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High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva

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ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

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Histological analysis of ATDC5 chondrogenic differentiation. (A) Alcian Blue staining of sections from ATDC5 cell 3D cultures grown in proliferative medium (PM, upper panels) and in differentiation medium (DM, lower panels), in the presence of 50 µM Dipy or DMSO. Scale bars: 100 μm (10×) and 50 μm (20×). (B) Histological and immunohistochemical analysis of ATDC5 cells cultured as alginate spheres grown in DM in the presence of DMSO or 50 µM Dipy (upper and lower panels, respectively). Scale bars: 25 μm.
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DMM023929F3: Histological analysis of ATDC5 chondrogenic differentiation. (A) Alcian Blue staining of sections from ATDC5 cell 3D cultures grown in proliferative medium (PM, upper panels) and in differentiation medium (DM, lower panels), in the presence of 50 µM Dipy or DMSO. Scale bars: 100 μm (10×) and 50 μm (20×). (B) Histological and immunohistochemical analysis of ATDC5 cells cultured as alginate spheres grown in DM in the presence of DMSO or 50 µM Dipy (upper and lower panels, respectively). Scale bars: 25 μm.

Mentions: ATDC5 cells were induced to develop 3D pellets in the presence of differentiating medium (DM), with and without Dipy (50 µM). After 3 weeks of culture, pellets were embedded in paraffin, and histological sections stained with Alcian Blue to verify the deposition of glycosaminoglycans typical of the cartilage extracellular matrix. As shown in Fig. 3A (left panels), compared to what was observed in proliferative medium (PM), pellets grown in DM are characterized by the presence of cells with peculiar morphology, with typical lacunae embedded in the extracellular matrix. By contrast, pellets grown in the presence of Dipy, both in PM and DM, showed the presence of small and undifferentiated cells (Fig. 3A, right panels).Fig. 3.


High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva
Histological analysis of ATDC5 chondrogenic differentiation. (A) Alcian Blue staining of sections from ATDC5 cell 3D cultures grown in proliferative medium (PM, upper panels) and in differentiation medium (DM, lower panels), in the presence of 50 µM Dipy or DMSO. Scale bars: 100 μm (10×) and 50 μm (20×). (B) Histological and immunohistochemical analysis of ATDC5 cells cultured as alginate spheres grown in DM in the presence of DMSO or 50 µM Dipy (upper and lower panels, respectively). Scale bars: 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920148&req=5

DMM023929F3: Histological analysis of ATDC5 chondrogenic differentiation. (A) Alcian Blue staining of sections from ATDC5 cell 3D cultures grown in proliferative medium (PM, upper panels) and in differentiation medium (DM, lower panels), in the presence of 50 µM Dipy or DMSO. Scale bars: 100 μm (10×) and 50 μm (20×). (B) Histological and immunohistochemical analysis of ATDC5 cells cultured as alginate spheres grown in DM in the presence of DMSO or 50 µM Dipy (upper and lower panels, respectively). Scale bars: 25 μm.
Mentions: ATDC5 cells were induced to develop 3D pellets in the presence of differentiating medium (DM), with and without Dipy (50 µM). After 3 weeks of culture, pellets were embedded in paraffin, and histological sections stained with Alcian Blue to verify the deposition of glycosaminoglycans typical of the cartilage extracellular matrix. As shown in Fig. 3A (left panels), compared to what was observed in proliferative medium (PM), pellets grown in DM are characterized by the presence of cells with peculiar morphology, with typical lacunae embedded in the extracellular matrix. By contrast, pellets grown in the presence of Dipy, both in PM and DM, showed the presence of small and undifferentiated cells (Fig. 3A, right panels).Fig. 3.

View Article: PubMed Central - PubMed

ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

No MeSH data available.


Related in: MedlinePlus