oa Characterization of Early Porcine Aortic Valve Disease

In calcific aortic valve disease (CAVD), pathological differentiation of valve interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. We have shown that VIC pathological differentiation in vitro is sensitive to matrix stiffness. We therefore hypothesized that focal changes in the stiffness of the fibrosa with CAVD progression correlate with alterations in the extracellular matrix (ECM) and pathological cell phenotypes in vivo. Twenty-four male Yorkshire pigs were fed either a control or experimental diet supplemented with 12% lard and 1.5% cholesterol for 2 or 5 months (n = 6 per group). Effective moduli were measured in focal regions of the fibrosa (n = 295) and ventricularis (n = 142) layers of intact right coronary aortic valve leaflets by micropipette aspiration. Histology was conducted within radial center sections and selected focal test locations within the fibrosa from each leaflet to characterize local ECM and cell phenotypes. Early stage disease was induced by the experimental diet, as evidenced by more significant proteoglycan-rich lesions (~10 - 200 µm thick) onlayed on the fibrosa (p < 0.05, controlling for genetic variability). Interestingly, lipid deposition was seen in only 28% of regions with early lesions, and 15% of regions without early lesions. No osteoblastic or macrophage cells were present in control or experimental valves, with only slight myofibroblast presence at the base of some 5 month lesions. Chondrogenic Sox9-positive cells were observed extensively and were positively correlated with proteoglycan content (p < 0.0001). Regions within the fibrosa were significantly stiffer than those in the ventricularis in both control and experimental valves (p <0.001). Although there were no significant differences between the control and experimental overall tissue moduli at these early time points, modulus heterogeneity did increase with disease. Furthermore, lesions in the fibrosa had lower moduli than non-lesion fibrosa locations (p < 0.06). These soft lesions contained significantly more proteoglycan (p <0.001) and Sox9 expression (p = 0.014) than normal fibrosa tissue. In conclusion, ECM remodelling can occur in a porcine model of early CAVD in the absence of lipid deposition, inflammatory cells, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich lesion and chondrogenic cell presence. Mechanically soft proteoglycan-rich lesions may support chondrogenic VIC differentiation, providing new insights into early CAVD pathogenesis.