Distribution Of Autoantibody Against α₁-Adrenoceptor In Sera From Patients With Primary Hypertension And Its Role In Vascular Activities In Rats

BackgroundHuman essential hypertension is a complex systemic disorder which is considered to be determined by complex interactions between genetic predisposition and environmental factors, and is the characteristic of sustained blood pressure elevation. Hypertension, which is considered as the most important independent risk factor of other cardiovascular diseases, is becoming an increasingly common health problem worldwide. In the middle and advanced stage of hypertension, long-term surge of blood flow with high pressure results in endarterium mechanically damage and lipid deposition in the wall of blood vessel, thereby form lipid plaque and induce arterial sclerostenosis. In addition, long term of repeated arterial spasm, especially arteriolosclerosis, induce ischemic pathologic changes in some important organs such as heart, brain and kidney. The pathophysiology of hypertension is complicated and enigmatic. Despite the great development in prevention, detection and therapeutic action of hypertension, blood pressure control is still by no means TsatisfactoryT in a great proportion (about 70%) of patients, and hypertension and its concomitant risk factors remain uncontrolled, furthermore, the disease and the happening of its complication can't be fundamentally reversed and prevented, indicating that there remains some unknown factors involving in the pathophysiological progress of hypertension.In 1994, Fu et al for the first time reported that the concentration of circulating autoantibodies directed against the second extracellular loop ofαB1B-AR (αB1B-AA) had been found to be increased in patients with malignant hypertension. This finding attracts more attention of the role of immunologic factor in the development of hypertension. Subsequently, the autoantibodies were demonstrated to exert agonist-like effect, such as a positive chronotropic effect on isolated neonatal rat cardiomyocytes, and stimulating their activities via activation of L-type calcium channels in both 10-day-old embryonic chick and 20-week-old human fetal heart cells. Interestingly, it showed no desensitization phenomenon, which was different with the agonist, indicating that long-term stimulation of this autoantibody may make the corresponding receptor activation. Recent studies demonstrated that the active immunization with peptide corresponding to the second extracellular loop ofαB1B-adrenoceptor produced cardiovascular pathophysiological changes of hypertension—cardiac hypertrophy and deposition of vascular interstitial collagen in vivo, and promoted proliferation and increased the expression of c-jun in rat vascular smooth muscle cells (VSMC) in vitro; meanwhile, removal ofαB1B-AA in sera of patients with primary hypertension by immunoabsorption result in blood pressure lower with decreased level ofαB1B-AA lasting for 6 months. These results indicated that excess activity ofα₁-AA might be related with some pathophysiological changes of hypertension, and may play a role in immunological mechanism during the progress of blood pressure elevation.α₁-Adrenoceptor (α₁-AR) belongs to the superfamily of G protein-coupled-receptors (GPCR), which is the largest superfamily of membrane receptor protein in human. GPCR is composed of 300-400 amino acid residues in the structure, and there are three extracellular and three intracellular loops. All the three extracellular loops ofα₁-AR are expressed on the cardiovascular system, and have opportunity to get in touch with immune system. However, whether the other two extracellular loops possess corresponding immunogenicity, and further stimulate the organism to produce the respective autoantibody remains unknown. If so, their respective clinical significance is worthy more attention. However, the study on the autoantibody against the first and third extracellular loop ofα₁-AR was rarely reported, and the correlation between the level of the autoantibody and elevated blood pressure was not analyzed so far, and it is premise of the study on the role of anti-α₁-adrenoceptor in the development of hypertension to resolve these problems.Althoughα₁-AR extensively distributed in human body and mediated its biological action such as vasoconstriction, heart contractility, cardiac remodeling and VSMC proliferation, it is