【作者】 王博；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 临床医学（心血管外科）， 2016， 博士

【摘要】 研究背景和目的主动脉瓣钙化及其进展导致的钙化性主动脉瓣狭窄与关闭不全是一种严重危害人类健康的疾病,随着人口老龄化、生活水平的提高、平均寿命的延长,其发病率呈不断上升趋势。目前认为,瓣膜钙化并非简单的钙磷被动沉积过程,而是类似动脉粥样硬化,由多种细胞和信号蛋白介导的主动性调控病理过程。主要发病机制包括：内皮细胞损伤、脂蛋白沉积、慢性炎症反应、细胞外基质重构等。然而,目前对于钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)尚无有效的药物干预措施,手术或介入治疗换瓣仍是治疗首选。由于患者年龄普遍较大,且多合并有高血压、糖尿病等其他基础疾病,无疑增加了手术风险。因此,对主动脉瓣钙化发病机制进行深入探讨,寻求新的防治策略迫在眉睫。内质网应激(endoplasmic reticulum stress, ER stress)是由于各种病理性刺激如钙调节异常、同型半胱氨酸代谢及氧化应激过程出现紊乱,引起的内质网应激级联反应,主要包括蛋白质合成暂停,内质网应激蛋白表达和细胞凋亡三个主要过程。内质网应激广泛存在于神经退行性病变、血管粥样硬化及血管钙化等疾病的病理过程,且本课题组前期实验证明内质网应激在瓣膜钙化的过程中具有明显的促钙化作用。终末期氨基糖基化受体(receptor for advanced glycation end products, RAGE)激活后可导致内质网应激反应在上皮细胞的相关研究中已被证实,淀粉样蛋白β(Amyloid β,Aβ)作为RAGE的配体,能够刺激RAGE受体并激活其下游的炎症相关信号通路,相关研究证明在动脉粥样硬化的血管以及钙化瓣膜组织中均发现Aβ蛋白的存在。因此,我们推测在钙化瓣膜中Aβ能够通过激活RAGE受体诱导内质网应激反应从而发挥促钙化作用。在本研究中,我们为了明确Aβ/RAGE通路激活后能否通过诱导内质网应激反应促进主动脉瓣钙化开展了以下工作。首先,通过对比人与动物钙化瓣膜中Aβ/RAGE通路与内质网应激相关蛋白表达的差异,确定上述过程在瓣膜钙化过程中是否激活;其次,构建双敲基因动物模型,观察特异性敲除小鼠RAGE基因后Aβ/RAGE通路、内质网应激、成骨及炎症相关因子表达的差异；第三,在体外实验中通过构建原代瓣膜问质细胞钙化模型,并予以各种不同干预,利用免疫蛋白印迹、免疫组化等分子生物学技术,详细阐述Aβ/RAGE通路、内质网应激及瓣膜钙化三者之间相互作用机制。实验方法与结果体内实验设计：取CAVD患者瓣膜作为钙化组。急性主动脉夹层行Bentall手术患者瓣膜作为对照组进行组织学切片染色,所有涉及人体标本均符合医学伦理学要求,经过华中科技大学伦理委员会批准且通过患者知情同意并签字。与对照组瓣膜相比,钙化组瓣膜组织内皮缺损并出现成纤维细胞样改变,成骨因子BMP2和Runx2表达增多。内皮是瓣膜的第一道屏障,成纤维样改变通常被认为是正常瓣膜向瓣膜钙化的过渡状态,瓣膜的成骨分化是导致钙化的最重要因素。因此,上述结果成功验证所取钙化组瓣膜符合钙化瓣膜特点。同时,Aβ、RAGE受体与内质网应激相关蛋白C/EBP homologous protein(CHOP)和protein kinase-like ER kinase(PERK)的表达在钙化组瓣膜中均上调,提示上述蛋白可能参与到瓣膜钙化的过程,且免疫荧光染色证实Ap与RAGE受体特异性结合,说明Aβ能通过RAGE受体发挥作用。动物实验中正常对照组选用C57/B6J背景小鼠,瓣膜钙化造模组选用ApoE-/-小鼠,干预组选用ApoE-/-RAGE-/-双敲基因小鼠。各组小鼠均选取8周龄,体重30g左右雄性小鼠。小鼠进入SPF培养室后先给予正常饮食1周,待小鼠适应环境且行心脏彩超检查无异常后,三组均开始给予西式饮食喂养。上述西式饮食及ApoE-/-小鼠由北京华阜康公司提供；ApoE-/-RAGE-／-双敲除小鼠的构建由南京大学模式动物中心提供技术支持；SPF级动物实验室由华中科技大学实验动物中心提供。Control组、ApoE-/-组和ApoE-/-RAGE-/-组三组动物模型均于西式饮食干预前以及干预24周之后,在异氟烷(2.5%)全麻下行超声检测。超声设备型号为GE-vivid 7,探头频率采用18-38MHz。从SPF级动物实验室领出小鼠前,提前12小时给予小鼠禁食禁水处理,防止饮食饮水对后续血糖血脂检测结果的干扰。小鼠用脱毛膏脱毛后置于麻醉罐中,当小鼠心率缓慢降至300-350bpm范围时经胸行超声检测,主要收集主动脉瓣口血流速度、主动脉瓣口直径及小鼠心功能相关指标,主动脉瓣膜开口面积(AVA)的计算方法参照连续方程法,所有超声数据由同一超声科医师收集。与Control组相比,ApoE-/-组超声显示主动脉瓣口峰速明显增高,最高值可达20mm/s;形态学染色显示瓣膜形态增厚明显,且茜素红染色显示钙结节所占培养板比例明显增多；免疫组化检测显示BMP2与Runx2表达明显增高；内质网应激相关蛋白CHOP与PERK明显上调。上述结果均说明西式饮食成功诱导ApoE-/-小鼠瓣膜钙化,且内质网应激可能在瓣膜钙化过程中发挥作用,本部分结果与人体标本中上述指标的改变相一致。与ApoE-/-组相比,ApoE-/-RAGE-/-组心脏超声检测显示主动脉瓣口峰速明显降低；形态学染色可见瓣膜形态增厚不明显,且钙结节比例明显下降；免疫组化检测BMP2与Runx2表达明显减少；内质网应激相关蛋白CHOP和PERK明显下调；巨噬细胞标记物CD68表达减少。上述结果提示,特异性敲除RAGE基因延缓了小鼠主动脉瓣膜钙化的进展,抑制了内质网应激反应及炎症细胞浸润。我们进一步检测ApoE-/-RAGE-/-组小鼠血糖血脂代谢情况,与ApoE-/-组相比数据无统计学差异。上述结果进一步提示RAGE基因敲除延缓瓣膜钙化的过程与血糖血脂的改变无关,我们推测可能是通过抑制内质网应激反应、成骨分化以及炎症反应实现。Ap/RAGE通路、内质网应激反应与瓣膜钙化相互影响的机制将在体外实验中进行阐述。细胞实验部分：对照组瓣叶取自急性主动脉夹层行Bentall术者。采用胶原酶消化法获得原代瓣膜间质细胞,选用2-5代细胞进行实验干预。在不同浓度梯度Aβ40(0μM, 25μM,50μM,100μM)干预下对主动脉瓣膜间质细胞(aortic valve interstitial cells,AVICs)进行孵育,通过钙化培养基建立体外钙化模型,观察RAGE受体与成骨蛋白的表达,结果显示RAGE受体、BMP2与Runx2的表达随Aβ40浓度的递增而增加。通过siRNA干扰技术抑制RAGE受体的表达,免疫蛋白印迹结果显示Aβ40诱导BMP2与Runx2表达的作用明显减弱；Aβ40诱导内质网应激相关蛋白CHOP与PERK的表达也明显减少。上述结果表明AP40/RAGE通路作为上游蛋白分子能够诱导成骨因子表达以及内质网应激反应。下一步将探讨成骨因子的表达与内质网应激相互作用的机制。取不同时间点,采用Aβ40 (50μM)对细胞进行孵育,观察ERK1/2、NF-κB和p38 MAPK蛋白磷酸化程度,免疫蛋白印迹显示ERK1/2和NF-κB的磷酸化程度在一定时间内随Aβ40干预时间增加而增加,p38 MAPK磷酸化程度无明显改变。ERK1/2和NF-κB磷酸化能诱导成骨因子及炎症因子的表达,因此我们推钡Aβ40/RAGE通路通过激活内质网应激产生级联反应促进ERK1/2与NF-κB磷酸化,进而影响瓣膜钙化。为了验证这一推测,我们通过内质网应激特异性抑制剂Tauroursodeoxycholic acid (TUDCA)干预细胞,在Aβ40孵育下观察ERK1/2与NF-κB磷酸化程度,免疫蛋白印迹结果显示随TUDCA浓度增高,Aβ40诱导的ERK1/2与NF-κB磷酸化水平递减。通过ERK1/2与NF-κB磷酸化特异性抑制剂PD 98059与BAY 11-7082抑制剂干预,观察BMP2与Runx2蛋白的表达,免疫蛋白印迹结果显示BMP2与Runx2的表达受到不同程度的抑制。上述结果提示Aβ40/RAGE通路能够诱导内质网应激反应并产生级联反应,激活ERKl/2与4F-κB磷酸化最终改变成骨因子的表达。通过siRNA干扰技术,抑制RAGE受体的表达,实时定量PCR结果显示Aβ40诱导IL-1β、TNF-α和MCP-1基因表达的效应显著降低。说明Aβ40/RAGE通路诱导产生的免疫炎症反应可能参与到瓣膜钙化的过程。结论本实验中,我们首先证实了CAVD患者主动脉瓣与动物瓣膜钙化模型中Aβ40/RAGE通路与内质网应激反应被激活；与造模组相比,在动物体内特异性敲除RAGE基因后瓣膜钙化程度及内质网应激反应均减轻。体外实验则进一步证明Aβ40与RAGE受体,内质网应激反应及成骨分化之间的相互作用机制。本文详细阐述了Aβ40/RAGE通路诱导内质网应激反应并产生级联反应,激活ERK1/2与NF-κB磷酸化并最终改变成骨因子表达的过程。更多还原

【Abstract】 AbstractAmyloid beta (Aβ) peptides enhance the activation of receptor for advanced glycation end products (RAGE) in immune-inflammatory diseases. RAGE exhibits several effects in the setting of numerous cardiovascular events. We hypothesized that the Aβ/RAGE pathway was involved in the pathogenesis of aortic valve (AV) calcification, and RAGE knockout intervention could attenuate AV calcification in ApoE-/-RAGE-/- mice via inhibiting the endoplasmic reticulum(ER) stress. To test our hypothesis, the expression of Aβ,RAGE, pro-osteogenic markers and ER stress relative protein were examined in human calcific AVs. We found that the expression of those proteins was significantly increased. These results were confirmed in the ApoE-/- mice model of AV calcification as well. RAGE knockout intervention significantly attenuated AV calcification, and simultaneously decreased Aβ40 accumulation, ER stress, pro-osteogenic markers expression and macrophage infiltration. In cultured human aortic valve interstitial cells (AVICs), we found that Aβ40 induced ER stress, inflammatory response, osteoblastic differentiation and calcification, largely dependent on RAGE signaling pathway. However, pretreatment with siRNA RAGE significantly suppressed RAGE-mediated production of MCP-1, IL-1β, TNF-a and pro-osteogenic factors. In addition, ER stress inhibitor (TUDCA) decreased the phosphorylation of both NF-κB and ERK1/2, the downstream signaling cascades of RAGE. Our data provide the first evidence that Aβ4O induced ER stress to promote inflammation and osteoblastic differentiation of AVICs via RAGE, thus promoting AV calcification, and that RAGE knockout intervention attenuated AV calcification via inhibiting the pathway in vitro and in vivo.Materials and MethodsChemicals and reagentsThe following antibodies were used:Aβ40 (Abcam ab110888), Runx2 (Abcam ab76956), bone morphogenetic protein2 (BMP2) (Abcam ab82511), RAGE (Abcam ab3611), phosphorylated extracellular-regulated