| BackgroundCalcific aortic valve disease(CAVD)is a common cardiovascular disease in the elderly.In developed countries,one-third of people over age 60 have echocardiographic or radiological evidence of aortic valve calcification,and 10%of them exhibit CAVD.In China,a study has found the overall prevalence of CAVD in people more than 45 years of age is 12.8%.The global prevalence of CAVD is expected to substantial increase given the projected increase in the world’s population and increase in aging population.Currently,there is no pharmacological intervention capable of delaying or halting CAVD progression.There is only surgical or percutaneous interventional valve replacement to cure severe aortic stenosis.The survival time of patients with symptomatic severe aortic stenosis is significantly shortened.CAVD threatens patients’ life and brings serious medical burden to all over the world.The pathogenesis of CAVD remains not well understood.Hence there is of foremost importance to delineate and understand the underlying mechanisms responsible for the development of CAVD.This disease was originally thought to be a degenerative disease associated with aging.However,it is becoming increasingly apparent that it is an active and regulated process,involving inflammation,lipid accumulation,extracellular matrix remodeling,osteogenic activities and calcification.The calcific aortic valve disease is similar to atherosclerosis in risk factors,symptoms and pathogenesis.Many studies have confirmed that inflammation plays an important role in the process of aortic valve calcification and most scholars believe that CAVD is a chronic inflammatory disease.The truths that oral infections bacteria exist in calcific stenotic aortic valve lesions and inoculation of experimental rabbits with the oral bacteria induce calcific aortic valve disease speculate that chronic infection and inflammation plays an important role in the pathogenesis and progression of aortic valve calcification.The pathogenesis study of atherosclerosis have found that bacteria and virus infection is closely related to atherosclerosis.Many viruses such as herpes virus,cytomegalovirus,EB virus and so are involved in atherosclerosis.At present,study about virus infections and aortic valve calcification is few and the effect and pathophysiological mechanism of virus infection on aortic valve is unclear.Exogenous double strand RNA(dsRNA)is produced by viruses during replication and can be recognized by pattern recognition receptors to induce immune and inflammation to defence virus infection.Endogenous dsRNA is produced from overlapping convergent transcription of distinct protein-coding genes and is released from damaged or necrotic cells.Indeed,extracellular nucleic acids,including double-stranded forms,act as endogenous molecule to modulate the inflammatory responses associated with myocardial ischemia-reperfusion injury,autoimmune diseases,renal failure,diabetes mellitus and neurodegenerative disease.The role of exogenous or endogenous dsRNA in aortic valves during the process of calcification and sclerosis is unknown.Toll-like receptor 3(TLR3)is a major sensor for exogenous and endogenous dsRNA.Toll-like receptors(TLRs)family is pattern recognition receptors for pathogen-associated molecular patterns and damage-associated molecular patterns.In addition to playing a key role in host defense against danger,activation of TLRs has been linked to the pathogenesis of many inflammatory and autoimmune diseases.TLR3 expresses immune and non-immune cells and localizes on cell surface and/or in intracellular endosome.TLR3 recognizes endogenous and exogenous dsRNA,and then utilizes TIR domain containing adaptor inducing interferon-β(TRIF)to activate interferon regulatory transcription factor 3(IRF3)and nuclear factor κB(NF-κB)and mitogen-activated protein kinases(MAPKs)for signal transduction to nuclei,and control of gene transcription and protein expression involving in the physiological and pathological processes.Aortic valve interstitial cells(AVICs),the main cellular component of the aortic valve,play an important role in the pathogenesis of calcific aortic stenosis.In this regard,atherogenic factors and mechanical forces