Monocytes Augment Inflammatory Responses In Human Aortic Valve Interstitial Cells Via β2-integrin/ICAM-1-mediated Signaling

BackgroundCalcific aortic valve disease(CAVD)is a common acquired valve disease in the elderly.At present,no pharmacological intervention can significantly improve CAVD.Patients with advanced progressive CAVD may develop into aortic stenosis,heart failure,and even shock and death,which seriously threats human health and life.CAVD is a slow progressive disease.Chronic inflammation in the valve tissue plays a crucial role in CAVD progression.Monocyte infiltration and macrophage accumulation are found in diseased aortic valves.Soluble extracellular matrix(ECM)proteins can act as damage-associated molecular patterns(DAMPs)to induce pathophysiological processes associated with the CAVD progression in aortic valve interstitial cells(AVICs).While studies have shown that human AVICs exhibit inflammatory response to soluble matrilin-2,the impact of monocyte on this response remains unclear.The purpose of this study was to explore the mechanism by which monocyte enhances human AVIC inflammatory responses to soluble matrilin-2 via cell-cell interaction.MethodsHuman AVICs were isolated from normal aortic valve tissues and were cultured alone or cocultured with THP-1 monocytes.The culture systems were stimulated with soluble recombinant matrilin-2 at different concentration and time gradients,and the effects of matrilin-2 on AVIC inflammatory indexes and pro-inflammatory pathways were detected by immunoblotting and ELISA.Immunofluoresscent staining was used to observe the cell-cell interaction in the co-culture system.Then we neutralized β2-integrin and/or ICAM-1 with a specific antibody to study the role of β2-integrin and ICAM-1 in matrilin-2-induced cell interaction and AVIC inflammatory responses.In addition,we inhibited YAP and NF-κB with specific inhibitors to study the effects of and the link between YAP and NF-κB in AVIC inflammatory responses to matrilin-2.Finally,treated AVICs with human recombinant CD 18 to simulate the interaction between monocyte β2-integrin and AVICs,the expression of inflammation and YAP,and the phosphorylation of NF-κB in AVICs were detected under the stimulation of matrilin-2,with or without ICAM-1 neutralizing antibody,to further explore the role of β2-integrin and ICAM-1 in mediating proinflammatory signaling from monocytes to AVICs.ResultsMatrilin-2 dose-and time-dependently induced the expression of ICAM-1 and IL-6 in AVICs.Stimulation with 1 μg/mL of matrilin-2 for 48 hours can increase the levels of ICAM-1 and IL-6 in AVICs,and the expression of β2-integrin in monocytes,but matrilin-2 had no effect on monocyte production of ICAM-1 or IL-6.In the co-culture system,AVIC expression of ICAM-1 and IL-6 following an exposure to matrilin-2 was further enhanced by the presence of monocytes,and monocytes significantly adhered to the AVICs,Neutralization of β2-integrin or ICAM-1 decreased the number of monocytes adhering to AVICs and suppressed the expression of ICAM-1 and IL-6 induced by matrilin-2.Compared to monocultured AVICs,the levels of YAP and phosphorylated NF-κB in co-cultured AVICs upon matrilin-2 stimulation were also up-regulated by the presence of monocytes.Inhibition of YAP or NF-κB markedly reduced the inflammatory production in AVICs.Furthermore,treating monocultured AVICs with recombinant CD 18 had an effect of enhancing the expression of ICAM-1,IL-6 and YAP,as well as the phosphorylation of NF-κB induced by matrilin-2,while neutralization of ICAM-1 suppressed the effects.ConclusionsMonocyte β2-integrin interacts with AVIC ICAM-1 to augment AVIC inflammatory responses to soluble matrilin-2,among which YAP/NF-κB axis may be one of the key signaling pathways that transform β2-integrin/ICAM-1 signals into AVIC inflammatory cascade.Infiltrated monocytes in valve tissue may enhance AVIC sensitivity to proinflammatory damage-associated molecular patterns through cell-cell interaction with AVICs to promote valvular inflammation.