in vascular system where it distributed with more density and plays more remarkable effect. Therefore, we supposed thatα₁-AA with agonist-like effect may have direct vasoconstrictive effect, and if so, whether the high level ofα₁-AA is of pathologic significance of vascular lesion. Meanwhile, the vascular endothelium function impaired under long-term effect of high blood pressure, whetherα₁-AA play the similar vasoconstriction on isolated arteries from normotensive and spontaneously rats. So far, we emphasized to explore the direct and acute effect ofα₁-AA on heart and vascular smooth muscle, and lack the direct evidence thatα₁-AA could induce vasoconstriction in arteries (especially small resistant arteries).The elucidation of these problems is the key element for recognizing the role and mechanism ofα₁-AA in the progress of hypertension, and provides experimental foundation for high level ofα₁-AA existence in the sera of patients with hypertension. As mentioned above,α₁-AR distributed extensively all over the body. In the cardiovascular system, it exits not only in the VSMC but also presents on the membrane of vascular endothelium, which plays an important role in the regulation of vasomotor function. Due to theα₁-AA possessed agonist-like effect, we bring the hypothesis thatα₁-AA can bind toα₁-AR existed in vascular endothelium when it flows in the vessels with blood circulation. However, these presumptions need to be testified. It is known to all that as soon as the vascular endothelium injury in vivo, many vasoactive substances (eg. platelet and blood coagulation factor and so on) aggregated, then initiated subsequent release reaction. These pathological processes are beneficial to the formation of atherosclerotic plaque. However, the mechanism of vascular endothelium damage remains largely unknown. Therefore, it will conduce to reveal the mechanism of vascular pathological changes in hypertension by obtaining the direct evidence thatα₁-AA involved in the hypertension-induced endothelium injury.In conclusion, we design this research project as follows: (1) to examine the distribution of autoantibodies direct against the three extracellular loops ofα₁-AR respectively by clinical epidemiological investigation, and further analyze the correlation between the level of the antibody and the blood pressure to identify the clinical significance of the autoantibody; (2) according to the experiments above, the blood pressure-related autoantibody type was identified, then observe the direct effect of the autoantibody on the conduit artery and small resistant arteries and the potential mechanism, such as: firstly, to observe the direct effect ofα₁-AA on vasoconstriction in isolated thoracic arteries and small resistant arteries from important organs (coronary artery, middle cerebral artery, renal artery and mesenteric artery); then the effect ofα₁-AA on intracellular CaP2+P concentration in cultured rat vascular smooth muscle cell was examined by confocal microscopy; to identify whetherα₁-AA exhibited the same characteristic of vasoconstriction in normotensive and hypertensive state, the isolated thoracic aorta from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats were used, and further testify the negative regulatory role of endothelium and NO inα₁-AA-induce vasoconstriction; to observe the direct damage effect ofα₁-AA on cultured endothelium cell; we can obtain the direct evidence for the effect ofα₁-AA on VSMC and endothelium cell and the regulatory role of endothelium and NO inα₁-AA-induce vasoconstriction in hypertension state from the second part; (3) observe the pressor response by acute administration withα₁-AA, and the changes of blood pressure, responsiveness to pressor agent and endothelium injury level in vivo by establishing model by active immunization with the peptide corresponding to the second extracellular loop ofα₁-AR; meanwhile, detect the plasma endothelin-1 content as indicator of the endothelium injury (our previous study has demonstrated that long-term ofα₁-AA can induce vascular smooth muscle cell transconformation). This study may contribute to clarify the involvement ofα₁-AA in the vascular dysfunction and endothelium injury during the development of hypertension.The hypothesis that we intend to confirm are as follows:α₁-AA may be related with the process of hypertension via affecting the vascular function and structure. (1) The high