can stimulate AVICs to produce cytokines,growth factors,matrix proteins and osteogenic mediators.Our previous studies have demonstrated that AVICs can produce cytokines and differentiate into osteoblast-like cells when TLR2/4 stimulation.Currently,the effect of dsRNA and TLR3 on aortic valve calcification and the influence of them on AVICs are unknown.Polyinosinic-polycytidylic acid[poly(I:C)]is a synthetic analog of dsRNA and has been used widely in in vivo and in vitro studies to determine the effect of dsRNA.Several studies show that poly(I:C)induces the production of cytokines and stimulation of human mesenchymal stromal cells with poly(I:C)promotes their osteogenic differentiation potential.So we choose poly(I:C),the mimic of dsRNA,to treat human AVICs and observe the expression of inflammatory and osteogenic mediators and calcium deposition and nodule formation in AVICs.We also elucidate the possible signaling mechanism involved in these effects.The research findings will provide theoretical basis for potential target to prevent and control the calcific aortic valve disease.ObjectiveThe study objective is to determine the effect of dsRNA and TLR3 on human AVICs to induce the inflammatory and osteogenic responses and in vitro formation of calcium deposits,and to elucidate the mechanisms of these effects.Method1.AVICs were isolated from normal human valves and cultured.Cells of passage 4 to 6 were used for this study.Cells were subcultured on plates and treated when they reached 80%to 90%confluence.2.AVICs were exposed to poly(I:C)for 2 hours to 24 hours.The levels of IL-6,IL-8 and MCP-1 in cell cutured supernatant were analyzed with ELISA.The expression of ICAM-1,BMP-2,TGF-β1 and ALP was analyzed with western immunoblotting.3.AVICs were exponsed to poly(I:C)prolonged for 2 weeks.ALP activity was analyzed with ALP activity staining and calcium deposit formation was analyzed with Alizarin red S staining.4.AVICs were exposed to poly(I:C)for 30 minutes to 24 hours.The phosphorylation of NF-κB p65 and ERK1/2 was analyzed with western immunoblotting.5.AVICs were treated with TLR3 siRNA or TRIF siRNA for 72 hours to knockdown of TLR3 and TRIF expression.AVICs were exposed to poly(I:C)for 2 hours to 24 hours.The phosphorylation of NF-κB p65 and ERK1/2 and the expression of ICAM-1,BMP-2,ALP and TGF-β1 were analyzed with western immunoblotting.The levels of MCP-1,IL-6 and IL-8 were analyzed with ELISA.6.AVICs were exposed to poly(I:C)for 24 hours with or without the inhibitors of NF-κB or ERK1/2 prior to 1 hour.The levels of MCP-1,IL-6 and IL-8 were analyzed with ELISA and the expression of ICAM-1,BMP-2,ALP and TGF-β1 was analyzed with western immunoblotting.7.AVICs were exposed to poly(I:C)for 24 hours with or without NF-κB p50 migration inhibitor(SN50)prior to 1 hour.The levels of MCP-1,IL-6 and IL-8 were analyzed with ELISA and the expression of ICAM-1 was analyzed with western immunoblotting.The expression of ICAM-1 induced by LPS with SN50 was as positive control.8.AVICs were exposed to poly(I:C)or LPS for 30minutes to 24 hours and the expression of NF-κB p50 was analyzed with western immunoblotting.AVICs were exposed to poly(I:C)or LPS for 2 hours and 4 hours and NF-κB p50/p65 heterodimer was examined with co-immunoprecipitation.9.AVICs were exposed to poly(I:C)or LPS for 2 hours and 4 hours with or without SN50 prior to 1 hour and translocation of NF-κB p50 and p65 into nucleus was analyzed with immunofluorescence staining.Results1.Poly(I:C)(0.5 μg/mL-2.5 μg/mL)induced the protein expression of ICAM-1,BMP-2,TGF-β1 and ALP with dose-response fashion in human AVICs.Poly(I:C)(2.5 μg/mL)upregulated expression of ICAM-1,BMP-2,TGF-β1 and ALP respectively about 11 times,2.4 times,2.1 times and 1.8 times compared with control(P<0.05).Poly(I:C)induced the massive release of IL-6,IL-8 and MCP-1 in cultured supernatant of AVICs:IL-6(134.279±19.661 pg/mL VS 668.317±71.411 pg/mL),IL-8(10.198±6.363 pg/mL VS 1345.647±339.309 pg/mL),MCP-1(254.893±58.556 pg/mL VS 782.355±123.391pg/mL)(P<0.05).2.The level of IL-1β induced by poly(I:C)had no significant difference with the untreated control(P>0.05).The level of IL-1β in culture media was fairly low(31 pg/mL or lower).3.Prolonged