P38 MAP Kinase in Valve Interstitial Cells is Activated by Angiotensin II or Nitric Oxide/Peroxynitrite, but Reduced by Toll-Like Receptor-2 Stimulation

Background and aim of the study: The involvement of p38 MAPK in mediating factors that may produce aortic valve disease is unknown. Angiotensin II (Ang II) has been implicated in the development of aortic stenosis through either the generation of free radicals and/or the modulation of inflammatory responses. A variety of proinflammatory factors utilize Toll-like receptors, and these may also play a role in the development of aortic valve disease. Methods: Valve interstitial cells (VICs) were cultured from porcine aortic valves. Cells were treated with Ang II, 3-morpholinosydnonimine (SIN-1), which liberates NO and superoxide anion generating peroxynitrite, or the lipopetide Toll-like receptor-2 (TLR-2) agonist Pam3CSK4 Results: In response to Ang II (1 mu M), MAPK phosphorylation levels were increased by 3.5-fold after 15 min, peaked at 4.6-fold after 60 min, and decreased to 1.9-fold greater than control after 120 min of treatment. In response to SIN-1, phosphorylation levels were increased progressively throughout the 90 min of treatment and were significantly (p <0.05) twofold (1.9 +/- 0.3) greater than control or native p38 MAPK (2.3 +/- 0.4) after 90 min. SB202190, a relatively selective inhibitor of the p38 alpha MAPK isoform, reduced SIN-1-induced p38 MAPK phosphorylation. In contrast, there was a rapid and marked decline in phosphorylated p38 MAPK, in response to Pam3CSK4 that was evident at 30 min; after 90 min, the p38 MAPK level was 85% lower than baseline. Conclusion: p38 MAPK is present in VICs, and is activated by Ang II. Peroxynitrite similarly increased p38 MAPK phosphorylation, which suggests that these two factors involve similar pathways in their effect on VICs. Alternatively, peroxynitrite may be involved in the pathway by which Ang II activates p38 MAPK. The dramatic reduction in p38 MAPK phosphorylation by TLR-2 stimulation excludes a role for this receptor type in mediating Ang II or peroxynitrite effects, and suggests that inflammatory factors that act through TLR-2 to dephosphorylate p38 MAPK utilize pathways different from Ang II or peroxynitrite, to produce their effect on the aortic valve.