Human myxomatous mitral valves exhibit focal expression of cartilage-related proteins Corresponding

Results and Conclusions: Myxomatous, but not normal, mitral valves demonstrated sharp focal areas that were abundant in aggrecan, type II collagen, and Sox9. These focal areas co-localized with areas of myxomatous pathologic change on Movat staining. Some cells in these areas had a round and hypertrophic morphology reminiscent of chondrocytes. Runx2 was only weakly present in normal and myxomatous mitral valves. These findings suggest a focal pathologic process in MMVD that mimics chondrogenesis. Carla M. R. Lacerda, Ph.D. a, Holly B. MacLea, D.V.M. , Colleen G. Duncan, D.V.M., Ph.D. , John D. Kisiday, Ph.D., E. Christopher Orton, D.V.M., Ph.D. a


Introduction
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 [1,2]. 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) [3,4]. 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].  [6][7][8]. 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 cartilagerelated and bone-related markers including aggrecan (cartilage-abundant PG core), type II collagen (cartilagespecific collagen), Sox9 (chondrogenic transcription factor), and Runx2 (osteogenic transcription factor) in normal and myxomatous human mitral valves.

Tissue collection and histology:
Surgically-excised myxomatous mitral valves (n The thickness of valve leaflets was measured in 3 locations throughout the samples with calipers. Myxomatous classification was determined by histological analyses by a pathologist and previous methods used by our group [11]. Tissues were fixed with 10% formalin, (Continued on page 23) embedded in paraffin, cut into 4 µm sections, and mounted on glass slides. Each valve was stained with hematoxylin-eosin and modified Movat pentachrome stain for histopathologic evaluation. Valves were evaluated for morphologic changes including PG/GAG accumulation, disruption of collagen bundles, elastin fragmentation, and loss of normal valve architecture.

Immunohistochemistry:
Immunohistochemistry was performed to determine the presence of Runx2, Sox9, type II collagen and aggrecan. Immunohistochemistry staining was performed as per previously published methods [12].

Data analysis:
Slides were photographed at low-magnification and areas of positive aggrecan staining were quantified using color thresholding in the ImageJ software [13].

Study population:
The median ages of the normal control group (n

Pathology:
The  Figure 1G). Type II collagen staining was not present in normal mitral valves. Type II collagen is the prototype collagen for cartilage and is considered specific for cartilaginous tissues [15]. Co-localization of type II collagen and aggrecan constitutes further evidence for a focal pathologic process that mimics chondrogenesis in myxomatous mitral valves.   stream 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 compression) within the valve apparatus [20,24]. 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].  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 ex- and CAVD apparently differ in their pathology remain to be determined.