Distinct endothelial pathomechanisms drive arterio-venous malformation in Alk1 or Smad4 loss-of-function conditions

Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant disorder associated with mutations in the bone morphogenetic protein (BMP) pathway. Inherited heterozygous loss of function mutations and acquired loss of heterozygosity in either Alk1, Eng, or Smad4 lead to the development of arteriovenous malformations (AVMs), which trigger local vessel instability, hypoxia and vessel leakage or rupture. Current models assume common cellular pathomechanisms culminating in altered endothelial cell (EC) shape regulation, directional migration and proliferation control as a consequence of deficient BMP pathway signaling in ECs under the influence of blood-flow mediated shear stress. Here we report that loss of Alk1 or Smad4 surprisingly triggers very distinct endothelial phenotypes, signaling dynamics and transcriptional changes in ECs, both in vitro and in vivo. EC behavior in both in vivo and mosaic flow cultures illustrate that cells deficient in Smad4 effectively migrate against the direction of fluid shear, from veins to arteries, whereas cells lacking Alk1 fail to polarize and migrate against flow. Our data suggest that AVMs triggered by Smad4 mutations occur through hyperpruning of capillaries, thus precipitating flow in a single shunt, whereas AVMs caused by Alk1 mutation grow by cell accumulations close to the vein, as well as the persistence of a hyperdense plexus that drives nidus formation. We propose that the cellular pathomechanisms leading to AVM formation are not the same if the upstream BMP receptor Alk1, or the downstream common transcription factor Smad4 are mutated, raising the prospect for urgently needed, mechanism-based, therapeutic avenues that need to be tailored to correcting the specific pathomechanism. Overall design: In order to understand the how SMAD4 and Alk1 loss of function affects the transcriptome of endothelial cells exposed to laminar shear stress, we combined siRNA knockdown and flow exposure over time. Human umbilical venous endothelial cells (HUVECs) which were treated with following siRNAs: siCTRL, siSMAD4, siAlk1, and exposed to 4 or 16 hours of laminar fluid shear stress. Static controls were also collected for each condition. In addition, wildtype HUVECs were also used as a reference control, as well as a double knockdown of siAlk1+siSMAD4 (siDouble) as an additional control. In total, there are 5 treatment groups - WT, siCTRL, siSMAD4, siAlk1, siDouble; and 3 flow groups - static, 4h, 16h. Samples were collected from 3 independent experiments.