kinase1/2 (p-ERK1/2) (Immunoway yp1197), total ERK1/2(Immunoway YT1625), phosphorylated p38 mitogen-activatedprotein kinase (p-p38 MAPK) (Immunoway yp0338), total p38 MAPK (Immunoway YT3513), phosphorylated NF-κB (p-NF-κB) (Immunoway yp0191), CD68 (Abcam ab201340) and total NF-κB (Immunoway YM3053), CHOP(Immunoway yp13811) and PERK(Immunoway yp16566). β-actin was used for normalisation. All chemicals and reagents used for cell isolation and culture were purchased from Hyclone. The lipofectamine 2000 transfection reagent and other transfection-related reagents were from Invitrogen. RIPA Lysis and Extraction Buffer with protease and phosphatase inhibitor cocktails for Western blotting were from Thermo Fisher Scientific. All other chemicals and reagents, including Aβ40 (A1075; purity 99%) and baicalin (CAS Number:21967-41-9; purity 98%), PD 98059, BAY 11-7082 were obtained from Beyotime Biotechnology Co. Both Aβ40 and the baicalin solution were generated as described previously [21,22].Human aortic valves collectionThis study complied with the Declaration of Helsinki, and was approved by the Review Board of Union Hospital and Tongji Medical College; all patients provided written informed consent. Calcific AV leaflets were obtained intraoperatively from 25 patients undergoing aortic valve replacement in Union Hospital due to severe CAVD. The relative thin and "normal" AV leaflets were collected from 10 age-matched patients undergoing Bentall surgery due to acute aortic dissection. The tissue samples were obtained and kept frozen in liquid nitrogen until use.Experimental animal modelAll animal experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animal study was approved by the Institutional Animal Research Committee of Tongji Medical College. Eight-week-old male ApoE-/-mice on C57BL/6J background (n=11) weighing 25-30g and their wild-type littermates (n=10) and ApoE-/-RAGE-/-mice on C57/6J background (n=11) were purchased from Nanjing University (China) and housed at the animal care facility of Tongji Medical College, at 25℃, under a 12h/12h light-dark cycle. AV calcification was generated using a 24-week protocol as described previously [23]. After initial quarantine, all the mice were normally assigned into three groups:control group (n=10), C57BL/6J mice receiving a high fat (HF) diet containing 0.15% cholesterol and 19.9% fat; ApoE group (n=11), ApoE-/-mice receiving the HF diet; ApoE-/-RAGE-/-group (n=11), ApoE-/-RAGE-/-mice receiving the HF diet.Echocardiography and tissue processingTransthoracic echocardiography was performed at baseline and the end of 24-week treatment respectively. The mice were sedated with 2.5%-isofluorane anesthesia. The images were acquired using a 18-38 MHZ phased-array probe (MS400) connected to a Vevo 1100 Imaging system. The echocardiographic data and related calculations were collected and performed by an experienced operator blinded