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

Thoracic aortic dissection (TAD) is associated with a high mortality rate. Despite the existence of different mouse models for TAD, the underlying disease mechanisms remain elusive. Treatment options are limited and mainly consist of surgical repair at critical diameters as current pharmacological interventions are unable to stop disease progression. In humans, loss of function (LOF) of SMAD3 and SMAD6 impair vascular homeostasis, increasing the risk for TAD. We developed a zebrafish model for thoracic aortic dissection/rupture by targeting both ohnologs of smad3 and smad6. We discovered an increased diameter of the ventral aorta in smad3a-/-;smad3b-/- double knockout zebrafish larvae, while smad6a-/-;smad6b-/- double knockout zebrafish have a reduced diameter associated with early mortality. Surprisingly, the 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 showed medial elastolysis and aortic dissections and ruptures at sites exposed to high hemodynamic stress. RNA-sequencing of qKO larvae indicated a profile of reduced negative regulation of proteolysis and upregulation of melanogenesis, a previously unaddressed pathway in this pathology. We confirmed that pharmacological modulation of tyrosinase has an effect on aortic morphology. Our study shows that dysregulation of SMAD3/6-dependent signaling has an important impact on thoracic aortic homeostasis, and that using the qKO zebrafish model, thus far the only known model of aortic dissection and rupture in this species, can identify novel pathophysiological pathways leading to TAD. Overall design: For RNA extraction, the Rneasy® minikit (Qiagen; Hilden, Germany) was used following manufacturers guidelines. One experiment was performed using TruSeq. RNA sequencing was performed on five pools, each containing five smad3a-/-;smad3b-/-;smad6a-/-;smad6b-/- quadruple knockout (qKO) zebrafish or wt control cousins. Only samples with RNA integrity number (RIN) values higher than 9 were considered. All other experiments were performed using Quantseq. For quantseq sample 1-10, RNA sequencing was performed on five pools, each containing three smad3a-/-;smad3b-/-;smad6a-/-;smad6b-/- quadruple knockout (qKO) adult aortas and five pools of three wt control cousins aortas. For quantseq sample 11-20, RNA sequencing was performed on five pools, each containing three smad3a-/-;smad3b-/- double knockout (DKO) zebrafish or wt control cousins. For quantseq sample 21-30, RNA sequencing was performed on five pools, each containing three smad6a-/-;smad6b-/- double knockout (DKO) zebrafish or wt control cousins.