Cardiac remodeling after long-term stimulation by antibodies against the alpha1-adrenergic receptor in rats

Although autoantibodies against the alpha1-adrenergic receptor which had been found in hypertensive patients had agonist-like activity as phenylephrine, the effects of these antibodies on cardiac remodeling have not been known. In this paper, the models with agonist-like activity of antibodies to alpha1-adrenergic receptor were made by immunized Wistar rats using synthesized peptides of alpha1A-adrenergic receptor and raised for 1 year, and the excited antibodies against the alpha1-adrenergic receptor which could elevate the free Ca2+ in isolated adult rat cardiomyocytes had been existed throughout the experiments after immunization. In immunized rats, despite that systolic blood pressure (SBP) had no difference with normal control, the hypertrophy of heart and cardiomyocytes was observed, the collagen deposition in heart interstitium increased, and c-jun expression and matrix metalloproteinase (MMP)-2 mRNA expression and activity in heart had increased. The results suggested that antibodies against the alpha1-adrenergic receptor could induce cardiac remodeling and maybe play a particular role in hypertension.     

抗α1-肾上腺素能受体抗体对培养大鼠主动脉平滑肌细胞增殖的影响

目的：观察抗α1-肾上腺素能受体自身抗体对培养平滑肌细胞增殖的影响，并探讨其可能机制。 方法： 用酶消化法培养大鼠平滑肌细胞及用亲和层析的方法纯化抗α1-肾上腺素能受体抗体，用BrdU掺入法检测平滑肌细胞的增殖率，用RT-PCR及Western blotting法检测c-jun、c-fos的表达。 结果： 抗α1-肾上腺素能受体抗体可以显著增加VSMC的增殖，并随时间的延长及抗体滴度的增加而增强，并可增加c-jun mRNA和蛋白的表达，其作用与NE相似，均显著高于正常对照IgG，并均可被α1-肾上腺素能受体阻滞剂prazosin阻断；而该抗体对c-fos的表达无显著增加。 结论： 抗α1-肾上腺素能受体抗体可以显著增加培养大鼠平滑肌细胞的增殖，并可增加c-jun的表达，在高血压血管重塑中可能发挥一定作用。     

肾性高血压大鼠与心脏α1肾上腺素能受体自身抗体的关系

目的:研究肾性高血压大鼠发病与心脏α₁肾上腺素能受体自身抗体的关系。方法:利用手术的方法建立肾性高血压大鼠模型,观察心脏α₁肾上腺素能受体自身抗体与肾性高血压发病的关系,同时对自身抗体生物活性进行分析。结果:在狭窄左肾动脉两周后,大鼠血浆中心脏α₁肾上腺素能受体自身抗体的阳性率和滴度与术前相比均明显升高,并且可持续到12周,以后逐渐下降|同时心脏α₁肾上腺素能受体自身抗体对培养的乳鼠心肌细胞的跳动具有激动剂样效应。结论:心脏α₁肾上腺素能受体自身抗体可能通过作用使外周血管阻力增加,从而促进肾性高血压大鼠心肌肥厚。     

eNOS基因体外转染对大鼠颈总动脉球囊损伤后血管平滑肌细胞增殖规律的影响

目的将内皮型一氧化氮合酶(endothilialnitircoxidesynthase,eNOS)基因,通过基因工程技术转染至体外培养的大鼠颈总动脉球囊损伤后的血管平滑肌细胞中,观察其对血管平滑肌细胞增殖规律的影响。方法三月龄雄性Wastar大鼠20只,分为正常对照组、空白对照组、pcDNA3.1(-)转染组和pcDNA3.1-eNOS转染组,每组各5只。除正常对照组外15只Wastar大鼠,施行经皮冠状动脉腔内成形术(PTCA)。术后一月,处死所有20只大鼠,取出左侧颈总动脉,以组织块贴壁法培养损伤后血管平滑肌细胞,采用脂质体载体Fugene6介导真核表达载体pcDNA3.1-eNOS和空载体pcDNA3.1(-)分别转染eNOS转染组和pcDNA3.l(-)转染组的平滑肌细胞,以RT-PCR法、原位杂交法和免疫荧光法检测eNOS基因的表达;以MTT法检测细胞增殖程度。结果在转染pcDNA3.l-eNOS基因的损伤后血管平滑肌细胞中可以检测出eNOSmRNA和蛋白的表达,且其细胞增殖程度显著低于未转染eNOS基因者(P<0.05)。结论eNOS基因体外转染可以明显抑制大鼠颈总动脉球囊损伤后血管平滑肌细胞的增殖活性。     

Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction

This study was conducted to determine the following: (1) whether matrix metalloproteinase-1 (MMP-1) is increased in systemic vessels of preeclamptic women, (2) whether this increase might be mediated by neutrophils, and (3) whether MMP-1 could be responsible for vascular dysfunction. Omental arteries and plasma were collected from healthy pregnant and preeclamptic women. Omental arteries were evaluated for gene and protein expression of MMP-1, collagen type 1α, tissue inhibitor of metalloproteinase-1, and vascular reactivity to MMP-1. Gene and protein expression levels were also evaluated in human vascular smooth muscle cells (VSMCs) co-cultured with activated neutrophils, reactive oxygen species, or tumor necrosis factor α. Vessel expression of MMP-1 and circulating MMP-1 levels were increased in preeclamptic women, whereas vascular expression of collagen or tissue inhibitor of metalloproteinase-1 were down-regulated or unchanged. In cultured VSMCs, the imbalance in collagen-regulating genes of preeclamptic vessels was reproduced by treatment with neutrophils, tumor necrosis factor α, or reactive oxygen species. Chemotaxis studies with cultured cells revealed that MMP-1 promoted recruitment of neutrophils via vascular smooth muscle release of interleukin-8. Furthermore, MMP-1 induced vasoconstriction via protease-activated receptor-1, whose expression was significantly increased in omental arteries of preeclamptic women and in VSMCs co-cultured with neutrophils. Collectively, these findings disclose a novel role for MMP-1 as a mediator of vasoconstriction and vascular dysfunction in preeclampsia.     

Immunization strategies for the production of rat monoclonal anti-idiotope antibodies

Anti-idiotypic antibodies are powerful reagents for the study of immunoregulation, and have potential interest as vaccines against tumors and infectious diseases. Three immunization strategies for the production of rat monoclonal anti-idiotope antibodies have been compared in this paper. Male Wistar rats were immunized i.p. and at multiple subcutaneous sites with 750 micrograms of purified monoclonal antibody against Plasmodium falciparum for three times and subsequently boosted by (1) intraperitoneal injection with 750 micrograms of the immunogen, (2) intravenous inoculation with 400 micrograms of the IgG, and (3) intrasplenic immunization with 200 micrograms of the idiotype. With the intraperitoneal boost method, the frequency of hybrids with anti-idiotope activity was 0.3-0.9% with 62.8-85.2% of the seeded wells containing hybrids. In the intravenous boost group, the percentage of hybrids demonstrating anti-idiotope activity increased to 11.0-13.3% with 80.2-97.9% of the hybrid efficiency. When immunized by the intrasplenic boost route, the frequency of anti-idiotope hybrids generated rose to 12.9-16.4% with 82.3-96.6% of the hybrid efficiency. There was no obvious effect of the boost immunizing methods on the generation of rat monoclonal anti-mouse IgG antibodies. These results indicated that the multiple-site immunization followed by intravenous or intrasplenic boost injection was an appropriate immunizing method for the production of monoclonal anti-idiotope antibodies.     

降钙素基因相关肽对血管平滑肌表型转化和增殖的影响

目的 探讨降钙素基因相关肽(CGRP)对大鼠血管平滑肌细胞(VSMCs)表型转化和增殖的影响以及它们之间的关系.方法 取大鼠主动脉先体外培养8d后再贴块培养(VSMCs);对照组直接用贴块法培养细胞.实验分为CGRP作用组和无CGRP作用组.用5-BrdU标记平滑肌细胞增殖变化;RT-PCR检测HRG-1和SM22α表达变化.结果 血管经体外培养8d后再贴壁培养的平滑肌细胞可见大量棕黄色标记增殖的细胞核,HRG-1和SM22α mRNA表达明显减少;而CGRP作用组标记的血管平滑肌增殖细胞明显减少,HRG-1和SM22α mRNA表达明显上调.结论 CGRP对VSMCs增殖有抑制作用并同时可使VSMCs从合成型向收缩型转化.     