to the assignments, as previously described. The flow velocity of the aortic valve was evaluated via continuous wave Doppler.After final transthoracic echocardiography assessments, the animals were euthanized via intravenous injection of a lethal dose of sodium pentobarbital (100mg/kg); their hearts were rapidly harvested, rinsed in PBS, fixed in 4% paraformaldehyde, and embedded in paraffin for subsequent morphological and histochemical analysis.Histopathology and immunohistochemistry stainingThe AVs were cut into 4-μm slices and stained with haematoxylin and eosin (H&E), as well as alizarin red. Immunohistochemical staining was performed as previously described. The sections were then incubated with antibodies against Ap (1:200), AP40 (1:300), RAGE (1:200), BMP2 (1:200), Runx2 (1:200) and CD68 (1:300). Image-Pro Plus (Media Cybernetics) was utilized to determine the quantitative results (positive staining area/total AV area) for 2 sections from each AV leaflet; at least 2 of the 3 AV leaflets from each animal were analyzed. The investigators performing the analysis were blinded to the study groups.Cell culture and treatmentHuman AVICs were isolated from the AV leaflets of patients undergoing Bentall surgery due to acute aortic dissection using collagenase digestion method as previously described. In brief, the AV leaflets were digested in DMEM containing 1 mg/ml collagenase type I at 37℃ for 30 min. After removal of endothelial cells by vortex, the leaflets were further digested with a fresh solution of 1 mg/ml collagenase medium for 6-8 h at 37℃. Repeated aspiration was performed to break up the aortic valves, and the cells were subsequently collected via centrifuge. The AVICs were cultured in DMEM with penicillin G, streptomycin, amphotericin B, and 10% FBS in an incubator with a 5% CO2 at 37℃. Passage 3-6 was used for all experiments. After 70-80% confluence, cells were serum-starved overnight and incubated with the indicated concentrations of either Aβ40 (OμM,25 μM,50 μM,100 μM) or TUDCA (1 μM,2 μM) for 72 hours. If needed, pharmacological reagents, including 40 uM PD 98059 (Beyotime Biotechnology),10 μM BAY 11-7082 (Beyotime Biotechnology), were added 1 h prior to the addition of Aβ40 or Baicalin. For mineralization experiments, AVICs were seeded in 24-well plates. After 70% confluence, cells were incubated with indicated interventions in DMEM supplemented with conditional medium (CM):5% FBS,2 mmol/L β-glycerophosphate (Sigma-Aldrich),100 nmol/L dexamethasone (Sigma-Aldrich), and 50 μg/ml ascorbic acid (Sigma-Aldrich) for 14 days.siRNA knock-down of RAGE expressionThe small interfering RNAs (siRNAs) against RAGE were a pool of three sequences provided by Ribobio, China, which were designed against RAGE. Scrambled siRNA duplexes with nonspecific sequences were used as negative control. After 60-70% confluence, AVICs were transfected with scrambled or RAGE siRNAs