坎地沙坦阻断血管紧张素Ⅱ介导的原代急性髓样白血病细胞增殖的作用及机制

目的: 观察血管紧张素Ⅱ 1型受体(AT1R)拮抗剂坎地沙坦抑制血管紧张素Ⅱ(Ang Ⅱ)介导的原代急性髓样白血病(AML)细胞增殖的作用及机制。方法: MTT法观察Ang Ⅱ对原代AML细胞、正常骨髓单个核细胞增殖的影响以及坎地沙坦和AT2R拮抗剂 PD123319对AngII促原代AML细胞增殖的拮抗作用; Western blotting法观察坎地沙坦和PI3K抑制剂对原代AML细胞Akt磷酸化的影响。结果: AngII能剂量和时间依赖性促进原代AML细胞增殖(P<0.05),而对正常骨髓无此作用。坎地沙坦随浓度和时间依赖性阻断Ang II作用下白血病细胞增殖(P<0.05)。PI3K抑制剂可抑制Ang II促进原代AML细胞的增殖(P<0.05),坎地沙坦能明显下调Ang II增加原代AML细胞Akt的磷酸化水平(P<0.05)。结论: 坎地沙坦通过抑制PI3K/Akt信号转导途径抑制Ang II/AT1R介导的白血病细胞增殖。     

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Essential role for Smad3 in angiotensin II‐induced tubular epithelial–mesenchymal transition

Angiotensin II (Ang II) is a key mediator of chronic kidney disease, in which epithelial–mesenchymal transition (EMT) is a critical process mediated by the TGFβ/Smad signalling pathway. The present study examined the specific role of Smads in Ang II‐induced EMT in vitro and in vivo. We found that Ang II signalled through the receptor of AT1, not AT2, to activate Smad2/3 and induce EMT in a normal rat tubular epithelial cell line (NRK52E). Activation of Smads by Ang II was attributed to degradation of an inhibitory Smad7, which was mediated by the AT1‐Smurf2‐dependent ubiquitin degradation mechanism because blockade of AT1 receptor or knockdown of Smurf2 inhibited Smad7 loss, thereby reducing Smad2/3 activation and EMT in response to Ang II. In contrast, over‐expression of Smad7 inhibited Ang II‐induced Smad2/3 activation and EMT in NRK52E cells and in a rat model of remnant kidney disease. Moreover, knockdown of Smad3, not Smad2, attenuated Ang II‐induced EMT. In conclusion, Ang II activates Smad signalling to induce EMT, which is mediated by a loss of Smad7 through the AT1‐Smurf2‐dependent ubiquitin degradation pathway. Smad3, but not Smad2, may be a mediator of EMT, while Smad7 may play a protective role in EMT in response to Ang II. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

ERK1/2及JNK参与DL0805抑制血管紧张素Ⅱ引起的大鼠离体胸主动脉环收缩

目的研究Rho激酶抑制剂DL0805对血管紧张素Ⅱ(AngⅡ)引起的大鼠离体胸主动脉环收缩反应的影响及其可能的机制。方法测定离体血管张力观察大鼠胸主动脉环收缩反应,Western blot检测大鼠离体胸主动脉环ERK1/2和JNK蛋白磷酸化,和AngⅡ1型受体(AT1R)蛋白表达水平。结果 DL0805(10、25和50μmol/L)浓度依赖性地抑制AngⅡ(100 nmol/L)引起的内皮完整或去内皮的大鼠离体胸主动脉环收缩(P<0.01,P<0.001),DL0805(25和50μmol/L)抑制AngⅡ(100 nmol/L)诱导的ERK1/2和JNK的活化(P<0.05,P<0.01和P<0.001),但DL0805(5、25和50μmol/L)对AngⅡ刺激的血管环AT1R蛋白表达水平无显著影响。结论 DL0805抑制AngⅡ引起的大鼠离体胸主动脉环收缩,其机制可能与其抑制AngⅡ诱导的ERK1/2和JNK活化有关。     

