USE OF RETINOIC ACID RECEPTOR (RAR) AGONISTS FOR REVERSING, PREVENTING, OR DELAYING CALCIFICATION OF AORTIC VALVE

Abstract

Aortic valve calcification is a condition in which calcium deposits form on the aortic valve in the heart. These deposits can cause narrowing at the opening of the aortic valve. This narrowing can become severe enough to reduce blood flow through the aortic valve—a condition called aortic valve stenosis. The inventors have shown that retinoic acid decreases calcification and osteoblast-like phenotype in valvular interstitial cells (VICs). More particularly, RARα activation reduces calcification and osteoblast-like phenotype in VIC. On the contrary, ALDH1A1 inhibition increases calcification and osteoblast-like phenotype in VIC. Thus the results prompt to consider that use or retinoic acid receptor (RAR) agonists would be suitable for the reversing, preventing or delaying calcification of the aortic valve.

Claims

1. A method of reversing, preventing, or delaying calcification of an aortic valve in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a retinoic acid receptor (RAR) agonist.

2. The method of claim 1 wherein the aortic valve is a diseased aortic valve.

3. The method of claim 2 wherein the patient suffers from a calcific aortic valve disease.

4. The method of claim 1 wherein the aortic valve is an implanted bioprosthetic valve.

5. The method of claim 4, wherein the step of administering prevents degeneration of the implanted bioprosthetic valve.

6. The method of claim 4 wherein the implanted bioprosthetic valve was implanted either surgically or after transcatheter aortic valve implantation (TAVI).

7. The method of claim 1 wherein the RAR agonist is a RARα agonist.

8. The method of claim 7 wherein the RARα agonist is selected from the group consisting of TTNPB, tamibarotene, 9-cis-retinoic acid, trans-retinoic acid, AGN193836, Ro 40-6055, CD666, and 4-[[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)amino]carbonyl]benzoic acid (AM80).

9. A method of preparing an aortic bioprosthetic valve, comprising, during preparation, entrapping an amount of a RAR agonist within cusps off the aortic bioprosthetic valve, wherein the amount is sufficient to prevent calcification of the aortic bioprosthetic valve after implantation.

10. An aortic bioprosthetic valve comprising an amount of a RAR agonist sufficient to prevent calcification of the aortic bioprosthetic valve after implantation.

11. The aortic bioprosthetic valve of claim 10 wherein the RAR agonist is entrapped into cusps of the aortic bioprosthetic valve.

12. The aortic bioprosthetic valve of claim 10, wherein the amount of the RAR agonist is sufficient to prevent stenosis of the aortic bioprosthetic valve.

Description

FIGURES

[0020] FIG. 1: Retinoic acid decreases calcification and osteoblast-like phenotype

[0021] A: Calcification ratio of VIC treated with retinoic acid (1; 10; 100 or 1000 nM) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. B RUNX2 and relative mRNA expression in VIC treated with retinoic acid (1; 10; 100 or 1000 nM) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. Data are presented as mean+SEM, *: p<0.05

[0022] FIG. 2: Retinoids reduce calcification and osteoblast-like phenotype in a RAR activation dependent manner

[0023] A: Calcification ratio of VIC treated with retinoic acid, Arotinoid acid (TTNB, RAR agonist) or SR11237 (RXR agonist) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. B and C: RUNX2 and MSX2 relative mRNA expression in VIC treated with retinoic acid, Arotinoid acid (TTNB, RAR agonist) or SR11237 (RXR agonist) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. Data are presented as mean+SEM, *: p<0.05

[0024] FIG. 3: RARα activation reduces calcification and osteoblast-like phenotype in VIC

[0025] A: Calcification ratio of VIC treated with Arotinoid acid (TTNB, RAR agonist), AM80 (RARα agonist), CD2314 (RARβ agonist) or CD437 (RARγ agonist) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. B and C: RUNX2 and MSX2 relative mRNA expression in VIC treated with Arotinoid acid (TTNB, RAR agonist), AM80 (RARαagonist), CD2314 (RARβ agonist) or CD437 (RARγ agonist) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. Data are presented as mean+SEM, *: p<0.05

[0026] FIG. 4: ALDH1A1 inhibition increases calcification and osteoblast-like phenotype in VIC

[0027] A: Calcification ratio of VIC treated with 4-Diethylaminobenzaldehyde 25 or 50 nM, (DEAB, ALDH inhibitor) in pro-calcifying medium for 7 days. Pro-calcifying medium condition is set at 1. B: RUNX2 relative mRNA expression in VIC treated with DEAB in procalcifying medium for 7 days.

