Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase

Abstract

Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

Keywords: aortic valve sclerosis; bicuspid aortic valve; endothelial nitric oxide synthase.

Fig. 1.

Inhibition of valve interstitial cell (VIC) activation and expression of matrix metalloproteinase-9 (MMP-9) by valve endothelial cells (vlvECs) in vitro. A: culture well micrographs demonstrating immunostaining for α-smooth muscle actin (α-SMA, green) and Cell Tracker-tagged endothelial cells (red). B and C: percentage of VICs with positive immunostaining for α-SMA. D and E: normalized expression of MMP-9. TCPS, tissue culture polystyrene, Young's modulus = ∼1 GPa; Gel, photoactivatable hydrogel, Young's modulus = 28 kPa; l-NAME, N^G-nitro-l-arginine methyl ester; Indo, indomethacin. *P < 0.05 for VICs vs. VICs + vlvECs.

Fig. 2.

Profibrotic signaling in vitro. A–D: quantitative PCR for expression of collagen-1 or collagen-3. E and F: Western blotting for phospho (p)-Smad2 and total (t)-Smad2. TCPS Young's modulus = ∼1 GPa gel photo-activatable hydrogel, Young's modulus = 28 kPa. *P < 0.05 vs. VICs alone.

Fig. 3.

Fibrosis in the aortic valve. Masson's trichrome stain demonstrates total collagen content (blue) in bicuspid (B) and trileaflet (T) valves from endothelial nitric oxide synthase knockout (eNOS^−/−) and wild-type (WT) mice at 6 and 18 mo of age. *P < 0.05 vs. WT; N = 9 and 5 for 6 and 18 mo, respectively; T eNOS^−/−, N = 13, 6; B eNOS^−/−, N = 4, 4. Scale bar = 300 μm.

Fig. 4.

Collagen-1 and collagen-3 in the aortic valve. 3,3′-Diaminobenzidine (DAB) immunostains backstained with hematoxylin. Negative control denotes DAB staining without primary antibody. WT, N = 4 and 4 for 6 and 18 mo, respectively; T eNOS^−/−, N = 4, 4; B eNOS^−/−, N = 4, 4. *P < 0.05 vs. WT. Scale bar = 300 μm.

Fig. 5.

Calcification in the aortic valve. Small areas of calcification (Alizarin Red, arrows) in a bicuspid aortic eNOS^−/− valve at age 6 mo and more calcification in all groups at age 18 mo. Planimetry of Alizarin Red-positive staining indicates significant increase in calcification in bicuspid valves from 6- and 18-mo-old eNOS^−/− mice, compared with trileaflet valves from WT or eNOS^−/− mice. WT, N = 9 and 5 for 6 and 18 mo, respectively; T eNOS^−/−, N = 13, 6; B eNOS^−/−, N = 4, 4. *P < 0.05 vs. WT; †P < 0.05 vs. T eNOS^−/−. Scale bar = 300 μm.

Fig. 6.

Osterix in the aortic valve. Osterix was increased in bicuspid valves from 6-mo-old, but not 18-mo-old, eNOS^−/− mice (N = 4–6). AF autofluorescence; Osx osterix staining. *P < 0.05 vs. WT. Scale bar = 300 μm.

Fig. 7.

Proapoptotic signaling in the aortic valve. A: immunostaining for activated caspase-3 in valves from mice at 6 mo of age. Positive staining (arrows) was observed near sites of cusp attachment. B: group data; N = 4–6. *P < 0.05 vs. WT; †P < 0.05 vs. T eNOS^−/−. Scale bar = 300 μm.

Fig. 8.

Lipid deposition in the aortic valve. A: there was minimal staining with Oil Red-O in all groups at age 6 mo and increased lipids in all groups at age 18 mo. B: planimetry of Oil Red-O positive staining indicates that even though lipid deposition in the aortic valve of these normocholesterolemic mice was small, it was significantly higher in trileaflet and bicuspid eNOS^−/− valves compared with WT valves at age 6 mo. There was no significant difference at age 18 mo. *P < 0.05 vs. WT. WT, N = 9 and 5 for 6 and 18 mo, respectively; B eNOS^−/−, N = 4, 4; T eNOS^−/−, N = 13, 6. Note differences in scale in 6- and 18-mo-old mice. Scale bar = 300 μm.

Fig. 9.

Aortic valve function. A and B: aortic cusp separation (ACS). C: transaortic valve gradient in an eNOS^−/− mouse, determined by invasive hemodynamic assessment. D: group data for transvalvular gradient; N = 2 and 6 for WT at 6 and 18 mo, respectively; N = 4 and 6 for eNOS^−/−. E: absence of significant aortic regurgitation, as assessed by MRI (N = 15). *P < 0.05 vs. WT.

Fig. 10.

Proximal aorta. Aortic root diameters, at the sinuses of Valsalva, and at the sinotubular junction, were similar in 18-mo-old WT mice and eNOS^−/− mice with either trileaflet or bicuspid aortic valve. WT, N = 9 and 5 at 6 and 18 mo, respectively; T eNOS^−/−, N = 13, 6; B eNOS^−/−, N = 4, 4.