肾内血管紧张素系统在高血压性肾损害中的作用

目的:探讨血管紧张素II(AngII)及血管紧张素II受体(AT₁R)在缺血性肾损害时肾脏局部的作用。方法:采用放射免疫法及逆转录-聚合酶链反应(RT-PCR)技术,检测双肾动脉狭窄时大鼠血浆和肾脏的AngII含量和AT₁RmRNA的表达。结果:缺血性肾损害时大鼠血浆及肾组织AngII水平均高于对照组,P <0.0 5,而肾组织AT₁RmRNA的表达也较对照组高,P <0.0 1。结论:在双肾动脉狭窄缺血性肾损害时存在AngII及其AT₁R的异常,它们可参与肾脏损害作用.     

Pigment epithelium-derived factor (PEDF) blocks angiotensin II-induced T cell proliferation by suppressing autocrine production of interleukin-2

Angiotensin II (Ang II) elicits numerous inflammatory-proliferative responses in vascular cells, thereby being involved in atherosclerosis. We have previously shown that pigment epithelium-derived factor (PEDF) blocks the Ang II-induced endothelial cell activation, thus suggesting that PEDF may play a role in atherosclerosis. However, effects of PEDF on T cell activation, another key steps of atherosclerosis, remain to be elucidated. In this study, we examined whether PEDF could inhibit the Ang II-induced MOLT-3 T cell proliferation in vitro and the way that it might achieve this effect. Ang II significantly stimulated DNA synthesis in MOLT-3 T cells, which was inhibited by PEDF, olmesartan, an Ang II type I receptor blocker, an anti-oxidant N-acetylcysteine (NAC), or antibodies directed against IL-2. PEDF or NAC suppressed gene expression of interleukin-2 (IL-2) in Ang II-exposed MOLT-3 T cells. Furthermore, PEDF blocked the Ang II-induced reactive oxygen species (ROS) generation and NADPH oxidase activity in MOLT-3 T cells. These results demonstrate that PEDF inhibits the Ang II-induced T cell proliferation by blocking autocrine production of IL-2 via suppression of NADPH oxidase-mediated ROS generation. Blockade by PEDF of T cell activation may become a novel therapeutic target for atherosclerosis.     

Effect of an angiotensin II type 1 receptor blocker on caveolin-1 expression in prostate cancer cells

Introduction

Caveolin-1, the major structural protein of caveolae, interacts directly with the AT1 receptor. The biological functions of caveolin-1 in cancer are compound, multifaceted, and depend on cell type, tumour grade and cancer stage. The AT1-R-caveolin complex in caveolae may coordinate angiotensin II (Ang II) induced signalling. The aim of this study was to determine the effect of the angiotensin II receptor type 1 blocker candesartan on caveolin expression in human metastatic prostate adenocarcinoma cells PC-3.

Material and methods

WST-1 and BrdU assays were used as indicators of cell viability and proliferation after angiotensin II and/or candesartan stimulation. Real-time RT–PCR and western blot were used to study the effect of Ang II and/or candesartan on the expression of Cav-1 and AT1-R in PC-3 cells

Results

We found that the expression of caveolin-1 mRNA in the PC-3 cells treated with CV was significantly decreased in comparison with the control (2.9 ±0.17, 4.7 ±0.6, p < 0.05), whereas a higher caveolin-1 mRNA expression was observed in those after Ang II treatment (6.0 ±0.43, 4.7 ±0.6, p < 0.05). Protein analysis indicate that the expression of caveolin-1 protein in the PC-3 cells treated with candesartan was significantly decreased when compared with the control (0.69 ±0.05, 1.6 ±0.12, p < 0.05), whereas higher caveolin-1 protein expression was observed after Ang II treatment (2.5 ±0.20, 1.6 ±0.12, p < 0.05).

Conclusions

These results provide new information on the action of candesartan and may improve the knowledge about AT1 receptor inhibitors, which can be potentially useful in prostate cancer therapy.     

