The Crosstalk Between Monocyte And Aortic Valve Interstitial Cell Promotes The Development And Progression Of Calcific Aortic Valve Disease

BackgroundCalcific aortic valve disease(CAVD)is the most prevalent heart valve disorder in the elderly.The pathobiology of CAVD involves valvular fibrosis and calcification associated with chronic valvular inflammation.Further,inflammatory response in aortic valve interstitial cells(AVICs),the predominant cell in aortic valve,elevates aortic valve fibrogenic and osteogenic activitie.Recent studies suggest that valvular inflammation associated with monocyte infiltration promotes CAVD progression.However,the pro-inflammatory and pro-fibrocalcific communication between monocytes and AVICs,and the underlying mechanism are unclear.We hypothesized that monocytes up-regulate AVIC inflammatory activity and promote the fibrogenic and osteogenic activities.This study sought to characterize the interaction between monocytes and AVICs and to elucidate the underlying mechanism.Part 1 Monocytes enhance the inflammatory response in aortic valve interstitial cells through paracrine effectObjectTo investigate pro-inflammatory communication of activated monocytes with AVICs and the mechanism by which monocytes modulate the inflammatory activity in AVICs.Method1.AVICs and monocytes co-culture:AVICs and monocytes were co-cultured(the ratio of AVICs to monocytes is 1 to 3).Pam3CSK4 in 0.03 μg/ml was applied to co-cultured AVICs and monocytes for 24 h.AVICs and monocytes were lysed together.Immunoblotting was applied to analyze ICAM-1 and VCAM-1 levels in AVICs and monocytes.ELISA was applied to measure MCP-1 levels in the medium.2.Condition medium:The condition medium from Pam3SCK4(0.1μg/ml)treated monocytes was collected as Pam3 CM.The condition medium from non-treated monocytes was collected as Ctrl CM.3.Condition medium treatment:AVICs were treated with Pam3CM,Ctrl CM and 0.03 μg/ml Pam3SCK4 for 24 h.Immunoblotting was allied to analyze ICAM-1,VCAM-1 MyD88 and TLR2.ELISA was applied to measure MCP-1 level in the medium.For time trail test,AVICs were treated with Pam3 CM for 4,8,12 and 24 h.Immunoblotting and immunofluorescent staining were applied to analyze TLR2 expression.4.TLR2 inhibition or knockdown:TLR2 inhibitor CU CPT 22 was applied to AVICs before Pam3 CM treatment for 24 h.AVICs were transfected with TLR2 shRNA prior to Pam3 CM treatment for 24 h.Immunoblotting was used to analyze TLR2,ICAM-1 and VCAM1 levels in AVICs.MCP-1 level in the medium was measured by ELISA.5.Cytokines and chemokines analysis:Quantitative Multiplex ELISA Arrays was applied to measure 18 cytokines and chemokines levels in Pam3 CM and Ctrl CM.ELISA was used to further confirm TNF-α level in Pam3 CM and Ctrl CM.6.TNF-α neutralization:Pam3CM was incubated with TNF-α neutralizing antibody for 1 h before added to AVICs for 24 h.Immunoblotting was applied to analyze TLR2,ICAM-1 and VCAM-1 levels in AVICs.7.To test the pro-inflammatory effect of TNF-α:AVICs were treated with TNF-αrecombinant protein in 1 or 2 ng/ml.Immunoblotting was applied to analyze TLR2,ICAM1 and VCAM-1 levels in AVICs.Result1.When AVICs and monocytes were co-culture,TLR2 agonist Pam3CSK4 in 0.03pg/ml can up-regulate the expression of ICAM-1 and VCAM-1,and increase the production of MCP1.2.AVICs expressed higher levels of ICAM-1,VCAM-1 and MCP-1 under Pam3 CM treatment,while Ctrl CM or Pam3CSK4 in 0.03 μg/ml has no effect on ICAM-1,VCAM1 and MCP-1 production in AVICs.3.TLR2 inhibitor,CU CPT 22 can suppress the up-regulation of ICAM-1,VCAM-1 and MCP-1 induced by Pam3 CM in AVICs.4.Pam3 CM induced the up-regulation of TLR2 in AVICs.When the up-regulation of TLR2 was abolished by TLR2 shRNA,the effect of Pam3 CM on increasing ICAM-1 and VCAM-1 expression was suppressed.5.Pam3 CM has higher levels of