Multiple cell trypes, incluiding melanocytes, contribute to elastogenesis in the developing murine aortic valve
Elastic fibers are required for expansion and recoil during contraction and relaxation of vessels and heart valves. The aortic valve (AoV) extracellular matrix is generated and maintained by valvular interstitial cells (VICs), a heterogeneous population of cells derived from a mixture of developmental precursors such as endocardium, neural crest and second heart field. Although most studies have focused on VICs that exhibit a fibroblastic phenotype, significant subpopulations present with neuronal, glial, smooth muscle and melanocytic phenotypes. Relatively little is known about which sub population of VICs regulate and produce elastic fibers, though elastin (Eln) abnormalities result in congenital AoV defects and Eln degradation initiates AoV diseases. The present study establishes the timing ofElnexpression in the murine AoV. We performed RT-qPCR and found thatElnpeaks at late embryogenesis (embryonic day 17.5) and early postnatal (P) stages and decreases to low levels in adulthood. Using spatial transcriptomics in postnatal day 3 AoV, we segregated AoV cells from other cardiac cells and demonstrated thatElnexpression correlates with that of genes known to regulate elastogenesis, including the common smooth muscle cell marker - Acta2. By combining RNAscopein situhybridization with immunofluorescence, we confirmed that VICs that expressElnco-express alpha smooth muscle actin. Additionally, some of theEln/alpha smooth muscle actin expressing cells also express melanocytic markers. As previously reported in adult mice, we show a relationship between AoV pigment and elastic fiber patterning during early postnatal stages and further show that melanocytes may play a critical role in elastogenesis. Our results indicate that in the murine AoVElnis produced during early postnatal stages by cells that co-express phenotypic markers of various cell types, including smooth muscle actin and melanocytic specific genes. Overall design: To investigate the cell types and relevant mechanisms of elastinogenesis in the developing aortic valve. Mouse aortic valve tissues from postnatal day 3 and day 30 aged mice were snap frozen, OCT embedded, and sectioned using a cryostat. We used the 10xGenomics Visium Spatial Gene Expression kit to determine spatially encoded gene expression profiles on the tissues.