Publications by authors named "Jacques-Antoine Haefliger"

70 Publications

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

FASEB J 2020 06 22;34(6):8234-8249. Epub 2020 Apr 22.

Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37 ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37 mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.
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http://dx.doi.org/10.1096/fj.202000257RDOI Listing
June 2020

Impaired SMAD1/5 Mechanotransduction and Cx37 (Connexin37) Expression Enable Pathological Vessel Enlargement and Shunting.

Arterioscler Thromb Vasc Biol 2020 04 6;40(4):e87-e104. Epub 2020 Feb 6.

From the Department of Cardiovascular Sciences, Centre for Molecular and Vascular Biology (H.M.P., A.T., N.C., A.Z., E.A.V.J.), KU Leuven, Belgium.

Objective: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for , addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions.

Conclusions: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.
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http://dx.doi.org/10.1161/ATVBAHA.119.313122DOI Listing
April 2020

Connexin37-Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II -Mediated Hypertension.

J Am Heart Assoc 2019 04;8(8):e010823

1 Department of Medicine University of Lausanne Switzerland.

Background Gap junction channels made of Connexin37 (Cx37) are expressed by aortic endothelial and smooth muscle cells of hypertensive mice, as well as by the renin-secreting cells of kidneys. Methods and Results To decipher whether Cx37 has any role in hypertension, angiotensin II (Ang II ) was infused in normotensive wild-type and Cx37-deficient mice (Cx37-/-). After 2 to 4 weeks, the resulting increase in blood pressure was lower in Cx37-/- than in wild-type mice, suggesting an alteration in the Ang II response. To investigate this possibility, mice were submitted to a 2-kidney, 1-clip procedure, a renin-dependent model of hypertension. Two weeks after this clipping, Cx37-/- mice were less hypertensive than wild-type mice and, 2 weeks later, their blood pressure had returned to control values, in spite of abnormally high plasma renin levels. In contrast, Cx37-/- and wild-type mice that received N-nitro-l-arginine-methyl-ester, a renin-independent model of hypertension, featured a similar and sustained increase in blood pressure. The data indicate that loss of Cx37 selectively altered the Ang II -dependent pathways. Consistent with this conclusion, aortas of Cx37-/- mice featured an increased basal expression of the Ang II type 2 receptors ( AT 2R), and increased transcripts levels of downstream signaling proteins, such as Cnksr1 and Ptpn6 ( SHP -1). Accordingly, the response of Cx37-/- mice aortas to an ex vivo Ang II exposure was altered, since phosphorylation levels of several proteins of the Ang II pathway ( MLC 2, ERK , and AKT ) remained unchanged. Conclusions These findings provide evidence that Cx37 selectively influences Ang II signaling, mostly via a modulation of the expression of the Ang II type 2 receptor.
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http://dx.doi.org/10.1161/JAHA.118.010823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507190PMC
April 2019

Intravesical Ty21a Vaccine Promotes Dendritic Cells and T Cell-Mediated Tumor Regression in the MB49 Bladder Cancer Model.

Cancer Immunol Res 2019 04 29;7(4):621-629. Epub 2019 Jan 29.

Department of Urology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

Preclinical data show that intravesical instillation of Ty21a/Vivotif, a commercial vaccine against typhoid fever, is an effective alternative option to standard Bacillus Calmette-Guérin (BCG) immunotherapy for non-muscle-invasive bladder cancer (NMIBC). Here, we characterized the inflammatory effects of Ty21a on the bladder and investigated the immune mechanisms underlying tumor regression toward the use of this bacterial vaccine in NMIBC patients. MB49 bladder tumor-bearing mice had significantly improved survival after intravesical instillations of Ty21a doses of 10 to 10 colony-forming units. By IHC and morphology, both BCG and Ty21a instillations were associated with bladder inflammation, which was decreased with the use of low, but effective doses of Ty21a. Flow-cytometry analysis showed a significant infiltration of T cells, natural killer (NK) cells, and myeloid cells, compared with controls, after a single dose of Ty21a, whereas this was only observed after multiple doses of BCG. The induced myeloid cells were predominantly neutrophils and Ly6CCD103 dendritic cells (DC), the latter being significantly more numerous after instillation of Ty21a than BCG. infection of human leukocytes with Ty21a, but not BCG, similarly significantly increased DC frequency. CD4 and CD8 T cells, but not NK cells nor neutrophils, were required for effective bladder tumor regression upon Ty21a treatment. Thus, the generation of antitumor adaptive immunity was identified as a key process underlying Ty21a-mediated treatment efficacy. Altogether, these results demonstrate mechanisms behind intravesical Ty21a therapy and suggest its potential as a safe and effective treatment for NMIBC patients.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0671DOI Listing
April 2019

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

PLoS One 2018 28;13(9):e0204045. Epub 2018 Sep 28.

Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States of America.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204045PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161854PMC
March 2019

Evaluating intimal hyperplasia under clinical conditions.

Interact Cardiovasc Thorac Surg 2018 09;27(3):427-436

Department of Vascular Surgery, Lausanne University Hospital, Lausanne, Switzerland.