TNF-α,and the neutralization of TNF-α abolished the effect of Pam3 CM on up-regulating TLR2 expression in AVICs and suppressed the increase of ICAM-1 and VCAM-1 in AVICs.6.TNF-α in 1 or 2 ng/ml can up-regulate TLR2 expression in AVICs,but TNF-α in these doses failed to induce ICAM-1 and VCAM-1 production.ConclusionThis study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory response.The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α secreted from activated monocytes.Infiltrated monocytes in aortic valve tissue may contribute to valvular inflammation associated with CAVD progression by rendering AVICs hypersensitive to proinflammatory stimuli.Part 2 Cytokines from activated monocytes promote fibrocalcific activity in aortic valve interstitial cellsObjectTo determine the effect of activated monocytes on AVIC fibrocalcification,identify the responsible factors from activated monocytes and elucidate the molecular mechanism underlying the effect of monocyte.Method1.Condition medium:The condition medium from Pam3SCK4(0.1 μg/ml)treated monocytes was collected as Pam3 CM.The condition medium from non-treated monocytes was collected as Ctrl CM.2.Analyzing collagen and calcium deposition:AVICs were treated with Pam3 CM for 14 days and Picro-sirius red was applied to analyze collagen deposition.AVICs were culture in pro-osteogenic medium following Pam3 CM treatment for 10 days.Alizarin red was applied to analyze calcium deposition.3.Pam3 CM treatment:AVICs were treated with Pam3 CM,Ctrl CM and 0.03 μg/ml for 48 h.Immunoblotting was allied to analyze collagen-Ⅰ,collagen-Ⅳ,MMP-2,MMP-9,ALP and Runx2 levels in AVICs.4.TNF-α and RANTES neutralization:Pam3 CM was treated TNF-α or RANTES neutralizing antibodies for 1 h before added to AVICs for 48 h.Immunoblotting was applied to analyze collagen-I,MMP-2 and ALP levels in AVICs.5.To analyze the activation of MAP kinase and NF-κB signaling pathway:phosphorylation of p38,ERK1/2,JNK and NF-κB were analyzed by immunoblotting after Pam3 CM treatment.Pam3 CM was treated TNF-α and/or RANTES neutralizing antibody for 1 h before added to AVICs,and phosphorylation of p38,ERK1/2,JNK and NF-κB were analyzed by immunoblotting.6.To test the effect of JNK and NF-κB pathway:SP600125 and Bay 11-7082 were applied to AVICs for 1 h to suppress the activation of JNK and NF-κB pathway respectively following Pam3 CM for 48 h.Immunoblotting was allied to analyze collagen-I,MMP-2 and ALP levels in AVICs.Result1.Pam3 CM induced AVICs collagen and calcium deposition,while Ctrl CM or Pam3CSK4 in 0.03 μg/ml has no effect on collagen and calcium deposition.2.Pam3 CM up-regulate the expression of the osteogenic mediator ALP,and fibrogenic mediators collagen-I and MMP-2 in AVICs.3.Pam3 CM has higher levels of TNF-α and RANTES.Neutralizing TNF-α attenuated the effect of Pam3 CM on the production of ALP,collagen-I and MMP-2,while neutralizing RANTES reduced the effect of Pam3 CM on collagen-I and MMP-2 production without an effect on ALP production.4.Pam3 CM induced the activation of NF-κB and MAP kinase in AVICs.Neutralizing TNFa attenuated phosphorylation of NF-κB and JNK,while neutralizing RANTES suppressed phosphorylation of JNK.5.NF-κB mediated the effect of Pam3 CM on ALP production while JNK mediated the effect of Pam3 CM on collagen-Ⅰ and MMP-2 production in AVICs.ConclusionThis study demonstrates that Pam3 CM increases fibrocalcification in AVIC and increase the production of osteogenic and fibrogenic mediators in AVICs.In Pam3 CM,TNF-α mediates the osteogenic mediator production through NF-κB pathway.Both TNF-α and RANTES contribute to the up-regulation of fibrogenic mediators induced by Pam3 CM through JNK pathway in AVICs.This study suggests that infiltrated monocytes may promote CAVD progression through promoting aortic valve fibrocalcification...