The age-adjusted prevalence of valvular heart disease is estimated at 2.5% in the USA, and its prevalence increases sharply after 65 years of age due to the predominance of degenerative etiologies 12. Degenerative valvular heart disease accounts for about 63% of native valvular heart disease. The most common manifestations of degenerative valvular disease are myxomatous mitral valve disease (MMVD) and calcific aortic valve disease (CAVD) 1. While both conditions are considered degenerative, MMVD and CAVD differ significantly in their functional manifestation and pathology. From a hemodynamic standpoint the most typical manifestation of MMVD is valve regurgitation, whereas the most common manifestation of CAVD is valve stenosis. Pathologically MMVD is characterized by gross leaflet thickening with chordae lengthening or rupture, net degradation of the extracellular matrix (ECM), and exuberant deposition of proteoglycan (PG)/glycosaminoglycan (GAG) 34. On the other hand, CAVD is characterized by leaflet immobility, lipid accumulation, progressive leaflet fibrosis and calcification 5. The reasons for these divergent manifestations of valvular degeneration in the mitral and aortic valve are currently not understood.

Research in heart valve development has suggested several commonalities between the pathways regulating this process and chondrogenesis, which in turn might have important roles in adult tissue remodeling. Heart valvulogenesis appears to be highly conserved among vertebrates and is governed by developmental regulatory pathways consisting of receptor-based signaling, transcription factors, and downstream structural genes 6. Developmental regulatory pathways governing valvulogenesis also control development of cartilage, bone, and tendon 6. Specific shared regulatory pathways include Notch (atrialis/ventricularis & elastic artery development), BMP-Sox9-aggrecan (spongiosa & chondrogenesis), Wnt-periostin (fibrosa & osteogenesis), and FGF-scleraxis-tenascin (chordae tendineae & tendon development). These observations, coupled with pathologic similarities between degenerative valve disease and osteogenesis in the case of CAVD or chondrogenesis in the case of MMVD have led to a working hypothesis that degenerative valve diseases may be recapitulating osteogenesis or chondrogenesis through shared regulatory pathways 678. Recent studies have shown that calcific aortic valves express bone-related markers including Runx2, osteocalcin, osteopontin, and others 9,10. These observations support a hypothesis that calcific aortic valves undergo active osteogenesis, but much less is known about MMVD.

In this study, we tested the hypothesis that myxomatous mitral valves might be undergoing a pathologic process that mimics chondrogenesis. To address this hypothesis we evaluated presence of cartilage-related and bone-related markers including aggrecan (cartilage-abundant PG core), type II collagen (cartilage-specific collagen), Sox9 (chondrogenic transcription factor), and Runx2 (osteogenic transcription factor) in normal and myxomatous human mitral valves.

The cartilage-related markers studied here interconnect during the earlier phase of chondrogenesis, which chondrocytes are proliferating. These chondrocytes strongly up-regulate the expression and activity of the transcription factor Sox9, inducing important downstream targets such as type II collagen and aggrecan 17. In addition, cells with high levels of Sox9 have the ability of down-regulating Runx2 by directing its degradation, thus preventing or delaying osteogenesis 18.

Proteoglycans consist of a core protein with a variable number of covalently bound GAG side-chains 19. Principal PG cores in normal mitral valves include decorin, biglycan, and versican; and not surprisingly these and other PG cores are more abundant in myxomatous mitral valves 20,21, 22,23. Aggrecan is the prototypic PG core in cartilage 19. Aggrecan has one of the largest PG core proteins and approximately 100 GAG side-chains of varying composition depending on the species 19. This structure makes aggrecan highly hydroscopic and largely gives cartilage its unique biomechanical properties to resist compressive forces. Expression of aggrecan has been previously reported in developing heart valves 8, but is not known to be present in adult mitral valves. This study demonstrated discrete focal expression of aggrecan in myxomatous, but not normal, mitral valves. These focal areas of aggrecan expression correlated strongly with focal areas of myxomatous pathology in myxomatous mitral valves based on Movat staining, but aggrecan was not present in all areas of myxomatous change. The spatial distribution of PG and GAG profiles in mitral valves is not uniform and is likely influenced by different mechanical forces (e.g. tension vs. compression) within the valve apparatus 20,21,22,23,24. Interstitial cells in aggrecan-positive areas often exhibited a rounded hypertrophic morphology reminiscent of chondrocytes that has been reported previously in human myxomatous mitral valves 9.

