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ACTIVATED PLATELETS PROMOTE THE PROGRESSION OF CALCIFIC AORTIC VALVE STENOSIS
Abstracts
166 JMJD3 REGULATES THE EXPRESSION OF AUTOTAXIN
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Featured Research Presenter Trainee Research Award Finalist - Basic Science
D Argaud, M Boulanger, P Mathieu Québec, Québec BACKGROUND:
Autotaxin (ATX), which is encoded by the ENPP2 gene, is known to be a strong promoter of inflammation. The circulating level of ATX is increased in obese patients and promotes the development of calcific aortic valve disease. However, the regulation of ATX expression remains elusive. The objective of this study was to examine the molecular mechanism involved in the regulation of ATX expression. METHODS AND RESULTS: HEK293T cells were treated with 100 ng/ml of LPS for 6 hours and functional characterization of gene expression and promoter activation were carried out. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-q-PCR) has been used to study the binding of p65, a member of NF-kB family, on ATX promoter and to evaluate histone-related modifications. Then several co-immunoprecipitations (co-IP) were performed to investigate the interactome of new ATX regulators. In reporter assay, LPS increased the activity of a construction containing the promoter region of ATX. LPS also promoted the expression of mRNA encoding for ATX, which was negated by Bay 117085, an inhibitor of IkB kinase (IKK), and was robustly increased by the overexpression of p65. Following a treatment with LPS, we observed by using ChIP-q-PCR an enrichment of p65 at two kB consensus sequences in the ATX promoter and in one site near TSS. In reporter assay, the deletion of these two kB consensus sequences decreases the p65-mediated activation. After LPS treatment, the level of H3K27me3, a histone repressive mark, was decreased. Knockdown of H3K27me3 demethylase JMJD3 with small interfering RNA (siRNA) prevented LPS-mediated expression of ATX. Moreover, in ChIP-q-PCR experiments we observed an enrichment of JMJD3 to ATX promoter and through the gene body following LPS treatment. Simultaneously, the recruitment of RNA polymerase II increased and phosphorylation occurs principally on Ser2. Co-IP experiments suggested that JMJD3 and RNA polymerase IIinteracts with LPS treatment. Knockdown experiments showed that JMJD3 is crucial for the recruitment of RNA polymerase II to the promoter of ATX. CONCLUSION: LPS promotes the expression of ATX in HEK293T cells through a NF-kB pathway and an epigenetic mechanism involving H3K27me3 and JMJD3.JMJD3 may represent a suitable target to modulate ATX-mediated inflammation.
167 ACTIVATED PLATELETS PROMOTE THE PROGRESSION OF CALCIFIC AORTIC VALVE STENOSIS R Bouchareb, M Boulanger, M Ghada, M Nsaibia, F Hadji, A Dahou, L Tastet, Y Messaddeq, B Arsenault, P Pibarot, Y Bossé, A Marette, P Mathieu Québec, Québec BACKGROUND: Calcific aortic valve stenosis (CAVS) is a chronic disorder characterized by ectopic mineralization. Studies have shown that circulating platelets are activated during CAVS. However, whether platelets are actively involved into the pathobiology of CAVS is presently unknown. We have recently showed that autotaxin (ATX), which is secreted by valve interstitial cells (VICs), promotes the production of lysophosphatidic acid (LPA) with proosteogenic activity. We hypothesized that platelets could interact with ATX and promote the production of LPA and the osteogenic transition of VICs. METHODS: Explanted human and mouse [LDLR-/- apoB100/ 100 IGFII (IGFII)] calcified aortic valves (AVs) were analysed by scanning electron microscope (SEM). We investigated in vitro the mechanisms by which platelets promote VIC mineralization. These results were validated in a mouse model of CAVS. RESULTS: SEM analyses revealed the presence of activated platelets expressing GPIIbIIIa at the surface of calcified AVs (human and mouse) where the endothelium was activated or denuded. A treatment of VICs with activated human platelets significantly increased the mineralization compared to control. The mineralization was accompanied by an overexpression of osteogenic markers (RUNX2, BGLAP, ALP, and BMP2). Treatment of VICs with activated platelets increased significantly the enzymatic activity of ATX. Accordingly, ATX inhibition by HA130 or a siRNA significantly decreased platelet-induced VIC mineralization. In addition, blocking LPA receptors (LPARs) also inhibited platelet-induced calcification. In binding assay, ATX secreted by VICs interacted with platelets leading to the production of LPA. Inhibition of GPIIbIIIa prevented the interaction of ATX with platelets and the mineralization of VIC cultures. In IGFII mice, the intravenous injection of activated platelets accelerated the development of CAVS,
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Canadian Journal of Cardiology Volume 33 2017
whereas a Lpar1-3 blocker prevented the rise of transaortic velocities. CONCLUSION: Activated platelets are present in mineralized AVs and interact with ATX in promoting an osteogenic program. This work has potential clinical implications since it suggests that the pharmacological manipulation of ATXplatelet axis could prevent the mineralization of the AV.
