| 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. |
PDF Full Text Request