level and high positive rate ofα₁-AA against the second extracellular loop existed in the sera of patients with primary hypertension, which have positive relation with elevated blood pressure;α₁-AA displayed direct vasoconstriction on conductive arteries and small resistant arteries from important organs, in which the increased intracellular CaP2+P involved, and may show different contractive effects on arteries from different originals;α₁-AA-induced vasoconstriction enhanced in the thoracic aortic rings from SHR than in WKY rats, and dysfunctional endothelium and decreased availability of NO may be involved in it;α₁-AA itself showed direct damage effects on cultured endothelial cells; (3)α₁-AA may exhibit different effect on blood pressure by acute and long-term stimulation; long-term stimulation ofα₁-AA may induce hyperresponsiveness to the vasoconstrictor and decreased relaxation, even vascular endothelium injury. All of these changes might be related with blood pressure elevation, decreased organ blood flow and other pathological alteration in patients with primary hypertension.SECTION 1 Screening of Autoantibodies againstα₁-Adrenoceptor in Sera from Patients with Primary Hypertension and the Potential SignificanceObjective:In this study, peptides corresponding to the first, second and third extracellular loops of humanα₁-AR were synthesized respectively as antigen, and the immunoglobulin fractions of sera from 86 normotensives and 73 patients with primary hypertension were detected for the presence of autoantibodies against the three extracellular loops ofα₁-adrenoceptor (anti-α₁-EC_I, anti-α₁-EC_II and anti-α₁-ECBIIIB) by ELISA to observe the distribution of the autoantibodies againstα₁-AR and confirm correlation between the level of autoantibody and elevated blood pressure. The association between the level ofα₁-AA and blood pressure was also analyzed to explore the potential significance of existence ofα₁-AA.Methods:1. Patients recruitment and evaluation1.1 The hypertensive patient (PHT) group included 73 primary hypertensive patients who met the 1999 diagnostic criteria of World Health Organization (WHO) for hypertension. Inclusion criteria were systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg. Blood pressure was measured manually with a standard mercury sphygmomanometer three times, with the patients seated after a 5 min resting period. None of the patients had been treated with anα₁-blocker orαB2B-agonist during a period of at least 3 months before blood collection. All hypertensive patients were on treatment with one or two antihypertensive drugs (including diuretics, beta-adrenoceptor blockers, calcium antagonists, angiotensin II type I receptor blockers, and angiotensin-converting-enzyme inhibitors). Exclusion criteria included: diagnosis of secondary hypertension, acute/chronic renal or endocrine diseases; any autoimmune disease; concurrent life-threatening illness of severe illness requiring extensive systemic treatment.1.2 The normotensive control group (NT) included 86 healthy subjects randomly selected from the same community, with 90 mmHg 1-AA was measured by enzyme-linked immunosorbent assay (ELISA) as we used previously, and the results were expressed as optical density (OD) values. We also calculated positive/negative (P/N) ratio [(specimen OD ? blank OD) / (negative control OD ? blank control OD)] of each sample, and those samples with a P/N value at least 2.1 were considered asα₁-AA positive.4. Statistical analysisThe chi-square test, rank sum test,unpaired student's t test, analysis of variance were calculated using the SPSS 13.0 software. In all cases a P-value of <0.05 was considered statistically significant.Results1. Distribution of autoantibodies against the first, second and third extracellular loop ofα₁-AR in sera of of patients with primary hypertension and healthy normotensive subjects 1.1 Positive rate of anti-α₁-AR in the sera of patients with primary hypertension and healthy normotensive subjectsIn this study, sera from 73 patients with primary hypertension and 86 normotensive controls were collected for detection of autoantibodies againstα₁-adrenoceptors. As shown in Figure 1, the autoantibodies against each extracellular loops of the humanα₁-AR were detected in the sera of hypertensive patients and healthy normotensive controls, and the positive frequency of them were calculated according to the formula described