| BackgroundIn the past three decades, cardiovascular diseases （CVD） have become a ubiquitous cause ofmorbidity and a leading contributor to mortality worldwide. Metabolic syndrome （MetS）, whichis defined as concomitant disorders of central obesity, lipid and glucose metabolism, and highblood pressure, is considered to be closely related to an increased risk of CVD. Therefore,clarifying the etiology and mechanisms of MetS has become an imperative task for the medicalprofession. To our knowledge, complex interactions between genes, atherogenic diet, sedentarylifestyle and environmental factors could lead to MetS. However, growing evidences havehighlighted the fetal origin of adult disease （FOAD） hypothesis, which proposes that adult-onsetMetS and CVD are also closely associated with the intrauterine and initial extrauterine stages oflife. Multiple insults, including maternal exposure to harmful factors, inadequate placentalperfusion, and/or poor/excess maternal nutrition, may result in intrauterine growth restriction（IUGR）, with extenuating consequences upon adult health. However, the etiology andpathogenesis of FOAD are complex and not fully understood presently.The placenta is the place where the fetus can absorb nutrients. It is generally considered thatplacental dysfunction may be the key initial step for FOAD. There are many risk factors that candestroy the placenta. However, there are also many unknown adverse factors that need to befurther investigated. Recently, evidence that the autoantibody against the angiotensin II type1receptor （AT1-Ab）, first detected in nearly all women with severe preeclampsia and a smallnumber of normotensive pregnant women, might lead to IUGR has emerged. This autoantibodyspecifically recognizes the functional epitope of the second extracellular loop of the AT1receptor（amino acid residues165-191, AT1R-ECII）, possessing AT1receptor agonist-like pathologicaleffects. Previous studies demonstrated that AT1-Ab might cause impaired placental perfusion viavaried mechanisms, such as minimizing trophoblast invasion, increasing placental cell apoptosis,and inducing placental vasoconstriction in vitro. The onslaughts above may contribute topathological placental injury, limiting intrauterine fetal growth and maturation. However, there isa lack of more direct evidence demonstrating how the antibody affects offspring development.Our study aimed to elucidate whether babies born to AT1-Ab-positive mothers would displayabnormal birth weights. If so, would these babies show increased susceptibility to MetS in there middle age, and what are the underlying mechanisms?To demonstrate how the antibody affects offspring development and the potentialmechanisms, we needed to answer the following questions:（1） Whether maternal AT1-Ab cantransfer to the offspring, if so, what is the potential path?（2） The biological properties of theAT1-Ab within offspring require further elucidations. Most importantly, whether the antibodiesmight exert negative effects upon fetal birth weights has not been fully known.（3） Can offspringof AT1-Ab-positive mothers have increased susceptibility to MetS upon maturation? If so,whether these metabolic abnormalities result from intrauterine and/or extrauterine exposure toAT1-Ab? As MetS has become one of the major public-health challenges worldwide, therapeuticintervention to block the adverse effects of AT1-Abs during pregnancy, or preventive actions（e.g., a low-sugar, low-calorie diet） taken by descendants of AT1-Ab-positive pregnant women,may be novel strategies in the battle against MetS and its cardiovascular complications.Part One The transport characteristics of AT1-Ab in mother rats and theeffects of maternal antibodies on offspring birth weightObjectiveTo establish the pregnant rat models with AT1receptor antibodies （AT1-Ab） by activeimmunization and observe the transport characteristics of AT1-Ab in the mother rats and theeffects of maternal antibodies on offspring birth weight.Materials and methods1AnimalsHealthy AT1-Ab-negative female Wistar rats weighing0.18-0.20kg （6-8weeks old） wereselected.2Methods2.1PeptideThe peptide corresponding to the sequence of the second extracellular loop of the human AT1receptor （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,95%purity） wassynthesized by GL Biochem （Shanghai） Ltd.2.2AT1-Ab-positive pregnant rat modelWistar rats which met the requirements were randomly divided into two groups: immunized group and vehicle group.