Angiopoietin-2 Inhibition Rescues Arteriovenous Malformation in a Smad4 Hereditary Hemorrhagic Telangiectasia Mouse Model.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in four TGFβ pathway members, including the central transcriptional mediator of the TGFβ pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVM), which can hemorrhage leading to stroke, aneurysm or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood and the TGFβ downstream effectors responsible for HHT-associated AVM formation are currently unknown.

METHODS: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and ChIP-sequencing experiments on BMP9 stimulated endothelial cells (ECs) and isolated ECs from a Smad4 inducible, EC specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the Angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We utilized monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development.

RESULTS: Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, while Tek levels are decreased resulting in an overall reduction in Angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4 deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4.

CONCLUSIONS: Our studies provide a novel mechanism whereby loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of HHT alleviated these vascular phenotypes further implicating ANGPT2 as an important TGFβ downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of HHT symptoms, such as AVMs.