Calcific aortic valve disease (CAVD) is a chronic, progressive inflammatory disease. Soluble extracellular matrix (ECM) proteins can function as damage-associated molecular patterns (DAMPs) and may play a role in the progression of CAVD. Matrilin-2 is an ECM protein and has been found to up-regulate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho is an anti-aging protein that is recently found to have an anti-inflammatory effect. The impact of matrilin-2 and Klotho on AVICs inflammatory response is unclear. This study is to test the hypothesis that matrilin-2 induces the inflammatory response in human AVICs and to explore the anti-inflammatory potential of Klotho for suppression AVIC inflammation. Methods and Results: Human AVICs isolated from normal valves were treated with recombinant matrilin-2 (2.0 μg/ml). Matrilin-2 caused NF-κB-dependent increase in the levels of ICAM-1, MCP-1 and IL-6. In addition, matrilin-2 induced rapid activation of PKR through Toll-like receptor (TLR) 2 and 4. Treatment with PKR inhibitors, 2-AP or C13H8N4OS, prior to matrilin-2 stimulation, abrogated NF-κB phosphorylation and intranuclear translocation. Inhibition of PKR abolished the production of inflammatory mediators induced by matrilin-2 in human AVICs. Further experiments using recombinant Klotho revealed that Klotho (0.5 μg/ml) suppressed the activation of PKR and NF-κB, and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. Conclusion: This study demonstrates that soluble matrilin-2 induces the inflammatory response in human AVICs through a TLR-PKR-NF-κB signaling cascade and that Klotho is capable of suppressing human AVICs inflammatory response to a soluble ECM protein. The novel findings of this study indicate that soluble ECM proteins may fuel the progression of CAVD by inducing aortic valve inflammation and that Klotho has the potential for suppression of such inflammation.