IGF1-mediated mesenchymal-endothelial transition as a potential regulatory target in calcific aortic valve disease

Background: Aortic valve interstitial cells (VICs) are considered a highly plastic heterogeneous mesenchymal cell population. Although previous studies have demonstrated their potential to differentiate into myofibroblasts, osteoblasts, chondrocytes, and adipocytes, their ability to differentiate into endothelial cells has not been confirmed. Notably, normal aortic valve tissue is avascular, but in the diseased valves of patients with calcific aortic valve disease (CAVD), we often find cells with an endothelial phenotype, but the origin of these cells is unclear.

Methods: We performed paraffin section immunofluorescence analysis on calcified aortic valves from 16 patients with CAVD. Primary VICs and valve endothelial cells (VECs) were isolated from normal porcine aortic valves. Endothelial cell-related functional assays, including in vivo Matrigel plug assay and in vitro tube formation assay, demonstrated the acquisition of endothelial functions by VICs. Transcriptome Sequencing, western blot, qPCR, and immunofluorescence were used to identify the molecular mechanisms underlying mesenchymal-endothelial transition (MEndT). We constructed a CAVD mouse model by wire injury using Col1a2-CreERT: R26R-tdTomato mice. Finally, our results were further validated using publicly available single-cell Sequencing data related to CAVD.

Results: We identified a rarely discussed population of CD31-positive and α-SMA-negative non-vascular structural cells in the calcified aortic valves. Following osteogenic stimulation, we demonstrated through in vitro and in vivo experiments that VICs showed increased expression of endothelial-associated markers and acquired endothelial cell-related functions. We demonstrated that the IGF1-PI3K-AKT-HIF pathway mediates MEndT. Furthermore, in the CAVD mouse model, we confirmed that intraperitoneal injection of recombinant mouse IGF1 protein significantly increased the proportion of endothelial cells of mesenchymal origin in the aortic valve and alleviate disease progression; conversely, intraperitoneal administration of the IGF1R inhibitor BMS-536924 reversed this effect. Analysis of single-cell Sequencing data from two different CAVD datasets revealed that as valve interstitial cells differentiate toward an osteogenic lineage, they also enrich biological processes related to endothelial cell function.

Conclusions: We provide the first evidence that VICs have the capability to undergo endothelial transition under calcific conditions and that MEndT occurs in aortic valve of the CAVD mouse model. These findings reveal the presence of MEndT in CAVD, offering a potential therapeutic target.

Calcific aortic valve disease; IGF1; Mesenchymal-endothelial transition.