（1） Immunized group rats were actively immunized with synthetic AT1R-ECIIpeptide every twoweeks. After8weeks, immunized female rats were mated with normal male rats（non-immunized）. During pregnancy and nursing periods, female rats received immunizationmonthly. Caudal vein blood was drawn before each immunization, and sera were collected andstored at40°C to monitor antibody generation using the ELISA method.（2） Vehicle group rats were treated with a mixture of Freund’s complete/incomplete adjuvant（without antigen） and saline in an identical manner.2.3To determine the pathways of AT1-Ab in the mother ratsUsing the above-mentioned pregnant rats model:（1） The20-day pregnant （in labor） rats wereintraperitoneally anesthetized with10%chloral hydrate （3ml/kg）, and caesarean operationscommenced. We collected fetal rat serum and placenta to detect the AT1-Ab through ELISAmethod and the immunohistochemical method.（2） During lactation, mother rats’ milk wascollected from the stomachs of newborns. The AT1-Ab content of the milk was determined byELISA. In the cross-feeding experimental protocol, the immunized group and vehicle groupmother rats that delivered on the same day were paired. Their newborns were separated bygender. In one pair of mother rats, male newborns were fed by their mother, and femalenewborns were exchanged for cross-feeding by the opposite group’s mother. There were foursubgroups: the vehicle group male newborn rats; the immunized group female newborn rats fedby vehicle mother rats; the immunized group male newborn rats; the vehicle group femalenewborn rats fed by immunized mother rats. After seven days of feeding, serum AT1-Ab levelswere compared among the subgroups.2.4To culture the vascular smooth muscle cell （VSMC）Thoracic aortic smooth muscle cells were cultured by the explant method.2.5Determination of intracellular free Ca2+by confocal laser technologyIntracellular free Ca2+concentration in VSMC was monitored utilizing Fluo-3/AM, a commonfluorescent Ca2+indicator. Briefly, the fourth generation of cells were cultured in a specialconfocal dish with PBS solution and then loaded with Fluo-3/AM （10mol/L diluted indimethyl sulfoxide） at37oC for60mins. After being washed three times with PBS solution toremove unhydrolyzed indicator, fluorescence in cells was measured using a confocal microscope.The excitation wavelength was488nm, and the emission wavelength was522nm. Ten cells ineach group were randomly selected for image analysis. The changes of intracellular Ca2+were defined as the difference between the fluorescence intensity at each recording time and thefluorescence value at baseline.2.6Preparation of thoracic aorta ringsWhen the rats were anesthetized, thoracic aortas were quickly removed and placed in ice-cold4-（2-hydroxyethyl）-1-piperazineethanesulfonic acid （HEPES） solution （in mM: NaCl144.0, KCl5.8, MgCl2ï¹'6H2O1.2, CaCl22.5, Glucose11.0, Hepes5.0. pH7.4）. The surrounding tissue wascleaned and the aortas were cut into rings of3-4mm in length. The rings were suspended onsteel hooks in tissue baths containing10ml of HEPES solution bubbled with100%O2andmaintained at37°C. The changes in isometric force were recorded by a PowerLab system （ADInstruments, Australia）.Results1Successful establishment of AT1-Ab-positive pregnant rat models by active immunizationTo explore the transfer capacity characteristics of AT1-Ab, AT1-Ab-positive female rats’ modelwas established by active immunization. The results from ELISA showed that AT1-Ab could bedetected in sera from female rat at the2ndweek