In the United States, aortic valve disease (AoVD) is estimated to affect 2% of the population and the prevalence increases in the aged [1
]. Stenotic AoVD involves reduced valve cusp movement with narrowing of the effective valve opening and is often characterized by pathological calcification [3
]. Recently, a National Heart Lung and Blood Institute working group stated that calcific aortic valve disease (CAVD) is an actively regulated disease process and recommended that further understanding of the molecular pathways involved in the progression of CAVD is necessary [5
]. The natural history of AoVD is progressive and often necessitates aortic valve (AoV) replacement surgery using either a mechanical or bioprosthetic valve [6
]. Unfortunately, replacement valves are associated with long-term complications. Anticoagulation therapy is necessary with mechanical replacement valves to avoid thromboemboli, and bioprosthetic valves are not durable and often require additional surgeries [6
]. Molecular markers of AoVD progression, especially early AoVD pathogenesis, are presently unknown. In this study, the molecular characteristics of pediatric and adult diseased stenotic AoVs are examined. The goal of these studies is to define specific molecular indicators of valve pathogenesis that could lead to the development of early medical treatment to halt disease progression and prevent the need for surgery.
The mesenchymal progenitor cells, comprising the early endocardial cushions from which the heart valves develop, share a molecular profile with early chondrogenic mesenchymal precursor cells [8
]. Transcription factors, including Twist1, Sox9, and Msx2, and the signaling molecule BMP2 (bone morphogenetic protein 2), are key factors in both early valve and bone development [10
]. The mesenchymal progenitor cells of the endocardial cushions are the precursors of the mature valvular interstitial cells (VICs), which are the primary cellular component of adult valves [19
]. After birth, there is a sharp decrease in the number of proliferating VICs and healthy mature valves are composed primarily of quiescent VICs with little to no cell proliferation or synthetic activity [19
]. During disease, there is VIC disarray and clusters of VICs are activated, expressing myofibroblast markers and undergoing cell proliferation, ultimately leading to heterogeneous abnormalities in matrix composition [22
]. The study of gene expression changes in activated VICs likely will provide mechanistic insights into valve pathogenesis at the molecular level. Presently, it is unclear how VIC activation leads to AoVD and whether it plays a role in early pathogenesis.
During valve maturation, the valve extracellular matrix (ECM) becomes stratified with increased collagen organization and increased elastic fiber deposition [20
]. The mature aortic valves are composed of three ECM layers, the collagen-rich fibrosa layer, the ventricularis layer consisting primarily of elastic fibers, and the middle spongiosa layer with loosely arranged proteoglycans [21
]. Histologically, both pediatric and adult diseased AoVs have increased ECM production and aberrant remodeling, leading to a loss of valve cusp stratification and increased valve cusp thickness [21
]. Importantly, the onset of valve calcification is unusual before the fifth decade of life; however, calcification tends to occur a decade earlier in cases where the underlying AoV is congenitally malformed [28
]. Interestingly, pediatric stenotic AoVs, the vast majority of which are malformed, do not typically calcify [28
]. In contrast, pathologic calcification is commonly found in adult stenotic AoVD [31
]. A hallmark study describing gene expression changes in adult CAVD reported that an osteoblast-like gene expression profile was associated with valve calcification [32
]. Historically, valve calcification was presumed to be strictly dystrophic (passive), however, a growing body of literature suggests that genes associated with chondrogenesis and osteogenesis are expressed by activated VICs in adult CAVD [22
The goal of this study is to compare pediatric diseased AoVs that are not calcified with adult CAVD specimens as a means to define molecular indicators of early and late AoVD and specific markers of pathological calcification. Evaluation of gene expression using markers of early valve mesenchymal progenitors, chondrogenic precursors, and osteogenic differentiation in pediatric and adult diseased AoVs will provide insight into the molecular basis for AoVD progression from non-calcified disease valves to late stage calcified valves. In this study, we observe that activated VICs in pediatric and adult diseased AoVs share expression of valve mesenchymal progenitor and chondrogenic precursor markers, whereas expression of osteogenic differentiation markers is specific to adult CAVD.