Mice knockdown for angiopoietin‐like 2 spontaneously develop aortic valve stenosis

High circulating levels of angiopoietin‐like 2 (ANGPTL2), a pro‐inflammatory protein that plays a role in tissue remodelling, are associated with carotid wall thickening, great artery stiffening, coronary artery disease and heart failure. ANGPTL2 is therefore considered as a predictive marker of cardiovascular diseases. Cardiac valve leaflet thickening and calcification lead to aortic valve stenosis (AVS), the most common valve disease for which there is currently no effective pharmacological treatment. Whether ANGPTL2 regulates aortic valve (AoV) structure and function is unknown. Our aim was to investigate the consequences of knockdown (KD) of ANGPTL2 on AoV structure, function and related molecular pathways in mice. Hypothesis ANGPTL2‐KD is beneficial to the cardiovascular system and prevents age‐related aortic valve leaflet thickening and dysfunction. Methods All the experiments were performed in ANGPTL2‐KD mice and wild‐type (WT) littermates of both sexes. The AoV function was explored by high frequency echocardiography. The AoV leaflet fibrosis was assessed in cardiac histological sections stained with Masson's Trichrome. Gene and protein expressions were determined by quantitative RT‐PCR in dissected fresh AoV leaflets and by immunofluorescence in cardiac histological sections, respectively. ANGPTL2 protein expression was measured by Western Blot in valve interstitial cells (VICs) cultured from AoV of WT mice. Results Contrary to our hypothesis, echocardiography evaluation of AoV function revealed that in young (2‐month old, mo) ANGPTL2‐KD mice (5 males and 5 females), 8/10 had severe AVS characterised by a ~50% reduction in valve area (p<0.001) and by thickened AoV leaflets (193±60 vs . 140±40 μm, p<0.01) compared to WT mice. The severity of the AVS was similar among male and female mice and did not worsen in adult mice (7‐mo). Surprisingly, at 2‐mo, KD of ANGPTL2 induced AoV leaflet fibrosis in female mice (+47%, n≥4, p<0.05) but not in males (+5%, n=3). Analysis of gene expression in freshly isolated AoV leaflets showed an increase (+130%) of the pro‐calcifying gene bmp2 , and a decrease (−38%) of notch1 signalling – known to participate to the structural development and homeostasis of AoV leaflets – including its downstream targets hey1 and hey2 (−60% and −43%, respectively; p<0.05 vs . WT). The reduction of activated‐Notch1 expression in AoV leaflets was also observed at the protein level (−28%, n=6, p<0.05). We also confirmed that ANGPTL2 is expressed in VICs from WT mice. Conclusion Our data reveal for the first time that KD of ANGPTL2 is deleterious to AoV structure by activating the calcification‐related gene bmp2 and repressing the key Notch1 pathway, leading to severe valvular disease in mice. This new mouse model of spontaneous AVS could lead to a better understanding of the molecular events that trigger its onset. Support or Funding Information This work was funded by grants from the Canadian Institutes of Health Research (MOP 133649) and by the Foundation of the Montreal Heart Institute. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .