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Extracellular ATP is a central signaling molecule in inflammatory responses. Pannexin1 (Panx1) channels release ATP in a controlled manner and have been implicated in various inflammatory pathologies, but their role in atherogenesis remains elusive. Using atherosclerosis-susceptible mouse models with ubiquitous deletion of Panx1 (Panx1 −/− Apoe −/− ) or with Cre recombinase-mediated deletion of Panx1 in endothelial cells and monocytes (Tie2-Cre Tg Panx1 fl/fl Apoe −/− ; Panx1 del Apoe −/− ), we identified a novel role for Panx1 in the lymphatic vasculature. Atherosclerotic lesion development in response to high-cholesterol diet was enhanced in Panx1 del Apoe −/− mice, pointing to an atheroprotective role for Panx1 in endothelial and/or monocytic cells. Unexpectedly, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but Panx1 −/− Apoe −/− mice displayed reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these Panx1-deficient mice. Mechanistically, Panx1 −/− Apoe −/− mice showed impairment of lymphatic vessel function with decreased drainage of interstitial fluids and reduced dietary fat absorption. Thus, the detrimental effect of Panx1 deletion in endothelial and/or monocytic cells during atherogenesis is counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice. Collectively, our findings unveil a pivotal role of Panx1 in linking lymphatic function to lipid metabolism and atherosclerotic plaque development.

Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 mu M L-NAME), blockade of ETA-and ETB-receptors (10 mu M bosentan) and stimulation of the endothelium with 0.001-10 mu M acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. L-NAME (1) increased arterial contractile responses to K+ (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 mu M) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of L-NAME during contractions induced by either 10 mu M PHE or 40 mM K+ were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.

Background Hemostasis is a tightly regulated physiological process to rapidly induce hemostatic plugs at sites of vascular injury. Inappropriate activation of this process may lead to thrombosis, i.e. pathological blood clot formation in uninjured vessels or on atherosclerotic lesions. ATP release through Pannexin1 (Panx1) membrane channels contributes to collagen-induced platelet aggregation in vitro. Objective To investigate the effects of genetic and pharmacological inhibition of Panx1 on hemostasis and thrombosis in vivo. Results Bleeding time after tail clipping was increased by 2.5-fold in Panx1−/− mice compared to wild-type controls, suggesting that Panx1 deficiency impairs primary hemostasis. Wire myography on mesenteric arteries revealed diminished vasoconstriction in response to phenylephrine or U446619 in Panx1−/− mice. Mice with platelet-specific deletion of Panx1 (Panx1PDel) displayed 2-fold longer tail bleeding times than Panx1fl/fl controls. Moreover, venous thromboembolism (VTE) after injection of collagen/epinephrine in the jugular vein was reduced in Panx1−/− and Panx1PDel mice. Panx1PDel mice also showed reduced FeCl3-induced thrombosis in mesenteric arteries. BrilliantBlue-FCF, a Panx1 channel inhibitor, decreased collagen-induced platelet aggregation in vitro, increased tail bleeding time and reduced VTE in wild-type mice. Furthermore, we developed a specific Panx1 blocking antibody targeting a Panx1 extracellular loop, which reduced ATP release from platelets in vitro. Treating wild-type mice with this antibody increased tail bleeding time and decreased VTE compared to control antibody. Conclusions Panx1 channel deletion or inhibition diminishes clot formation during hemostasis and thrombosis in vivo. Blocking Panx1 channels may be an attractive strategy for modulating platelet aggregation in thrombotic disease.

Endothelial arginase 1 was ablated to assess whether this prevents hyperglycemia-induced endothelial dysfunction by improving arginine availability for nitric oxide production. Endothelial Arg1-deficient mice (Arg1-KOT ie2) were generated by crossing Arg1fl/fl (controls) with Tie2Cretg/− mice and analyzed by immunohistochemistry, measurements of hemodynamics, and wire myography. Ablation was confirmed by immunohistochemistry. Mean arterial blood pressure was similar in conscious male control and Arg1-KOT ie2 mice. Depletion of circulating arginine by intravenous infusion of arginase 1 or inhibition of nitric oxide synthase activity with L-NG-nitro-arginine methyl ester increased mean arterial pressure similarly in control (9 ± 2 and 34 ± 2 mmHg, respectively) and Arg1-KOT ie2 mice (11 ± 3 and 38 ± 4 mmHg, respectively). Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Arg1-KOT ie2 and control animals by wire myography. Diabetes was induced in 10-week-old control and Arg1-KOT ie2 mice with streptozotocin, and vasomotor responses were studied 10 weeks later. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in normoglycemic control and Arg1-KOT ie2 mice. The relaxing response to acetylcholine was dependent on the availability of extracellular l-arginine. In the diabetic mice, arterial relaxation responses to endothelium-dependent hyperpolarization and to exogenous nitric oxide were impaired. The data show that endothelial ablation of arginase 1 in mice does not markedly modify smooth muscle and endothelial functions of a resistance artery under normo- and hyperglycemic conditions.

