Potential Therapeutic Effects Of Angiopoietin-2 Inhibition On Rescuing Hereditary Hemorrhagic Telangiectasia Associated Cerebrovascular Defects

Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular disease characterized by arteriovenous malformations (AVMs), which are abnormal, enlarged connections between arteries and veins that are prone to hemorrhage. AVMs often form in the brain and can lead to stroke. Mutations in endoglin (Eng), activin receptor-like kinase 1 (Alk1) or smad-related protein 4 (Smad4) cause HHT. Endothelial cell (EC) specific knock out of Alk1, Eng, or Smad4 in mice mimics HHT vascular defects, including AVM formation. We showed that Smad4 directly repressed transcription of the critical pro-angiogenic factor Angiopoietin-2 (Ang2) in ECs and that in Smad4-HHT mice, Ang2 /ANG2 levels are robustly and ectopically increased in ECs. By inhibiting Ang2, we prevented and rescued AVMs and normalized retinal vasculature. Since Smad4 is a central transcription factor in the TGF-β signaling pathway and a downstream effector of the Eng and Alk1 co-receptors, we hypothesized that Ang2 inhibition may universally normalize HHT vascular phenotypes. Methods: Blue latex dye perfusion methods were used to visualize and characterize cerebrovascular defects in HHT mouse models (n=5~7). RNA-seq was performed on isolated brain ECs (iBECs) to further define molecular phenotypic characteristics (n=5). In situ hybridization on brain slices (n=3) and ELISA on whole brain lysate (n=3~9) were performed to confirm elevated Ang2 RNA and protein levels. Ang2 inhibitor was delivered via intraperitoneal injection, followed by cerebrovascular assessments (n=5~9). Results: We found that brain AVMs are present in Smad4-, Alk1-, and Eng-HHT mutants, with all models displaying elevated Ang2 levels. RNA-seq of iBECs in Smad4-, Alk1-, and Eng-HHT models reveals 5502, 5307, and 1659 differentially expressed genes in respective mutants compared to controls. Data set comparisons revealed common, increased pro-angiogenic genes, which helped to define the HHT-specific angiogenic profile. Moreover, Ang2 inhibition decreased Smad4-HHT’s brain vessel diameter and Alk1-HHT’s brain vessel density, and prevented brain AVM formation in Eng-HHT mice. In conclusion, Ang2 inhibition partially normalized cerebrovascular defects in Smad4- and Alk1-HHT mice, and completely in Eng-HHT mice.