Unidirectional Shear Stress-Operated NOTCH/CXCR4 Molecular Switch Controls Semilunar Valve Maturation and Collagen...

Much is known about the genetic regulation of early valvular morphogenesis, but mechanisms governing later fetal valvular remodeling remain unclear. Hemodynamic forces strongly influence morphogenesis, but it is unknown whether or how they interact with valvulogenic signaling programs. Apparent side-specific expression of valvulogenic programs motivates the hypothesis that shear stress pattern-specific endocardial signaling directs the remodeling and maturation of valve leaflets. Here, we aim to determine how local hemodynamic stress regulates the maturation of fetal semilunar heart valves.

We identified strong ventricularis-specific expression of endocardial NOTCH1 and mesenchymal CXCR4 (C-X-C chemokine receptor type 4) during fetal valve stages. Valve cell-type specific conditional Notch and Cxcr4 mouse deletions were generated and analyzed in vivo consequences, which were then tested directly using ex vivo chick endocardial cells and valve organoids via gain and loss of function approaches. Samples were then quantitatively analyzed via histology, immunohistochemistry, and qRT-PCR (quantitative real-time polymerase chain reaction).

We established that unidirectional laminar shear stress regulates CXCR4 via endocardial NOTCH signaling through upregulation of CXCR4 ligand SDF1 (stromal cell-derived factor 1). Global deletion and endocardium-derived mesenchymal cell-specific deletion of Cxcr4 both resulted in hyperproliferative and thickened outflow tract valves. In addition, conditional ablation of Cxcr4 also revealed that it promotes matrix remodeling and tissue compaction through inhibition of BMP (bone morphogenetic protein) and WNT signaling programs.

High-magnitude unidirectional laminar shear stress is transduced by endocardial cells, turning on a NOTCH1/CXCR4 molecular switch. This switch stops the valve mesenchymal growth program by inhibiting WNT/BMP. Simultaneously, it also orchestrates valve condensation, mesenchymal cell differentiation, and ECM (extracellular matrix) remodeling. Taken together, our findings identify a novel molecular switch controlled by local hemodynamic cues that directs valve maturation robustly in a side-specific manner.