Early mechanisms of aortic failure in a zebrafish model for thoracic aortic dissection and rupture

Thoracic aortic aneurysm and dissection (TAAD) associates with a high mortality rate. Despite the existence of different mouse models for TAAD, the underlying disease mechanisms remain elusive. Treatment options are limited and mainly consist of surgical repair at critical aortic diameters as current pharmacological interventions are unable to stop disease progression. In humans, loss of function (LOF) of SMAD3 and SMAD6 impairs vascular homeostasis, increasing the risk for TAAD. We developed a zebrafish model for thoracic aortic dissection/rupture by targeting both ohnologs of smad3 and smad6 . At 10 days post fertilization, we found an increased diameter of the ventral aorta in smad3a −/−; smad3b −/− double knockout zebrafish, while smad6a −/−; smad6b −/− double knockout zebrafish have a reduced aortic diameter associated with early mortality. We discovered that a smad3a −/−; smad3b −/−; smad6a −/−; smad6b −/− quadruple knockout (qKO) zebrafish model is viable and survives to adulthood, although exposure to stress leads to sudden death. Histological analysis of the adult ventral aorta shows medial elastolysis, aortic dissections and ruptures at sites exposed to high biomechanical stress. RNA-sequencing of 5 days post fertilization qKO zebrafish indicates a profile of reduced negative regulation of proteolysis and upregulation of melanogenesis, a previously unaddressed pathway in this pathology. We confirm that pharmacological modulation of tyrosinase, the enzyme responsible for the production of melanin, influences aortic morphology. Overall, the qKO mutant, thus far the only known zebrafish model of thoracic aortic dissection and rupture, reveals novel SMAD3/6-dependent pathways that impact thoracic aortic homeostasis, in this way opening avenues for the development of novel treatments in TAAD. ### Competing Interest Statement The authors have declared no competing interest. * AA3 : aortic arch 3 AA4 : aortic arch 4 BMPs : bone morphogenetic proteins Co-SMAD : common mediator SMAD CPM : counts per million DEG : differentially expressed genes DKO : double knockout dpf : days post fertilization ECM : extracellular matrix FDR : false discovery rate GDFs : differentiation factors GO : gene ontology GRCz11 : Genome Reference Consortium Zebrafish Build 11 GSEA : gene set enrichment analysis ID : intellectual disability I-SMAD : inhibitory SMAD KEGG : Kyoto Encyclopedia of Genes and Genomes LDS3 : Loeys-Diets syndrome type 3 LOF : loss of function mpf : months post fertilization MR : magnetic resonance PSI : Paul Scherrer Institut PTU : 1-phenyl-2-thiourea treatment qKO : smad3a−/−;smad3b−/−;smad6a−/−;smad6b−/− quadruple knockout RO : reverse osmosis R-SMAD : receptor-regulated SMAD SKO : single knockout smad3a/b DKO : smad3a−/−;smad3b−/− double knockout smad6a/b DKO : smad6a−/−;smad6b−/− double knockout TAA : thoracic aortic aneurysm TAAD : thoracic aortic aneurysm and dissection TAD : thoracic aortic dissection TEM : transmission electron microscopy TGF-β : transforming growth factor β ZIRC : Zebrafish International Research Center