Canadian Institutes of Health Research (CIHR)
Canadian Cardiovascular Society (CCS) Moderated Presentations OUTCOMES WITH ACUTE CORONARY SYNDROME Sunday, October 22, 2017 168 CLINICAL OUTCOMES OF ST-ELEVATION MYOCARDIAL INFARCTION PATIENTS PRESENTING TO NON-PCI CENTERS TREATED WITH FIBRINOLYSIS COMPARED TO PRIMARY PCI: AN ANALYSIS FROM THE VANCOUVER COASTAL HEALTH AUTHORITY STEMI PROGRAM A AlKhodair, J Cairns, C Fordyce, M Perry-Arnesen, M Mackay, W Tocher, J Singer, T Lee, G Wong Vancouver, British Columbia BACKGROUND:
Fibrinolytic therapy is a viable option for patients with ST-elevation myocardial infarction (STEMI) who present to a non-PCI capable hospital (NPCICH) and cannot be transferred for primary PCI (PPCI) in a timely fashion. Comparisons of outcomes between patients receiving fibrinolysis and those transferred for PPCI could yield data of value in choosing the best option for patients presenting to NPCICHs. METHODS: We retrospectively analyzed STEMI patients >18 years of age who presented to a NPCICH within the Vancouver Coastal Health Authority between 2007-2014 and who received either on-site reperfusion or were transferred for PPCI.
Timely fibrinolysis was defined as first medical contact (FMC) to needle time < 30 min; timely PPCI was defined as FMC to balloon time < 120 min. We compared the composite of inhospital mortality, heart failure, cardiogenic shock or major bleeding for patients in the following groups: timely vs delayed fibrinolysis, timely vs delayed PPCI and timely fibrinolysis vs delayed PPCI, using logistic regression analysis to adjust for clinically important baseline variables. We excluded patients who presented with cardiogenic shock (n¼24), pre-hospital cardiac arrest (n¼9) and who received no reperfusion therapy. RESULTS: We identified 699 eligible patients (198 on-site fibrinolysis, 501 PPCI). Timely reperfusion was achieved in 20% of fibrinolysis patients and 42% of patients transferred for PPCI. There were no significant differences in patient characteristics between patients receiving timely vs delayed reperfusion irrespective of the reperfusion strategy, apart from older age in those receiving delayed PPCI (mean 62.3yrs vs 66.4, P < 0.001). The primary outcome was less frequent with both timely vs delayed fibrinolysis (15.4% vs 21.8%, P¼0.375) and timely vs delayed PPCI (9.2% vs 19.6%, P¼0.002). (Table 1). In an adjusted logistic regression analysis, delayed reperfusion with fibrinolysis was associated with an increased risk of the composite end point compared to timely reperfusion with both fibrinolysis (OR 1.79 95% CI 1.07- 3.00) and PPCI (OR 1.80, 95% CI 1.12-2.89), respectively. Although the primary endpoint was numerically lower amongst patients with timely fibrinolysis compared to delayed PPCI, this association lost significance after an adjusted analysis (OR 0.98, 95% C.I. 0.34-2.77). CONCLUSION: A significant proportion of STEMI patients who present to NPCICHs do not receive timely fibrinolysis or PPCI. Delayed reperfusion is associated with worse outcomes irrespective of reperfusion strategy, although the primary outcome was the lowest with timely PPCI. Additional interventions are warranted to improve the timeliness of both pharmacological and mechanical reperfusion for NPCICHs.
