Influence of Mechanical Environment on Aortic Valve Calcification, Thickening, Osteogenic marker Expression and ECM composition: Findings from an Ex Vivo Model

Abstract

Aortic stenosis (AS) involves narrowing of the aortic valve due to calcification and leaflet thickening. Calcifications occur in areas of high mechanical stress, suggesting that mechanical environment regulates calcification, but direct evidence is limited. This study investigates the role of mechanical stress in aortic valve calcification, thickening, osteogenic marker expression, signaling pathway activation and extracellular matrix (ECM) composition using an ex vivo calcification model (Miniature Tissue Culture System, MTCS). 

Whole mouse hearts were cultured in the MTCS with the aortic valve in a continuous open or closed position, exposed to low (300 µL/min; LF) or high (3000 µL/min; HF) flow rates in calcifying medium (3mM phosphate buffer) for one week. After culture, the hearts were processed for histological analyses. 

Calcification was observed only in valves cultured in a closed position at HF. The calcification extent correlated with aortic annulus diameter (R²=0.86, p<0.0001). The percentage of RUNX1/2/3-positive cells was lowest in open valves at LF (p<0.05), while the ALP-positive area was highest in closed valves at LF (p<0.05). The percentage of COX2-positive cells was highest in closed valves at HF (p<0.0001). The aggrecan-positive area was lowest in the aortic root of closed valves at HF (p<0.05). Activation of SMAD1/5/9 signaling was increased in open valves compared with closed valves (p<0.05).The collagen 1-positive area was highest in closed valves at LF (p<0.05).

Mechanical environment significantly influences aortic valve calcification, osteogenic marker expression, signaling pathway activation and ECM composition, offering insights into AS pathogenesis and potential therapeutic strategies.