in the method section. Compared with the normotensive controls, the frequency of anti-α₁-EC_I and anti-α₁-EC_II was found to be significantly higher in hypertensive patients (anti-α₁-EC_I: 32.8% vs. 8.1%; anti-α₁-EC_II: 34.2% vs. 10.4%, P<0.01). Whereas no statistical difference in the frequency of anti-α₁-ECBIIIB was seen between patients with primary hypertensive patients and normotensive subjects (2.7 % vs. 3.5%,P>0.05).1.2 Levels of autoantibodies against three extracellular loops of the humanα₁-AR in patients with primary hypertension and healthy normotensive subjectsThe level of autoantibodies against the three extracellular loops of the humanα₁-AR in sera from patients with primary hypertension and normotensive controls are shown in Figure 2 as expressed by OD values Compared to normothensive group, levels of anti-α₁-ECBI Band anti-α₁-EC_II were statistically significant higher in patients with primary hypertension (anti-α₁-EC_I: 0.25±0.06 vs. 0.16±0.02, P<0.01; anti-α₁-EC_II: 0.30±0.07 vs. 0.14±0.03, P<0.01; anti-α₁-EBIIIB: 0.16±0.05 vs. 0.15±0.03, P>0.05). Of the three autoantibodies (anti-α₁-EC_I, anti-α₁-EC_II and anti-α₁-EBIIIB), levels of both anti-α₁-ECB1 Band anti-α₁-EC_II were higher than that of anti-α₁-ECBIIIB in primary hypertensive patients, indicating that both the first and second extracellular loop than the third loops ofα₁-AR possessed significantly higher antigenicity.1.3 Titers of autoantibodies against three extracellular loops of the humanα₁-AR in patients with primary hypertension and healthy normotensive subjectsThe titers of the autoantibodies were also calculated by the formula described in the method section, and the titer of both anti-α₁-EC_I and anti-α₁-EC_II were statistically significant higher in patients with primary hypertension, however, there is no difference between the two groups in the titer of anti-α₁-EBIIIB(anti-α₁-EC_I: 1:148.6±8.7 vs. 1:20.9±2.1, P<0.01; anti-α₁-EC_II: 1:168.6±6.3 vs. 1:24.2±1.8, P<0.01; anti-α₁-EBIIIB: 1:22.7±2.3 vs. 1:25.4±3.7, P>0.05)as shown in Figure 3.2. Association of elevated blood pressure with the level of autoantibodies against theα₁-AR There was a positive correlation between elevated blood pressure (including systolic and diastolic blood pressure) and the level of anti-α₁-EC_II in both normotensive subjects and primary hypertensive patients as shown in Figure 4. Figure 4A and 4B showed the correlation between the level ofα₁-AA and systolic (SBP) and diastolic blood pressure (DBP) in both normotensives and patients with primary hypertension (SBP: RP2P= 0.25, P< 0.01; DBP: RP2P= 0.51, P< 0.01). Figure 5A and 5B showed the correlation between the level ofα₁-AA and SBP and DBP in normotensives and patients with primary hypertension, respectively (SBP: RP2P= 0.14, P< 0.01 in primary hypertensive patients, RP2P= 0.08, P< 0.01 in normotensive subjects; DBP: RP2P= 0.36, P< 0.01 in primary hypertensive patients; RP2P= 0.15, P< 0.01 in normotensive subjects). However, there is no significant correlation between blood pressure and the level of anti-α₁-ECBâ… B(SBP: RP2P= 0.0021, P>0.05 in primary hypertensive patients, RP2P= 0.0007, P>0.05 in normotensive subjects; DBP: RP2P= 0.0099, P>0.05 in primary hypertensive patients; RP2P= 0.0416, P>0.05 in normotensive subjects) as shown in Figure 6 and 7.3. The effect of antihypertensive medicine on the development ofα₁-AATo observe whether the kind of antihypertensive medicine affect the development ofα₁-AA, the medication of both antibody-positive and antibody-negative patients in the hypertensive group was summarized (Table 3). The results demonstrated that there was no difference in medication with each kind of antihypertensive drugs between anti-α₁-EC_II-positive and anti-α₁-EC_II-negative patients.Conclusion1. Higher level and positive rate of anti-α₁-EC_I and anti-α₁-EC_II existed in the sera of patients with primary hypertension, indicating that immunological factor may be involved in the process of hypertension, and the two kind of antibody might be related to the pathophysiology of hypertension;2. The level of anti-α₁-EC_II exhibited positive correlation with systolic and diastolic blood pressure, suggesting that anti-α₁-EC_II might be relevant with blood pressure elevation in patients with primary hypertension.3. The data showed that the kind of clinical medication did not affect the production of anti-α₁-EC_II; however, further clinical sample