Connexins and pannexins form connexons, pannexons and membrane channels, which are critically involved in many aspects of cardiovascular physiology. For that reason, a vast number of studies have addressed the role of connexins and pannexins in the arterial and venous systems as well as in the heart. Moreover, a role for connexins in lymphatics has recently also been suggested. This review provides an overview of the current knowledge regarding the involvement of connexins and pannexins in cardiovascular physiology.

BACKGROUND Atherosclerosis is a chronic inflammatory disease of large to medium sized arteries and is the main underlying cause of death worldwide. The lymphatic vasculature is critical for processes that are intimately linked to atherogenesis such as the immune response and cholesterol metabolism. However whether lymphatic vessels truly contribute to the pathogenesis of atherosclerosis is less clear despite increasing research efforts in this field. DESIGN PubMed and Ovid MEDLINE databases were searched. In addition key review articles were screened for relevant original publications. RESULTS Current knowledge about lymphatic vessels in the arterial wall came from studies that examined the presence and location of such vessels in human atherosclerotic plaque specimens as well as in a variety of arteries in animal models for atherosclerosis (e.g. rabbits dogs rats and mice). Generally three experimental approaches have been used to investigate the functional role of plaque associated lymphatic vessels; experimental lymphostasis was used to investigate lymphatic drainage of the arterial wall and more recently studies with genetic interventions and/or surgical transplantation have been performed. CONCLUSIONS Lymphatic vessels seem to be mostly present in the adventitial layer of the arterial walls of animals and humans. They are involved in reverse cholesterol transport from atherosclerotic lesions and arteries with a dense lymphatic network seem naturally protected against atherosclerosis. Lymphangiogenesis is a process that is an important part of the inflammatory loop in atherosclerosis. However how augmenting or impeding the distribution of lymphatic vessels impacts disease progression remains to be investigated in future studies. This article is protected by copyright. All rights reserved.

SummaryPannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific 10Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1–400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1–400A allele. Conversely, the frequency of Panx1–400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1–400C channels compared with Panx1–400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1–400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1–400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

The endothelium, a monolayer of cells that lines blood vessels, acts as a physical barrier between circulating blood and vascular smooth muscle cells. The purpose of this chapter is to provide a general overview on the structural heterogeneity of the endothelium. Moreover, the most important physiological functions of the vascular endothelium in blood vessels are discussed. More detailed insights into the pathogenesis of specific diseases, including atherosclerosis and hypertension, are provided in other chapters of this book.

// Jean-Francois Denis 1, * , K.E. Ludwig Scheckenbach 1, * , Anna Pfenniger 1, 2 , Merlijn J. Meens 1 , Rob Krams 3 , Lucile Miquerol 4 , Steven Taffet 5 , Marc Chanson 6 , Mario Delmar 7 and Brenda R. Kwak 1, 2 1 Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland 2 Department of Medical Specializations - Cardiology, University of Geneva, Geneva, Switzerland 3 Department of Bioengineering, Imperial College, London, UK 4 Aix-Marseille University, CNRS UMR 7288, Developmental Biology Institute of Marseille, Marseille, France 5 Department of Microbiology, SUNY Upstate Medical University, Syracuse, NY, USA 6 Departments of Pediatrics and of Cell Physiology and Metabolism, Geneva University Hospitals and University of Geneva, Geneva, Switzerland 7 The Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Brenda R. Kwak, email: Brenda.KwakChanson@unige.ch Keywords: atherosclerosis, endothelium, Cx40, shear stress, IκBα Received: November 08, 2016 Accepted: February 27, 2017 Published: March 22, 2017 ABSTRACT Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter ( Cx40 +/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro . Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, Tie2Cre Tg Cx40 fl/fl Apoe -/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.