血管紧张素II及其受体在血管平滑肌细胞迁移中的作用

目的:探讨血管紧张素II(AngII)及其受体(ATRs)在局部血管损伤后血管平滑肌细胞(VSMC)迁移中的作用及其机制。方法:以体外培养VSMC为基础,采用细胞化学和改良Boyden'schamber的方法,观察AngⅡ干预VSMC后AngII受体的表达、VSMC迁移能力的变化、肌动蛋白纤维丝的动态组装变化,并探讨AT₁R拮抗剂、AT₂R拮抗剂对上述观测指标的影响。结果:AngII10^-7mol/L可以刺激VSMC发生迁移,该作用是通过影响VSMC内应力纤维动态组装而实现的;AngII干预VSMC后可使AT₁R表达上调,随着作用时间延长AT₁R表达水平下降。AT₁R拮抗剂可下调AT₁R表达。AngII通过AT₁R的介导发挥其影响VSMC迁移能力的生物学效应。AT₂R对此无明显影响。结论:AngII通过AT₁R介导来调节VSMC内肌动蛋白微丝的动态组装,进而改变VSMC的迁移能力,从而发挥其介导VSMC迁移的生物学效应。     

Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 μg/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs. the regulation of β- and γ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT(1) receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.     

血管紧张素Ⅱ受体在压力超负荷致左室肥大中的作用

目的:探讨血管紧张素Ⅱ受体(ATRs)在压力超负荷致左室肥大中的作用。 方法:采用大鼠腹主动脉缩窄模型,通过放免法测心肌组织血管紧张素Ⅱ(Ang Ⅱ)含量,放射性配基结合分析法检测心肌组织ATRs及其亚型的变化。结果:手术组AngⅡ含量显著增高,与左室重量指数(LVMI)呈正相关(r=0.8066,P<0.01)。ATRs最大结合容量 (Bmax) 显著高于对照组(P＜0.01),但两组之间的平衡解离常数(kd)、血管紧张素Ⅱ1型受体(AT₁R)和血管紧张素Ⅱ2型受体(AT₂R)之间的比例无显著差异。非肽类AT₁R 拮抗剂Irbesartan可显著抑制Ang Ⅱ的升高和左室肥大,非肽类AT₂R 拮抗剂CGP42112A则无此作用。结论: 压力超负荷时心肌组织ATRs上调,Ang Ⅱ致左室肥大的作用主要由AT₁R介导。     

Pregnancy: Atrial natriuretic peptide antagonizes the contractile effect of angiotensin II in the human uterine artery

The regulatory peptides angiotensin II (Ang II) and atrial natriureticpeptide (ANP) are believed to play important roles in the pathogenesisof pre-eclampsia. The interactions between Ang II, ANP and noradrenaline(NA) were studied in vitro on the human uterine artery. BothAng II and NA contracted the isolated vessel in a concentration-dependentway. At high doses a decrease in the contractile force inducedby Ang II but not NA was encountered. ANP inhibited the smoothmuscle activity elicited by Ang II, resulting in a dextroshiftof the concentration-response curve, and a decrease in bothE_max (the maximum contractile response) and pD2 (the negativelogarithm of the agonist concentration inducing 50% of the E_max)for Ang II. The results might indicate a specific antagonismbetween Ang II and ANP, probably at the post-receptor level.ANP did not induce any significant change in pD₂ of the concentration-responsecurve for NA. Only at the highest dose of ANP (10^–7 M)was E_max depressed. Thus, the results only indicate a weak antagonisticrelationship between NA and ANP in the human uterine artery.     

