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ROLE OF LONG NON-CODING RNAS IN THE REGULATION OF THE NOTCH PATHWAY: IMPLICATION FOR CALCIFIC AORTIC VALVE STENOSIS
Abstracts
promote the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. METHODS: We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. RESULTS: In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/- receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gas vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated cells increased the NPP1 promoter activity by 6.5-fold. CONCLUSION: The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gas and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD.
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determined. Long non-coding RNAs (Long ncRNAs) may be involved in the control of the Notch pathway. METHODS: We analyzed the gene expression pattern of valvular tissues (controls and CAVS) using microarrays. Furthermore, we explored the role of a Long ncRNA on the mineralization of VICs. Signaling of the Notch pathway was investigated by promoter luciferase activity and by analyzing mRNA expression levels of target genes. RESULTS: A transcriptomic study revealed that a Long ncRNA is overexpressed in CAVS. This result was confirmed by qPCR in a larger group of aortic valve tissues (n¼152). Silencing and overexpression with a vector encoding this Long ncRNA decreased and increased the mineralization of VICs, respectively. Overexpression of this Long ncRNA led to lower activity of the Notch1 promoter, which resulted in a substantial decrease in the cleaved intracellular portion of Notch1 receptor (NICD). The transfection of the Long ncRNA in VICs decreased significantly the activity of the Rbp-jk response element located downstream of NICD and resulted in the overexpression of bone morphogenetic protein 2 (BMP2) and Runx2, two target genes repressed by Rbp-jk responsive element. In a rescue experiment in VICs, the transfection of a vector encoding NICD prevented the mineralization induced by the Long non-coding RNA. CONCLUSION: Overexpression of a Long ncRNA in CAVS promoted a dysfunction of the Notch signaling pathway and induced the osteoblastic transition of VICs. To our knowledge, this is the first study to report that a Long ncRNA plays a crucial role in Notch signaling and the development of CAVS.
373 QUINAZOLIN-4-PIPERIDIN-4-METHYL SULFAMIDE COMPOUNDS ARE SPECIFIC INHIBITORS OF HUMAN NPP1 AND PREVENT CALCIFICATION OF VALVE INTERSTITIAL CELLS EE Shayhidin, E Forcellini, M Boulanger, A Mahmut, S Dautrey, X Barbeau, P Lague, J Sévigny, J Paquin, P Mathieu Québec, Québec
372 ROLE OF LONG NON-CODING RNAS IN THE REGULATION OF THE NOTCH PATHWAY: IMPLICATION FOR CALCIFIC AORTIC VALVE STENOSIS F Hadji, M Boulanger, S Guauque-Olarte, R Bouchareb, Y Bossé, P Mathieu Québec, Québec BACKGROUND: Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease. The disease process is largely driven by an abnormal mineralization of leaflets. The Notch pathway is involved in the osteogenic transition of valve interstitial cells (VICs) and play an important role in CAVS. However, the molecular processes involved in the regulation of Notch signaling in CAVS remain to be
BACKGROUND:
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role into several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack of potency and specificity. Quinazolin-4-piperidin-4-methyl sulfamide (QPS) derivatives have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. In the present series of experiment, we have evaluated the efficacy of two derivatives, QPS1-2, to inhibit human NPP1 and we have evaluated the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate induced mineralization of human primary aortic valve interstitial cells (VICs).
promote the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. METHODS: We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. RESULTS: In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/- receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gas vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated cells increased the NPP1 promoter activity by 6.5-fold. CONCLUSION: The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gas and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD.
S245
determined. Long non-coding RNAs (Long ncRNAs) may be involved in the control of the Notch pathway. METHODS: We analyzed the gene expression pattern of valvular tissues (controls and CAVS) using microarrays. Furthermore, we explored the role of a Long ncRNA on the mineralization of VICs. Signaling of the Notch pathway was investigated by promoter luciferase activity and by analyzing mRNA expression levels of target genes. RESULTS: A transcriptomic study revealed that a Long ncRNA is overexpressed in CAVS. This result was confirmed by qPCR in a larger group of aortic valve tissues (n¼152). Silencing and overexpression with a vector encoding this Long ncRNA decreased and increased the mineralization of VICs, respectively. Overexpression of this Long ncRNA led to lower activity of the Notch1 promoter, which resulted in a substantial decrease in the cleaved intracellular portion of Notch1 receptor (NICD). The transfection of the Long ncRNA in VICs decreased significantly the activity of the Rbp-jk response element located downstream of NICD and resulted in the overexpression of bone morphogenetic protein 2 (BMP2) and Runx2, two target genes repressed by Rbp-jk responsive element. In a rescue experiment in VICs, the transfection of a vector encoding NICD prevented the mineralization induced by the Long non-coding RNA. CONCLUSION: Overexpression of a Long ncRNA in CAVS promoted a dysfunction of the Notch signaling pathway and induced the osteoblastic transition of VICs. To our knowledge, this is the first study to report that a Long ncRNA plays a crucial role in Notch signaling and the development of CAVS.
373 QUINAZOLIN-4-PIPERIDIN-4-METHYL SULFAMIDE COMPOUNDS ARE SPECIFIC INHIBITORS OF HUMAN NPP1 AND PREVENT CALCIFICATION OF VALVE INTERSTITIAL CELLS EE Shayhidin, E Forcellini, M Boulanger, A Mahmut, S Dautrey, X Barbeau, P Lague, J Sévigny, J Paquin, P Mathieu Québec, Québec
372 ROLE OF LONG NON-CODING RNAS IN THE REGULATION OF THE NOTCH PATHWAY: IMPLICATION FOR CALCIFIC AORTIC VALVE STENOSIS F Hadji, M Boulanger, S Guauque-Olarte, R Bouchareb, Y Bossé, P Mathieu Québec, Québec BACKGROUND: Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease. The disease process is largely driven by an abnormal mineralization of leaflets. The Notch pathway is involved in the osteogenic transition of valve interstitial cells (VICs) and play an important role in CAVS. However, the molecular processes involved in the regulation of Notch signaling in CAVS remain to be
BACKGROUND:
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role into several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack of potency and specificity. Quinazolin-4-piperidin-4-methyl sulfamide (QPS) derivatives have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. In the present series of experiment, we have evaluated the efficacy of two derivatives, QPS1-2, to inhibit human NPP1 and we have evaluated the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate induced mineralization of human primary aortic valve interstitial cells (VICs).