using Lipofectamine 2000 and Opti-MEM according to manufacturer’s recommendations, and changed the medium after 6 h. After 72 h, the cells were harvested for analysis of protein expression.Western blotting analysisThe tissue samples and the cells were both homogenized in RIPA Lysis and Extraction Buffer with protease and phosphatase inhibitor cocktails following manufacturer’s instructions. Western blotting was performed as previously described [12]. The following primary antibodies were used:Aβ4O (1:1000 dilution), A042 (1:1000 dilution), RAGE (1:1000 dilution), BMP2 (1:1000 dilution), Runx2 (1:1000 dilution), p-ERK1/2 (1:500 dilution),total ERK1/2 (1:1000 dilution), p-p38 MAPK (1:500 dilution), total p38 MAPK (1:1000 dilution), p-NF-κB (1:500 dilution) and total NF-κB (1:1000 dilution), CHOP (1:500 dilution), PERK (1:500 dilution). β-actin was used as normalization for total protein or cytosolic protein determination. In the phosphorylation induction assay, indicated total protein expression levels were used for normalization. Bands were quantified by densitometry using Quantity One Software (Bio-Rad, Hercules, CA).Real-Time polymerase chain reaction (PCR) RNA analysisReal-time PCR was used to detect the expression of mRNAs encoding interleukin (IL)-1β, tumor necrosis factor-a (TNF-a), monocyte chemoattractant protein-1 (MCP-1). RNA was isolated and reverse transcribed to cDNA as previously described. Real-time PCR assays were carried out using a SYBR_Premix Ex TaqTM (Takara) on a Step One Plus TM Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Primers were as folIows:IL-1β (F:5’-ATA AGC CCA CTC TAC ACC T3’,R:5’-ATT GGC CCT GAA AGG AGA GA-3’), TNF-a (F:5’-CCA ATG GCA GAG TGG GTA TG-3’,R:5’-TGA AGA GGA CCT GGG AGT AG-3’), MCP-1 (F:5’-GTC ACC AGC AGC AAG TGT C-3’, R: 5’-CCA GGT GGC TTA TGG AGT C-3’), p-actin (F:5’-GAC CTG ACC GAC TAC CTC-3’, R:5’-GCT TCT CCT TGA TGT CCC-3’). Results were normalized to p-actin expression and analyzed by the AACt method.Statistical analysisThe data are represented as means+standard deviation. Parameters were evaluated via either Student’s t-test or the one-way ANOVA, followed by LSD a post hoc multiple comparison test. P-values less than 0.05 were considered statistically significant.ResultsAβ/RAGE pathway and ER stress are up-regulated in human calcific AVsImmunohistochemical staining was performed to detect the expression of Aβ, RAGE, BMP2, Runx2, CHOP and PERK in calcific and normal AVs respectively. We observed the abundant presence of Aβ in calcific AVs compared to the normal ones, implicating the potential role of Aβ in AV calcification. As Aβ40 enhanced the action of RAGE agonist, we next evaluated the staining of RAGE. RAGE expression was obviously augmented in calcific AVs. Both BMP2, Runx2, CHOP and PERK have been verified to correlate with AV calcification.RAGE knockout intervention attenuates HF-diet-induced AV calcification in