Effect of angiotensin-(1-7) and angiotensin II on the proliferation and activation of human endometrial stromal cells in vitro

Recent studies have shown that angiotensin II (Ang II) or angiotensin-(1-7) [Ang-(1-7)] has effect on the proliferation and activation of a variety of cells, however, the exact mechanisms that the role of Ang II or Ang-(1-7) in human endometrial stromal cell (ESCs) remains elusive. Here we demonstrated that Ang II could promote proliferation and activation of ESCs, up-regulated the expression of a-SMA, TGF-β1 and IGF-I, increased the secretion of extracellular matrix [Type I collagen (Col I) and fibronectin (FN)] of ESCs; Ang-(1-7) could inhibit Ang II induced the proliferation and activation of ESCs, down-regulated the expression of a-SMA, TGF-β1 and IGF-I, decreased the secretion of extracellular matrix (Col I and FN) of ESCs. These findings suggest that Ang-(1-7) can inhibits Ang II induced the proliferation of ESCs, Ang-(1-7) can inhibits the Ang II induced activation of ESCs and decreases secretion of Col I and FN by suppressing TGF-β1 and IGF-I expression.     

CX3CL1/CX3CR1 Axis Contributes to Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation and Inflammatory Cytokine Production

Angiotensin II (Ang II) dysregulation has been determined in many diseases. The CX3CL1/CX3CR1 axis, which has a key role in cardiovascular diseases, is involved in the proliferation and inflammatory cytokine production of vascular smooth muscle cells (VSMCs). In this study, we aim to explore whether Ang II has a role in the expression of CX3CL1/CX3CR1, thus contributing to the proliferation and pro-inflammatory status of VSMCs. Cultured mouse aortic VSMCs were stimulated with 100 nmol/L of Ang II, and the expression of CX3CR1 was assessed by western blot. The results demonstrated that Ang II significantly up-regulated CX3CR1 expression in VSMCs and induced the production of reactive oxygen species (ROS) and the phosphorylation of p38 MAPK. Inhibitors of NADPH oxidase, ROS, and AT1 receptor significantly reduced Ang II-induced CX3CR1 expression. Targeted disruption of CX3CR1 by transfection with siRNA significantly attenuated Ang II-induced VSMC proliferation as well as down-regulated the expression of proliferating cell nuclear antigen (PCNA). Furthermore, CX3CR1-siRNA suppressed the effect of Ang II on stimulating Akt phosphorylation. Besides, the use of CX3CR1-siRNA decreased inflammatory cytokine production induced by Ang II treatment. Our results indicate that Ang II up-regulates CX3CR1 expression in VSMCs via NADPH oxidase/ROS/p38 MAPK pathway and that CX3CL1/CX3CR1 axis contributes to the proliferative and pro-inflammatory effects of Ang II in VSMCs.     

Renin angiotensin system modulates mTOR pathway through AT2R in HIVAN

Mammalian target of rapamycin (mTOR) has been reported to contribute to the development of HIV-associated nephropathy (HIVAN). We hypothesized that HIV may be activating renal tissue mTOR pathway through renin angiotensin system (RAS) via Angiotensin Receptor Type II receptor (AT2R). Renal tissues of Vpr transgenic and Tg26 (HIVAN) mice displayed enhanced phosphorylation of mTOR and p70S6K. Aliskiren, a renin inhibitor attenuated phosphorylation of both mTOR and p70S6K in renal tissues of HIVAN mice. Interestingly, Angiotensin Receptor Type I (AT1R) blockade did not modulate renal tissue phosphorylation of mTOR in HIVAN mice; on the other hand, AT2R blockade attenuated renal tissue phosphorylation of mTOR in HIVAN mice. In vitro studies, both renin and Ang II displayed enhanced mouse tubular cell (MTC) phosphorylation of p70S6K in a dose dependent manner. HIV/MTC also displayed enhanced phosphorylation of both mTOR and p70S6K; interestingly this effect of HIV was further enhanced by losartan (an AT1R blocker). On the other hand, AT2R blockade attenuated HIV-induced tubular cell phosphorylation of mTOR and p70S6K, whereas, AT2R agonist enhanced phosphorylation of mTOR and p70S6K. These findings indicate that HIV stimulates mTOR pathway in HIVAN through the activation of renin angiotensin system via AT2R.     

