Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Interaction Between ALK1 Signaling and Connexin40 in the Development of Arteriovenous Malformations.

OBJECTIVE: To determine the role of Gja5 that encodes for the gap junction protein connexin40 in the generation of arteriovenous malformations in the hereditary hemorrhagic telangiectasia type 2 (HHT2) mouse model. APPROACH AND RESULTS: We identified GJA5 as a target gene of the bone morphogenetic protein-9/activin receptor-like kinase 1 signaling pathway in human aortic endothelial cells and importantly found that connexin40 levels were particularly low in a small group of patients with HHT2. We next took advantage of the Acvrl1(+/-) mutant mice that develop lesions similar to those in patients with HHT2 and generated Acvrl1(+/-); Gja5(EGFP/+) mice. Gja5 haploinsufficiency led to vasodilation of the arteries and rarefaction of the capillary bed in Acvrl1(+/-) mice. At the molecular level, we found that reduced Gja5 in Acvrl1(+/-) mice stimulated the production of reactive oxygen species, an important mediator of vessel remodeling. To normalize the altered hemodynamic forces in Acvrl1(+/-); Gja5(EGFP/+) mice, capillaries formed transient arteriovenous shunts that could develop into large malformations when exposed to environmental insults. CONCLUSIONS: We identified GJA5 as a potential modifier gene for HHT2. Our findings demonstrate that Acvrl1 haploinsufficiency combined with the effects of modifier genes that regulate vessel caliber is responsible for the heterogeneity and severity of the disease. The mouse models of HHT have led to the proposal that 3 events-heterozygosity, loss of heterozygosity, and angiogenic stimulation-are necessary for arteriovenous malformation formation. Here, we present a novel 3-step model in which pathological vessel caliber and consequent altered blood flow are necessary events for arteriovenous malformation development.

Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by vascular malformations. Many affected individuals develop recurrent nosebleeds, which can severely affect their quality of life and are clinically difficult to treat. We report here that treatment with thalidomide reduced the severity and frequency of nosebleeds (epistaxis) in the majority of a small group of subjects with HHT tested. The blood hemoglobin levels of the treated individuals rose as a result of reduced hemorrhage and enhanced blood vessel stabilization. In mice heterozygous for a null mutation in the Eng gene (encoding endoglin), an experimental model of HHT, thalidomide treatment stimulated mural cell coverage and thus rescued vessel wall defects. Thalidomide treatment increased platelet-derived growth factor-B (PDGF-B) expression in endothelial cells and stimulated mural cell activation. The effects of thalidomide treatment were partially reversed by pharmacological or genetic interference with PDGF signaling from endothelial cells to pericytes. Biopsies of nasal epithelium from individuals with HHT treated or not with thalidomide showed that similar mechanisms may explain the effects of thalidomide treatment in humans. Our findings demonstrate the ability of thalidomide to induce vessel maturation, which may be useful as a therapeutic strategy for the treatment of vascular malformations.