A disease-inspired <i>in vitro</i> model of aortic valve stenosis to investigate the drivers of endothelial–mesenchymal transition

Calcific aortic valve disease (CAVD) is the most prevalent heart valve disorder worldwide. Despite its growing clinical burden, there are currently no pharmacological treatments available to prevent or reverse disease progression; transcatheter or surgical valve replacement remains the only therapeutic option. In this study, we developed a disease-inspired <i>in vitro</i> model to investigate how mechanical and biochemical cues, specifically reduced tensile stress and enrichment of hyaluronic acid (HA) within extracellular matrices (ECM), influence valvular endothelial cell biology, both hallmark features of CAVD. To achieve this, we developed a hydrogel-based model, incorporating different concentrations of HA to mimic healthy, mild, and moderate stages of CAVD. Valvular endothelial cells were cultured on these ECM conditions and subjected to cyclic stretch at 0%, 10%, and 20%, representing static (no mechanical stimulation), physiological (healthy), and pathological (hypertensive) conditions, respectively. We found that HA enrichment within the ECM, combined with reduced stretch intensity, induced an intermediate endothelial-to-mesenchymal transition (EndMT) phenotype, as evidenced by increased expression of vWF, CD31, αSMA, and MMP9. Notably, the application of 10% and 20% cyclic stretch mitigated the pro-EndMT effects of HA enrichment. Additionally, HA enrichment and stretch intensity influenced the expression of ICAM-1, an inflammatory marker, and subsequent THP-1 monocyte adhesion. Under static conditions, HA deposition alone did not significantly affect cell adhesion. However, 10% stretch reduced ICAM-1 expression and monocyte adhesion, whereas at 20% stretch, high HA concentrations enhanced monocyte adhesion. To validate our <i>in vitro</i> findings, we analysed aortic valve tissues from CAVD patients and healthy donors. CAVD valves showed the increased CD31, MMP9, and α-SMA expression, reduced vWF levels, and heightened cellular infiltration, confirming key inflammatory and structural changes observed <i>in vitro</i>. This work establishes a physiologically relevant <i>in vitro</i> platform to study CAVD progression and highlights the importance of ECM composition and mechanical loading in modulating endothelial biology and immune interactions.