APOE-NOTCH Axis Governs Elastogenesis During Human Cardiac Valve Remodeling

Abstract Background Valve remodeling is a complex process involving extracellular matrix organization, development of trilaminar structures, and physical elongation of valve leaflets. However, the cellular and molecular mechanisms regulating valve remodeling and their roles in congenital valve disorders remain poorly understood. Methods Semilunar valves and atrioventricular valves from healthy and age-matched human fetal hearts with pulmonary stenosis (PS) were collected. Single-Cell RNA-sequencing (scRNA-seq) was performed to determine the transcriptomic landscape of multiple valvular cell subtypes in valve remodeling and disease. Spatial localization of newly-identified cell subtypes was determined via immunofluorescence and RNA in situ hybridization. The molecular mechanisms mediating valve development was investigated utilizing primary human fetal heart valve interstitial cells (VICs) and endothelial cells (VECs). Results scRNA-seq analysis of healthy human fetal valves identified a novel APOE + elastin-producing VIC subtype (Elastin-VICs) spatially located underneath VECs sensing the unidirectional flow. Knockdown of APOE in fetal VICs resulted in significant elastogenesis defects. In pulmonary valve with PS, we observed decreased expression of APOE and other genes regulating elastogenesis such as EMILIN1 and LOXL1 , as well as elastin fragmentation. These findings suggested the crucial role of APOE in regulating elastogenesis during valve remodeling. Furthermore, cell-cell interaction analysis revealed that JAG1 from unidirectional VECs activates NOTCH signaling in Elastin-VICs through NOTCH3. In vitro Jag1 treatment in VICs increased elastogenesis, while similar observations were found in VICs co-cultured with VECs in the presence of unidirectional flow. Notably, we found that the JAG1-NOTCH3 signaling pair was drastically reduced in the PS valves. Lastly, we demonstrated that APOE is indispensable for JAG1-induced NOTCH activation in VICs, reinforcing the presence of a synergistic intrinsic and external regulatory network involving APOE and NOTCH signaling that is responsible for regulating elastogenesis during human valve remodeling. Conclusion scRNA-seq analysis of human fetal valves identified a novel Elastin-VIC subpopulation, and revealed mechanism of intrinsic APOE and external NOTCH signaling in regulating elastogenesis during cardiac valve remodeling. These mechanisms may contribute to deciphering the pathogenesis of elastin malformation in congenital valve diseases. Clinical Perspective What Is New? High-resolution single-cell transcriptome atlas generated from healthy human fetal heart valves and valves affected by pulmonary stenosis during the early phase of valve remodeling prior to birth. A unique subset of valve interstitial cells (VICs) that produce elastin (Elastin-VICs) was identified. Elastin-VICs specifically located underneath the valve endothelial cells (VECs) sensing unidirectional flow, and played a crucial role in elastin maturation via the expression of APOE. Elastin-VICs communicated with adjacent VECs via the JAG1-NOTCH signaling, facilitating elastin formation and valve remodeling. What Are the Clinical Implications? Elastin-VICs from patient valvular tissues with Pulmonary Stenosis exhibit decreased APOE-NOTCH signaling and elastin fragmentation. Direct targeting of APOE and NOTCH signaling could be a novel approach to promote elastin fiber formation and valve remodeling in patients with valvular defects.