Effect of active immunization against angiotensin II type 1 (AT1) receptor on hypertension & arterial remodelling in spontaneously hypertensive rats (SHR).

BACKGROUND & OBJECTIVES: a0 ngiotensin II receptor type 1 (AT1) is known to be involved in the pathogenesis of hypertension. t0 his study was undertaken to explore the effect of active immunization against AT1 receptor on blood pressure and small artery remodelling in spontaneously hypertensive rat (SHR). METHODS: Male SHR and Wistar rats aged two months were actively immunized with different peptides (ATR12185ͱͲATR10014 and ATR12181) corresponding to particular sequences of rat AT1 receptor, while another SHR group was given losartan (10 mg/kg/day) orally once a day. Anti-AT1 receptor antibodies were detected by ELISA and blood pressure was measured. The effect of the antibodies on the artery and vascular smooth muscle cells (VSMCs) proliferation was studied. RESULTS: all immunized animals produced antibodies against the particular peptides. The systolic blood pressure was decreased in the SHR immunized with peptide-ATR12181 compared with the control. However, no changes were observed in the SHR immunized with other two peptides. The Wistar rats immunized with the three peptides did not show any changes in blood pressure. The media/lumen area ratio of the mesenteric artery was reduced in SHR immunized with ATR12181 and similar to that of the SHR treated with losartan. The antibody from SHR immunized with ATR12181 had no effect on the proliferation of VSMC. But it could inhibit the proliferation caused by angiotensin II and its effect at the titre of 1:40 was similar to that of 1µmol/l losartan. INTERPRETATION & CONCLUSIONS: Our findings demonstrated that the antibody from SHR immunized with ATR12181 had the effect of reducing blood pressure and target organ protection similar to losartan. Active immunization against AT1 receptor may be a promising strategy in future for the treatment of hypertension.

No evidence of coronary plaque stabilization by allopurinol in patients with acute coronary syndrome.

BACKGROUND: Allopurinol, a xanthine inhibitor that lowers uric acid concentration, has been proven to reduce inflammation and oxidative stress in patients with cardiovascular disease. However, it is unknown whether these beneficial effects translate into favorable plaque modification in acute coronary syndromes (ACS). This study aimed to investigate whether allopurinol could improve coronary plaque stabilization using coronary computed tomography angiography (CCTA). METHODS: This was a prospective, single-center, randomized, double-blind clinical trial began in March 2019. A total of 162 ACS patients aged 18-80 years with a blood level of high-sensitivity C-reactive protein (hsCRP) â€‹> â€‹2 â€‹mg/L were included. The subjects were randomly assigned in a 1:1 ratio to receive either allopurinol sustained-release capsules (at a dose of 0.25 â€‹g once daily) or placebo for 12 months. The plaque analysis was performed at CCTA. The primary efficacy endpoint was the change in low-attenuation plaque volume (LAPV) from baseline to the 12-month follow-up. RESULTS: Among 162 patients, 54 in allopurinol group and 51 in placebo group completed the study. The median follow-up duration was 14 months in both groups. Compared with placebo, allopurinol therapy did not significantly alter LAPV (-13.4 â€‹± â€‹3.7 â€‹% vs. -17.8 â€‹± â€‹3.6 â€‹%, p â€‹= â€‹0.390), intermediate attenuation plaque volume (-16.1 â€‹± â€‹3.0 â€‹% vs. -16.2 â€‹± â€‹2.9 â€‹%, p â€‹= â€‹0.992), dense calcified plaque volume (12.2 â€‹± â€‹13.7 â€‹% vs. 9.7 â€‹± â€‹13.0 â€‹%, p â€‹= â€‹0.894), total atheroma volume (-15.2 â€‹± â€‹3.2 â€‹% vs. -16.4 â€‹± â€‹3.1 â€‹%, p â€‹= â€‹0.785), remodeling index (2.0 â€‹± â€‹3.9 â€‹% vs. 5.4 â€‹± â€‹3.8 â€‹%, p â€‹= â€‹0.536) or hsCRP levels (-73.6 [-91.6-17.9] % vs. -81.2 [-95.4-47.7] %, p â€‹= â€‹0.286). CONCLUSIONS: Our findings suggest that allopurinol does not improve atherosclerotic plaque stability or inflammation in ACS.

Enhancement of autophagy by simvastatin through inhibition of Rac1-mTOR signaling pathway in coronary arterial myocytes.

