Background: Aortic valve stenosis (AS) is one of the most common valve diseases in the western world. In our previous results, we could show that mice with global endothelial nitric oxide synthase knock out (eNOS KO), a genotype characterised by endothelial dysfunction, develop a spontaneous AS in aging mice. The underlying events on the cellular level in the aortic valves are still elusive.

Hypothesis: We hypothesise that spontaneous AS in global eNOS KO mice is caused by fibrosis, calcification and accompanied by inflammatory processes of the aortic valve mimicking human disease. It was our aim to investigate histological markers of fibrosis, calcification and inflammation in aortic valves of 12-month-old eNOS KO mice compared with age-matched wildtype (wt) mice or wt mice 4 weeks after sham or wire injury surgery (WI) to induce aortic valve stenosis at the extent comparable to eNOSko. 

Methods: Global eNOS KO mice were housed for 12 months and male 12-week-old C57Bl/6 (wt) mice were subjected to either wire injury of the aortic valve (WI) to induce a moderate aortic valve stenosis, or a sham surgery. Echocardiographic analysis of the aortic valve was performed four weeks after WI and in 12-months-old eNOSko compared to sham and baseline to verify development of aortic valve stenosis. Cryosections of aortic valves of 12-month-old eNOS KO mice and wt were stained with a) hematoxylin and eosin (H&E), b) anti-CD68, c) anti-α-SMA or anti-vimentin and d) anti-BMP2 or anti-RUNX2 antibodies. From images of H&E stained whole valves, the diameter of each leaflet was measured. In the antibody-stained valves, number of antibody positive cells per total number of cells was calculated.

Results: In histological analysis 12-month-old eNOS KO mice showed an increased aortic valve leaflet thickness, comparable to that of mice four weeks after WI. While the number of CD68 positive macrophages was increased in aortic valves four weeks after WI, no increase in number of macrophages could be observed in the valves of eNOS KO mice. α-SMA and vimentin as markers of aortic valve fibrosis were increased in both mice after WI and in eNOS KO animals. In contrast, of the calcification markers only BMP2 was increased in aortic valves as well of mice after WI as of eNOS KO mice, while the expression level of RUNX2 was unchanged.

Conclusion: Fibrosis and calcification contribute to the spontaneous development of AS in eNOS KO mice, but, in contrast to the WI model, there is no inflammatory component in the aging eNOS KO AS model. This might reflect AS at different stages. On the other hand, it suggests different pathomechanisms behind the development of spontaneous and induced AS, which requires further analysis. These results point out, that besides mechanical stress also disturbances in enzyme expression (e.g. eNOS), result in AS via different pathomechanisms and might serve as potential therapeutic targets.