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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.

We read with interest the article by Tu et al1 demonstrating that genetic loss of Bmp9 (bone morphogenetic protein 9) protects mice from hypoxia-induced pulmonary hypertension and that inhibition of BMP9 with anti-BMP9 or the BMP9/BMP10 ligand trap (ALK1 [activin receptor-like kinase 1] extracellular domain) partly protects rodents from the progression of pulmonary hypertension. These results are at odds with the human genetic evidence showing that loss-of-function mutations in GDF2/BMP9 play a causal role in heritable pulmonary arterial hypertension (PAH).2 Loss-of-function mutations in the endothelial receptors for BMP9, BMPR2,3 and ALK14 are also important causes of heritable PAH. Furthermore, patients with portopulmonary hypertension exhibit extremely low levels of plasma BMP9.5 In that article, administration of the ligand trap, ALK1-Fc (ALK-1 fused to the IgG Fc domain), exacerbated hypoxia-induced pulmonary hypertension in mice, which directly contradicts similar experiments performed by Tu et al.1 Moreover, therapeutic administration of exogenous BMP9 reverses pulmonary hypertension in the monocrotaline and sugen/hypoxia rat models of pulmonary hypertension and a genetic mouse model of BMPR2 deficiency.6 Notably, Tu et al1 did not examine whether BMP9 (or BMP10) administration affected the course of disease. Thus, the results presented by Tu et al1 appear counterintuitive in the context of human and rodent data that support the view that therapeutic enhancement of the BMP9/BMPR2/ALK1 signaling axis in PAH patients is beneficial.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-β receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC 50 (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.