BACKGROUND/AIMS: In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs) to thereby exert their beneficial effects in atherosclerosis. METHODS AND RESULTS: Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR) activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries. CONCLUSION: Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth of these cells.

Attenuation by statins of membrane raft-redox signaling in coronary arterial endothelium.

Membrane raft (MR)-redox signaling platforms associated with NADPH oxidase are involved in coronary endothelial dysfunction. Here, we studied whether statins interfere with the formation of MR-redox signaling platforms to protect the coronary arterial endothelium from oxidized low-density lipoprotein (OxLDL)-induced injury and from acute hypercholesterolemia. In cultured human coronary arterial endothelial cells, confocal microscopy detected the formation of an MRs clustering when they were exposed to OxLDL, and such MR platform formation was inhibited markedly by statins, including pravastatin and simvastatin. In these MR clusters, NADPH oxidase subunits gp91(phox) and p47(phox) were aggregated and were markedly blocked by both statins. In addition, colocalization of acid sphingomyelinase (ASM) and ceramide was induced by OxLDL, which was blocked by statins. Electron spin resonance spectrometry showed that OxLDL-induced superoxide (O2(.-)) production in the MR fractions was substantially reduced by statins. In coronary artery intima of mice with acute hypercholesterolemia, confocal microscopy revealed a colocalization of gp91(phox), p47(phox), ASM, or ceramide in MR clusters. Such colocalization was rarely observed in the arteries of normal mice or significantly reduced by pretreatment of hypercholesterolemic mice with statins. Furthermore, O2(.-) production in situ was 3-fold higher in the coronary arteries from hypercholesterolemic mice than in those from normal mice, and such increase was inhibited by statins. Our results indicate that blockade of MR-redox signaling platform formation in endothelial cell membrane may be another important therapeutic mechanism of statins in preventing endothelial injury and atherosclerosis and may be associated with their direct action on membrane cholesterol structure and function.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4.

OBJECTIVE: Vascular smooth muscle cell (VSMC) differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension, atherosclerosis, and restenosis. MicroRNA-146a (miR-146a) has been proven to be involved in cell proliferation, migration, and tumor metabolism. However, little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells (ESCs). This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs. METHODS: Mouse ESCs were differentiated into VSMCs, and the cell extracts were analyzed by Western blotting and RT-qPCR. In addition, luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed. Finally, C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs, and immunohistochemistry, Western blotting, and RT-qPCR assays were carried out on tissue samples from these mice. RESULTS: miR-146a was significantly upregulated during VSMC differentiation, accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin (SMαA), smooth muscle 22 (SM22), smooth muscle myosin heavy chain (SMMHC), and h1-calponin. Furthermore, overexpression of miR-146a enhanced the differentiation process in vitro and in vivo. Concurrently, the expression of Kruppel-like factor 4 (KLF4), predicted as one of the top targets of miR-146a, was sharply decreased in miR-146a-overexpressing ESCs. Importantly, inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs. In addition, miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors, including serum response factor (SRF) and myocyte enhancer factor 2c (MEF-2c). CONCLUSION: Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

Distinct Features of Gut Microbiota in High-Altitude Tibetan and Middle-Altitude Han Hypertensive Patients.

Indigenous animals show unique gut microbiota (GM) in the Tibetan plateau. However, it is unknown whether the hypertensive indigenous people in plateau also have the distinct gut bacteria, different from those living in plains. We sequenced the V3-V4 region of the gut bacteria 16S ribosomal RNA (rRNA) gene of feces samples among hypertensive patients (HPs) and healthy individuals (HIs) from 3 distinct altitudes: Tibetans from high altitude (3600-4500 m, n = 38 and 34), Hans from middle altitude (2260 m, n = 49 and 35), and Hans from low altitude (13 m, n = 34 and 35) and then analyzed the GM composition among hypertensive and healthy subgroups using the bioinformatics analysis, respectively. The GM of high-altitude Tibetan and middle-altitude Han HPs presented greater α- and ß-diversities, lower ratio of Firmicutes/Bacteroidetes (F/B), and higher abundance of beneficial Verrucomicrobia and Akkermansia than the low-altitudes HPs did. The GM of high-altitude Tibetan and middle-altitude HIs showed greater α-diversity and lower ratio of F/B than the low-altitudes HIs did. But, ß-diversity and abundance of Verrucomicrobia and Akkermansia among different subgroups of HIs did not show any differences. Conclusively, the high-altitude Tibetan and middle-altitude Han HPs have a distinct feature of GM, which may be important in their adaptation to hypertension in the plateau environments.

