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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.

No MeSH data available.


Chronic Dipy treatment decreases BMP2-induced HO lesions and calcium deposition. (A) Hematoxylin and eosin (H&E) staining showing lesions in muscle injected with BMP-2 and cardiotoxin in mice treated with vector or Dipy for 10 or 21 days. No lesions appear in muscle damaged without BMP. Scale bars: 1 mm. (B) Alizarin Red S staining showing calcium deposition in tissue sections of muscle injected with BMP2 and cardiotoxin in mice treated with the vehicle or Dipy for 21 days. Scale bars: 500 μm. (C) Graph representing the relative quantification of the Alizarin-Red-S-positive area. Data represent mean±s.e.m. *P≤0.05 (n=3, Dipy versus control).
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DMM023929F7: Chronic Dipy treatment decreases BMP2-induced HO lesions and calcium deposition. (A) Hematoxylin and eosin (H&E) staining showing lesions in muscle injected with BMP-2 and cardiotoxin in mice treated with vector or Dipy for 10 or 21 days. No lesions appear in muscle damaged without BMP. Scale bars: 1 mm. (B) Alizarin Red S staining showing calcium deposition in tissue sections of muscle injected with BMP2 and cardiotoxin in mice treated with the vehicle or Dipy for 21 days. Scale bars: 500 μm. (C) Graph representing the relative quantification of the Alizarin-Red-S-positive area. Data represent mean±s.e.m. *P≤0.05 (n=3, Dipy versus control).

Mentions: Ossicle formation and HO volume were evaluated by μCT scan after 10 (n=6 for each group, Fig. S5 protocol A) and 21 (n=11 for each group, Fig. S5 protocol A) days of treatment. After 10 days of treatment, we observed highly variable volumes of HO (mineralized volume, mm3) in control mice and no significant difference in HO volume was observed in treated mice compared to controls (Fig. 6A,B). By contrast, after 21 days of treatment, μCT scans showed a significant reduction of HO volume in mice treated with Dipy compared to controls (Fig. 6C,D). Histological analysis revealed that HO lesions (Fig. 7A) in treated mice were reduced, possibly due to a delay in maturation. In particular, Toluidine Blue staining indicated a reduced deposition of cartilage matrix, also at 10 days of treatment (Fig. S6), whereas Alizarin Red staining at 21 days and quantification of the area of calcium deposition showed a decrease in lesions of Dipy-treated mice (Fig. 7B,C), in agreement with the μCT scan results.Fig. 6.


High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva
Chronic Dipy treatment decreases BMP2-induced HO lesions and calcium deposition. (A) Hematoxylin and eosin (H&E) staining showing lesions in muscle injected with BMP-2 and cardiotoxin in mice treated with vector or Dipy for 10 or 21 days. No lesions appear in muscle damaged without BMP. Scale bars: 1 mm. (B) Alizarin Red S staining showing calcium deposition in tissue sections of muscle injected with BMP2 and cardiotoxin in mice treated with the vehicle or Dipy for 21 days. Scale bars: 500 μm. (C) Graph representing the relative quantification of the Alizarin-Red-S-positive area. Data represent mean±s.e.m. *P≤0.05 (n=3, Dipy versus control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920148&req=5

DMM023929F7: Chronic Dipy treatment decreases BMP2-induced HO lesions and calcium deposition. (A) Hematoxylin and eosin (H&E) staining showing lesions in muscle injected with BMP-2 and cardiotoxin in mice treated with vector or Dipy for 10 or 21 days. No lesions appear in muscle damaged without BMP. Scale bars: 1 mm. (B) Alizarin Red S staining showing calcium deposition in tissue sections of muscle injected with BMP2 and cardiotoxin in mice treated with the vehicle or Dipy for 21 days. Scale bars: 500 μm. (C) Graph representing the relative quantification of the Alizarin-Red-S-positive area. Data represent mean±s.e.m. *P≤0.05 (n=3, Dipy versus control).
Mentions: Ossicle formation and HO volume were evaluated by μCT scan after 10 (n=6 for each group, Fig. S5 protocol A) and 21 (n=11 for each group, Fig. S5 protocol A) days of treatment. After 10 days of treatment, we observed highly variable volumes of HO (mineralized volume, mm3) in control mice and no significant difference in HO volume was observed in treated mice compared to controls (Fig. 6A,B). By contrast, after 21 days of treatment, μCT scans showed a significant reduction of HO volume in mice treated with Dipy compared to controls (Fig. 6C,D). Histological analysis revealed that HO lesions (Fig. 7A) in treated mice were reduced, possibly due to a delay in maturation. In particular, Toluidine Blue staining indicated a reduced deposition of cartilage matrix, also at 10 days of treatment (Fig. S6), whereas Alizarin Red staining at 21 days and quantification of the area of calcium deposition showed a decrease in lesions of Dipy-treated mice (Fig. 7B,C), in agreement with the μCT scan results.Fig. 6.

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.