De novo expression of Kv6.3 contributes to changes in vascular smooth muscle cell excitability in a hypertensive mice strain

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage-dependent ion channels (mainly Ca²⁺ and K⁺ channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage-dependent K⁺ channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed-specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real-time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed-specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 ( Kcng3 ) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch-clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.     

Weight gain and food intake in corticotropin releasing factor immunized Zucker rats

In Zucker obese rats (fa/fa) there are disturbances in the regulation of ACTH and corticosterone. In addition, beta-endorphin concentrations are higher in the pituitary and hypothalamus in obese than in lean rats. Since ACTH and beta-endorphin are thought to be controlled by corticotropin releasing factor (CRF), these effects may be due to abnormalities in CRF regulation. This possibility was investigated by immunizing rats against CRF. Obese rats immunized against CRF developed higher titer antibodies than lean rats. Hypothalamic CRF concentrations were higher in CRF-immunized obese but not lean rats compared with those of control rats, suggesting that compensation for sequestration of peripheral CRF developed in obese rats. In obese, but not lean rats, immunization against CRF decreased weight gains during weeks 1-4 and increased gains during weeks 9-12 and food intakes were decreased during weeks 5-8 compared with those for obese rats immunized against bovine serum albumin (BSA). Adrenal glands weighed 30% less in both obese and lean rats immunized against CRF compared with those immunized against BSA. These responses to immunization against CRF occurred even though plasma, hypothalamic and pituitary concentrations of ACTH and beta-endorphin were unaffected at the end of the study.     

雌激素对大鼠血管平滑肌细胞囊泡素-1基因表达的影响

目的:探讨雌激素对血管平滑肌细胞囊泡素-1基因表达的影响。方法:取Wistar雌性大鼠,分为3组:假手术组,卵巢切除后皮下埋植雌激素组 (OVX+E组)及卵巢切除后皮下埋植安慰剂组(OVX+V组)。用药2周后处死大鼠,剥离主动脉平滑肌组织,提取总RNA进行半定量RT-PCR分析,检测雌激素对囊泡素-1(caveolin-1)基因表达的影响。为进一步明确雌激素是否直接调节血管平滑肌细胞caveolin-1基因表达,又采用100 nmol/L 17β-雌二醇(17β-E₂)处理培养的大鼠血管平滑肌细胞24 h,通过Northern blot分析检测雌激素对细胞caveolin-1 mRNA表达的影响。结果:OVX+E组大鼠主动脉平滑肌组织caveolin-1基因表达量明显高于OVX+V组,17β-E₂处理的培养细胞中caveolin-1基因mRNA表达量高于未用药的培养细胞。 结论:雌激素可促进血管平滑肌细胞caveolin-1基因表达,反映了雌激素心血管作用机理的一个方面。     

Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis.

Interleukin-1 (IL-1) has been implicated as a regulatory protein in the development and clinical sequelae of atherosclerosis. To determine which cells in the atherosclerotic plaque synthesize IL-1 in situ, the authors evaluated histologic sections of iliac arteries from cynomolgus monkeys using probes for IL-1 alpha and beta. A polyclonal antibody to IL-1 alpha and beta was used to determine if proteins were concomitantly produced. The predominant cells expressing IL-1 alpha and beta mRNA were foam cells in the intima. Adherent leukocytes and vascular smooth muscle cells (VSMCs) expressed mRNA for IL-1 alpha. Microvascular endothelium expressed mRNA for both IL-1 alpha and beta. IL-1 proteins were located frequently in cells expressing IL-1 mRNA. These results indicate that endothelium and VSMCs, in conjunction with macrophages, serve as localized sources of IL-1 protein synthesis. These findings suggest that vascular cells may contribute directly to the pathogenesis of atherosclerotic vascular disease by actively secreting potent biologic mediators that modify vascular and immune cell function.     