ApoE/RAGE-/-miceTransthoracic echocardiography demonstrated that the ApoE-/-mice fed with high-fat diet for 24 weeks developed AV calcification. Continuous wave Doppler was used to measure the peak aortic jet velocity. Compared with the controls, the transvalvular systolic velocity in ApoE-/-mice was increased markedly. However, the ApoE-/-RAGE-/-mice given RAGE knockout intervention showed significant reduction in transvalvular systolic velocity, compared with the ApoE-/-mice.Histological staining was performed with the AV paraffin sections. We observed increased AV leaflet thickness via HE staining in the ApoE-/-mice compared with the controls. The RAGE knockout intervention treated mice exhibited minor AV leaflet thickness. AV calcification was assessed using alizarin red staining. Calcium deposits in the leaflets were increased in the ApoE-/-group. In contrast, only mild calcification was observed in the ApoE-/-RAGE-/-mice. Additionally, we measured serum lipid levels and observed that RAGE knockout intervention had no influence on serum lipid levels.RAGE knockout intervention prevents HF diet-induced activation of Aβ, RAGE,pro-osteogenic factors and ER stress proteins in ApoE-/-RAGE-/- miceWe next evaluated the immunohistochemical staining of Aβ in AV leaflets of the ApoE-/-RAGE-/- mice, and found that it was present at higher levels compared with the controls, in agreement with our observations in human AVs. In parallel with the attenuated AV calcification, Aβ aggregation was also significantly decreased in ApoE-/- RAGE-/- group. Furthermore, the expression of RAGE and the two pro-osteogenic factors (BMP2 and Runx2) and ER stress relative proteins (CHOP and PERK) were also examined. These parameters were significantly increased in the ApoE-/- mice and decreased in the ApoE-/-RAGE-/- mice.Aβ40 induces the osteoblastic differentiation and calcification of AVICs through RAGE signaling pathwayOur in vivo data showed that Aβ40 aggregation as well as RAGE activation was involved in AV calcification. Previous studies have demonstrated that Aβ40 enhanced the activation of RAGE. However, it was unknown whether Aβ40 could promote the osteoblastic differentiation and calcification of AVICs through RAGE. To confirm this, we incubated human AVICs with different concentrations of Aβ40. We then examined expression of RAGE and several typical osteogenic biomarkers. The expression of RAGE, BMP2 and Runx2 significantly increased following Aβ40 stimulation, in a dose-dependent manner. And prolonged exposure to Aβ40 enhanced calcium deposits in AVICs. To further validate this, siRNA targeting RAGE was introduced. RAGE siRNA specifically and efficiently knocked down expression of RAGE, down-regulated expression of BMP2 and Runx2, as well as inhibited calcium deposition. Together, these results indicated that RAGE mediated the osteoblastic differentiation and calcification of AVICs induced