Features of valve sclerosis include disorganization of collagen bundles, loss of valve cusp stratification, and increased activity of ECM remodeling enzymes (35
). (). AoV disease is a narrowing of the valve opening, which often includes calcification of the valve cusps () (3
). Valve calcification occurs primarily in the fibrosa layer, in regions of greatest mechanical stress, and calcium deposits often protrude on the aortic surface of the valve (37
). Two patterns of calcification have been described. The first beginning at the valve hinge, where the cusp attaches to the aortic wall, and the second initiating along the line of coaptation, the point at which the AoV cusps meet during diastole (38
). Histologically, the majority of valve calcification is thought to be dystrophic (passive) however, cartilaginous nodules and mature lamellar bone have also been described, suggesting an active calcification process has been underappreciated (39
The valvular interstitial cells (VIC) are the main cellular constituents of the mature valves and contribute to valve homeostasis as well as valve pathogenesis. The VIC in a healthy valve arise from valve progenitor cells of the developing endocardial cushions, are primarily quiescent, and are important for maintaining normal valve structure and function () (40
). Conversely, in a diseased valve, the VIC become activated to a myofibroblast state and express the marker alpha smooth muscle actin () (20
). Other cell populations including infiltrating immune cells and resident valve stem cells have also been described in diseased AoV (39
). Quiescent VIC do not proliferate whereas activated VIC have been shown to undergo cell proliferation (42
). There is accumulating evidence that the activated, proliferating VIC initiate a transcriptional program common to heart valve progenitors and osteogenic processes.
Recent studies report the expression of transcription factors associated with valve and bone formation in human diseased AoV. In limb morphogenesis, Msx2 is important for the proliferation of osteogenic progenitor cells and for bone and cartilage formation through the regulation of the canonical Wnt signaling pathway (15
). Likewise, in human diseased AoV, Msx2 expression is increased in calcified areas associated with increased Wnt signaling and cell proliferation (21
). Similarly, Twist1 is expressed in the osteoblast progenitors, where it inhibits osteoblast differention while promoting chondrogenesis (12
). In human diseased AoV, Twist1 expression is apparent in small clusters of cells near regions of calcification and increased cell proliferation (Wirrig and Yutzey, unpublished). These studies support a role for Twist1 in promoting VIC proliferation during AoV disease. Sox9 is expressed in the mesenchyme of the developing limbs and is important for differentiation of the chondrocyte lineage and for the expression of ECM genes characteristic of cartilage, such as collagen 2a1
). In diseased AoV, Sox9 expression is increased in the VIC, consistent with a role in cell proliferation and ECM gene regulation (20
). Combined, this evidence supports the idea that transcription factors involved in early valve formation and chondrogenesis are abnormally activated in AoV disease. However, it is not known if this is part of a valve repair process or a maladaptive pathologic mechanism.
In addition to factors involved in chondrogenesis, transcription factors associated with endochondral ossification are reported to have increased expression in diseased aortic valves. Runx2 (RUNT-related transcription factor 2) is expressed strongly in osteoblasts during bone development, is important for osteoblast maturation, and regulates the expression of osteogenic ECM genes including collagen 10
). Osterix (transcription factor Sp7) functions downstream of Runx2 and is necessary for bone formation, osteoblast differentiation, and expression of osteocalcin (47
). Studies on human diseased AoV demonstrate increased expression of Runx2 and Osterix in the VIC surrounding areas of calcification (20
). Furthermore, increased expression of Runx2 target genes collagen 10
have also been described in stenotic AoV disease (35
). Therefore, the increased expression of Osterix, Runx2, and the downstream bone matrix proteins collagen 10 and osteocalcin, is evidence for an active osteogenic process in AoV disease. Progress towards a better understanding of the molecular mechanisms underlying calcific AoV disease is being made, however significant gaps still exist in the areas of VIC activation, valve stem cell contributions, and initiation of an osteogenic gene expression profile.