Mutations in the gap junction protein connexin47 (Cx47) are associated with lymphedema. However, the role of Cx47 in lymphatic pathophysiology is unknown. We demonstrate that Cx47 is expressed in lymphatic endothelial cells by whole-mount immunostaining and qPCR. To determine if Cx47 plays a role in lymphatic vessel function we analysed Cx47-/- mice. Cx47-deficiency did not affect lymphatic contractility (contractile amplitude or frequency) or lymphatic morphology (vessel diameter or number of valves). Interstitial fluid drainage or dendritic cell migration through lymphatic vessels was also not affected by Cx47-deficiency. Cx47 is dispensable for long-chain fatty acid absorption from the gut but rather promotes serum lipid handling as prolonged elevated triglyceride levels were observed in Cx47-deficient mice after oral lipid tolerance tests. When crossed with Apolipoprotein E-deficient (Apoe-/-) mice, LDL-cholesterol was decreased in young Cx47-/-Apoe-/- adults as compared to Apoe-/- mice, which was inverted later in life. Finally, advanced atherosclerotic plaques in thoracic-abdominal aortas of 15 months-old mice tended to be larger in Cx47-/-Apoe-/- mice. These plaques contained fewer macrophages but similar amounts of T lymphocytes, collagen and lipids than plaques of Apoe-/- mice. In conclusion, Cx47 is expressed in lymphatic endothelium and seems modestly implicated in multiple aspects of lymphatic pathophysiology.

Connexins (Cxs) form a family of transmembrane proteins comprising 21 members in humans. Cxs differ in their expression patterns, biophysical properties and ability to combine into homomeric or heteromeric gap junction channels between neighbouring cells. The permeation of ions and small metabolites through gap junction channels or hemichannels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. Among others, Cx37, Cx40, Cx43, Cx45 and Cx47 are found in heart, blood and lymphatic vessels. Mutations or polymorphisms in the genes coding for these Cxs have not only been implicated in cardiovascular pathologies but also in a variety of other disorders. While mutations in Cx43 are mostly linked to oculodentodigital dysplasia, Cx47 mutations are associated with Pelizaeus-Merzbacher-like disease and lymphoedema. Cx40 mutations are principally linked to atrial fibrillation. Mutations in Cx37 have not yet been described, but polymorphisms in the Cx37 gene have been implicated in the development of arterial disease. This review addresses current knowledge on gene mutations in cardiovascular Cxs systematically and links them to alterations in channel properties and disease.

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (

Endothelial cells (ECs) of healthy arteries express high levels of connexin40 (Cx40), but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is known to be atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. Here, we investigate the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around the right common carotid artery of mice expressing eGFP controlled by the Cx40 promoter. We found that Cx40 expression is down-regulated in regions of oscillatory shear stress (0.45±0.13, p=0.04), but is conserved in regions of high and low laminar shear stress (1.45±0.48 and 0.85±0.11, N=8) by en face immunofluorescence on Cx40 +/eGFP mice. Using a high-throughput phage display, we retrieved a consensus binding motif for the intracellular regulatory C-terminus of Cx40 (Cx40CT), i.e. HS[I,L,V][K,R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5 to 16 of IκBα, a member of the protein family that inhibit the nuclear translocation of NFκB. In vitro binding of this peptide was confirmed by crosslinking and by en face proximity ligation assay targeting IκBα and Cx40 on rat carotid arteries. Nuclear translocation of NFκB in mouse ECs in response to activation by TNFα was enhanced after reduction of Cx40 with siRNA. Transfection of Cx40 or Cx40CT in HeLa cells demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced of NFκB nuclear translocation. Finally, we observed an exaggarated induction of atherosclerotic plaques by LLSS or OSS in Apoe −/− mice with endothelial-specific deletion of Cx40. In conclusion, our data show a novel functional interaction between IκBα and Cx40 that may be relevant for the control of NFκB activation by shear stress in development of atherosclerotic disease. The author hereby declares no conflict of interest

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) μm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) μm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 μm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) μm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 μm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 μm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 μm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 μm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

