Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling

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.

Figure 1

Alizarin Red staining analysis. A: Representative Alizarin Red S (ARS) stained hydrogels in control (Ctrl), OGM, and co-culture (VIC+VEC) conditions. Scale bar = 200 μm. B: Quantification of calcium deposition via ARS absorbance in 3D hydrogel VIC/VEC co-culture, n = 6. Each measurement is relative to VIC control. Values are expressed as relative dye absorbance and are means ± SEM. ^∗P < 0.05 between treatment groups (effect of cell type); ^∗∗P < 0.05 within groups (effect of media).

Figure 2

IHC and real-time PCR analysis. A: IHC of α-SMA (green) expression in 3D PAVIC and PAVIC+PAVEC gels. Scale bars: 50 μm. B: Real-time PCR shows the effect of OGM on the expression of αSMA (ACTA), osteocalcin (BGLAP), Runx-2 (RUNX2), and eNOS (NOS3) in constrained 3D PAVIC and PAVIC+PAVEC gels (n = 6). Values are expressed as fold expression changes and are means ± SEM. ^∗P < 0.05 between treatment groups (effect of cell type); ^†P < 0.05 within groups (effect of media).

Figure 3

PAVIC gel analysis. A: Quantitative tissue calcification via relative absorbance of ARS in constrained 3D PAVIC gels in control and OGM, as well as OGM treated with 1 μmol/L DETA-NO. Osteocalcin (BGLAP) (B) and Runx-2 (RUNX2) (C) expression in constrained 3D PAVIC gels treated with OGM and 1 μmol/L DETA-NO. D: Comparison of the effect of different concentrations of l-NAME on the on calcium deposition within co-culture gels via ARS staining. Osteocalcin (BGLAP) (E) and Runx-2 (RUNX2) (F) expression in constrained 3D co-culture gels treated with OGM and l-NAME. ^∗P < 0.05 between treatment groups (effect of cell type); ^†P < 0.05 within groups (effect of media).

Figure 4

Alizarin Red–positive nodule analysis. Comparison of Alizarin Red–positive nodules per high-powered field (hpf) formed on the ventricular and aortic sides of intact valve cusps before (baseline) and after 3 weeks of incubation in control medium (Ctrl), OGM, or OGM supplemented with 100 μmol/L l-NAME (n = 4). ^∗P < 0.05 within groups (effect of media); ^†P < 0.05 between treatment groups (effect of cell side). Images within the graph are representative histological sections of ARS-positive nodules on aortic valve cusps placed in OGM.

Figure 5

eNOS expression in human valves. A: Immunofluorescent staining for eNOS (red) on the ventricular and aortic sides of normal and calcified human valves. Arrows indicate eNOS expression. Scale bars: 20 μm. B: Quantitative measure of eNOS expression on the ventricular and aortic side of normal and calcified human valves (measured area of expression per length). ^∗P < 0.05. C: IHC cGMP (red) on healthy porcine aortic valves on both fibrosa and ventricularis sides of the valve cusps. D: Accumulation of cGMP in valve cusps following exposure of the aortic or ventricular sides of the aortic valve to their respective patterns of flow. ^∗P < 0.05. E: IHC for osteocalcin and α-SMA on porcine leaflets exposed to OGM with sGC inhibitor (ODQ) and sGC activator (BAY). Scale bars: 100 μm. F: Quantification of ARS-positive nodules formed with OGM supplemented with ODQ and BAY. ^∗P < 0.05