Rat Aortic Valve Interstitial Cells

Glycosaminoglycans Affect Endothelial to Mesenchymal Transformation, Proliferation, and Calcification in a 3D Model of Aortic Valve Disease

CAVD involves multifaceted processes in the valve microenvironment, resulting in matrix reorganization, calcification, and functional impairment. Current valve replacements do not biologically mimic native valves.

In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). After 14 days in culture, samples were tested for early ALP activity and late-stage calcification using ARS assays. All conditions showed ALP activity below 0.11 mg p-nitrophenol/mg protein, with insignificant differences between GAG conditions and the 1.5 mg/mL collagen-only controls. The collagen stiffness control at 2.2 mg/mL was notably higher than both GAG conditions, indicating stiffness plays a greater role in early disease stages than GAG presence (Fig. 1A). Alizarin Red S staining identified calcific nodule formation in hydrogels, with stained hydrogels imaged (Fig. 1D–G) and analyzed via ImageJ. Plate reader quantification showed that increased stiffness (2.2 mg/mL collagen) promoted calcification, with 20 mg/mL CS significantly enhancing matrix mineralization, while HA did not (Fig. 1B). Stain quantification matched the stained area quantification in ARS images (Fig. 1C).

Fig. 1. Chondroitin sulfate promotes calcific nodule formation and glycosaminoglycans (GAGs) do not produce increased alkaline phosphatase (ALP) activity (Bramsen J A, Alber B R, et al., 2022).

Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

BAV, a result of abnormal leaflet fusion during development, involves complex interactions among neural crest and endocardial derivatives. Nos3's expression is critical for valve development due to its sensitivity to hemodynamic signals, and its dysregulation is linked to BAV. Despite recent advances, the etiology of BAV is poorly understood. Odelin et al. have recently shown that Krox20 is expressed in endothelial and cardiac neural crest derivatives that normally contribute to aortic valve development and that lack of Krox20 in these cells leads to aortic valve defects including partially penetrant BAV formation.

Odelin et al. used compound heterozygous Krox20+/?;Nos3+/? mice to assess BAV malformations and conducted in vivo and in vitro analyses to demonstrate Krox20's regulatory effect on Nos3. Laforest et al. identified conserved GATA binding sites in the murine ?1.5-kb Nos3 promoter and showed that Gata5 enhances Nos3 promoter activity via these sites. Based on this, they examined the Nos3 promoter for conserved Krox20 binding sites. Bioinformatics analysis revealed two conserved Krox20-binding sites (K1 and K2) in the Nos3 proximal promoter (Fig. 2A). EMSA experiments showed that Krox20 binds to these sites with higher affinity for K2 (Fig. 2B). Luciferase assays indicated that Krox20 overexpression in Cos7 cells increased transcriptional activity of the ?1500 bp region up to 2.5-fold, but not the ?265 bp promoter (Fig. 2C). Mutation of the K2 site abolished Krox20-mediated activation of the ?1500 bp promoter, confirming its importance. ChIP experiments on E13.5 OFT and ventricle tissues showed that Krox20 binds specifically to the A region of the Nos3 promoter in OFT (Fig. 2D). Additionally, Krox20 transfection in rat aortic valve interstitial cells (AVICs) activated Nos3 expression (Fig. 2E). These findings identify Krox20 as a Nos3 promoter activator and suggest that reduced Nos3 expression may contribute to BAVs in Krox20 mutant mice.

Fig. 2. Krox20 promotes the transcriptional activity of the Nos3 proximal promoter (Odelin G, Faure E, et al., 2019).