Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells

[Background]: Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. [Methods]: TLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity. [Results]: Interstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity. [Conclusions]: Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.This study was supported by Red Recava from Instituto de Salud Carlos III, and grants from CNIC-Translational CNIC-09-2007, Junta de Castilla y León HUV03A08, BIO39/VA28/10 and GR230; grants from Plan Nacional de Salud y Farmacia SAF2006/08031 (co-funded by FEDER-FSE2000/ 2006), and SAF2009/08692. MIFP is a fellow from the FPI Program, Ministerio de Ciencia y Tecnología. A.I.D. is a researcher from the Miguel Servet Program, Instituto de Salud Carlos III.Peer Reviewe