Shear Stress Regulates C-type Natriuretic Peptide Gene Expression by Valvular Endothelial Cells in a Sex-Dependent Manner

Aortic valve disease initiates on the fibrosa (aorta-facing) side of the valve leaflets, whereas the ventricularis side is disease-protected. Spatially predictable disease correlates with side-specific hemodynamics and gene expression: fibrosa-side valvular endothelial cells (VECs) experience low magnitude, oscillatory shear stress and have lower expression of pathological inhibitors, whereas ventricularis VECs experience higher magnitude shear stress and have a more protective phenotype. Among the protective genes higher expressed on the ventricular side, C-type natriuretic peptide (CNP) stands out as an endogenous inhibitor of fibrotic differentiation of aortic valve interstitial cells and valvular disease in vivo. However, shear stress regulation of CNP in aortic valves has not been characterized. To this end, porcine VECs were subjected to prescribed shear stresses in vitro. Steady, high magnitude shear stress and ventricular side-specific shear stress waveforms upregulated CNP gene (NPPC) expression in a KLF2-dependent manner. NPPC was upregulated by shear stress in VECs isolated from both the fibrosa and ventricularis sides, but with greater sensitivity in fibrosa-side cells. Notably, while KLF2 expression was upregulated with shear stress in both male and female VECs, NPPC was upregulated only in male VECs. Side- and sex-specific CNP expression patterns observed in vitro were also observed in immunohistochemically-stained human aortic valves. This work positions CNP as a biomechanically regulated factor in aortic valve homeostasis and pathogenesis, and provides insights into the factors regulating sex differences in valvular disease.