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A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

Kerr G, Sheldon H, Chaikuad A, Alfano I, von Delft F, Bullock AN, Harris AL - Angiogenesis (2015)

Bottom Line: K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses.In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition.Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford, OX3 7DQ, UK.

ABSTRACT
Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

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Related in: MedlinePlus

K02288 treatment results in a hypersprouting phenotype. a Endothelial spheroids were embedded in a fibrin gel and treated with either 1 μM K02288 or 100 ng/mL ALK1-Fc. The vehicle DMSO was used as a control. Ten spheroids per condition were photographed at ×10 magnification on day 2 or day 6 after embedding (representative examples are shown). b The number and length of sprouts were quantified on day 2 after embedding using ImageJ software. Experiments were carried out at least three times, and error bars represent SEM. Data were analysed using a one-way ANOVA; **p < 0.01, ***p < 0.001
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Fig3: K02288 treatment results in a hypersprouting phenotype. a Endothelial spheroids were embedded in a fibrin gel and treated with either 1 μM K02288 or 100 ng/mL ALK1-Fc. The vehicle DMSO was used as a control. Ten spheroids per condition were photographed at ×10 magnification on day 2 or day 6 after embedding (representative examples are shown). b The number and length of sprouts were quantified on day 2 after embedding using ImageJ software. Experiments were carried out at least three times, and error bars represent SEM. Data were analysed using a one-way ANOVA; **p < 0.01, ***p < 0.001

Mentions: We next analysed the effects of K02288 in 3D culture models of angiogenesis. We used a hanging drop assay in which endothelial cells are collected as spheroids and embedded in a fibrin gel. In this assay, treatment with K02288 resulted in a hypersprouting phenotype (Fig. 3a), with an increase in both the number and the length of vessels formed after 2 days (Fig. 3b). Hypersprouting was also observed upon treatment with ALK1-Fc, consistent with the work of Larrivée et al. [5] and in agreement with the expected cooperation between BMP9 and Notch signalling [5, 6]. Interestingly, Cunha et al. [27] have also reported an inhibitory effect of ALK1-Fc, perhaps reflecting that sprouting is both a highly dynamic and context dependent process.Fig. 3


A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

Kerr G, Sheldon H, Chaikuad A, Alfano I, von Delft F, Bullock AN, Harris AL - Angiogenesis (2015)

K02288 treatment results in a hypersprouting phenotype. a Endothelial spheroids were embedded in a fibrin gel and treated with either 1 μM K02288 or 100 ng/mL ALK1-Fc. The vehicle DMSO was used as a control. Ten spheroids per condition were photographed at ×10 magnification on day 2 or day 6 after embedding (representative examples are shown). b The number and length of sprouts were quantified on day 2 after embedding using ImageJ software. Experiments were carried out at least three times, and error bars represent SEM. Data were analysed using a one-way ANOVA; **p < 0.01, ***p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4363482&req=5

Fig3: K02288 treatment results in a hypersprouting phenotype. a Endothelial spheroids were embedded in a fibrin gel and treated with either 1 μM K02288 or 100 ng/mL ALK1-Fc. The vehicle DMSO was used as a control. Ten spheroids per condition were photographed at ×10 magnification on day 2 or day 6 after embedding (representative examples are shown). b The number and length of sprouts were quantified on day 2 after embedding using ImageJ software. Experiments were carried out at least three times, and error bars represent SEM. Data were analysed using a one-way ANOVA; **p < 0.01, ***p < 0.001
Mentions: We next analysed the effects of K02288 in 3D culture models of angiogenesis. We used a hanging drop assay in which endothelial cells are collected as spheroids and embedded in a fibrin gel. In this assay, treatment with K02288 resulted in a hypersprouting phenotype (Fig. 3a), with an increase in both the number and the length of vessels formed after 2 days (Fig. 3b). Hypersprouting was also observed upon treatment with ALK1-Fc, consistent with the work of Larrivée et al. [5] and in agreement with the expected cooperation between BMP9 and Notch signalling [5, 6]. Interestingly, Cunha et al. [27] have also reported an inhibitory effect of ALK1-Fc, perhaps reflecting that sprouting is both a highly dynamic and context dependent process.Fig. 3

Bottom Line: K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses.In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition.Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford, OX3 7DQ, UK.

ABSTRACT
Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

Show MeSH
Related in: MedlinePlus