BACKGROUND AND PURPOSE Calcitonin gene-related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin-1 (ET-1) from ETA receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G-protein βγ subunits (Gβγ) in the arterial effects of CGRP receptor stimulation. EXPERIMENTAL APPROACH To test the hypothesis that instead of α subunits of G-proteins (Gαs), Gβγ mediates the effects of CGRP receptor activation, we used (i) rat isolated mesenteric resistance arteries (MRA), (ii) pharmacological modulators of cyclic nucleotides; and (iii) low molecular weight inhibitors of the functions of Gβγ, gallein and M119. To validate these tools with respect to CGRP receptor function, we performed organ bath studies with rat isolated MRA, radioligand binding on membranes from CHO cells expressing human CGRP receptors and cAMP production assays in rat cultured VSMC. KEY RESULTS In isolated arteries contracted with K+ or ET-1, IBMX (PDE inhibitor) increased sodium nitroprusside (SNP)- and isoprenaline (ISO)- but not CGRP-induced relaxations. While fluorescein (negative control) was without effects, gallein increased binding of [125I]-CGRP in the absence and presence of GTPγS. Gallein also increased CGRP-induced cAMP production in VSMC. Despite these stimulating effects, gallein and M119 selectively inhibited the relaxing and anti-endothelinergic effects of CGRP in isolated arteries while not altering contractile responses to K+ or ET-1 or relaxing responses to ISO or SNP. CONCLUSION AND IMPLICATIONS Activated CGRP receptors induce cyclic nucleotide-independent relaxation of VSMC and terminate arterial effects of ET-1 via Gβγ.

Introduction: Mutations in GJC2, which encodes connexin47 (Cx47), are associated with lymphedema. Moreover, Cx47 mRNA was recently found in human lymphatic endothelial cells (LECs). Hypothesis: Lymphatic endothelial Cx47 has a role in lymphatic physiology and pathology. Methods: Confocal microscopy and qPCRs were used to assess expression of Cx47. Lymphatic drainage was studied by subcutaneous Evans Blue injections in the footpad of 12 week-old ApoE -/- or Cx47 eGFP/eGFP ApoE -/- mice. Atherosclerosis was studied in 15 months-old ApoE -/- or Cx47 eGFP/eGFP ApoE -/- mice. Results: Cx47 was expressed in lymphangions of mesenteric collecting vessels in Cx47 eGFP/eGFP Prox1-mOrange2 mice as assessed by microscopy and its expression was confirmed by qPCR on mesentery, gut and skin mRNA of ApoE -/- but not Cx47 eGFP/eGFP ApoE -/- mice. Interestingly, lymphatic drainage was enhanced in Cx47-deficient mice (n=10/11, p-/- and Cx47 eGFP/eGFP ApoE -/- mice on regular chow. These studies showed that Cx47-deficiency does not affect the size or composition (e.g. lipid, collagen or macrophage content) of aortic plaques in the aortic root. However, plaque development in the thoracic-abdominal aorta of 15 months-old mice tended to be larger in Cx47 eGFP/eGFP ApoE -/- mice (21±5 vs 30±4 % lipid area, n=10, p=0.08). Unexpectedly, total cholesterol (TC) and LDL, but not HDL, triglycerides or free fatty acids, were increased in serum of Cx47 eGFP/eGFP ApoE -/- mice as compared to ApoE -/- serum (TC: 472±24 vs 647±18 mg/dL, n=10, peGFP/eGFP ApoE -/- mice may, partially, compensate for increased serum TC and LDL thereby preventing exuberant atherosclerotic plaque formation despite increased serum lipid levels. Conclusions: In conclusion, Cx47 is expressed in LECs of the lymphangion and seems implicated in multiple aspects of lymphatic (patho)physiology.

Objective Shear stress patterns influence atherogenesis and plaque stability; low laminar shear stress (LLSS) promotes unstable plaques whereas oscillatory shear stress (OSS) induces more stable plaques. Endothelial connexin37 (Cx37) expression is also regulated by shear stress, which may contribute to localization of atherosclerotic disease. Moreover, Cx37 reduces initiation of atherosclerosis by inhibiting monocyte adhesion. The present work investigates the effect of Cx37 on the phenotype of plaques induced by LLSS or OSS. Methods Shear stress-modifying casts were placed around the common carotid artery of ApoE −/− or ApoE −/− Cx37 −/− mice, and animals were placed on a high-cholesterol diet for 6 or 9 weeks. Atherosclerotic plaque size and composition were assessed by immunohistochemistry. Results Plaque size in response to OSS was increased in ApoE −/− Cx37 −/− mice compared to ApoE −/− animals. Most plaques contained high lipid and macrophage content and a low amount of collagen. In ApoE −/− mice, macrophages were more prominent in LLSS than OSS plaques. This difference was reversed in ApoE −/− Cx37 −/− animals, with a predominance of macrophages in OSS plaques. The increase in macrophage content in ApoE −/− Cx37 −/− OSS plaques was mainly due to increased accumulation of M1 and Mox macrophage subtypes. Cx37 expression in macrophages did not affect their proliferation or their polarization in vitro . Conclusion Cx37 deletion increased the size of atherosclerotic lesions in OSS regions and abrogated the development of a stable plaque phenotype under OSS in ApoE −/− mice. Hence, local hemodynamic factors may modify the risk for adverse atherosclerotic disease outcomes associated to a polymorphism in the human Cx37 gene.