Collagen is a prominent component of the ECM of heart valves, particularly the lamina fibrosa. The most abundant collagen types in adult heart valves are types I and III 25,26. Despite net degradation and disarray of collagen in MMVD, types I and III collagen are more abundant in myxomatous mitral valves 25. Type II collagen is the most abundant collagen in cartilage and its expression is considered largely specific for cartilaginous tissues 27. Type II collagen is expressed in the heart during the development of endocardial cushions, but not in the adult heart 6. In this study, focal presence of type II collagen in aggrecan-abundant areas of myxomatous change constitutes further compelling evidence for a chondrogenic process in myxomatous mitral valves. Type II collagen has been previously identified in adult calcific aortic valves, largely associated with areas of osteogenic transformation 10. This finding supports the notion that the osteogenic process in CAVD recapitulates endochondral bone formation wherein bone formation progresses through a process of ossification of a cartilaginous template 9.

The transcription factor Sox9 is typically expressed in chondroprogenitors, periarticular chondrocytes and proliferating chondrocytes. Runx2, which serves as a positive regulatory factor in chondrocyte maturation, is expressed in hypertrophic chondrocytes and throughout ossification stages. Thus, their regulatory mechanisms follow a temporal pattern during endochondral ossification. 28,29. A variety of signaling pathways promote their regulation 30 with chondrogenesis being favored by high Sox9 and low Runx2 expression and osteogenesis being favored by low Sox9 and high Runx2 expression 29. In this study, we observed strong focal presence of Sox9 in myxomatous mitral valves that co-localized with focal areas of aggrecan and type II collagen positive staining. This suggests that focal chondrogenesis in myxomatous mitral valves may be mediated in part through the Sox9 transcription factor. Bone morphogenetic protein (BMP) and transforming growth factor beta (TFGβ) are members of the same signaling superfamily. Both are known to regulate Sox9 in late-stage chondrogenesis 3132, 33. Increased abundance of TGFβ has been demonstrated in canine myxomatous mitral valves suggesting a role for TGFβ in a chondrogenic-like pathology in degenerative mitral valve , possibly through regulation of Sox9 34, 35,36. BMP2 induces Sox9 and aggrecan expression in developing heart valve progenitor cells 37. BMP-Sox9 is postulated to mediate valvular heart diseases through recapitulation of shared developmental pathways that regulate valvulogenesis and chondrogenesis but this hypothesis has not been yet confirmed 6,7,8. Lastly, studies have demonstrated direct cross-talk between BMP and Wnt canonical signaling during chondrogenesis 38,39. Possible roles for BMP or Wnt signaling in degenerative valvular heart disease remain to be elucidated.

Strong Runx2 expression has been demonstrated in calcific aortic valves and has thus been implicated in an osteogenic pathologic process in CAVD 9,10. Runx2 was not strongly expressed in either normal or myxomatous mitral valves suggesting that it does not play a significant role in MMVD. Sox9 expression has also been demonstrated in calcific aortic valves 9,10. Cells that express Sox9 can be progenitors for a variety of cell types, including chondrocytes and osteoblasts 40. During skeletogenesis, bone formation occurs through endochondral ossification of a cartilage template. Endochondral ossification proceeds through an early phase of Sox9 expression during chondrocyte proliferation and a late phase of Runx2 expression during chondrocyte hypertrophy and bone formation 28. Thus, it can be suggested that CAVD disease mimics an endochondral osteogenesis process, whereas MMVD disease recapitulates chondrogenesis only.

Limitations of this study are the low relatively low number of normal mitral valves available for study and the fact that they were not perfectly age-matched to the myxomatous mitral valve group. Despite the low sample number of the normal control, statistical significance was reached in frequency positive straining for chondrogenic markers because of their virtual absence from normal mitral valves. The lack of perfect age-matching between the normal control group and the myxomatous group raises the possibility that age could influence expression of chondrogenic makers. The absence of availability of posterior leaflets in the normal group is also a limitation. However, we did not observe differences between anterior and posterior leaflets in the myxomatous group.

In conclusion, focal co-expression of aggrecan, type II collagen, and Sox9, but not Runx2, suggest a pathologic process that mimics chondrogenesis in human myxomatous mitral valves. The signaling pathways that mediate this process and the reasons why MMVD and CAVD apparently differ in their pathology remain to be determined.