Abstract Body: Background: Tumor Necrosis Factor-alpha (TNFα) is a key inflammatory cytokine involved in calcific aortic valve disease (CAVD). Following mechanical stress, activated monocytes, macrophages induce TNFα signaling in aortic valve (AoV). The activated TNFα binds to its own receptors, TNFR1 and TNFR2, in which TNFR1 has a death domain likely to cause cell death in AoV. Subsequently, the release of extracellular vesicles (calcium and inorganic phosphates) following inflammatory process results in valve calcification and stenosis.
Hypothesis: Blocking TNFα-mediated inflammation by genetic knockout of Tnfr1/Tnfr2 slowdown CAVD progression in mice.
Methods and Results: Using a mouse model of CAVD with deletion of Ldlr, low-density lipoprotein receptor, we compared the disease progression in mice fed with standard and high fat diet. To get better understanding on the role of TNFα in CAVD, Ldlr^-/- mice (3 months old) on the TNFR1 and TNFR2 knockout background, were also fed with high fat diet for 3 months (Ldlr^-/-; Tnfr1^-/-: Tnfr2^-/-). Aortic valve function was analyzed by using echocardiogram and it showed decreased AoV velocity and increased ejection fraction in Ldlr^-/-; Tnfr1^-/-: Tnfr2^-/- compared to Ldlr^-/-; Tnfr1^+/-: Tnfr2^+/- group. The pathological changes such as fibrosis, calcium and collagen deposition seen under routine and specialized histological staining methods were reduced in TNF receptors knockout group. Next, using customized designed for PCR arrays, we examined the expression of 62 candidate genes involved in inflammation, apoptosis, extracellular matrix remodeling or aortic valve stenosis pathways. The top downregulated genes in the Ldlr^-/-; Tnfr1^-/-: Tnfr2^-/- group compared to Ldlr^-/-; Tnfr1^+/-: Tnfr2^+/- group are (Col3a1, MMp7, Klk1) whereas the top upregulated gene are (Igf1r and MMp14). We are currently validating these findings by using qPCR and immunofluorescence.
Conclusion: Our findings reveal blocking TNFα signaling receptors downregulate the genes related to inflammatory cytokine activation and aortic valve stenosis in the Ldlr knockout mice fed with high fat diet. Blockage of TNFα likely reduces apoptosis and inflammation while improves extracellular matrix remodeling to slowdown CAVD progression. Our findings thus suggest a new therapeutic strategy for CAVD.