Search

Patients with a congenital bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, have an increased risk of developing thoracic aneurysms and aortic dissection. The mechanisms underlying BAV-associated aortopathy are poorly understood. This study examined BAV-associated aortopathy in Nos3−/− mice, a model with congenital BAV formation. A combination of histological examination and in vivo ultrasound imaging was used to investigate aortic dilation and dissections in Nos3−/− mice. Moreover, cell lineage analysis and single-cell RNA sequencing were used to observe the molecular anomalies within vascular smooth muscle cells (VSMCs) of Nos3−/− mice. Spontaneous aortic dissections were found in ascending aortas located at the sinotubular junction in ∼13% of Nos3−/− mice. Moreover, Nos3−/− mice were prone to developing aortic dilations in the proximal and distal ascending aorta during early adulthood. Lower volumes of elastic fibres were found within vessel walls of the ascending aortas of Nos3−/− mice, as well as incomplete coverage of the aortic inner media by neural crest cell (NCC)-derived VSMCs. VSMCs of Nos3−/− mice showed downregulation of 15 genes, of which seven were associated with aortic aneurysms and dissections in the human population. Elastin mRNA was most markedly downregulated, followed by fibulin-5 expression, both primary components of elastic fibres. This study demonstrates that, in addition to congenital BAV formation, disrupted endothelial-mediated nitric oxide (NO) signalling in Nos3−/− mice also causes aortic dilation and dissection, as a consequence of inhibited elastic fibre formation in VSMCs within the ascending aorta.

Lipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the cardiovascular development of Lrp2 KO mice between embryonic day 10.5 (E10.5) and E15.5, applying morphometry and immunohistochemistry, using antibodies against Tfap2α (neural crest cells), Nkx2.5 (second heart field), WT1 (epicardium derived cells), tropomyosin (myocardium) and LRP2. The Lrp2 KO mice display a range of severe cardiovascular abnormalities, including aortic arch anomalies, common arterial trunk (persistent truncus arteriosus) with coronary artery anomalies, ventricular septal defects, overriding of the tricuspid valve and marked thinning of the ventricular myocardium. Both the neural crest cells and second heart field, which are essential for the lengthening and growth of the right ventricular outflow tract, are abnormally positioned in the Lrp2 KO. This explains the absence of the aorto-pulmonary septum, which leads to common arterial trunk and ventricular septal defects. Severe blebbing of the epicardial cells covering the ventricles is seen. Epithelial-mesenchymal transition does occur; however, there are fewer WT1-positive epicardium-derived cells in the ventricular wall as compared to normal, coinciding with the myocardial thinning and deep intertrabecular spaces. LRP2 plays a crucial role in cardiovascular development in mice. This corroborates findings of cardiac anomalies in humans with LRP2 mutations. Future studies should reveal the underlying signaling mechanisms in which LRP2 is involved during cardiogenesis.

The bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, belongs to the most prevalent congenital heart diseases in the world, occurring in 0.5-2% of the general population. We aimed to understand how changes in early cellular contributions result in BAV formation and impact cardiovascular outflow tract development. Detailed 3D reconstructions, immunohistochemistry and morphometrics determined that, during valvulogenesis, the non-coronary leaflet separates from the parietal outflow tract cushion instead of originating from an intercalated cushion. Nos3−/− mice develop a BAV without a raphe as a result of incomplete separation of the parietal outflow tract cushion into the right and non-coronary leaflet. Genetic lineage tracing of endothelial, second heart field and neural crest cells revealed altered deposition of neural crest cells and second heart field cells within the parietal outflow tract cushion of Nos3−/− embryos. The abnormal cell lineage distributions also affected the positioning of the aortic and pulmonary valves at the orifice level. The results demonstrate that the development of the right and non-coronary leaflets are closely related. A small deviation in the distribution of neural crest and second heart field populations affects normal valve formation and results in the predominant right-non-type BAV in Nos3−/− mice.