EXAMPLE

Methods

[0028] Modulation of retinoic acid signaling in pro-calcifying conditions

[0029] VIC were treated for 7 days in a medium composed of DMEM with 10% FBS, supplemented with ascorbic acid (283 μM, Sigma Aldrich), Sodium Phosphate (3 mM, Sigma Aldrich) and dexamethasone (100 nM, Stem Cell technologies). VIC were treated with retinoic acid (RA: 1; 10; 100; 1000 nM), TTNPB (RAR pan agonist, 1 μM) AM80 (RARα specific agonist, 1 μM), CD2314 (RARβ specific agonist, 1 μM), CD437 (RARγ specific agonist, 1 μM) or 4-Diethylaminobenzaldehyde (DEAB, ALDH inhibitor, 25, 50 nM).

[0030] Calcification Measurement

[0031] After 7 days of treatment, VIC were rinsed twice in PBS then fixed in paraformaldehyde 4% for 15 minutes. After washing with deionized water, calcium deposits were stained with an alizarin red solution (1%, pH 6.38) for 10 minutes. Then, cells were washed with deionized water and staining was fixed with absolute ethanol. Quantification is performed according to Gregory et al method. Briefly, cells were incubated with acetic acid 10% for 30 minutes under smooth shaking. Cells were then transferred to a microtube and vortexed for 30 seconds. Mineral oil was added to each tube and samples were heated for 10 minutes à 85° C. and cooled in ice for 5 minutes. After centrifugation (15 minutes, 16.000 g), supernantants are collected and 200 μl of ammonium hydroxide are added. Samples are then read at 405 nm in a microplate.

[0032] Quantitative PCR

[0033] RNA was extracted from VICs after homogenization in TRIzol reagent (Life Technologies), following the Chomczynski and Sacchi protocol. For gene expression analysis, 2 micrograms of total extracted RNA were reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Life Technologies) in a total volume of 20 μl, according to manufacturer's procedures. Samples were incubated at 25° C. for 10 min, followed by 2 h at 37° C. The obtained cDNA were stored at −20° C. Quantitative PCR was performed using an ABI PRISM 7000 Detection System (Life Technologies). Four microliters of reverse transcription product were added to 16 μl of a mix consisting of 10 μl of master mix containing Taq polymerase, MgCl2, and dNTPs (Life Technologies), 1 μl solution containing sense and antisense primers and TaqMan® probe specific for the gene of interest and 5 μl of water. The expressions of the transcripts were normalized by Abelson (ABL) mRNA expression, used as an endogenous reference gene. The relative quantification of the transcripts was performed using the cycle threshold (Ct) comparative method and calculated with the formula 2—ΔΔCt. For each sample, detection of PCR products was performed separately and in duplicate. ABL (Hs01104728_m1), RUNX2 (Hs00231692_m1) and MSX2 (Hs00741177_m1) commercial assays were purchased from Life Technologies.

[0034] Statistical Analyses

[0035] All data are reported as mean ±SEM. A Student t-test for continuous variables were used. For all analysis, a p-value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 20.0 for Windows (SPSS, Inc., Chicago, Ill.).

Results

[0036] The results are depicted in FIGS. 1-4. FIGS. 1A and 1B show that retinoic acid decreases calcification and osteoblast-like phenotype. FIGS. 2A-2C show that retinoids reduce calcification and osteoblast-like phenotype in a RAR activation dependent manner. Moreover, FIGS. 3A-3C show that: RARα activation reduces calcification and osteoblast-like phenotype in VIC. Finally, FIGS. 4A and 4B show that ALDH1A1 inhibition increases calcification and osteoblast-like phenotype in VIC.

REFERENCES

[0037] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0038] 1. Nishimura R A, Otto C M, Bonow R O et al (2014) 2014 AHA/ACC guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 63(22):e57-185. doi:10.1016/j.jacc.2014.02.536

[0039] 2. Yetkin E, Waltenberger J (2009) Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 135(1):4-13. doi:10.1016/j.ijcard.2009.03.108

[0040] 3. Lindman B R, Clavel M A, Mathieu P et al (2016) Calcific aortic stenosis. Nat Rev Dis Primers 2:16006. doi:10.1038/nrdp.2016.6

[0041] 4. Taylor P M, Batten P, Brand N J, Thomas P S, Yacoub M H (2003) The cardiac valve interstitial cell. Int J Biochem Cell Biol 35(2):113-118

[0042] 5. Liu A C, Joag V R, Gotlieb A I (2007) The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol 171(5):1407-1418. doi: 10.2353/ajpath.2007.070251

[0043] 6. Cloyd K L, El-Hamamsy I, Boonrungsiman S et al (2012) Characterization of porcine aortic valvular interstitial cell ‘calcified’ nodules. PLoS ONE. doi:10.1371/journal.pone.0048154