Abstract 4136901: Mechanosensitive channel protein Piezo1 synergizes with TLR4 to enhance osteogenic responses in human aortic valve interstitial cells

Background: Calcific aortic valve disease (CAVD), a common heart valve disease in the elderly, has a complex pathogenesis. Abnormal blood flow shear can be regarded as an important factor in the development of CAVD, and a key molecule that responds to mechanical stress is the mechanosensitive ion channel protein Piezo1; however, the role of Piezo1 in CAVD has not been clarified. Methods and results: We collected CAVD patients and their age- and sex-matched controls, and compared the differences in the protein expression levels of Piezo1 and TLR4 between the two using Western blot and other techniques, and found that the expression of Piezo1 and TLR4 proteins was elevated in the CAVD group. We gave LPS or Piezo1 agonist Yoda1 to stimulate aortic valve mesenchymal stromal cells, respectively, and the Western blot results showed that ALP and Runx2 expression were elevated after LPS and Yoda1 stimulation, and immunofluorescence and alizarin red staining showed similar results. Inhibition of TLR4 or Piezo1 attenuated the osteogenic differentiation of AVICs. Cellular immunofluorescence results showed that TLR4 could be transported from the Golgi to the cell membrane under Yoda stimulation, and Golgi-cell membrane transport of TLR4 was attenuated after Rab3b knockdown by siRNA. After first pre-stimulation with Yoda1 and then stimulation with LPS, Western blot and immunofluorescence results showed that the protein levels of ALP and Runx2 as well as NF-κB phosphorylation were to be significantly increased. Conclusion: Stimulation of Piezo1 and TLR4 can enhance the osteogenic response of AVICs. Stimulation of Piezo1 enhances the transport of TLR4 from the Golgi to the cell membrane via Rab3b, and Piezo1 synergistically enhances the osteogenic response of AVICs with TLR4, thus promoting the development of CAVD.