P5988Non-classical Ly6C-low monocytes contribute to aortic valve stenosis development in mice
Authors: Niepmann S T , Weissheit C , Kleiner J , Kurts C , Nickenig G , Zimmer S 
Published: October 2019

Abstract

Introduction
Aortic valve stenosis (AS) is one of the most common valvular diseases in the western world. Once patients with AS develop symptoms mortality increases up to 50% over two years. For many years AS was believed to be a passive degenerative process. Recent findings suggest a more complex pathomechanism with a strict molecular and cellular regulation involving local inflammation and invasion of immune cells. Specifically, pro-inflammatory macrophages have been shown to play an important role in disease progression. On the other hand, monocyte counts in the peripheral blood of patients with severe AS are significantly decreased and there is an inverse correlation between valve orifice area and circulating monocyte count. In order to elucidate the exact function of pro-inflammatory classical (Ly6C-high) and non-classical (Ly6C-low) monocytes in disease initiation processes, we conducted a series of experiments in a model of murine AS.

Methods
In this study 10–12 weeks old male C57BL6/J or CX3CR1-deficient mice were investigated. For AS induction, a coronary springwire was introduced into the left ventricle, pushed and rotated over the aortic valve under echocardiographic guidance. In sham operated mice the wire was only inserted into the left carotid artery without manipulation of the aortic valve. Development of AS was confirmed via weekly ultrasound examinations. Immune cells in the aortic valve were quantified using flow cytometry and immunofluorescence microscopy.

Results
Ultrasound analysis after two weeks confirmed the development of AS in the injured wildtype mice. Transaortic peak velocity levels were significantly increased compared to sham operated mice. Flow cytometry of explanted aortic valves showed a strong cellular immunoreaction in the stenotic valves. The amount of anti-inflammatory Ly6C-low monocytes was significantly increased compared to sham-mice, whereas the number of pro-inflammatory Ly6C-high monocytes remained stable. Interestingly CD-4 positive T-cells were augmented in stenotic aortic valves. Immunofluorescence confirmed Ly6C-low monocytes infiltration in the valve, in close proximity to the endothelial cells on the ventricular and the aortic side. The expression of recruitment and adhesion markers CD11b and CX3CR1 were significantly increased on the surface of the Ly6C-low monocytes of AS mice. To investigate the role of Ly6C-low monocytes in AS development, CX3CR1 deficient mice, which are not able to recruit non-classical monocytes via CX3CR1, were subjected to aortic valve wire injury. After two weeks, peak velocity levels in CX3CR1−/− mice remained nearly unchanged and were significantly decreased compared to wildtype mice.

Conclusion
Our data suggest that non-classical Ly6Clow monocytes play a crucial role in AS development in mice. Therefore, they may be targets for future therapeutic interventions.

Acknowledgement/Funding
S.T.N. was funded by Else-Kröner-Fresenius-Foundation of the Medical Faculty of the University of Bonn