Proteomic Characterization of Extracellular Matrix Regulation in Human Aortic Valve Development and Disease

We present the first report of the collagen protein interactome in human aortic valve (AV) development and disease. AV disease affects up to 13% of the world population. As no therapies are available, patients must “watch and wait” until surgical valve replacement is necessary to prevent heart failure. Extracellular matrix (ECM) forms the basis for correct valve function but alterations of the ECM scaffold during development and disease are not well defined. Here, we report ECM protein regulation from a cohort of 26 valvular tissues in four pathological categories: normal functioning AVs, pediatric end-stage congenital aortic valve stenosis (CAVS), adult end-stage fibrocalcific aortic valve stenosis (FAVS), and a pulmonic Ross operation valve. A novel proteomic method was used to target ECM proteins, primarily collagens, for sequence analysis from FFPE 5-μm thin tissue sections. Collagens represented 19/49 proteins identified (39%); A total of 46/49 ECM proteins (94%) were implicated in direct interactions in collagen synthesis, regulation or modification. Data suggested dynamic regulation of collagen types during age and disease, e.g., Col15a1 was found only in normal AV, whereas Col4a1 was reported only in patients younger than 5 years. Upstream regulators of the collagen interactome included COLQ1, IGFBP2, and SPARC. Detailed peptide sequence analysis reported 70 specific collagen peptide sequences containing up to multiple hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis on HYP peptides reported differential regulation across patient categories – i.e., Col3a1 peptides show 52% increased HYP in CAVS compared to age matched normal valve. Interestingly, the pulmonic Ross valve represented unique collagen HYP signatures distinct from all other aortic valve tissues. Tissue from pediatric end-stage CAVS patients showed a higher percentage of hydroxylated peptides compared to non-hydroxylated peptides (33%) as compared to normal (25%) or adult end-stage FAVS (16%). We anticipate that the completed study will be useful to targeted therapies aiming to inhibit fibrosis and ECM remodeling, and may better inform engineered options for valve replacement.