氯血红素对大鼠血管平滑肌细胞血红素氧合酶1表达的影响

目的:观察氯血红素(Hm)对体外培养的大鼠血管平滑肌细胞(VSMCs)血红素氧合酶1(HO-1)表达的影响,以探讨内源性一氧化碳(CO)抑制AngⅡ诱导的VSMCs增殖的分子机制。方法:体外培养Wistar大鼠主动脉VSMCs,用AngⅡ诱导其增殖,分别用血红素氧合酶1(HO-1)的诱导剂和阻断剂,以诱导和阻断HO-1的表达。通过逆转录聚合酶链式反应(RT-PCR)及蛋白印迹杂交(Westernblot)检测HO-1mRNA和蛋白质的表达,酶联免疫法检测培养上清液中碳氧血红蛋白(COHb)含量,四唑氮(MTT)比色法检测VSMCs增殖。结果:Hm显著增加VSMCsHO-1mRNA及蛋白质的表达及培养上清液中COHb含量,同时VSMCs增殖活力被显著抑制。结论:HO-1mRNA及蛋白表达增加是内源性CO抗VSMCs增殖的分子生物学基础。提示HO-CO信号系统在以VSMCs增殖为特征的心血管病的发生和发展中具有重要作用。     

Alpha1-adrenoceptors in the guinea pig thoracic aorta.

In the present study, we tried to determine which alpha1-adrenoceptor subtypes are involved in the guinea pig thoracic aorta by using in vitro functional analysis. In first, we tried to estimate the pA2 values of some key alpha1-adrenoceptor antagonists (prazosin, 5-methylurapidil, WB4101, BMY7378 and tamsulosin) against responses to norepinephrine in the thoracic aorta of guinea pigs. The concentration-response curves of norepinephrine were rightward shifted by the presence of prazosin, 5 methylurapidil, WB4101, BMY7378 and tamsulosin. The pA2 values for these antagonists against norepinephrine were 7.83, 7.78, 8.20, 5.73 and 9.57, respectively. In second, we tried to compare the estimated pA2 values obtained in the present study with reported pKi and pA2 values for cloned and native alpha1-adrenoceptor subtypes. In rabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract which were proposed to contain the putative alpha1L-adenoceptor, we obtained the good correlation for the pA2 values reported in these tissues with pA2 values estimated in guinea pig thoracic aorta. Moreover, regression lines were close to the line of identity. These results suggest that the alpha1-adenoceptors mediating contraction of guinea pig thoracic aorta are similar pharmacologically to the putative alpha1L-adenoceptor subtype in rabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract. As a final point, guinea pig thoracic aorta may be able to use as a tool to develop the new alpha1-adrenoceptor antagonist which is therapeutically advantageous in the treatment of urinary tract obstruction (e. g., in benign prostatic hyperplasia).     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响

背景：一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。 目的：观察内皮型一氧化氮合酶 (endothelial nitric oxide synthase,eNOS)基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。 方法：建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。 结果与结论：AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组(P < 0.01)。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

Dynamics of dopamine and norepinephrine contents in the dorsal hippocampus of rats during immunization with dopamine conjugate

The dynamics of dopamine and norepinephrine contents in the dorsal hippocampus of rats immunized with a dopamine-BSA conjugate was studied during immobilization stress by mean of the microdialysis technique. Immunization with dopamine conjugate was accompanied by intensive production of antibodies against dopamine in rat blood and a tendency toward an increase in dopamine content in the dorsal hippocampus even in the basal state (before stress exposure). Under stress conditions, dopamine content in the dorsal hippocampus of immunized rats significantly increased. In control rats, stress was accompanied by a significant increase in norepinephrine content in the dorsal hippocampus. The observed peculiarities in dopamine and norepinephrine contents in the dorsal hippocampus of rats immunized with a dopamine conjugate were typical of active animals more resistant to emotional stress. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 374–377, April, 2007     

Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor alpha subunit extracellular domain 1-210.

