The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzymelinked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.

Background Calcific aortic stenosis is a chronic inflammatory disease, and aortic valve interstitial cells (AVICs) play an important role in valvular inflammation. While AVICs from stenotic aortic valves exhibit an augmented response to Toll-like receptor 4 (TLR4) stimulation, the underlying mechanism is unclear. This study tested the hypothesis that an excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the inflammatory response to lipopolysaccharide (LPS) in AVICs of stenotic valves. Methods and Results Human AVICs were isolated from normal and stenotic leaflets. NF-κB activation, and production of IL-8, MCP-1 and ICAM-1 were analyzed following treatment with LPS. The role of Notch1 in the inflammatory response was determined using inhibitor, siRNA and specific ligand. Cells from diseased valves produced greater levels of chemokines and ICAM-1 that are associated with enhanced NF-κB activation. Interestingly, diseased cells exhibited augmented Jagged1 release and Notch1 activation after TLR4 stimulation. Inhibition and silencing of Notch1 each resulted in greater suppression of the TLR4-induced inflammatory response in diseased cells. Conversely, activation of Notch1 with a specific ligand Jagged1 enhanced the LPS-induced inflammatory response in normal AVICs. Further, Notch1 intracellular domain was co-immunoprecipited with IKK following LPS stimulation, and inhibition of Notch1 abrogated the difference in the level of NF-κB activation between diseased and normal cells. Conclusion Notch1 enhances the inflammatory response to TLR4 stimulation in human AVICs through modulating NF-κB activation. Excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the TLR4 response in AVICs of stenotic valves.