGs from offsprings in immunized group). IgGs fractions from control group newborn rats had no effect on vasoconstrictive response (Fig 6).8. The level of ET-1 was increased in newborn ratsET-1 is one of the important indicators for reflecting endothelial injury. We detected the serum of offsprings at one week old and found that, compared with control group, the concentration of ET-1 was markedly increased in newborn rats in immunized group. There was significant difference between them (Fig.8).Conclusion:1. AT1-AA can transport passively from immunized mater to the fetal rats;2. AT1-AA can passively transfer through placenta and milk;3. AT1-AA from pregnant mother belongs to subclass 2a and 2b of IgG, which identified the characteristics of passive transportation of AT1-AA;4. AT1-AA in fetal rats obtained through passive transportation remains displays AT1 receptor agonist-like effects, which are consistent to that of maternal antibody. |
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