Side-Specific Endothelial-Dependent Regulation of Aortic Valve Calcification

Abstract

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC-VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology.

Integrated Bioinformatics Analysis Predicts the Key Genes Involved in Aortic Valve Calcification: From Hemodynamic Changes to Extracellular Remodeling

“…Oxidative stress has been shown to participate in the development of CAVD (Miller et al 2008;Kennedy et al 2009;Li and Förstermann 2013) and be associated with local hemodynamic changes (Richards et al 2013). NOS3, commonly known as eNOS, has long been known for its protective effects against aortic valve inflammation and calcification (Clapp et al 2004;Kennedy et al 2009).…”

“…4B). According to the literature, NOS3 is a mechano-sensitive gene that is regulated by the flow-sensitive KLF2 transcription factor (SenBanerjee et al 2004;Richards et al 2013). The findings suggest that there is a close relationship between hemodynamics and endothelial oxidative stress in the development of CAVD.…”

Shear-Sensitive Genes in Aortic Valve Endothelium

“…The ventricularis experiences unidirectional laminar shear stress as blood is ejected from the ventricle to the aorta. The fibrosa experiences disturbed flow and oscillatory shear stress, as shown in Figure 2B, which have been directly linked to onset and progression of CAVD (6,40,104). Shear stress regulates endothelial cell function: the cells sense changes in shear stress to determine systemic changes, such as hypertension.…”

“…Bosse et al (14) studied the role of NO in aortic valve calcification using a coculture system of endothelial cells and aortic valve interstitial cells (AVICs) and found that endothelial cells secrete NO, which is absorbed by AVICs, preventing calcification through regulation of the Notch1 signaling pathway in AVICs. Richards et al (104) found that in both calcified and noncalcified human aortic valves, the ventricularis side exhibited a threefold higher expression of eNOS compared with the fibrosa. Interestingly, the calcified valves presented lower values of eNOS expression on both sides compared with healthy aortic valves.…”

Comparison of Mesenchymal Stem Cell Source Differentiation Toward Human Pediatric Aortic Valve Interstitial Cells within 3D Engineered Matrices

“…Bound Alizarin red S dye was then released from the gels using 10% acetic acid, followed by neutralization with 10% ammonium hydroxide. 36 The absorbance of the dye solution was measured at 405 nm.…”