Heart valve cells mediate extracellular matrix (ECM) remodeling during postnatal valve leaflet stratification, but phenotypic and transcriptional diversity of valve cells in development is largely unknown. Single cell analysis of mouse heart valve cells was used to evaluate cell heterogeneity during postnatal ECM remodeling and leaflet morphogenesis. The transcriptomic analysis of single cells from postnatal day (P)7 and P30 murine aortic (AoV) and mitral (MV) heart valves uncovered distinct subsets of melanocytes, immune and endothelial cells present at P7 and P30. By contrast, interstitial cell populations are different from P7 to P30. P7 valve leaflets exhibit two distinct collagen-and glycosaminoglycan-expressing interstitial cell clusters, and prevalent ECM gene expression. At P30, four interstitial cell clusters are apparent with leaflet specificity and differential expression of complement factors, ECM proteins and osteogenic genes. This initial transcriptomic analysis of postnatal heart valves at single cell resolution demonstrates that subpopulations of endothelial and immune cells are relatively constant throughout postnatal development, but interstitial cell subpopulations undergo changes in gene expression and cellular functions in primordial and mature valves.

Objective Calcific Aortic Valve Disease (CAVD) is the most prevalent type of heart valve disease, affecting ~2% of the US population. CAVD is characterized by the presence of calcific nodules resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that BMP signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane co-receptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho−/− mice. Approach & Results We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho−/− mice. In addition, cellular and ECM changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho−/− AoV. Likewise, osteogenic media (OM) treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the Bmpr1a receptor in Klotho−/− aortic VICs, as well as BMP pathway inhibition of OM-treated aortic VICs in vitro, results in the inhibition of AoV calcification. Discussion BMP signaling and osteochondrogenic gene induction are active in calcified Klotho−/− AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

ObjectivesCalcific aortic valve disease (CAVD) is a progressive disease ranging from aortic valve (AoV) sclerosis to AoV stenosis (AS), characterised by severe calcification with impaired leaflet function. Due to the lack of early symptoms, the pathological progression towards valve dysfunction is poorly understood. The early patterns of AoV calcification and altered extracellular matrix (ECM) organisation were analysed in individuals postmortem without clinical AS compared with clinical AS.MethodsHistological patterns of calcification and ECM organisation in postmortem AoV leaflets without clinical AS obtained from a tissue repository and surgical specimens obtained from individuals with clinical AS were compared with in vivo imaging prior to transcatheter AoV implantation.ResultsAoV calcification was detected in all samples from individuals >50 years old, with severity increasing with age, independent of known CAVD risk factors. Two distinct types of calcification were identified: ‘Intrinsic’, primarily found at the leaflet hinge of postmortem leaflets, accompanied by abnormal collagen and proteoglycan deposition; and ‘Nodular’, extending from the middle to the tip regions in more severely affected postmortem leaflets and surgical specimens, associated with increased elastin fragmentation and loss of elastin integrity. Even in the absence of increased thickening, abnormalities in ECM composition were observed in postmortem leaflets without clinical AS and worsen in clinical AS.ConclusionsTwo distinct phenotypes of AoV calcification are apparent. While the ‘nodular’ form is recognised on in vivo imaging and is present with CAVD and valve dysfunction, it is unclear if the ‘intrinsic’ form is pathological or detected on in vivo imaging.