Role of soluble endoglin in BMP9 signaling.

Endoglin (ENG) is a coreceptor of the transforming growth factor-β (TGFβ) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.British Heart Foundation, Cancer Genomics Centre Netherlands and from the Netherlands CardioVascular Research Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON PHEADRA)

Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis

Members of the transforming growth factor β (TGF-β) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-β and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-β and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-β and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-β family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis

Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents

Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and β4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature

Role of soluble endoglin in BMP9 signaling

Endoglin (ENG) is a coreceptor of the transforming growth factor-β (TGFβ) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases

<i>Eng</i>^−/− ESC-derived EBs plated on gelatin coated plates lack organized vessel structures.

<p>(A) PECAM-1 immunohistochemical staining of ESC-derived 11-day-old EBs plated on gelatin for 4 days displayed “organized”, “intermediate” or “dispersed” phenotype. (B) Quantification of wild type, <i>Eng</i>^+/−, and <i>Eng</i>^−/− ESC-derived 15-day-old EBs vascular phenotypes as they were defined in A.</p

<i>Eng</i> deficiency inhibits VEGF-induced sprouting of HUVEC spheroids.

<p>(A) Effect of shRNA-mediated depletion of <i>Eng</i> on VEGF-induced endothelial cell sprouting. HUVECs were transduced with lentivirus expressing endoglin shRNA overnight. HUVEC spheroids with deficient endoglin expression were embedded in collagen and stimulated with VEGF (50 ng/ml). A representative experiment is shown. (B) Quantitation of effects seen in (A). (C) Effect of TRC105 ENG antibody on VEGF-induced endothelial cell sprouting. HUVEC spheroids were embedded in collagen and stimulated with VEGF (50 ng/ml), TRC105 (10 µg/ml), or both overnight. As control antibody for experiments using TRC105, the Fc domain (MOPC-21) from Bio Express, West Lebanon, NH, was used. Pictures were taken by phase-contrast microscopy. Quantitative analysis of the mean total sprout length was performed on 10 spheroids per experimental group. <i>P</i>≤0.05. (D) VEGF-induced VEGFR2 phosphorylation at site 1175 and extracellular regulated kinase (ERK) mitogen activated protein (MAP) kinase phosphorylation was examined in shRNA-mediated <i>Eng</i> knockdown cells. Two bands were detected with the phosphor ERK MAPK antibodies with a molecular weight of 44 and 42 kDa; they represent ERK1 and ERK2 isoforms, respectively. Number sign (#) represents a background band, indicating the even loading for the experiment. Asterisks indicate the protein bands with expected size.</p

VEGF-induced angiogenesis is impaired in <i>Eng</i>^+/⁻ fetal metatarsal bones.

<p>Metatarsals of 17-day-old mouse fetuses were prepared from wild type and <i>Eng</i>^+/− mice, transferred to cell-culture plates, allowed to adhere, and then stimulated with VEGF (50 ng/ml). (A) Cultures were fixed and vessel-like structures were visualized by anti-PECAM-1 staining. Six bones were stimulated per experimental group and one representative picture of each group is shown. (B) VEGF addition stimulated the formation of vessel-like structures. No significant difference in the baseline vascular network formation was observed between wild type and <i>Eng</i>^+/− metatarsals. The induction of the vascular network of wild type metatarsals is significantly stronger than the network of <i>Eng</i>^+/− metatarsals. <i>P</i>≤0.05.</p