Disturbed nitric oxide signalling gives rise to congenital bicuspid aortic valve and aortopathy

ABSTRACT 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.


Fig. S2. Temporal distribution of spontaneous death events.
Wild type (n=103) and Nos3 -/-(n=133) in which spontaneous death was observed were examined for the chronologic distribution of death events. No significant (P >0.05) difference was observed between wild type and mutant populations using Mantel-Cox comparison of survival curves. with Sirius red to show collagen deposition in the media and adventitia of the ascending aorta. C-D: Sirius red staining of the embryonic aortic wall of (C) wild type and (D) Nos3 -/mice at stage E17.5. E-H: Volumetric quantification of collagen staining within the medial (E,G) as well as adventitial layers (F,H) of the adult and embryonic ascending aortic wall show no difference (P > 0.05) in the deposition of collagen between wild type and Nos3 -/mice. Ao: Aorta. Data are mean ± s.d. for n ≥ 3 mice per group. Scale bar: 50 µm h muscle cells populate the inner media of the scending aorta of Wnt1Cre;mTmG and .5. Neural crest derived vascular smooth muscle cells owing embryos of developmental age E17.5. Note that ced in neural crest derived VSMCs than VSMCs of ild type and ta.
Neural crest derived s he B: Transversal sections of th embryos at e cells (VSMCs) express both Wnt1Cr escent and B, but ote that expression of ACTA2 is more SMCs of different origin at E12.5 in both DAPI 50µm. Ao:
Neural crest derived smooth muscle cells populate the inner media of embryos at E12.5. Neural crest derived vascular smooth mus D: Fl and B, but showing embryos of developmental age E17.5. expression of ACTA2 is more pronounced in neural crest derived VSMCs than embryos. Nuclear staini ion normalized to Rpl32 and Gapdh. A-F: qPCR expression results of 6 month old wild type (N=5) and Rpl32 as well as Gapdh as reference genes. Statistical analysis were performed using a two tailed student T-test, * and ** indicate P<0.05 and P<0.01 respectively. A.U: Arbitrary Units. Data are mean ± sd.
F: qPCR expression results of 6 month old wild type (N=5) and Nos3 -/-(N=5) mice using as reference genes. Statistical analysis were performed using a two test, * and ** indicate P<0.05 and P<0.01 respectively. A.U: Arbitrary Units.
(N=5) mice using as reference genes. Statistical analysis were performed using a twotest, * and ** indicate P<0.05 and P<0.01 respectively. A.U: Arbitrary Units.  ELN_FWD   CCC ACC TCT TTG TGT TTC GC  ELN_REV  CCC AAA GAG CAC ACC AAC AAT  FBLN5_FWD  GTG CTT GGG GTT GGT TTT GA  FBLN5_REV  TCA GTT CCC CAT CTT TTG CCA  ACTA2_FWD  GCT ACG AAC TGC CTG ACG G  ACTA2_REV  TAG GTG GTT TCG TGG ATG CC  RPL32_FWD  CAC CAC TCA GAC CGA TAT GTG AAA A  RPL32_REV  TGT TGT CAA TGC CTC TGG GTT T  GAPDH_FWD TTG ATG GCA ACA ATC TCC AC GAPDH_REV CGT CCC GTA GAC AAA ATG GT