Loss of Syndecan-3 Drives a Pro-inflammatory, EndMT-prone Phenotype in Aortic Valve Endothelial Cells

The earliest events in calcific aortic valve disease (CAVD) occur at the valve endothelium, where the cells lining the leaflet undergo NF-kappaB (Nuclear Factor kappa B)-driven inflammatory activation and endothelial-to-mesenchymal transition (EndMT). The endothelial glycocalyx (EG) sets the threshold at which endothelial cells (ECs) respond to such cues, yet its composition and function in the aortic valve remain almost entirely uncharacterized. Here we define the cell-surface proteoglycan (CPG) landscape of human aortic valve endothelial cells (HAVECs) under inflammatory stimulation and identify syndecan-3 (SDC3) as a regulator of valve endothelial activation. Among the proteoglycans examined, SDC3 was highly expressed and inflammation-responsive. Silencing SDC3 in unstimulated HAVECs was sufficient to upregulate NF-kappaB pathway components and the leukocyte adhesion molecules vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin, and it amplified their induction upon TNF-alpha challenge. Pharmacological inhibition of IKKbeta; abolished this difference, placing SDC3 upstream of NF-kappaB, while SDC3 overexpression attenuated tumor necrosis factor-alpha (TNF-alpha)-induced VCAM1, the inverse of the knockdown effect. Loss of SDC3 further primed HAVECs toward a mesenchymal, EndMT-prone phenotype under transforming growth factor-beta 2 (TGF-beta2) stimulation, marked by increased smooth muscle protein 22-alpha (SM22alpha) and reduced platelet endothelial cell adhesion molecule (PECAM1/CD31). Together, these findings identify SDC3 as an endogenous restraint on the inflammatory and mesenchymal programs that characterize the endothelial phase of CAVD and establish the valve endothelial glycocalyx as a previously unappreciated yet potent regulator of aortic valve endothelial dysfunction. Preserving or augmenting SDC3 may warrant investigation as a means of holding valve endothelial cells in a quiescent state.