should be collected to confirm this conclusion. SECTION 2 The Vasoconstrictive Effects of Autoantibodies against the Second Extracellular Loop ofα₁-Adrenoceptor and the Regulatory Role of Endothelium and Nitric OxideObjective1. To determine whetherα₁-AA can cause vascular contraction directly, if so, further investigate the cellular receptors that mediate their vasoactivity.2. To detect whetherα₁-AA can induce intracellular Ca²⁺ increase in cultured vascular smooth muscle cell;3. To investigate whetherα₁-AA exhibited the similar vasocontractive characteristic on isolated thoracic artery rings from normotensive and hypertensive rats, and explore the alterations of endothelial modulation ofα₁-AA-induced contraction in hypertension.4. Furthermore, to observe the direct effects ofα₁-AA on vascular endothelial cell injury.Method1. Preparation of Immunoglobulin G:On the basis of a sera-positive response in an ELISA to peptides 192-218 of theα₁-AR, immunoglobulin fractions G (IgG) from the mixed sera of 25α₁-AA positive hypertensive patients were prepared by MabTrap Kit (Amersham Bioscience, Uppsala, Sweden). The IgG from mixed sera of 20 healthy normotensive subjects (n IgG) whoseα₁-AA was negative was prepared in an identical fashion and used as a control. The specificity and concentration of purified IgG was determined by the SDS-PAGE (Figure 8), Bicinchoninic Acid (BCA) Protein Assay (Pierce, USA) (Figure 9) and ELISA, respectively.2. The vasoconstrictive effect ofα₁-AA was determined in isolated rat thoracic aorta, coronary artery, renal artery, middle cerebral artery, and mesenteric artery by vascular tension recording technique. There are 6 groups as follows:(1)α₁-AA positive group(positive IgG, P-IgG)ï¼›(2)phenylephrine (PE), as positive control groupï¼›(3)α₁-AA negative group (negative IgG, N-IgG), as negative control groupï¼› (4)positive IgG + prazosin (selectiveα₁-AR antagonist) groupï¼›(5)positive IgG+α₁-Ag(the peptide corresponding to the second extracellular loop ofα₁-AR)ï¼›(6)α₁-Ag (the peptide corresponding to the second extracellular loop ofα₁-AR).3. Cytosolic CaP2+P changes in cultured vascular smooth muscle cell were determined by Fluo-3/AM and confocol microscopy.4. Primary culture of rat thoracic aorta smooth muscle cells were identified by immunofluoresce -nce technique.5. Vascular reactivity experiments were performed in segments of thoracic aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).6. LDH release, Caspase-3, 8, 9 activity, apoptosis index (aridine orange and propidium iodide staining, AO/PI staining) and DNA damage (single cell gel electrophoresis assay, SCGE) in cultured HUVEC were examined after administration ofα₁-AA (0.01P PμM-1μM) for 6h, 12h, 24h and 48h, respectively.Results1. Vasoconstrictive effect ofα₁-AA on isolated vascular rings.1.1α₁-AA induce isolated thoracic aortic rings in a dose-dependent mannerThe result demonstrates the effect ofα₁-AA in stimulating the contraction of rat thoracic aortic rings. Vascular rings were treated withα₁-AA (1.0μM) purified from mixed sera of 25α₁-AA positive hypertensive patients, and changes of the tension of the thoracic aorta rings were measured. The effects of purified IgGs obtained from mixed sera of 20 normotensive subjects on the isolated thoracic aorta rings were observed in the control group. Compared with control, 0.01, 0.1 and 1.0μMα₁-AA administration markedly increased the tension of thoracic aorta rings in a dose-dependent manner, and increased tension were 0.22±0.04 g, 0.85±0.11g and 1.83±0.17 g (Figure 10, P<0.05). The tension stimulated byα₁-AA was completely inhibited by prazosin (1.0P PμM), a selectiveα₁-AR blocker, or by preincubation with the peptides corresponding to the second extracellular loop ofα₁-AR (3.0μM) (Figure 11). This phenomenon indicates that the increased tension induced byα₁-AA is a result of the activation ofα₁-AR, with the second extracellular loop ofα₁-AR acting as an important binding site forα₁-AA. More interestingly, the contraction induced byα₁-AA of the vascular ring persisted for more than 2 h, exhibiting no-desensitization phenomena. 