AIM: Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice.METHODS AND RESULTS: Endothelium-selective Ass-deficient mice (Assfl/fl/Tie2Cretg/- = Ass-KOTie2) were generated by crossing Assfl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Ass-KOTie2 and control animals. At the age of 10 weeks, diabetes was induced in control and Ass-KOTie2 mice by streptozotocin injections. Vasomotor responses of diabetic animals were studied 10 weeks later. MAP was similar in control and Ass-KOTie2 mice. Depletion of circulating L-arginine by arginase 1 infusion or inhibition of NOS activity with L-NAME resulted in an increased MAP (10 and 30 mmHg, respectively) in control and Ass-KOTie2 mice. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in healthy control and Ass-KOTie2 mice. However, in diabetic Ass-KOTie2 mice, relaxation responses to acetylcholine and endothelium-derived NO (EDNO) were significantly reduced when compared to diabetic control mice.CONCLUSIONS: Absence of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KOTie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting endothelial function in diabetes.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18 F-FEDAA1106 and 2-deoxy-2-[ 18 F]fluoro-Dglucose ( 18 F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18 F-FEDAA1106 or 18 F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18 F-FEDAA1106 (p , .01) or FDG (p , .05). However, the 18 F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18 F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18 F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

SummaryUbiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial effects on progression and composition of atherosclerotic lesions. Cx43 is expressed in multiple atheroma-associated cells but its function in each cell type is not known. To examine specifically the role of Cx43 in immune cells, we have lethally irradiated low-density lipoprotein receptor-deficient mice and reconstituted with Cx43+/+, Cx43+/− or Cx43−/− haematopoietic fetal liver cells. Progression of atherosclerosis was significantly lower in aortic roots of Cx43+/− chimeras compared with Cx43+/+ and Cx43−/− chimeras, and their plaques contained significantly less neutrophils. The relative proportion of circulating leukocytes was similar between the three groups. Interestingly, the chemoattraction of neutrophils, which did not express Cx43, was reduced in response to supernatant secreted by Cx43+/− macrophages in comparison with the ones of Cx43+/+ and Cx43−/− macrophages. Cx43+/− macrophages did not differ from Cx43+/+ and Cx43−/− macrophages in terms of M1/M2 polarisation but show modified gene expression for a variety chemokines and complement components. In conclusion, titration of Cx43 expression in bone marrow-derived macrophages reduces atherosclerotic plaque formation and chemoattraction of neutrophils to the lesions.

Connexins form a family of transmembrane proteins that consists of 20 members in humans and 21 members in mice. Six connexins assemble into a connexon that can function as a hemichannel or connexon that can dock to a connexon expressed by a neighbouring cell, thereby forming a gap junction channel. Such intercellular channels synchronize responses in multicellular organisms through direct exchange of ions, small metabolites, and other second messenger molecules between the cytoplasms of adjacent cells. Multiple connexins are expressed in the cardiovascular system. These connexins not only experience the different biomechanical forces within this system, but may also act as effector proteins in co-ordinating responses within groups of cells towards these forces. This review discusses recent insights regarding regulation of cardiovascular connexins by mechanical forces and junctions. It specifically addresses effects of (i) shear stress on endothelial connexins, (ii) hypertension on vascular connexins, and (iii) changes in afterload and the composition of myocardial mechanical junctions on cardiac connexins.