The duplicated alpha subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rH alpha 1-210) corresponding to the proposed extracellular domain of the alpha subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human alpha subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rH alpha 1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's alpha subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.     

Post-translational modification of rat immunoglobulins synthesized in the Xenopus oocyte translation system

The post-translational modification of rat immunoglobulin synthesised in Xenopus laevis oocytes was studied. The major products of translation of rat spleen poly-(A) containing mRNA were found to be assembled 7S immunoglobulin molecules indicating extensive modification of primary translation products. The possibility that these immunoglobulin molecules might include antibodies of defined specificity was investigated using spleen mRNA from rats hyperimmunized with ferritin and keyhole limpet haemocyanin. The presence of antibodies to immunizing antigen in oocyte translation products was determined by affinity chromatography on Sepharose-antigen columns and the synthesis of Sepharose-antigen binding antibodies was observed, equivalent to 2.5-3% of total immunoglobulins. The oocyte produced antibodies were of the same immunoglobulin class as the circulating antibodies from the immunized rats.     

Low-affinity antibodies against collagen type II produced by lymph node cells are associated with pathology in collagen-induced arthritis in rats.

The relationship between the affinity of antibodies against type II collagen (CII) and arthritis was studied in rats immunized intradermally with bovine CII. Disease was associated with a higher mean titre of serum antibody and a lower mean functional antibody affinity (determined in a chaotropic dissociation assay) against both the immunizing bovine CII and homologous autoantigenic rat CII in comparison with the response in immunized rats that did not develop disease. The functional affinity of the antibodies present in the serum was found to correlate with that of antibodies produced in culture by cells from the lymph nodes draining the site of immunization with collagen. The reduction in mean functional affinity in the anti-collagen response may be the result of the increased production of antibodies of the lowest affinity and a consequent broadening of the affinity heterogeneity. It is proposed that production of low-affinity antibodies in the lymph nodes draining the site of immunization with collagen is important in the pathogenesis of collagen-induced arthritis in rats.     

Intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated Salmonella enterica serovar enteritidis antigen

Induction of intestinal mucosal immune responses against Salmonella enterica serovar enteritidis was studied by immunizing chickens with liposome-associated antigen. An ultrasonicated whole cell extract of the bacteria was used for immunizing antigen. Intraocular immunization induced serum IgA, IgG and IgM responses. Also, significant IgA and IgG antibodies were detected in the intestinal tract. Immunization with antigen alone induced only IgG response in the intestine. Salmonella enteritidis-specific antibody-secreting lymphocytes were detected in the spleen and lamina propria of the intestinal tract of immunized chickens. Immunoglobulin (Ig) fractions extracted from intestines of immunized chickens inhibited the adherence of S. enteritidis to cultured HeLa cells. These results indicate that intraocular immunization with liposome-associated S. enteritidis elicits specific antibody-producing lymphocytes in the intestinal tract, and that Ig secreted in the intestine inhibits adherence of the bacteria to intestinal epithelial cells, suppressing the spread of bacterial infection in the host.     

Prevention of experimental autoimmune cardiomyopathy in rabbits by receptor blockers

We investigated the effects of beta1-adrenoceptor blockade and M2-muscarinic receptor antagonist in rabbits which have developed dilated cardiomyopathy-like changes after immunization with the peptides from the second extracellular loop of human beta1-adrenoceptor (beta1-peptide) and M2-muscarinic receptor (M2-peptide). Ten rabbits, which were immunized with beta1-peptide once a month for one year, were treated with bisoprolol and 10 rabbits, which were immunized with M2-peptide, were treated with otenzepad. Although both groups treated with receptor blockade or antagonist showed an increased titer of anti-beta1-adrenoceptor or anti-M2-muscarinic receptor antibodies, myocardial damages were markedly less than those in beta1-peptide- or M2-peptide-immunized rabbits. This study indicates that anti-beta1-adrenoceptor and anti-M2-muscarinic receptor antibodies are of pathogenic importance in the development of human dilated cardiomyopathy, and that beta-adrenoceptor blockade, bisoprolol, and M2-muscarinic receptor antagonist, otenzepad, might be clinically useful for treatment of dilated cardiomyopathy.