166 JMJD3 REGULATES THE EXPRESSION OF AUTOTAXIN
S101
Featured Research Presenter Trainee Research Award Finalist - Basic Science
D Argaud, M Boulanger, P Mathieu Québec, Québec BACKGROUND:
Autotaxin (ATX), which is encoded by the ENPP2 gene, is known to be a strong promoter of inflammation. The circulating level of ATX is increased in obese patients and promotes the development of calcific aortic valve disease. However, the regulation of ATX expression remains elusive. The objective of this study was to examine the molecular mechanism involved in the regulation of ATX expression. METHODS AND RESULTS: HEK293T cells were treated with 100 ng/ml of LPS for 6 hours and functional characterization of gene expression and promoter activation were carried out. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-q-PCR) has been used to study the binding of p65, a member of NF-kB family, on ATX promoter and to evaluate histone-related modifications. Then several co-immunoprecipitations (co-IP) were performed to investigate the interactome of new ATX regulators. In reporter assay, LPS increased the activity of a construction containing the promoter region of ATX. LPS also promoted the expression of mRNA encoding for ATX, which was negated by Bay 117085, an inhibitor of IkB kinase (IKK), and was robustly increased by the overexpression of p65. Following a treatment with LPS, we observed by using ChIP-q-PCR an enrichment of p65 at two kB consensus sequences in the ATX promoter and in one site near TSS. In reporter assay, the deletion of these two kB consensus sequences decreases the p65-mediated activation. After LPS treatment, the level of H3K27me3, a histone repressive mark, was decreased. Knockdown of H3K27me3 demethylase JMJD3 with small interfering RNA (siRNA) prevented LPS-mediated expression of ATX. Moreover, in ChIP-q-PCR experiments we observed an enrichment of JMJD3 to ATX promoter and through the gene body following LPS treatment. Simultaneously, the recruitment of RNA polymerase II increased and phosphorylation occurs principally on Ser2. Co-IP experiments suggested that JMJD3 and RNA polymerase IIinteracts with LPS treatment. Knockdown experiments showed that JMJD3 is crucial for the recruitment of RNA polymerase II to the promoter of ATX. CONCLUSION: LPS promotes the expression of ATX in HEK293T cells through a NF-kB pathway and an epigenetic mechanism involving H3K27me3 and JMJD3.JMJD3 may represent a suitable target to modulate ATX-mediated inflammation.
167 ACTIVATED PLATELETS PROMOTE THE PROGRESSION OF CALCIFIC AORTIC VALVE STENOSIS R Bouchareb, M Boulanger, M Ghada, M Nsaibia, F Hadji, A Dahou, L Tastet, Y Messaddeq, B Arsenault, P Pibarot, Y Bossé, A Marette, P Mathieu Québec, Québec BACKGROUND: Calcific aortic valve stenosis (CAVS) is a chronic disorder characterized by ectopic mineralization. Studies have shown that circulating platelets are activated during CAVS. However, whether platelets are actively involved into the pathobiology of CAVS is presently unknown. We have recently showed that autotaxin (ATX), which is secreted by valve interstitial cells (VICs), promotes the production of lysophosphatidic acid (LPA) with proosteogenic activity. We hypothesized that platelets could interact with ATX and promote the production of LPA and the osteogenic transition of VICs. METHODS: Explanted human and mouse [LDLR-/- apoB100/ 100 IGFII (IGFII)] calcified aortic valves (AVs) were analysed by scanning electron microscope (SEM). We investigated in vitro the mechanisms by which platelets promote VIC mineralization. These results were validated in a mouse model of CAVS. RESULTS: SEM analyses revealed the presence of activated platelets expressing GPIIbIIIa at the surface of calcified AVs (human and mouse) where the endothelium was activated or denuded. A treatment of VICs with activated human platelets significantly increased the mineralization compared to control. The mineralization was accompanied by an overexpression of osteogenic markers (RUNX2, BGLAP, ALP, and BMP2). Treatment of VICs with activated platelets increased significantly the enzymatic activity of ATX. Accordingly, ATX inhibition by HA130 or a siRNA significantly decreased platelet-induced VIC mineralization. In addition, blocking LPA receptors (LPARs) also inhibited platelet-induced calcification. In binding assay, ATX secreted by VICs interacted with platelets leading to the production of LPA. Inhibition of GPIIbIIIa prevented the interaction of ATX with platelets and the mineralization of VIC cultures. In IGFII mice, the intravenous injection of activated platelets accelerated the development of CAVS,
S102
Canadian Journal of Cardiology Volume 33 2017
whereas a Lpar1-3 blocker prevented the rise of transaortic velocities. CONCLUSION: Activated platelets are present in mineralized AVs and interact with ATX in promoting an osteogenic program. This work has potential clinical implications since it suggests that the pharmacological manipulation of ATXplatelet axis could prevent the mineralization of the AV.
