Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs — including in HHT patient blood outgrowth ECs — and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

Background: Hereditary hemorrhagic telangiectasia ( HHT ) is a rare genetic vascular disorder caused by mutations in endoglin ( ENG ), activin receptor‐like kinase 1 ( ACVRL 1 ; ALK 1 ), or SMAD 4 . Major clinical symptoms of HHT are arteriovenous malformations ( AVM s) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD 4 mutations have been lacking. The goal of this study is to evaluate Smad4 ‐inducible knockout ( iKO ) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models. Methods and Results: Global Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4 ‐ iKO mice exhibited signs of gastrointestinal bleeding and AVM s in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVM s were detected on both distal and proximal retinal vasculature of Smad4 ‐ iKO s. Aberrant smooth muscle actin staining was observed in the initial stage AVM s and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVM s along the gastrointestinal tract and wounded skin. HHT ‐related phenotypes of Smad4 ‐ iKO s appeared to be comparable with those found in Alk1 ‐ iKO and Eng ‐ iKO mice. Conclusions: These data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4 ‐ iKO will be an alternative animal model of AVM research associated with HHT .

Rationale: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG , ALK1 , or SMAD4 . Since proteins from all 3 HHT genes are components of signal transduction of TGF-β (transforming growth factor β) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. Objective: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT. Methods and Results: We generated a novel mouse allele (ROSA26 ^Alk1 ) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26 ^Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1 - and Eng -inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng -iKO as well as Alk1 -iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1 -iKO models. Conclusions: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by the presence of arteriovenous malformation (AVM) in multiple organs. HHT is caused by mutations in genes encoding major constituents for transforming growth factor-β (TGF-β) family signaling: endoglin (ENG), activin receptor-like kinase 1 (ALK1), and SMAD4. The identity of physiological ligands for this ENG-ALK1 signaling pertinent to AVM formation has yet to be clearly determined. To investigate whether bone morphogenetic protein 9 (BMP9), BMP10, or both are physiological ligands of ENG-ALK1 signaling involved in arteriovenous network formation, we generated a novel Bmp10 conditional knockout mouse strain. We examined whether global Bmp10-inducible knockout (iKO) mice develop AVMs at neonatal and adult stages in comparison with control, Bmp9-KO, and Bmp9/10-double KO (dKO) mice. Bmp10-iKO and Bmp9/10-dKO mice showed AVMs in developing retina, postnatal brain, and adult wounded skin, while Bmp9-KO did not display any noticeable vascular defects. Bmp10 deficiency resulted in increased proliferation and size of endothelial cells in AVM vessels. The impaired neurovascular integrity in the brain and retina of Bmp10-iKO and Bmp9/10-dKO mice was detected. Bmp9/10-dKO mice exhibited the lethality and vascular malformation similar to Bmp10-iKO mice, but their phenotypes were more pronounced. Administration of BMP10 protein, but not BMP9 protein, prevented retinal AVM in Bmp9/10-dKO and endothelial-specific Eng-iKO mice. These data indicate that BMP10 is indispensable for the development of a proper arteriovenous network, whereas BMP9 has limited compensatory functions for the loss of BMP10. We suggest that BMP10 is the most relevant physiological ligand of the ENG-ALK1 signaling pathway pertinent to HHT pathogenesis.