after initial active immunization, and the level ofthe autoantibody quickly increased and reached the peak at8thweek （OD value:2.76±0.09vs.0.33±0.02, P<0.01, vs. vehicle group at the same time point） as shown in Fig.1A. However,AT1-Ab was not detected in the concurrent vehicle group rats, suggesting that the activeimmunization model was successfully established. In addition, the isotype of AT1-Ab belongedto IgG, but not IgM or IgA （Fig.1B and1C）. Then the AT1-Ab-positive female rats were matedwith health male rats.IgGs in the sera were purified by affinity chromatography. The specificity of purified IgGswas determined by the SDS-PAGE. The results showed that two straps of55KD and25KDappeared which represented one heavy chain and one light chain, respectively （Fig.1-2A）. TheIgG subclass of AT1-Abs in immunized rats was determined to be mostly IgG2b, with a minorpercentage belonging to the IgG2a subclass （Fig.1-2B）.2AT1-Ab can transfer across the placentaThe serum of fetal rats in labor （20days of gestation） was detected by ELISA. The content ofAT1-Ab was higher in the offspring （OD value was2.32±0.10） delivered by immunized motherrats than that in baby rats in the vehicle group （OD value was0.13±0.01）. There was astatistically significant difference （P<0.01, Fig.1-3A）. In order to track the line of AT1-Ab in theplacenta, we detected the antibody by immunohistochemistry. There was strong staining in the trophoblast of villi and vascular endothelial cells in the immunized group placenta, whichindicated the presence of AT1-Ab （Fig.1-3B）. There was no staining in the vehicle placenta （Fig.1-3B）.3Transfer of AT1-Ab via mother rats’ milkAfter breast-feeding to one week, the milk obtained from neonatal stomach in the immunizedgroup contained high titers of AT1-Ab （OD value was1.34±0.11vs.0.19±0.03, P<0.01, vs.vehicle group at the same period, Fig.1-4A）. The P/N value exceeded2.1, which means theAT1-Ab in the offspring was positive.Then the exchange-feed experiment was carried out to exclude interference to the milk in thestomach caused by many factors （such as saliva, gastric juice）. ELISA showed that, after7days,AT1-Ab was present in the female newborn rats of the vehicle group fed by immunized mother（OD value was2.60±0.05vs.0.16±0.03, P<0.01, vs. male newborn rats in the vehicle group, Fig.1-4B）. On the contrary, the level of AT1-Ab in the immunized group female newborn ratsdecreased after being fed by vehicle mother rats （OD value was1.04±0.12vs.2.62±0.08, P<0.01,vs. male newborn rats in the immunized group, Fig.1-4B）.4A significant increase of intracellular Ca2+levels in cultured VSMC was observed afterstimulation with IgG fractions from the immunized group newborn ratsThoracic aortic smooth muscle cells were cultured by explant method. Five days later, some cellsclimbed out from the edge of the tissue. After another five days, the VSMC covered the bottomof the culture bottle, which appeared as a spindle-or ribbon-like shape （Fig.1-5）. The cellactivity was measured by Guava PCA CytoSoft6.0.2software and the viability was more than91%（Fig.1-6）. Immunohistochemical results showed that the SM α-actin was positivelyexpressed in cultured cells, indicating that the cultured cells were VSMCs （Fig.1-7）.Fluo-3/AM was used as Ca2+indicator. The results showed that a significant increase ofintracellular Ca2+level in cultured VSMC was apparent after stimulation with IgG fractions fromthe immunized group newborn rats （1mol/L）. Maximal change of fluorescence occurred after1minute （P<0.01vs. vehicle IgGs）. Cellular preincubation with losartan （10mol/L） for20minutes caused markedly reduced and delayed Ca2+uptake, while vehicle IgGs （1mol/L） hadno effect upon intracellular Ca2+at any time （Fig.1-8）. The experiment suggested AT1-Ab mightpromote the intracellular calcium increase through the AT1receptor.5IgG fractions from AT1-Ab-positive newborn rats constricted rat thoracic aorta ringsAs shown in Fig.1-9,1mol/L IgG fractions isolated from the sera of AT1-Ab-positive newborn