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Funding Acknowledgements Type of funding sources: None. Introduction Atherosclerotic lesions preferentially develop in arterial regions exposed to disturbed blood flow, which are characterized by a dysfunctional proinflammatory endothelial cell (EC) phenotype. ECs release ATP in response to changes in wall shear stress (WSS), which subsequently regulates the inflammatory response. ATP can be released from cells in a controlled manner through Pannexin1 (Panx1) channels. Objective To study the expression of Panx1 in response to WSS and its role in the endothelium. Methods Human ECs (HUVECs or EA.hy926) were exposed to physiological high laminar shear stress (HLSS) and atheroprone oscillatory shear stress (OSS) for 48h using an orbital shaker. Panx1 transcript and protein levels were determined by qPCR and Western blot, respectively. mRNA was extracted for RNAseq. WSS-modifying casts were placed for 1 week around the carotid artery of mice and Panx1 expression was analyzed en face. Carotid WSS-modifying casts were also placed in Tie2CreTgPanx1fl/flApoE-/- and Panx1fl/flApoE-/- mice followed by 9 weeks high fat diet. CD68+ cells were quantified in OSS-induced atherosclerotic lesions. Results We found that Panx1 expression was increased in carotid regions exposed to OSS as compared to HLSS regions. These results were confirmed in vitro in HUVECs. In silico analysis of the promotor of Panx1 revealed binding sites for the WSS-sensitive transcription factors NF-kB and CREB. NF-kB and CREB mRNA levels were similar under OSS and HLSS, however NF-kB activation (phospho-NF-kB) was detected in ECs under OSS. Surprisingly, Panx1 mRNA level was not affected under these conditions, suggesting that the increased Panx1 protein observed under OSS may be due to decreased Panx1 degradation rather than to increased synthesis of the protein. Unbiased analysis of differential gene expression in ECs exposed to HLSS or OSS revealed 320 up-regulated and 353 down-regulated genes under OSS. Down-regulated genes included proteins involved in macro-autophagy. Inhibition of macro-autophagy in ECs by exposure to chloroquine for 6h increased the expression of glycosylated Panx1, suggestive for more Panx1 channels at the plasma membrane under these conditions. Finally, we observed that atherosclerotic lesions in OSS regions of Tie2CreTgPanx1fl/flApoE-/- mice contained more CD68+ cells than Panx1fl/flApoE-/- controls. Conclusion Endothelial Panx1 expression is upregulated in response to OSS. Absence of OSS effects on NF-kB, CREB and Panx1 mRNA revealed that the upregulation of Panx1 protein is not due to transcriptional effects of OSS. Expression of genes involved in the macro-autophagic process were down-regulated under OSS, and chemical inhibition of macro-autophagy augmented the plasma membranous form of Panx1. OSS-induced decrease in autophagic flux may enhance ATP release through Panx1 channels, thereby counterbalancing leukocyte recruitment in atherosclerosis. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: None. Background Intracranial aneurysm (IA) rupture is a common cause of hemorrhagic stroke. IAs are present in 3 to 5% of the general population and the annual rupture rate of an IA has been estimated to 1%. Once formed, an IA can rupture immediately, grow before rupturing, or remain stable. Predicting the evolution of an unruptured IA is very difficult. Indeed, the biology of the IA wall is complex and the precise biomechanical processes leading to aneurysm wall rupture are not yet known. While the contribution of wall shear stress in IA disease has been demonstrated in several studies, the role of cyclic circumferential stretch (CCS) remains poorly understood. Purpose The levels of CCS in saccular IAs are unknown but are generally considered very low or even absent. The goal of our study was to investigate the transcriptional response of endothelial cells (ECs) to absence of CCS and the potential impact on the endothelium function. Methods Human umbilical veins ECs (HUVECs) from 6 different donors were exposed to physiological CCS levels for cerebral arteries (6% stretch) or to aneurysmal CCS (0%) for 48h using the Flexcell strain unit FX-5000T, and unbiased transcriptomics were performed. The expression of selected genes was confirmed by qPCR. Results As a validation of our methodology, we verified the induction of eNOS phosphorylation after exposure of the ECs to physiological CCS. Stretched EC samples display a high similarity between each other while EC samples under aneurysmal CCS were spread-out on a multidimensional scaling plot. The differential gene expression of ECs exposed to aneurysmal or physiological CCS revealed 51 up-regulated genes and 49 down-regulated genes in absence of CCS. The gene set enrichment analysis identified 70 up-regulated and 38 down-regulated pathways in HUVECs exposed to aneurysmal CCS. Up-regulated pathways were involved in oxidative stress, angiogenic and inflammatory pathways, and the down-regulated pathways in proliferation and extracellular matrix-receptor interactions. We confirmed by qPCR the down-regulation by aneurysmal CCS of Gja4 and Gja5, genes coding for the gap junction proteins Connexin37 (Cx37) and Connexin40 (Cx40), respectively. Connexins (Cxs) are crucial proteins for endothelial homeostasis, vascular function, endothelial cell cycle regulation and inflammation, and their role in the context of IA disease will be further studied. Conclusion Our results indicate that physiological CCS prevents large deviation in ECs and regulate the expression of genes essential for maintaining vascular homeostasis. The down-regulation of endothelial Cx37 and Cx40 under aneurysmal CSS may contribute to the endothelial dysfunction associated with IA progression. Further investigation may help to better define the role of Cxs in aneurysmal disease and to understand the significance of absence of CCS in IA disease progression. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: None. Background Ischemic heart disease is the main cause of mortality worldwide. Ischemia occurs when blood flow to the myocardium is severely restricted. Reperfusion is crucial to avoid irreversible cell damage, which may lead to heart failure. No effective therapy is available in clinics to protect the heart from ischemia/reperfusion (I/R) injury. An inappropriate inflammatory response may worsen I/R injury. Thus, endothelial cells (ECs) are activated in response to I/R, which may drive an inflammatory response by releasing ATP through Pannexin1 (Panx1) channels. ATP acts further as a 'find me' signal for leukocytes. It is known that Panx1 channels open in response to oxygen and glucose deprivation –hallmarks of ischemia. Purpose We investigated the potential role of Panx1 in cardiac I/R injury. Methods Panx1 expression and function was detected by immunofluorescence, Western blot and ATP release assays. Cardiac function was measured in Langendorff-perfused hearts from WT or Panx1-/- mice and ex vivo subjected to 30min no-flow ischemia followed by 60min reperfusion with/without ischemic pre-conditioning (IPC; 3x5min). We induced in vivo I/R (30min/24h) by ligating the left coronary artery in WT, Panx1-/- mice, mice with Ly6G-Cre-mediated deletion of Panx1 and Panx1fl/fl controls. Infarct size, neutrophil recruitment and Troponin-I release were measured. Results Panx1 expression was detected in coronary and microvascular ECs, neutrophils and cardiomyocytes. After in vivo I/R, serum Troponin-I and infarct size were less pronounced in Panx1-/- mice, but neutrophil infiltration into the infarcted area was similar between Panx1-/- and WT mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in WT and Panx1-/- hearts was similar. However, Panx1-/- hearts showed enhanced left ventricle developed pressure and better recovery of cardiac contractility (dP/dtmax) and cardiac relaxation (dP/dtmin) after I/R. Both the time to onset of contracture (TTOC) and time to maximal contracture (TTMC) were delayed in Panx1-/- hearts. This improved cardiac recovery was abolished in Panx1-/- hearts subjected to IPC prior to I/R. Similarly, TTOC and TTMC did not vary between WT and Panx1-/- mice following IPC, suggesting altered mitochondrial function in Panx1-/- cardiomyocytes. Finally, Panx1 was found in mitochondria and mitochondrial ATP content was increased in Panx1-/- cardiomyocytes. Conclusion Panx1-/- mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and increased recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage. [ABSTRACT FROM AUTHOR]

Purpose: Apolipoprotein M (apoM) is a plasma lipoprotein that mainly associates with high-density lipoproteins (HDL) and that serves as a carrier of the bioactive lipid Sphingosine-1-Phosphate (S1P). Recent studies indicate that S1P binding to G-protein-coupled receptors, known as S1P-receptors, in the heart activates signalling pathways promoting cardiomyocyte survival, but downstream targets are largely unknown. Here, we investigate the putative role of the apoM-S1P axis in relation to cardioprotection against ischemia/reperfusion (IR) injury.Methods and Results: ApoM transgenic (Apom-Tg) mice, in which plasma S1P is increased by >250%, and wild-type (WT) mice were subjected to 30 min of left coronary artery ligation and 24 hrs reperfusion in vivo. We found a reduction of infarct size in Apom-Tg mice (15±1%) in comparison with WT mice (29±4%, N=8-9, p<0.01). In agreement, neutrophil infiltration into the infarcted area was lower in Apom-Tg mice (14.8±0.2% vs. 25.9±5.1 in WT, N=3, p<0.05). Interestingly, 5 min of S1P treatment at the onset of reperfusion reduced infarct size in response to 30 min of no-flow global ischemia (control: 23±3%, S1P-treated: 11±2%, N=5, p<0.05) in ex vivo Langendorff perfused hearts, suggesting that S1P exerts a direct protective effect on cardiomyocytes. Moreover, the sensitivity to ex vivo IR of Apom-Tg mice was not different from WT mice, further supporting that the cardioprotective effect observed in vivo is due to increased plasmatic S1P in these mice. To obtain further insight into the mechanism underlying S1P-induced cardioprotection, neonatal rat ventricular cardiomyocytes were treated for 5 min with S1P after pre-incubation with PKC kinase inhibitors or with specific antagonists of S1P receptors. We found by Western blot that S1P induced phosphorylation of the gap junction protein Connexin43 (Cx43) on Serine 368 by a PKC-dependent mechanism and that this phosphorylation was mediated by S1P2 and S1P3 but not by S1P1 receptors. Finally, 5 min of S1P treatment reduced gap junctional communication between cardiomyocytes (9±1 cells, N=29) in comparison to control conditions (15±2 cells, N=34, p<0.01), as assessed by dye coupling assay.Conclusion: Increased plasma apoM-S1P in mice protects the heart against IR injury. The molecular mechanism might involve reduced cardiomyocyte death by activation of S1P2 and S1P3 receptors, which leads to PKC-dependent phosphorylation of Cx43 and reduction of cell-to-cell coupling. [ABSTRACT FROM AUTHOR]