BACKGROUND: Endothelin-1 (ET1) is a potent vasoconstrictor peptide with pro-mitogenic and pro-inflammatory properties and is therefore of interest in the development of endothelial dysfunction, endothelium-dependent flow-related remodeling, and hypertension-related remodeling. ET1 can be formed through cleavage of big ET1 by endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP).METHOD: We investigated whether the dual NEP/ECE inhibitor SOL1 improves resistance artery function and structure in 12 weeks old spontaneously hypertensive rats (SHRs) and whether arterial structural responses to decreased (-90%) or increased (+100%) blood flow are impaired in young SHRs. To this end two groups of SHRs received chronic 4-week treatment at two different time points (4-8 and 8-12 weeks) prior to the experiment. We compared in-vitro effects of cyclo-oxygenase inhibition (1 μmol/l indomethacine), nitric oxide synthase inhibition (100 μmol/l N(ω)-L-nitro arginine methyl ester), and stimulation of the endothelium by 0.001-10 μmol/l acetylcholine (ACh) in isolated third-order mesenteric arteries of SHRs and aged-matched Wistar-Kyoto (WKY) rats.RESULTS: SOL1 had no effect on blood pressure in SHRs or WKY rats. ACh caused biphasic effects in mesenteric arteries of SHRs. The contractile component (endothelium-derived contractile factor) was absent in WKY and abolished by acute indomethacin administration or chronic SOL1 treatment. Endothelium-derived nitric oxide-type responses did not differ in both strains and were not influenced by SOL1 treatment. Endothelium-derived hyperpolarizing factor-type responses were severely impaired in SHRs as compared to WKY rats and were normalized by chronic SOL1 treatment. In first-order mesenteric arteries, outward flow-induced remodeling was impaired in SHRs. Chronic SOL1 treatment did not restore this response.CONCLUSION: Thus chronic SOL1 treatment during the development of hypertension in SHRs has no effect on blood pressure but improves several aspects of endothelium-dependent vasomotor responses but not arterial remodeling.

Atherosclerosis, a progressive disease of medium- and large-sized arteries, constitutes the major cause of death in developed countries, and is becoming increasingly prevalent in developing countries as well. The main consequences of atherosclerosis are myocardial infarction, cerebral infarction and aortic aneurysm. This inflammatory disease is characterised by specific intimal lesions where lipids, leukocytes and smooth muscle cells accumulate in the arterial wall over time. Risk factors for atherosclerosis can mainly be divided into two groups: i) risk factors induced by environment and behaviour (e.g., Western diet, smoking and sedentary lifestyle) and ii) genetic risk factors. Multiple epidemiological studies have associated a single nucleotide polymorphism (SNP) in the GJA4 gene, coding for connexin37 (Cx37), with increased risk for atherosclerosis and myocardial infarction. Connexins form gap junctions or hemi-channels that mediate an exchange of factors between i) the cytosol of two adjacent cells or ii) the cytosol and the extracellular environment, respectively. The GJA4 SNP codes for a proline-to-serine substitution at amino acid 319 in the regulatory C-terminus of the Cx37 protein, thereby altering basic and regulatory properties of its gap junction- and hemi-channels. In this review we discuss current evidence for mechanisms that link the GJA4 SNP to atherosclerosis or thrombus formation after plaque rupture.

Background: We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A(2), cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A(2). This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR. Methods and Findings: For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191 +/- 7 mmHg, losartan: 125 +/- 5 mmHg and hydralazine: 113 +/- 14 mmHg). The blood pressure lowering was associated with a 20-25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A(2). The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction. Conclusion: In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.

Slow dissociation of endothelin 1 from its endothelin A receptors is responsible for the long-lasting vasoconstrictor effects of the peptide. We showed recently that calcitonin gene-related peptide selectively terminates long-lasting contractile responses to endothelin 1 in isolated rat mesenteric arteries. Here we assessed whether the antiendothelinergic effect of calcitonin gene-related peptide is vascular bed specific and may terminate long-lasting pressor responses to exogenous and locally produced endothelin 1 in vivo. Regional heterogeneity of the calcitonin gene-related peptide/endothelin A receptor cross-talk was explored in arteries isolated from various rat organs. Endothelin A receptor-mediated arterial contractions were terminated by calcitonin gene-related peptide in rat mesenteric, renal, and spermatic arteries but not in basilar, coronary, epigastric, gastric, splenic, and saphenous arteries. Endothelin A receptor antagonism only ended endothelin 1-induced contractions in spermatic arteries. In anesthetized rats, instrumented with Doppler flow probes to record regional blood flows, long-lasting pressor and vasoconstrictor responses to an intravenous bolus injection of endothelin 1 or big endothelin 1 were transiently reduced by sodium nitroprusside (NO donor) but terminated by intravenously administered calcitonin gene-related peptide. In conscious rats, calcitonin gene-related peptide but not sodium nitroprusside terminated prolonged (>60-minute) pressor responses to endothelin 1 but not those to intravenous infusion of phenylephrine. In conclusion, pressor responses to circulating and locally produced endothelin 1 that are resistant to endothelin receptor antagonism and NO can be terminated by a regionally selective effect of calcitonin gene-related peptide. Calcitonin gene related peptide receptor agonism may represent a novel strategy to treat endothelin 1-associated cardiovascular pathologies. (Hypertension. 2011;58:99-106.) . Online Data Supplement