treatment with poly(I:C)for 2 weeks increased ALP activity and resulted in calcium deposit formation in human AVICs.The data from spectrophotometric analysis show calcium deposit induced by poly(I:C)increased about 4.5 times compared with control(P<0.05).4.Poly(I:C)induced quick and sustained phosphorylation of NF-κB p65 and ERK1/2.The level of phospho-NF-κB increased at 1 hour,attended the highest peak at 2 hours and remain elevated at 4 hours in AVICs after being exposed to poly(I:C)(P<0.05).The level of phospho-ERK1/2 increased at 30 mimutes,attended the highest peak at 2 hours and remain elevated at 8 hours exposed to poly(I:C)(P<0.05).5.Knockdown of TLR3 and TRIF with specific siRNA markedly decreased the expression of TLR3 and TRIF.The expression of TLR3 decreased 60%and the expression of TRIF almost dispeared(P<0.05).Silencing of TLR3 and TRIF essentially abrogated the phosphorylation of NF-κB p65 and ERK1/2 and the phosphorylation of NF-κB p65 and ERK1/2 at 2 hours decreased 2 and 4 times respectively compared with poly(I:C)alone(P<0.05).Knockdown of TLR3 and TRIF markedly reduced the expression of ICAM-1,BMP-2,TGF-β1 ALP and the level of IL-8,IL-6,MCP-1 induced by poly(I:C)in human AVICs(P<0.05).The level of IL-8 decreased but still higher than the untreat control when knockdown of TLR3(489.453±65.207 pg/mL VS148.021±52.088 pg/mL,P=0.024).The level of IL-8 decreased and has no difference with untreat control when knockdown of TRIF(P>0.05).6.Inhibition of NF-κB with IKK inhibitor markedly reduced the expression of BMP-2,TGF-β1 ALP and ICAM-1 and levels of IL-6,IL-8 and MCP-1 in cells exposed to poly(I:C)(P<0.05).7.Inhibition of ERK1/2 with the specific inhibitor only markedly reduced the expression of BMP-2,TGF-β1 ALP induced by poly(I:C)(P<0.05)but did not suppress the up-regulation of ICAM-1,IL-6,IL-8 and MCP-1 induced by poly(I:C)(P>0.05)..8.The NF-κB p50 migration inhibitor SN50 cannot inhibit the up-regulation of ICAM-1,IL-6,IL-8 and MCP-1 induced by poly(I:C)and even further increased the level of ICAM-l about 1.7 times and the level MCP-1(1702.690±135.736 pg/mL VS 1226.033±76.326 pg/mL)compared to poly(I:C)alone(P<0.05).On the contrary,SN50 can reduce the expression of ICAM-1 induced by LPS compared to LPS alone(P<0.05).9.NF-κB p50 was expressed in AVICs and there was no difference between exposed to poly(I:C)and exposed to LPS.The result of co-immunoprecipitation showed that NF-κB p65/p50 heterodimer exited in human AVICs,and after stimulation with poly(I:C)or LPS the heterodimer still exited.Moreover,there was no difference between exposed to poly(I:C)and exposed to LPS.The immunofluorescent staining results showed that poly(I:C)and LPS both induced quick translocation of NF-κB p50 and p65 subunits into nuclei.After pretreatment with SN50,the intranuclear localization of p50 induced by both poly(I:C)and LPS was suppressed,but the intranuclear localization of p65 induced by poly(I:C)still obviously existed,while the intranuclear localization of p65 induced by LPS almost disappeared.Conclusion1.DsRNA up-regulates the production of pro-inflammatory cytokines ICAM-1,IL-6,IL-8,MCP-1 and pro-osteogenic mediators BMP-2,TGF-β1,ALP in human AVICs.Prolonged exposition to dsRNA promotes ALP activity and calcium deposit formation in human AVICs.2.The pro-inflammatory effect of dsRNA is primarily mediated by TLR3-TRIF-NF-κB pathway and the pro-osteogenic effect is mediated by TLR3-TRIF-NF-κB and ERK1/2 pathways in human AVICs.3.DsRNA is incapable of activating the NLRP3 inflammasome in human AVICs.4.NF-κB dimmer complex activated by TLR3 is not the classical p65/p50 heterodimer.In this study,we firstly reveal that TLR3 activation by dsRNA in human AVICs up-regulates the expression of pro-inflammatory and pro-osteogenic mediators,and formation of calcium deposits.Further,we demonstrate the signaling mechanisms of these effects.Thus,TLR3-mediated AVIC inflammatory and osteogenic responses may contribute to the mechanism underlying CAVD progression.Understanding of the role of the innate immunoreceptors in CAVD may help in the development of pharmacological treatments for halting CAVD progression. |
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