The involvement of type 1a angiotensin II receptors in the regulation of airway inflammation in a murine model of allergic asthma

BACKGROUND: There has been increasing evidence suggesting the involvement of angiotensin II (Ang II) and type 1 Ang II receptors (AT1) in the pathogenesis of bronchial asthma. However, whether such an involvement would promote or suppress the pathophysiology of asthma is controversial. OBJECTIVE: The aim of this study was to investigate the role of AT1 in the development of allergic airway inflammation. METHODS: Agtr1a+/+ [wild-type C57BL/6 mice (WT)] and Agtr1a-/- mice [AT1a knockout mice (AT1aKO)] with a genetic background of C57BL/6 were systemically sensitized to ovalbumin (OVA), followed by OVA inhalation. OVA-specific IgE in serum obtained just before the inhalation was measured. Bronchoalveolar lavage (BAL) fluid and lung tissues were obtained at various time-points. Cell numbers and differentiation, and cytokine contents in BAL fluids were determined. Peribronchial accumulation of eosinophils and mucus inclusions in the bronchial epithelium were evaluated in lung tissues stained histochemically. Cell numbers and differentiation in BAL fluids of the mice were also determined after lipopolysaccharide (LPS) inhalation. RESULTS: The levels of OVA-specific IgE in AT1aKO were significantly higher than those in WT. The numbers of total cell, eosinophils and lymphocytes in BAL fluids 7 days after OVA inhalation in AT1aKO were significantly higher than those in WT. Airway inflammation in bronchial tissues in terms of eosinophil accumulation and mucus hypersecretion in AT1aKO was also stronger than in WT. The contents of IL-4, IL-5 and IL-13, but not IFN-gamma, in BAL fluids of AT1aKO were significantly higher than those of WT. In contrast, neutrophil accumulation in BAL fluids after LPS inhalation was significantly higher in WT than in AT1aKO. CONCLUSION: AT1a might be involved in the negative regulation of the development of allergic airway inflammation through polarizing the T-helper (Th) balance towards Th1 predominance. Therefore, it would be of clinical importance to investigate the effects of long-term administration of AT1 blockers on the Th1/Th2 balance in hypertensive patients with bronchial asthma.     

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ～2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01±0.10 ns) or Rab7 (2.11±0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78±0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09±0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.     

Angiotensin II up-regulates angiotensin I-converting enzyme (ACE), but down-regulates ACE2 via the AT1-ERK/p38 MAP kinase pathway

The recent discovery of the angiotensin II (Ang II)-breakdown enzyme, angiotensin I converting enzyme (ACE) 2, suggests the importance of Ang II degradation in hypertension. The present study explored the signaling mechanism by which ACE2 is regulated under hypertensive conditions. Real-time PCR and immunohistochemistry showed that ACE2 mRNA and protein expression levels were high, whereas ACE expression levels were moderate in both normal kidney and heart. In contrast, patients with hypertension showed marked ACE up-regulation and ACE2 down-regulation in both hypertensive cardiopathy and, particularly, hypertensive nephropathy. The inhibition of ACE2 expression was shown to be associated with ACE up-regulation and activation of extracellular regulated (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases. In vitro, Ang II was able to up-regulate ACE and down-regulate ACE2 in human kidney tubular cells, which were blocked by an angiotensin II (AT)1 receptor antagonist (losartan), but not by an AT2 receptor blocker (PD123319). Furthermore, blockade of ERK1/2 or p38 MAP kinases by either specific inhibitors or a dominant-negative adenovirus was able to abolish Ang II-induced ACE2 down-regulation in human kidney tubular cells. In conclusion, Ang II is able to up-regulate ACE and down-regulate ACE2 expression levels under hypertensive conditions both in vivo and in vitro. The AT1 receptor-mediated ERK/p38 MAP kinase signaling pathway may be a key mechanism by which Ang II down-regulates ACE2 expression, implicating an ACE/ACE2 imbalance in hypertensive cardiovascular and renal damage.