The paracrine signaling peptide endothelin-1 (ET1) is involved in cardiovascular diseases, cancer and chronic pain. It acts on class A G-protein-coupled receptors (GPCRs) but displays atypical pharmacology. It binds tightly to ET receptor type A (ET(A)) and causes long-lasting effects. In resistance arteries, the long-lasting contractile effects can only be partly and reversibly relaxed by low-molecular-weight ET(A) antagonists (ERAs). However, the neuropeptide calcitonin-gene-related peptide selectively terminates binding of ET1 to ET(A). We propose that ET1 binds polyvalently to ET(A) and that ERAs and the physiological antagonist allosterically reduce ET(A) functions. Combining the two-state model and the two-domain model of GPCR function and considering receptor activation beyond agonist binding might lead to better anti-endothelinergic drugs. Future studies could lead to compounds that discriminate between ET(A)-mediated effects of the endogenous isopeptides ET1, ET2 and ET3 and that become more effective when the activity of the endogenous endothelin system is elevated.

Background Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism. Methodology/Principal findings In isolated rat mesenteric resistance arteries, ETA-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ETA-antagonism, were observed to promote dissociation of pre-existing ET-1/ETA-receptor complexes. Conclusions Irreversible binding and activation of ETA-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.

Background— Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. Methods and Results— Here, we report that VEGF ∂/∂ mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. Conclusions— These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.

We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wire-myography, and pharmacological tools. ET(A)- and ET(B)-receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET(B)-receptors, NO-synthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET(A)-antagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 microM) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET(A)-mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K(+) channels.

Background— Smoothelins are actin-binding proteins that are abundantly expressed in healthy visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Their expression is strongly associated with the contractile phenotype of smooth muscle cells. Analysis of mice lacking both smoothelins ( Smtn-A/B −/− mice) previously revealed a critical role for smoothelin-A in intestinal smooth muscle contraction. Here, we report on the generation and cardiovascular phenotype of mice lacking only smoothelin-B ( Smtn-B −/− ). Methods and Results— Myograph studies revealed that the contractile capacity of the saphenous and femoral arteries was strongly reduced in Smtn-B −/− mice, regardless of the contractile agonist used to trigger contraction. Arteries from Smtn-A/B −/− compound mutant mice exhibited a similar contractile deficit. Smtn-B −/− arteries had a normal architecture and expressed normal levels of other smooth muscle cell–specific genes, including smooth muscle myosin heavy chain, α-smooth muscle actin, and smooth muscle-calponin. Decreased contractility of Smtn-B −/− arteries was paradoxically accompanied by increased mean arterial pressure (20 mm Hg) and concomitant cardiac hypertrophy despite normal parasympathetic and sympathetic tone in Smtn-B −/− mice. Magnetic resonance imaging experiments revealed that cardiac function was not changed, whereas distension of the proximal aorta during the cardiac cycle was increased in Smtn-B −/− mice. However, isobaric pulse wave velocity and pulse pressure measurements indicated normal aortic distensibility. Conclusions— Collectively, our results identify smoothelins as key determinants of arterial smooth muscle contractility and cardiovascular performance. Studies on mutations in the Smtn gene or alterations in smoothelin levels in connection to hypertension in humans are warranted.

Background Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. Results Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40+/− aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37−/− and Cx40−/− aortas but not in Cx40+/− aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40−/− and Cx40+/− aortas but not in Cx37−/− aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. Conclusions Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40. Electronic supplementary material The online version of this article (doi:10.1186/s12964-015-0110-1) contains supplementary material, which is available to authorized users.