rats induced significant vasoconstriction, similar to angiotensin II （AT1receptor agonist） of thesame concentration （contraction values,0.44±0.05g and0.49±0.07g, respectively）. Furthermore,the vasoconstrictive response was markedly blocked （0.05±0.02g, P<0.01vs. IgGs fromimmunized group offspring） by10mol/L losartan （AT1receptor blocker）. IgG fractions fromvehicle newborns demonstrated no vasoconstrictive effects, even at IgGs concentrations of5mol/L.6Abnormalities in weight status appeared at the early life stages in AT1-Ab-positive groupoffspringAT1-Ab-positive group newborn rats’ birth weights tended to be slightly less than that of thevehicle group, but not statistically significantly （P=0.06,6.21±0.09g vs.6.46±0.09g, Fig.1-10A）. Unexpectedly, immunized group offspring body weight values demonstrated muchgreater variability compared with the vehicle group. Fig.1-10B and1-10C reveals the bodyweights of both groups of3-week-old offspring, which suggests that there may be abnormalitiesin the development of fetuses.Summary1. AT1-Ab can transport from immunized mother rats to their offspring through placenta andmilk, and AT1-Ab in offspring rats still displays agonist-like effects;2. AT1-Ab may interfere with the body weight of offspring rats in their early life.Section two Increased susceptibility to metabolic syndrome in adult offspringof angiotensin type1receptor autoantibody-positive ratsObjectiveTo investigate whether middle-aged offspring of AT1-Ab-positive mothers are prone tometabolic disorder development and explore the potential mechanisms.Materials and methods1The establishment of AT1-Ab-positive female rat modelsSee the section one.2Glucose and lipid metabolism detectionFasting venous blood of rats were obtained. Fasting plasma glucose （FPG） and fasting insulin（FINS） were measured by the glucose oxidase-peroxidase method and radioimmunoassay （RIA）. The content of total cholesterol （TC） and triglycerides （TG） were detected by enzymecolorimetric method. High density lipoprotein cholesterol （HDL-C） level was measured with anautoanalyzer. The homeostasis model assessment-estimated insulin resistance （HOMA-IR） wasalso calculated （FPG×Fins/22.5）.3Blood pressure detectionThe blood pressure of rats was periodically determined with tail-cuff plethysmography.4The detection of change of fat in the liverLiver tissue was fixed with4%formaldehyde and conventional paraffin embedding wasconducted. The slides were stained by HE and the structure of liver tissue was observed under alight microscope.5The detection of serum adiponectin levelsELISA kit was used to detect total adiponectin levels in sera. The procedure was as follows:blank wells, standard wells and sample wells were set on96-well plates, respectively.100l ofsample diluent was added in blank wells,100l of standards and samples were added instandard wells and sample wells for2h at37°C, respectively. Then the liquid was discarded andthe wells were dried.100l of working fluid A was added to each well for1h at37°C. Afterwashing the wells three times,100l of working fluid B was added to each well for1h at37°C.After three washings,90l of substrate solution was added to each well and showed color in thedark at37°C. Reaction was terminated by added50l of stop solution. The absorbance valueswere detected at450nm using a microplate reader （Spectra Max Plus, Molecular Devices Corp,CA, USA）.Results1Increased serum insulin was observed in40-week-old immunized group offspring givennormal diet:The offspring of AT1-Ab-positive/negative pregnant rats were fed and raised in typical fashionuntil40weeks of age. No significant difference was observed between the two groups in regardsto fasting plasma glucose （FPG, P>0.05, Fig.2-1A） or blood pressure （P>0.05, Fig.2-1B）.However, fasting insulin in the offspring of AT1-Ab-positive mothers was greatly increased（23.9±1.1IU/ml vs.12.7±0.5IU/ml, P<0.01, Fig.2-1C）, and the homeostasis modelassessment for insulin resistance （HOMA-IR） was also elevated （5.67±0.21vs.2.32±0.15,P<0.01, Fig.2-1D）. 