The Cell-specific Engagement of Notch and Wnt Pathways in Calcific Aortic Valve Disease

Abstract

Objective: The Notch and Wnt pathways participate in calcific aortic valve disease (CAVD), but with conflicting findings to date. We hypothesize that Notch and Wnt pathway engagement during CAVD initiation are cell-type specific. Methods: Primary porcine aortic valvular endothelial (PAVEC) and/or interstitial (PAVIC) cells were cultured in a 3D spring mechanical constraint system or in 2D culture and challenged with osteogenic media (OGM) to induce calcification. DAPT and IMR-1 were used as Notch1 inhibitors, and lentiviral transduction was used to create constitutively-active b-catenin (B-cat+) PAVIC and doxycycline-inducible Notch1 intracellular domain (NICD+) PAVEC and PAVIC. Calcification was evaluated with alizarin red staining. Results: PAVIC treatment with Notch inhibitors DAPT and IMR-1 resulted in opposite calcific responses, and this correlated with active-form b-catenin signaling. Further interrogation using B-cat+ PAVIC showed increased calcification compared to wild-type (WT) cells. Treatment of B-cat+ PAVIC with DAPT, but not IMR-1, was able to reduce OGM-induced calcification to different extents in B-cat+ and WT cells. NICD overexpression was investigated in PAVIC and PAVEC. Culture of NICD+ PAVIC in 3D decreased calcification compared to controls, while NICD+ PAVIC and PAVEC co-culture increased calcification. Gene expression data in 2D NICD+ VEC found no difference in EndMT SNAI1/2 expression, and an increase in osteogenic genes Runx2, BMP2, SPARC, and SPP1 vs. WT controls. Conclusions: We demonstrate a divergence in Notch1 regulation of calcification with PAVIC DAPT and IMR-1 treatment with and without constitutively active b-catenin, and with NICD+ PAVIC monoculture vs. NICD+ PAVIC and PAVEC co-culture. Given our previous findings demonstrating early upregulation and later downregulation of Notch1 in VEC with TNFa-stimulus, our results are consistent with the hypothesis that Notch1 signaling has a dynamic and cell-specific influence over CAVD progression. Nuances in Notch1 and Wnt signaling could be vital to development of a CAVD therapeutic.