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

Abstract

Background: Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (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 (AVMs), 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 hereditary hemorrhagic telangiectasia–associated AVM formation are currently unknown. Methods: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation–sequencing experiments on BMP9 (bone morphogenetic protein 9)-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 used 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, whereas 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 the 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 hereditary hemorrhagic telangiectasia 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 hereditary hemorrhagic telangiectasia symptoms, such as AVMs.

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models

“…At this Smad1/5/8 response to ALK1-endoglin receptor activation by BMP9 (14)(15)(16). Consistent with this model, ALK1, endoglin, or Smad4 inactivation in mice and zebrafish leads to vascular defects, which include hypervascularization and AVMs (17)(18)(19)(20)(21)(22)(23)(24)(25). Downstream from ALK1-endoglin receptor loss of function, the exact pathways involved in HHT pathogenesis and ultimately, AVM development -i.e., the formation of direct shunts between an artery and a vein -remain incompletely understood (26).…”

Eye lymphatic defects induced by bone morphogenetic protein 9 deficiency have no functional consequences on intraocular pressure

“…In addition, the combined deletion of both ANGPT1/ANGPT2 induced severe defects in eye lymphatic vessel development 27 . A regulation of the expression of ANGPT2 by BMP9 and/or of its downstream signaling has recently been documented 51,52 . Thus, the possible existence of another crosstalk between BMP9 and ANGPT1, in eye lymphatic vessels, should be investigated in future studies.…”

Sirolimus plus nintedanib treats vascular pathology in HHT mouse models

“…Indeed, HHT-causing mutations decrease Smad1/5/8 response to ALK1-endoglin receptor activation by BMP9 (14)(15)(16). Consistent with this model, ALK1, endoglin, or Smad4 inactivation in mice and zebrafish leads to vascular defects, which include hypervascularization and AVMs (17)(18)(19)(20)(21)(22)(23)(24)(25). Downstream from ALK1-endoglin receptor loss-of-function, the exact pathways involved in HHT pathogenesis and ultimately, AVM development-i.e., the formation of direct shunts between an artery and a vein-remain incompletely understood (26).…”