2Development of MetS in adult offspring of the immunized group subjected to high-sugardiet challengeTo determine whether impaired insulin response in these animals may contribute to thedevelopment of MetS when fed with high-sugar food, additional experiments were performed. Ahigh-sugar diet （20%sucrose） was administered to the offspring during postnatal weeks40-48.FPG in the offspring of AT1-Ab-positive pregnant rats markedly increased at48weeks（6.35±0.25mmol/L vs. the vehicle group5.26±0.17mmol/L, P<0.01, Fig.2-2A）. Dyslipidemiaaccompanied the observed hyperglycemia. Compared to the vehicle group, elevated triglycerides（TG,6.61±0.34/L vs.2.02±0.11mmol/L, P<0.01, Fig.2-2B）, and decreased high-densitylipoprotein cholesterol （HDL-C,0.28±0.03mmol/L vs.0.48±0.03mmol/L, P<0.05, Fig.2-2C）were observed in the immunized group offspring, indicative of MetS. However, no significantdifference was observed between the two groups in regards to blood pressure （P>0.05, Fig.2-2D）.3Decreased insulin sensitivity probably plays an important role in increased susceptibilityto metabolic syndrome in adults.At48weeks, HE staining showed that the islet cell was hypertrophic and disordered in theoffspring of AT1-Ab-positive pregnant rats （Fig.2-3A）. Additionally, hepatic fat accumulationand lymphocyte infiltration were present in the immunized group offspring. Vehicle offspringmanifested grossly normal lobular architecture （Fig.2-3B）.Adiponectin is a fat cell factor with strong anti-diabetic and cardioprotective properties. It issignificantly reduced in patients with obesity and diabetes. To determine whether the offspringof AT1-Ab-positive pregnant rats may have altered adiponectin production, serum adiponectinlevels were determined. As summarized in Fig.2-3C, serum adiponectin levels decreasedsignificantly in the immunized group offspring compared to the vehicle group （3818±612g/Lvs.5837±678g/L, P<0.05, Fig.2-3C）. This result indicated that decreased adiponectin mayplay an important role in insulin resistance and metabolic syndrome.Summary1. The adult offspring of AT1-Ab-positive pregnant rats increased susceptibility to metabolicsyndrome;2. Decreased insulin sensitivity of target organs probably plays an important role in increasedsusceptibility to metabolic syndrome in adult rat offspring. Se ction three The effects of AT1-Ab on metabolic-related indicators in adultratsObjectiveTo establish the AT1-Ab-positive rat models by active immunization and observe the directeffects of AT1-Ab on glucose and lipid metabolisms in rats when excluding the interference ofpregnancy condition.Materials and Methods1Experimental subjectsTwenty healthy AT1-Ab-negative Wistar rats weighing0.18-0.20kg （male and female in half,6-8weeks old） were selected.2Methods2.1The long-term AT1-Ab-positive rat model was established by active immunizationTwenty healthy Wistar rats were randomly divided into two groups: immunized group andvehicle group. Rats in the immunized group were actively immunized with AT1R-ECIIfor40months according to the method described in section one in order to build the long-termAT1R-ECII-positive rat model.2.2Detection of the liver function, glucose and lipid metabolismRats should fast for12hours before blood collection. The liver function was examined byautomatic biochemical analyzer. Fasting blood glucose, TG and HDL-C were detected by themethod described in section two.2.3Endothelin-1（ET-1） assayAccording to kit instruction, the sera levels of ET-1were determined by double antibodysandwich ELISA.2.4Intercellular adhesion molecule1（ICAM-1） assayConfocal laser technology was used to detect ICAM-1. Rat thoracic aorta sections were cleanedand epitopes were re-exposed by microwave. Goats anti-rat antibody to ICAM-1was addedfollowed by incubation at4°C overnight; then the secondary antibody, FITC labeling rabbitsanti-goat IgG was added in a water bath at37°C; after washing with PBS5mins×3, confocallaser technology was used to observe pieces after glycerin sealing. The