1.2 Vasoconstrictive effect ofα₁-AA on isolated small resistance arteries.1.2.1 Vasoconstrictive effect of 1.0μMα₁-AA on small resistant arteryBoth PE andα₁-AA caused an increase in the contraction of the small artery studied (n=6; P<0.01; Figures 12A, 12B, 12C). All these effects were inhibited by prazosin (1.0μM). However, no effect was observed on mesenteric artery. Administration ofα₁-AA negative IgG purified from mixed sera of 20 normotensive subjects revealed no contractive effects on any type of small artery. Vasocontrictive effect of 1.0μMα₁-AA on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) can be inhibited by preincubated with prazosin (1.0μM) or the peptide corresponding to the second extracellular loop ofα₁-AR (3.0μM). Preincubation with 1.0μM prazosin or 3.0μM peptide corresponding to the second extracellular loop ofα₁-AR decreased the increased tension from 3.52±0.14 mN to 0.37±0.07mN and 0.38±0.15 mN in renal artery, from 0.79±0.11 mN to 0.12±0.06 mN and 0.11±0.07 mN in middle cerebral artery, from 0.67±0.05 mN to 0.08±0.03 mN and 0.06±0.02 mN in coronary artery. These experiments indicated that administration ofα₁-AA from hypertensive patients'show varying contractive effects on different isolated small vessel resistance.1.2.2α₁-AA induce concentration-dependent vasoconstriction on middle cerebral artery, coronary artery and renal arteryThe effect ofα₁-AA with different concentration from hypertensive patients on small resistance vascular ring (middle cerebral artery, coronary artery and renal artery) is shown in Figures 12D. 0.01, 0.1 and 1.0μMα₁-AA does-dependently increase the tension of isolated renal arteries (0.25±0.02 nM, 1.45±0.10 mN and 3.52±0.14 mN); 0.01, 0.1 and 1.0μMα₁-AA does-dependently increase the tension of isolated middle cerebral arteries (0.15±0.04 mN,0.45±0.07 mN and 0.79±0.11mN); and can also increase the tension of isolated coronary arteries in dose-dependent manner(0.13±0.03 nM, 0.34±0.04 mNå’Œ0.67±0.05 mN), which is similar to PE. No significant change was observed in N-IgG group. However,α₁-AA did not show any vasoconstrictive effect on isolated mesenteric arteries.2. Effects ofα₁-AA on variations in intracellular CaP2+P on cultured VSMCThe intracellular CaP2+P concentration of cultured VSMC significantly increased by stimulation with PE (1.0μM). Similar to phenylephrine,α₁-AA (1.0μM) can also evoke the cytosolic CaP2+P increased compared with N-IgG group, which can be inhibited by preincubated with prazosin (1.0μM) or peptide corresponding to the second extracellular loop ofα₁-AR for 30 min as shown in Figure 15. 3. Vasoconstrictive characteristic ofα₁-AA in rat isolated aortic rings from WKY and SHR and the modulation of endothelium and NO3.1 Contractile effects of phenyleprine andα₁-AA in rat isolated aortic rings from WKY and SHRPhenylephrine evoked concentration-dependent contraction in isolated thoracic arteries that was slightly greater in SHR than in WKY (Fig 16A).α₁-AA (similar to the selectiveα₁-AR agonist, phenylephrine) also induced concentration-dependent contractions greater in SHR than in WKY (Fig 16B). The mean values for 60 mM KCl contraction in aorta were 2.34±0.94g and 2.15±0.79g for WKY (n=24) and SHR (n=24), respectively.3.2 Decreased inhibitory effect of endothelium on the contractile response toα₁-AAEndothelium removal augmented the contractile response of isolated thoracic arteries to the selectiveα₁-AR agonist phenylephrine in WKY (Figure 17A) and SHR (Figure 17B), and it had similar effects onα₁-AA-induced contraction in WKY (Figure 17C) and SHR (Figure 17D), indicating that the endothelium play an inhibitory effect on the contractile response toα₁-AA. The ability of endothelium to depress the contractile response ofα₁-AA was found to be reduced in vessels from SHR as measured by the ratio EC50 endothelium intact/EC50 endothelium denuded (WKY vs. SHR; 2.05±0.39 and 1.14±0.18, respectively, P< 0.01). To clarify whether endothelium-dependent relaxation was different between WKY and SHR, the relaxing effects of ACh (an endothelium-dependent vasodilator) were studied. ACh showed concentration-dependent vascular relaxation of the precontraction in both WKY and SHR group. ACh-induced relaxation was greater in WKY than in SHR rings (as shown in Figure. 