Background: Calcification serves as a clinical marker of atherosclerotic plaque burden. Recent studies have also shed light on its intricate association with plaque vulnerability, likely depend on calcification structures and the surrounding environment. Asporin (ASPN) is a member of the small-leucine-rich proteoglycans that contains a unique and well-conserved stretch of aspartate (D) residues, which mimic the role of glycosaminoglycans. Via its D-repeat, ASPN can interact with calcium and several other extracellular matrix molecules, thus influencing collagen mineralization and TGF-β signaling. ASPN expression has been shown to be higher in asymptomatic compared to symptomatic atherosclerotic plaques, suggesting a potential protective role. However, the role of ASPN in maintaining plaque integrity remains unknown.

Purpose: Here we aimed to explore the role of ASPN in atherosclerosis, by investigating if ASPN was expressed in human carotid plaques and by determining if ASPN is associated with risk for postoperative cardiovascular events and death.

Methods: ASPN and TGF-β protein levels were measured by ELISA and Milliplex in 176 human plaque tissue homogenates obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque tissue sections were stained for ASPN, destabilizing (CD68, oil red O, glycophorin A) and stabilizing plaque components (Movat, α-actin). Plaque tissue RNA expression levels were assessed in 82 plaques using bulk RNA sequencing. Plaque levels of phosphate, elastin and collagen were measured using ELISA.

Results: ASPN protein levels were higher in plaques from asymptomatic patients compared to symptomatic patients (p=0.011). ASPN RNA levels correlated positively to RNA levels of the smooth muscle cell markers ACTA2, MYH11 and TAGLN. Moreover, protein levels correlated with plaque levels of phosphate (r=0.258, p=0.001), elastin (r=0.423, p<0.001), collagen (r=0.248, p=0.002) and stability factors like the TGF-β family members, TGF-β1 (r=0.176, p=0.035), TGF-β2 (r=0.581, p<0.001) and TGF-β3 (r=0.396, p<0.001). Furthermore, plaque ASPN protein concentration, correlated inversely with a histological plaque vulnerability index (r=-0.265, p=0.0029), which is ratio between the plaque area of destabilizing (macrophages, lipids, intra-plaque hemorrhage) and stabilizing plaque components (collagen and smooth muscle cells). Additionally, above median ASPN levels were associated with lower risk for postoperative cardiovascular events (p=0.008) and death (p=0.009).

Conclusion: Our findings suggest that elevated levels of ASPN might have a crucial role in maintaining plaque stability, affecting plaque calcification.