excitation wavelength ofFITC is495nm, and emission wavelength is519nm. 2.5Vacular cell adhesion molecule1（VCAM-1） detectionThe two-step immunohistochemical staining reagents were used to detect the expression ofVCAM-1in rat’s thoracic aorta endothelial cells.2.6The ultrastructure of coronary artery endothelial cells and thoracic aorta smoothmuscle cells were observed with transmission electron microscope （TEM）Heart tissue （1mm×1mm） was removed quickly and stored in2.5%glutaric dialdehyde for2hat4°C. After washing with phosphate buffer （pH7.4）, the sample was post-fixed in1%osmiumtetroxide for2h at4°C. Gradient dehydration by50%100%ethanol was performed, and thesample was then embedded in epoxies at37°C overnight. Ultrathin sections were cut at50nmand stained with lead citrate solution. Then a TEM was used for observing coronaryendothelium.2.7The expressions of SM α-actin, smooth muscle myosin heavy chain-2isoform（SM2-MyHC） and embryonic smooth muscle myosin heavy chain-B （SMemb） weredetected by immunohistochemical methodsThe rat thoracic aorta was separated, fixed with4%formalin, embedded by paraffin, and slicedto4m. After being dewaxed and hydrated, the slices were dipped in3%H2O2for5mins in thedark. High-pressure antigen retrieval followed. Afterwards, the primary antibody mouse anti-ratSM α-actin, rabbit anti-rat SM2-MyHC and mouse anti-rat SMemb antibody were addedseparately. The slices were incubated overnight at RT in a humidified chamber and washed withPBS three times. The secondary antibody was added and incubated for30mins at37°C Then theslices were washed with PBS for three times. Diaminobenzidine （DAB） color reagent was usedlast for staining. Intracytoplasmic brown granules were considered positive for target proteinpresence. Six visual fields were randomly viewed at each section; the picture was analyzed bythe Image-Pro Plu analysis system and the average OD was calculated.2.8Isolated the mesenteric smooth muscle cellsAfter anesthetization, the rat’s mesentery was separated and quickly removed to the pre-coolingTyrode’s salts solution. The second and third grade mesenteric artery was isolated under adissecting microscope. The artery was freed from surrounding tissues and cut into2-3mmlengths. The artery segments were removed into enzymes1（Papain1.0mg/ml, DTT0.68mg/ml,Albumin2.0mg/ml, diluted in Tyrode’s solution）, oscillated for7-10min in a water bath at37°C, and then changed into enzymes2（Collagenase F1.615mg/ml, diluted in Tyrode’s solution）and oscillated for3-4mins in a water bath at37°C. The zymolytic tissue was removed into the Tyrode’s salts solution without calcium to terminate the digestion until the cells with appropriaterefraction and smooth membrane were found under microscope. The cell suspension wasremoved on the coverslip and stored at4°C until use.2.9Single-channel current recordsThe single-channel currents were recorded with a patch-clamp amplifier （Japan CEZ-2300） andstored on a personal computer disk with an analog-to-digital converter （Digidata-1322A of Axoninstrument, USA）. Low-pass filter at1.0kHz was performed. The channel current amplitude wasfitted by a Gaussian curve. The pClamp version9software （Axon Instruments） was used for dataacquisition and analysis. The current amplitude （Am）, open probability （Po）, average open time（To）, and average close time （Tc） were recorded.Results1The building of long-term AT1-Ab-positive rat model by active immunization wassuccessfulThe titers of AT1-Abs in the sera of two rat groups were detected by using the ELISA method.As shown in Fig.3-1, active immunized Wistar rats generated increased serum levels ofAT1-Abs at the4thweek after initial immunization. Furthermore, the concentrations of AT1-Absmaintained a constant high level from the8thweek to the end of immunization （OD value at40weeks,1.41±0.36vs.0.33±0.09, P<0.01vs. vehicle group at the same time point）. However,AT1-Ab was not detected in the concurrent control, suggesting that the active immunizationmodels were successfully