by Aβ40.Aβ40/RAGE signaling pathway induces the the ER stressPreliminary data showed that alleviation of AV calcification and decreased Aβ40 accumulation as well as ER stress activation could be achieved by RAGE knockout intervention in the ApoE-/-RAGE-/-mice. We supposed that RAGE knockout intervention was able to prevent the activation of ER stress of AVICs induced by Aβ40. AVICs were pretreated with siRNA RAGE for 4-6 h and then stimulated with Aβ40 (50μM) for 72 h. As expected, siRNA RAGE down regulated the expression of CHOP and PERK, which was in line with the results in vivo. In addition, calcium deposition obviously decreased after TUDCA. This finding indicated that ER stress was downregulator of Aβ40/RAGE pathway.TUDCA inhibited the activation of RAGE downstream signaling pathwayWe stimulated AVICs in the presence of Aβ40(50 μM), with or without TUDCA. We then measured the phosphorylation levels of the downstream products of RAGE. Under basal conditions, the cultured AVICs exhibited low phosphorylation levels of NF-κB, ERK1/2 and p38 MAPK. However, treatment with Aβ40 significantly augmented the production of P-NF-κB and p-ERK1/2, but not p-p38 MAPK. The augmentation of p-NF-κB and p-ERK1/2 was blocked by TUDCA pre-treatment.We used Bay 11-7082 and PD 98059, inhibitors of NF-κB and ERK1/2 respectively, to confirm whether NF-κB and ERK1/2 signaling functionally mediated Aβ40-induced osteoblastic differentiation of AVICs. In the presence of Bay 11-7082 (10 μM) and PD 98059 (40μM), Aβ40-induced elevation of BMP2 and Runx2 was significantly inhibited as well as calcium deposition.RAGE knockout intervention protects against inflammation both in vitro and in vivoRAGE activation leads to persistent inflammation in a variety of cells. Aβ40 was also reported to induce macrophage activation in atherosclerosis. Given these findings, we sought to determine whether Aβ4O induces inflammatory response in AVICs through RAGE. We stimulated AVICs with Aβ4O(50μM) and measured inflammatory cytokine levels 24h later. Treatment with Aβ4Osignificantly augmented the production of MCP-1, IL-1β and TNF-a. This augmentation was blocked by RAGE knock-down pretreatment.We next evaluated whether RAGE knockout intervention exerted anti-inflammatory effect in AV leaflets in vivo. The ApoE-/- mice showed markedly increased macrophages infiltration and ER stress in AV leaflets, which was greatly attenuated in ApoE-/- RAGE-/-mice group. These results suggest that the pro-osteoblast and pro-inflammatory role of Aβ40/RAGE pathway promote AV calcification may be probably attributed to ER stress.ConclusionOur data provide the first evidence that Aβ40 induced inflammation and osteoblastic differentiation of AVICs via inducing ER stress signaling pathway, thus promoting AV calcification, and that RAGE knockout intervention attenuated AV calcification via inhibiting the pathway in vitro and in vivo.更多还原