Mineralization Of Valve Interstitial Cells Research Articles

Knockdown of CD44 expression decreases valve interstitial cell calcification in vitro.

The lack of pharmaceutical targets available to treat patients with calcific aortic valve disease (CAVD) necessitates further research into the specific mechanisms of the disease. The significant changes that occur to the aortic valves extracellular matrix (ECM) during the progression of CAVD suggests that these proteins may play an important role in calcification. Exploring the relationship between valve interstitial cells (VICs) and the ECM may lead to a better understand of CAVD mechanisms and potential pharmaceutical targets. In this study, we look at the effect of two ECM components, collagen and hyaluronic acid (HA), on the mineralization of VICs within the context of a two-dimensional, polyacrylamide (PAAM) model system. Using a novel, nondestructive imaging technique, we were able to track calcific nodule development in culture systems over a 3-wk time frame. We saw a significant increase in the size of the nodules grown on HA PAAM gels as compared with collagen PAAM gels, suggesting that HA has a direct effect on mineralization. Directly looking at the two known receptors of HA, CD44 and receptor for HA-mediated motility (RHAMM), and using siRNA knockdown revealed that a decrease in CD44 expression resulted in a reduction of calcification. A decrease in CD44, through siRNA knockdown, reduces mineralization on HA PAAM gels, suggesting a potential new target for CAVD treatment. NEW & NOTEWORTHY Our in vitro model of calcific aortic valve disease shows an interaction between the hyaluronic acid binding protein CD44 with the osteogenic factor OPN as a potential mechanism of aortic valve calcification. Using siRNA knockdown of CD44, we show an upregulation of OPN expression with a decrease in overall mineralization.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

AUTOTAXIN ENHANCES AORTIC VALVE MINERALIZATION THROUGH A NF-κB / IL-6 / BMP-2 DEPENDENT INFLAMMATORY PATHWAY

Calcific aortic valve disease (CAVD) is characterized by a progressive mineralization of the aortic valve. Recent studies of Mendelian randomization have revealed an association between lipoprotein (a) [Lp (a)] and CAVD. However, the mechanism by which the Lp (a) causes aortic valve interstitial cells (VICs) calcification remains unknown. Lp (a) carries oxidized phospholipids (OxPLs) with a high content in lysophosphatidylcholine (LPC). The enzyme autotaxin (ATX) converts LPC into lysophosphatidic acid (LysoPA). We investigated whether ATX-produced LysoPA could induce mineralisation. We documented the expression of ATX in control and CAVD valves. We investigated in vitro the mechanisms by which ATX promotes inflammation and mineralization of VICs. These results were then validated in a mouse model of CAVD. In CAVD, immunohistochemical stainings revealed the expression of ATX by VICs and ATX co-distributed with Lp (a). Moreover, ATX-specific enzyme activity confirmed the expression of ATX in CAVD. An interaction between ATX and Lp (a) was revealed by 'proximity ligation assay'. Also, we have detected a high level of ATX activity in Lp (a) purified fractions. On the other hand, we showed that ATX promotes inflammation and mineralization of the aortic valve by the activation of a NF-kB / IL-6 / BMP-2 dependent pathway. LysoPA injections, in mice LDLR - / - / ApoB 100/100 / IGFII fed with a HFHS diet, accelerated the development of CAVD compared with HFHS diet alone. Echocardiographic analyzes of these mice showed a two fold increase in the delta velocity in comparison with control mice. ATX is transported in the aortic valve by Lp (a) and is secreted by VICs. ATX and LysoPA promote inflammation and mineralization of the aortic valve and could therefore represent new therapeutic targets in CAVD.

Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells.

Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) 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 experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of 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). The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 μM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.

Innate and Adaptive Immunity in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is the most common heart valve disorder. CAVD is a chronic process characterized by a pathologic mineralization of valve leaflets. Ectopic mineralization of the aortic valve involves complex relationships with immunity. Studies have highlighted that both innate and adaptive immunity play a role in the development of CAVD. In this regard, accumulating evidence indicates that fibrocalcific remodelling of the aortic valve is associated with activation of the NF-κB pathway. The expression of TNF-α and IL-6 is increased in human mineralized aortic valves and promotes an osteogenic program as well as the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. Different factors, including oxidized lipid species, activate the innate immune response through the Toll-like receptors. Moreover, VICs express 5-lipoxygenase and therefore produce leukotrienes, which may amplify the inflammatory response in the aortic valve. More recently, studies have emphasized that an adaptive immune response is triggered during CAVD. Herein, we are reviewing the link between the immune response and the development of CAVD and we have tried, whenever possible, to keep a translational approach.

Abstract 16349: A Novel Class of Inhibitors That Are Specific to Human NPP1 Prevent Pathologic Mineralization of Valve Interstitial Cells

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. METHOD: We have investigated the potency and selectivity of QPS derivatives in inhibiting NPP1 by enzymatic activities. The biological effect of QPS derivatives was documented on the mineralization of valve interstitial cell cultures, apoptosis assay, as well as quantitative polymerase chain reaction. RESULTS: We documented that QPS1 derivative is a potent (67.9 ±5.3 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (ki 51.9±9.8 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolising enzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5’-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10μM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSION: We provide evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 that prevents pathologic mineralization in a cellular model.

Abstract 16348: Ecto-Nucleotidase-Derived Adenosine Promotes Mineralization Through A2a Receptor in Calcified Aortic Valves Disease

Background: Mineralization plays a crucial role in the development of calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases may promote the mineralization of valve interstitial cells (VICs). 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. The signaling pathway involving adenosine receptors was also studied. 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 Gαs 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 VICs 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 Gαs 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.

QUINAZOLIN-4-PIPERIDIN-4-METHYL SULFAMIDE COMPOUNDS ARE SPECIFIC INHIBITORS OF HUMAN NPP1 AND PREVENT CALCIFICATION OF VALVE INTERSTITIAL CELLS

s S245 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. 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 Bosse, P Mathieu

NPP1 AND 5’NUCLEOTIDASE PROMOTE THE MINERALIZATION OF THE AORTIC VALVE: ROLE OF A2A RECEPTOR AND CREB

Mineralization of the aortic valve is the major culprit in the development of calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases, which exert a control over extracellular nucleotides/nucleosides, may 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. 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. 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 Gαs 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. The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gαs 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.

USE AND OUTCOMES OF TRANSCATHETER AORTIC VALVE IMPLANTATION: Results OF A QUÉBEC-WIDE FIELD EVALUATION AND COMPARISON WITH THE TVT REGISTRY

s S245 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. 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 Bosse, P Mathieu

Elevated Expression of Lipoprotein-Associated Phospholipase A2 in Calcific Aortic Valve Disease: Implications for Valve Mineralization

This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. Invitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could be considered as a novel target in CAVD.