established. The remaining one immunized rat failing to produceAT1-Ab was excluded.2Slight dyslipidemia was present in the long-term AT1-Ab-positive rat modelsThere was no statistical difference in fasting blood-glucose （Fig.3-2A） and cholesterol （Fig.3-2B） between the two groups. The content of TG was slightly increased in the immunized groupcompared with the vehicle group （3.571±1.768mmol/L vs.1.977±0.644mmol/L, P<0.05, Fig.3-2C）. However, the level of HDL-C displayed a compensatory increase in the immunized group（1.118±0.136mmol/L vs.0.971±0.123mmol/L, P<0.05, Fig.3-2D）.3The liver functions in the AT1-Ab-positive rats were normalAt the end of active immunization （40weeks）, there was no significant elevation inglutamic-pyruvic transaminase （422.7±26.0vs.431.1±19.1, P>0.05, vs. vehicle group, Fig.3-3A）and glutamic-oxalacetic transaminease （156.9±16.1vs.149.3±34.0, P>0.05, vs. vehicle group,Fig.3-3B） between the two groups. 4The blood pressure did not rise in AT1-Ab-positive rat modelDuring the whole process of immunization, there was no hypertension in immunized group rats（Fig.3-4）.5Endothelium dysfunction presented in immunized group rats5.1ET-1increased in the sera of immunized group ratsAs detected by ELISA kit, a persistent increase of ET-1in the immunized group started from the12thweek till36thweek after initial immunization. Two peaks occurred at week12（27.33±3.50pg/ml） and week28（35.33±5.16pg/ml）, respectively. Both of them were significantly differentwith the vehicle group at the same time point （12.00±1.67pg/ml,12.00±2.90pg/ml, P<0.01,P<0.01）. The data were presented in Fig.3-5.5.2The AT1R-ECIIimmunization resulted in attenuated endothelium-dependentvasodilatation in ratsVascular dysfunction as evidenced by reduced acetylcholine-dependent aortic vasodilatation wasobserved in the immunized group which was introduced with human AT1R-ECIIfor40weeks.Thoracic aortas were pre-contracted by10-6mol/L norepinephrine, then endothelium-dependentvascular relaxation was performed using10-910-6mol/L acetylcholine. Compared with thevehicle group, the diastolic range significantly decreased in the immunized group rats （relaxationpercentage of the pre-contraction,50.64±6.25%vs.62.34±4.64%, P<0.05, vs. vehicle group, Fig.3-6A）.5.3The endothelium structure had no obvious changes in the long-term AT1-Ab-positiveratsAccording to the HE stain, there were no obvious structural changes in thoracic aorta of the twogroups’ rats at36weeks after initial immunization under microscope （Fig.3-7）5.4The arteria coronaria endothelium structure changed with long-term AT1-Ab-positiverat modelsCoronary artery structure in rats of the immunized group was determined by transmissionelectron microscopy. It was clear that penetrating vesicle-channels were shaped between thedouble-membrane of the capillary endothelial cells （）, and the number of pinocytotic vesiclesincreased （）（Fig.3-8A）. In addition, the cytoplasmic membranes of the vascular endotheliumeffervesced, and then the small vesicles were formed and shed. The shedding vesicle membranewas a lipid bilayer and the intramembrane component was cytoplasm （）（Fig.3-8B）, suggesting that the permeability of cardiac capillary endothelial cell membranes increased. Fig.3-8C and3-8D showed the lytic mitochondria （）.5.5The expressions of ICAM-1and VCAM-1increased in aorta endotheliocyte ofimmunized groupThe expression of endothelial ICAM-1was determined by laser scanning confocal microscopy.Compared with the vehicle group, enhanced fluorescence intensity was observed in immunizedrats at month9after initial immunization （Fig.3-9）. Consistent to the changes of ICAM,immunohistochemistry detection showed that the expression of VCAM-1was also increased inthe aortic endothelium of immunized group rats compared with the vehicle group （Fig.3-10）.Moreover, there were obvious infiltrations of lymphocytes around the thoracic aorta ofimmunized group rats （Fig.3-11）. These results indicated that inflammatory injury may beinduced after the long-term