Canadian Institutes of Health Research (CIHR)
Canadian Cardiovascular Society (CCS) Moderated Presentations OUTCOMES WITH ACUTE CORONARY SYNDROME Sunday, October 22, 2017 168 CLINICAL OUTCOMES OF ST-ELEVATION MYOCARDIAL INFARCTION PATIENTS PRESENTING TO NON-PCI CENTERS TREATED WITH FIBRINOLYSIS COMPARED TO PRIMARY PCI: AN ANALYSIS FROM THE VANCOUVER COASTAL HEALTH AUTHORITY STEMI PROGRAM A AlKhodair, J Cairns, C Fordyce, M Perry-Arnesen, M Mackay, W Tocher, J Singer, T Lee, G Wong Vancouver, British Columbia BACKGROUND:
Fibrinolytic therapy is a viable option for patients with ST-elevation myocardial infarction (STEMI) who present to a non-PCI capable hospital (NPCICH) and cannot be transferred for primary PCI (PPCI) in a timely fashion. Comparisons of outcomes between patients receiving fibrinolysis and those transferred for PPCI could yield data of value in choosing the best option for patients presenting to NPCICHs. METHODS: We retrospectively analyzed STEMI patients >18 years of age who presented to a NPCICH within the Vancouver Coastal Health Authority between 2007-2014 and who received either on-site reperfusion or were transferred for PPCI.
Timely fibrinolysis was defined as first medical contact (FMC) to needle time < 30 min; timely PPCI was defined as FMC to balloon time < 120 min. We compared the composite of inhospital mortality, heart failure, cardiogenic shock or major bleeding for patients in the following groups: timely vs delayed fibrinolysis, timely vs delayed PPCI and timely fibrinolysis vs delayed PPCI, using logistic regression analysis to adjust for clinically important baseline variables. We excluded patients who presented with cardiogenic shock (n¼24), pre-hospital cardiac arrest (n¼9) and who received no reperfusion therapy. RESULTS: We identified 699 eligible patients (198 on-site fibrinolysis, 501 PPCI). Timely reperfusion was achieved in 20% of fibrinolysis patients and 42% of patients transferred for PPCI. There were no significant differences in patient characteristics between patients receiving timely vs delayed reperfusion irrespective of the reperfusion strategy, apart from older age in those receiving delayed PPCI (mean 62.3yrs vs 66.4, P < 0.001). The primary outcome was less frequent with both timely vs delayed fibrinolysis (15.4% vs 21.8%, P¼0.375) and timely vs delayed PPCI (9.2% vs 19.6%, P¼0.002). (Table 1). In an adjusted logistic regression analysis, delayed reperfusion with fibrinolysis was associated with an increased risk of the composite end point compared to timely reperfusion with both fibrinolysis (OR 1.79 95% CI 1.07- 3.00) and PPCI (OR 1.80, 95% CI 1.12-2.89), respectively. Although the primary endpoint was numerically lower amongst patients with timely fibrinolysis compared to delayed PPCI, this association lost significance after an adjusted analysis (OR 0.98, 95% C.I. 0.34-2.77). CONCLUSION: A significant proportion of STEMI patients who present to NPCICHs do not receive timely fibrinolysis or PPCI. Delayed reperfusion is associated with worse outcomes irrespective of reperfusion strategy, although the primary outcome was the lowest with timely PPCI. Additional interventions are warranted to improve the timeliness of both pharmacological and mechanical reperfusion for NPCICHs.