Agonistic AT(1) receptor autoantibody increases in serum of patients with refractory hypertension and improves Ca(2+) mobilization in cultured rat vascular smooth muscle cells.

Agonistic AT(1) receptor autoantibodies (AT(1)-AAs) have been described in the patients with malignant hypertension or preeclampia. Furthermore, AT(1)-AAs were highly associated with refractory hypertension. Function of vascular smooth muscle cells (VSMCs) is important in the regulation of blood pressure. We investigated and compared the ability of angiotensin II (Ang II) and AT(1)-AAs to stimulate the intracellular calcium mobilization and cellular proliferation of rat VSMCs. Twenty-two patients with refractory hypertension, 24 patients with non-refractory hypertension and 37 normotensives were recruited. The serum of each patient was detected for the presence of AT(1)-AAs by ELISA. Ang II and the AT(1)-AAs from the sera of patients were used to stimulate rat VSMCs in vitro. AT(1)-AAs were detected in 10/22, 3/24 and 3/37 of patients with refractory hypertension, non-refractory hypertension and normotensives, respectively. AT(1)-AAs led the increase intracellular calcium mobilization in a dose-dependent manner and cellular proliferation of VSMCs just as Ang II. Both of these effects caused by AT(1)-AAs were blocked with losartan or a peptide corresponding to a part of the second extracellular loop of AT(1) receptor. Since AT(1)-AAs exhibited pharmacological activity in rat VSMCs just as Ang II, they might play a role in the elevation of peripheral vascular resistance and in vascular remodeling. And AT(1)-AAs were suggested to involve in resistance to antihypertensive therapy.

The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension.

BACKGROUND: Autoantibodies against angiotensin AT1 receptor have been discovered in patients with preeclampsia or malignant hypertension. Some studies have demonstrated that the autoantibodies are involved in the immunopathogenesis of hypertension and have an agonist effect similar to angiotensin II. METHODS: Autoantibodies against AT1 receptor were purified from sera of patients with primary hypertension by affinity chromatography. Proliferation of cultured rat vascular smooth muscle cells was detected by bromodeoxyuridine incorporation and activation of signalling molecules detected by Western blotting and electrophoretic mobility shift assay. RESULTS: The AT1-RAb caused a significant proliferation similar to the Ang II during first 24 hours. The levels of nuclear factor-kappaB (NF-kappaB), phosphorylated JAK2, phosphorylated STAT1 (pSTAT1) and phosphorylated STAT3 (pSTAT3) molecules were increased in response to the autoantibodies. In contrast, the activations of NF-kappaB and JAK-STAT were blocked by losartan, pyrrolidinedithiocarbamate (a specific inhibitor of NF-kappaB) and AG490 (a specific inhibitor of the JAK2 tyrosine kinase). The expressions of NF-kappaB, pSTAT1 and pSTAT3 reached peak levels at different times. Moreover, the relative densities of electrophoretic bands showed that activation of pSTAT3 was more significant than STAT1 induced by AT1-RAb. CONCLUSIONS: These results suggest that the autoantibodies against AT1 receptor have an agonist effect similar to Ang II in proliferation of VSMCs and the NF-kappaB and JAK-STAT proteins play essential roles. The effect is different from Ang II in that STAT3 is the main downstream activating molecule in JAK-STAT signalling pathway.

Target organ protection from a novel angiotensin II receptor (AT1) vaccine ATR12181 in spontaneously hypertensive rats.

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. It is evident that overactive renin-angiotensin systems play a central role in the development of hypertension and target organ damage associated with hypertension. We have previously found that a novel angiotensin II receptor (AT1) vaccine-ATR12181 attenuated the development of high blood pressure (BP) in spontaneously hypertensive rat (SHR) model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of target organ damage induced by hypertensive state. SHRs were immunized against a peptide (coded ATR12181) from the extracelluar portion of the AT1A receptor by repeated subcutaneous injections of peptide-tetanus-toxoid complex in combination with Freund's adjuvant. A 64 weeks long-term observation was performed. Repeated vaccinations resulted in the induction of anti-ATR12181 antibodies. At the end of observation, vaccinated SHRs manifested lower BP, decreased cardiac hypertrophy and attenuation of kidney injuries. mRNA levels of c-fos and c-jun in heart and kidneys were decreased in vaccinated SHRs. Since a self antigen was used, safety of vaccine was concerned. However, the signs of autoimmune diseases were not observed in the sections of heart and kidney. These data demonstrated that repeated immunization against a domain of the extracellular portion of the AT1 receptor was able to cause a target organ protection against hypertension. Active immunization against the AT1 receptor may be considered as a promising new strategy in the treatment of hypertension.