stimulation with AT1-Ab.6The thoracic aortic smooth muscle cells （VSMCs） phenotypes were changed in theAT1-Ab-positive rats.6.1The expressions of contractile phenotypic proteins decreased and the syntheticphenotypic protein increased in the immunized group rats’ thoracic aortaImmunohistochemistry detection showed that the expressions of contractile phenotypic proteinsSM α-actin （P<0.05, Fig.3-12） and SM2-MyHC （P<0.01, Fig.3-13） were down-regulated in thethoracic aortic smooth muscle cells of the immunized group rats. The synthetic phenotypicprotein SMemb was positive in the immunized group, while the protein was negative in thevehicle group （Fig.3-14）.In addition, the expression of SM2-MyHC in mesenteric artery of immunized group rats wasalso decreased, compared with vehicle group （P<0.01, Fig.3-15）6.2The thoracic aorta smooth muscle-dependent systolic and diastolic functions wereweakened in the AT1-Ab-positive group ratsAt40weeks after initial immunization, the contractile amplitude of the rats’ thoracic aortawithout endothelium responded to norepinephrine was significantly decreased in the immunizedgroup as compared with the vehicle group （Fig.3-16A）. The diastolic amplitude response tosodium nitroprusside was also impaired in the immunized group （Fig.3-16B）. 6.3The ultrastructural features of synthetic phenotype of VSMCs were revealed in theAT1-Ab-positive group rats’ thoracic aortas by transmission electron microscopyAt40weeks after initial immunization, transmission electron microscopy showed that thecytoplasmic myofilaments were dissolved （）（15000×, Fig.3-17A）. The nuclear of theVSMCs in AT1-Ab-positive rats’ thoracic aortic wall was approximately oval and depressed.The euchromatin, which showed deep electronic density （＊）, was dominant, while theheterochromatin, which showed low electron density, was relatively weak and distributed aroundthe edges of nuclear （☆）. The cytoplasmic rough endoplasmic reticulum of the VSMC becamemore intense and expanded, indicating that synthetic function of the VSMC was strong and thesecreted collagen fibers were increased （）（15000×, Fig.3-17B）.6.4The features of large conductance calcium-activated potassium channels （BKCa） in rats’mesenteric smooth muscle cellsThe BKCasingle-channel current in the smooth muscle cells of rat’s mesenteric artery （Fig.3-18）was recorded by cell-attached patch recording mode under140mmol/L symmetrichigh-potassium condition in room temperature. The bathing solution contained10-7mol/L Ca2+.Following with the elevated membrane potential, the amplitude of current and the openprobability of the BKCachannel were gradually increased （Fig.3-19A）. There is a good linearrelationship between the current and the voltage, and the conductance was194±2.9pS （n=10, Fig.3-19B）. Moreover, the currents of BKCachannel were sensitive to the intracellular Ca2+. Theopen probability of BKCawas significantly elevated upon higher intracellular Ca2+challenge. Thechannel activity of BKCawas completely blocked by the K+channel inhibitor TEA （1mmol/L,Fig.3-20A） and the selected BKCainhibitor IBTX （200nmol/L, Fig.3-20B）, which suggested thechannel we recorded was actually BKCa.6.5AT1-Ab-IgGs inhibited BKCachannel activity in smooth muscle cells through AT1receptorUnder the cell-attached patch recording mode, the activity of BKCachannel in smooth musclecells of mesenteric artery was significantly inhibited by100nM AT1-Ab-IgGs purified from theimmunized group rats in vitro. As the membrane potential was40mv, the channel openprobability and average channel open time were significantly decreased （Fig.3-21A, Table1）,the average close time were markedly extended （Fig.3-21A, Table1）, but the current amplitudehad no significant changes （Fig.3-21A, Table1）. However, the IgGs isolated from vehicle grouprat serum has no significant effect on activity of BKCachannel （Fig.3-21B, Table1）. As thesame as angiotensin II, the inhibition of AT1-Ab-IgGs on the VSMC BKCachannel was si... |
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