18).3.3 Effects of L-NAME and 1400W on phenyleprine andα₁-AA-induced aortic contractionInhibition of endothelium NOS by the non-selective NOS inhibitor, L-NAME (100μM) did not modify the basal arterial tension; however, L-NAME increased the contractile response by phenylephrine andα₁-AA in both SHR and WKY rats, respectively, indicating that the NO released from endothelium participated in the inhibitory effects onα₁-AA-induced contraction in both SHR and WKY. Surprisingly, inhibition of iNOS by 1400W (10μM) increased the contractile response in intact aortic rings from WKY rats (Figure 19A and Figure 19B); however, 1400W did not modify the phenylephrine andα₁-AA-induced contraction in SHR (Figure 19C and Figure 19D), indicating that it is eNOS but not iNOS mainly contribute to the negative modulation ofα₁-AA-induced contraction in SHR.4. Effect ofα₁-AA on HUVECs death4.1 Effect ofα₁-AA on HUVECs necrosis at different concentrationTo ensure that IgG fractions fromα₁-AA positive sera of hypertensive patients not only increases HUVECs apoptosis but also increases HUVECs necrosis, thus enlarging the HUVECs injury, lactate dehydrogenase (LDH) activity was measured. As summarized in Figure 21, an approximately 3.5 folds increase in LDH activity was observed in cultured HUVECs with 1.0μMhypertensive IgG compared with that in incubation of HUVECs with N-IgG, which was completely blocked by prazosin, anα₁-AR antagonist. The effect of hypertensive IgG was almost identical with that seen in PE-treated HUVECs. No effect was observed when the HUVECs were exposed to IgG fraction of 0.01μM.4.2 Effect ofα₁-AA on HUVECs apoptosis at different concentrationHUVECs death represents the total HUVECs injury caused by necrosis and apoptosis. As illustrated in Figure 22A, in vitro incubation of HUVEC with IgG fractions fromα₁-AA positive sera of hypertensive patients at the concentration of 0.01, 0.1 and 1.0μM (at 24 hours, the time was selected from our preliminary experiments, at this time, caspase 3 activation achieve culmination) resulted in dose-dependent caspase activation (1.28±0.17 mmol/h/mg protein, 1.85±0.09 mmol/h/mg protein and 2.87±0.11 mmol/h/mg protein) which identical with that seen in PE-treated HUVECs. However, pre-treatment with either prazosin or peptide corresponding to the second extracellular loop of theα₁ receptor before administration of IgG fractions fromα₁-AA positive sera of hypertensive patients completely inhibited caspase-3 release. Taken together, these results indicate that the apoptotic effect of IgG fraction isolated from theα₁-AA positive sera of hypertensive patients in HUVECs is mediated by activating the peptide corresponding to the second extracellular loop ofα₁-AR.Having demonstrated that hypertensive IgG elevated the activation of caspase-3 and increased apoptosis, we further determined the upstream pathway(s) via which hypertensive IgG increases caspase-3 activation. As summarized in Figure 23, addition of 0.01, 0.1 and 1.0μM IgG fractions fromα₁-AA positive sera of hypertensive patients markedly increased caspase-8 activation (0.25±0.03 mmol/h/mg protein, 0.40±0.05 mmol/h/mg protein and 0.58±0.03 mmol/h/mg protein). In contrast, administration of IgG fractions fromα₁-AA positive sera of hypertensive patients at concentration of 0.01 to 1.0μM failed to increase caspase-9 activation. These results demonstrated that IgG fractions fromα₁-AA positive sera of hypertensive patients increased HUVECs apoptosis by activating the extrinsic (i.e., death receptor-mediated), not intrinsic (i.e., mitochondrial-mediated), apoptosis pathway.Taken together, these results provided clear evidence that IgG fraction isolated from theα₁-AA positive sera of hypertensive patients at the concentration of 1.0μM causes significant HUVECs death by stimulating the peptide corresponding to the second extracellular loop of theα₁-AR.4.3 Effect of 1.0μMα₁-AA on HUVECs necrosis at different time pointsTo determine whetherα₁-AA may increase HUVECs death in a time-dependent fashion, we measured effect of IgG fractions fromα₁-AA positive sera of hypertensive patients at the concentration of 1.0μM on HUVECs death at different time points. As expected, LDH activity was markedly increased in incubation of HUVECs with hypertensive IgG in a time-dependent manner (Figure 21B). The LDH release began to increase at 12h, and reach the peak at 24h (about 3.5 fold increase). The effect can be inhibited bypreincubation with prazosin and peptide corresponding to the second extracellular loop ofα₁-AR.4.4 Effect of 1.0μMα₁-AA on HUVECs apoptosis at different time pointsTo our surprise, caspase-3 and caspase-8 activity began to increase at 12h, and achieved peak at 24h in incubation of HUVECs with hypertensive IgG (Figure 22B and 23B). However,α₁-AA had no effect on HUVECs caspase-9 activation (Figure 24). These results strongly suggest...