[Autoantibodies against alpha1 adrenergic receptor related with cardiac remodeling in hypertensive patients by clinical observation].

OBJECTIVE: To investigate the effects of autoantibodies against alpha(-) adrenergic receptor on cardiac remodeling in patients with hypertension. METHODS: Five hundred and fifty three patients with hypertension in our hospital were selected. The autoantibodies against alpha(1) adrenergic receptor in sera of donor were detected by ELISA, and the results of echocardiography were recorded. By multiple logistic regressions, the risk factors were analyzed on left ventricular enlargement of hypertension. RESULTS: The percentage of autoantibodies against alpha(1) adrenergic receptor positive was 32.3% (179/553). There were significant difference between the positive group and negative group on the ratio of left atrial enlargement (53.6%, 44.3%, respectively; P < 0.05) and left ventricular enlargement (12.8%, 6.1%, respectively; P < 0.01). The result of regression analysis demonstrated that 4 risk factors were related to left ventricular enlargement, including male, course of disease, heart rate (HR) and autoantibodies against alpha(1) adrenergic receptor in the serum (all P < 0.05). CONCLUSIONS: The autoantibodies against alpha(1) adrenergic receptor have a relationship with left ventricular enlargement of hypertension. Patients with the activity of autoantibodies against alpha(1) adrenergic might contribute to predict cardiac remodeling.

[Immune mechanism of cardiac remodeling induced by antibodies against to the alpha1-adrenergic receptor].

OBJECTIVE: To study the effects of autoantibodies against alpha1-adrenergic receptor on the cardiac remodeling and relevant mechanism. METHODS: Four-week-old male Wistar rats were immunized with synthesized second extracellular loop of alpha1-adrenergic receptor and raised for one year, with spontaneously hyperetensive rats (SHRs) and no-immunized Wistar rats of the same age as controls. Every one or two months blood was collected from the caudal vein to detect the level of serum antibodies to alpha1-adrenergic receptor by ELISA and the systolic blood pressure (SBP) and heart rate were measured. One year after the rats were killed and their hearts were taken out. The heart weight/body weight ratio, cardiac muscle cell cross-sectional area (CSA), interstitial collagen volume fraction (CVF) and the ratio of perivascular collagen area to vessel luminal area (PVCA) were calculated, the expression of alpha1-adrenergic receptor, c-fos and c-Jun in heart were measured by RT-PCR, Western blotting and immunohistochemistry. RESULTS: During the experiment the blood pressure of the SHRs were significantly higher than those of the immunization group and normal control group (both P < 0.01) without significant difference between the 2 latter groups. The heart weight/body weight ratio, CSA, CVF and PVCA of the immobilization group were 3.32 mg/g +/- 0.25 mg/g, 231 microm(2) +/- 11 microm(2), 5.40% +/- 0.66% and 1.89 +/- 0.62 respectively, all significantly higher than those of the normal control group (3.06 mg/g +/- 0.25 mg/g, 197 microm(2) +/- 19 microm(2), 3.22% +/- 0.15% and 0.86 +/- 0.17 respectively), but still significantly lower than those of the SHR group. Since the second week after immunization, the titre of antibody against of the immunization group began increase, peaked in the second and third months, and then decreased slowly, and remained at a high level by the end of experiment. The titre of antibody was not correlated with blood pressure. Hypertrophy of cardiac muscle cells and increase of sedimentation of collagen in stroma were seen in the hearts of the immunization group. RT-PCR showed that the expression of alpha1D-adrenergic receptor mRNA of the immunization group was 0.55 +/- 0.01, significantly lower than that of the normal control group (0.88 +/- 0.08, P < 0.05); the expression of c-jun mRNA in the immunization group was 0.82 +/- 0.02, significantly higher than that in the normal control group (0.42 +/- 0.07, P < 0.05); and there were no significant differences in the expressions of heart alpha1A- and alpha1B-afrenergic receptors and c-fos mRNAs between these 2 groups. Western blotting showed that the expression of c-jun protein in the immunization group was 6.24 +/- 2.13, significantly higher than that in the normal control group (2.55 +/- 0.58, P < 0.05); and there were no significant differences in the expressions of c-fos andalpha1A-adrenergic receptor proteins between these 2 groups. CONCLUSION: The antibodies against alpha1-adrenergic receptor up-regulates the expression of c-jun in cardiac muscle cells and interstitial fibroblast, which may be an immunologic mechanism of cardiac remodeling.

Autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension.

This study will explore the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension. Forty normotensives and 194 patients with hypertension were recruited for participation in this study. All patients accepted systemic combination drug treatment for antihypertension. According to the treatment results and the definition of refractory hypertension, the patients were divided into two groups: a refractory hypertension group and a non-refractory hypertension group. The epitope of the 2nd extracellular loop of type 1 angiotensin (AT1) receptor and alpha1-adrenergic receptor were synthesized and used as antigens to screen the autoantibodies against AT1-receptor and alpha1-adrenergic receptor by ELISA. The plasma renin activity and concentration of angiotensin II and catecholamine were also examined. The positive rates of the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension, 26.8% (52/194) and 25.3% (49/194), respectively, were higher than those in normotensives (7.5% and 5%)(p < 0.01). Further investigation showed that the frequencies of the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with refractory hypertension, 42.9% (42/98) and 36.7% (36/98), respectively, were higher than those in patients with non-refractory hypertension under systematic treatment (10.4% and 13.5%)(p < 0.01). The levels of circulating angiotensin II, catecholamine, proteinuria and serum creatine were also higher in the refractory hypertension group than in the non-refractory hypertension group. The findings showed that the frequencies of autoantibodies against AT1-receptor and alpha1-adrenergic receptor were higher in patients with hypertension, particularly in those with refractory hypertension, and that these autoantibodies might play a role in the pathogenesis of hypertension.

Candesartan versus imidapril in hypertension: a randomised study to assess effects of anti-AT1 receptor autoantibodies.

BACKGROUND: Anti-angiotensin II receptor subtype 1 (AT1 receptor) autoantibodies have previously been shown in sera of hypertensive patients. This study assessed whether anti-AT1-receptor autoantibody in serum is correlated with the efficacy of an AT1-receptor blocker (ARB; candesartan)-based regimen in hypertensive patients after 8 weeks of treatment. DESIGN: The Study of Optimal Treatment in Hypertensive Patients with Anti-AT1-Receptor Autoantibodies is a multicentre, randomised, blinded endpoint, open-label, parallel-group comparison clinical trial conducted in five centres in Wuhan, China. Treatment is designed as stepwise added-on therapy to reduce blood pressure (BP) < 140/90 mm Hg. 512 patients with moderate to severe primary hypertension were randomly assigned to an 8-week treatment with either ARB (candesartan)-based regimen (n=257) or ACE inhibitor (imidapril)-based regimen (n=255). RESULTS: Systolic and diastolic BP was reduced significantly in both treatment groups. The candesartan-based regimen achieved a significantly greater systolic BP reduction than imdapril (30.8 ± 10.3 vs 28.8 ± 10.3 mm Hg, p = 0.023). In those anti-AT1 receptor autoantibody-positive hypertensive patients, the mean systolic BP at baseline was higher than in the anti-AT1 receptor autoantibody-negative group (160.5 ± 16.5 vs 156.2 ± 17.7 mm Hg; p = 0.006). The mean BP reduction was greater in the candesartan-based regimen than the imidapril-based regimen (-35.4 ± 9.8/16.9 ± 6.9 vs -29.4 ± 9.8/14.2 ± 6.9 mm Hg; p = 0.000 and 0.002, respectively), and more patients on imidapril required add-on medications to achieve BP control (94% vs 86%; p=0.03). No correlation was observed between the titre of anti-AT1 receptor autoantibody and the efficacy of candesartan-based therapy. In those anti-AT1 receptor autoantibody-negative patients similar BP lowering was reached in the candesartan and the imidapril-based regimens. CONCLUSIONS: An ARB-based regimen is more effective in BP lowering than an ACE inhibitor-based regimen in the presence of anti-AT1 receptor autoantibodies. Trial registration number This trial has been registered at http://www.register.clinicaltrials.gov/ (identifier: NCT00360763).