Objectives: Open arterial revascularization using venous segments is frequently associated with the development of intimal hyperplasia (IH), leading to severe restenosis and graft failure. The lack of treatment to prevent this pathology is a major problem. Therefore, we generated a new porcine model, which closely mimics the clinical development of human IH, to test the therapeutic potential of candidate drugs.

Methods: A patch of jugular vein was sutured to the right common carotid artery of pigs, to expose the vein to haemodynamic conditions of the arterial bed. Four weeks after surgery, the operated vessels which received no further treatment (the control group) were compared with (i) contralateral, non-operated vessels (the healthy group); (ii) vessels of pigs that received a perivascular application of a drug-free microparticle gel (the placebo group) and (iii) vessels of pigs that perioperatively received the same gel loaded with 10-mg atorvastatin (the atorvastatin group).

Results: When compared with non-operated vessels, all operated segments displayed a sizable IH which was thicker in the venous patch than in the host artery. These alterations were associated with a thickening of the intima layer of both vessels in the absence of inflammation. The intima/media ratio has been significantly increased by 2000-fold in the vein patches. Perivascular application of atorvastatin did not prevent IH formation. However, the drug increased the adventitial neovascularization in the operated vessels.

Conclusions: The novel porcine model allows for monitoring IH formation under haemodynamic conditions which mimic clinical situations. It should facilitate the screening of innovative treatments to prevent restenosis.
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http://dx.doi.org/10.1093/icvts/ivy101DOI Listing
September 2018

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and HS Production.

Cell 2018 03;173(1):117-129.e14

Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address:

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (HS) production. HS mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.
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http://dx.doi.org/10.1016/j.cell.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901681PMC
March 2018

Targeting Cx40 (Connexin40) Expression or Function Reduces Angiogenesis in the Developing Mouse Retina.

Arterioscler Thromb Vasc Biol 2017 11 5;37(11):2136-2146. Epub 2017 Oct 5.

From the Department of Medicine (J.-A.H., F.A., L.H., L.L.G., F.A.) and Department of Urology (D.N.H.), Lausanne University Hospital, Switzerland; Department of Cell Physiology and Metabolism, University of Geneva Medical Center, Switzerland (P.M.); and Centre de Recherche Cardio-Thoracique de Bordeaux (INSERM U1045), Université de Bordeaux, France (E.G., F.A.).

Objective: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown.

Approach And Results: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice.

Conclusions: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.
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http://dx.doi.org/10.1161/ATVBAHA.117.310072DOI Listing
November 2017

Connexin37 reduces smooth muscle cell proliferation and intimal hyperplasia in a mouse model of carotid artery ligation.

Cardiovasc Res 2017 Jun;113(7):805-816

Department of Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), Laboratory of Experimental Medicine, c/o Department of Physiology, Bugnon 7a, 1005 Lausanne, Switzerland.

Aims: Intimal hyperplasia (IH) is an abnormal response to vessel injury characterized by the dedifferentiation, migration, and proliferation of quiescent vascular smooth muscle cells (VSMC) to form a neointima layer. Vascular connexins (Cx) are involved in the pathophysiology of various vascular diseases, and Cx43, the main Cx expressed in VSMC, has been shown to promote VSMC proliferation and IH. The aim of this study was to investigate the participation of another Cx, namely Cx37, in the formation of the neointima layer.

Methods And Results: Wild-type (WT) and Cx37-deficient (Cx37-/-) C57BL/6J mice were subjected to carotid artery ligation (CAL), a model of vessel injury and IH. The neointima developed linearly in WT until 28 days post surgery. In contrast, the neointima layer was almost absent 14 days after surgery in Cx37-/- mice, and twice as more developed after 28 days compared to WT mice. This large neointima formation correlated with a two-fold increase in cell proliferation in the media and neointima regions between 14 and 28 days in Cx37-/- mice compared to WT mice. The CAL triggered Cx43 overexpression in the media and neointima layers of ligated carotids in WT mice, and selectively up-regulated Cx37 expression in the media layer, but not in the neointima layer. The de novo expression of Cx37 in human primary VSMC reduced cell proliferation and P-Akt levels, in association with lower Cx43 levels, whereas Cx43 overexpression increased P-Akt levels.

Conclusion: The presence of Cx37 in the media layer of injured arteries restrains VSMC proliferation and limits the development of IH, presumably by interfering with the pro-proliferative effect of Cx43 and the Akt pathway.
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http://dx.doi.org/10.1093/cvr/cvx079DOI Listing
June 2017

Perivascular medical devices and drug delivery systems: Making the right choices.

Biomaterials 2017 06 28;128:56-68. Epub 2017 Feb 28.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, rue Michel Servet 1, CH-1211 Geneva 4, Switzerland. Electronic address:

Perivascular medical devices and perivascular drug delivery systems are conceived for local application around a blood vessel during open vascular surgery. These systems provide mechanical support and/or pharmacological activity for the prevention of intimal hyperplasia following vessel injury. Despite abundant reports in the literature and numerous clinical trials, no efficient perivascular treatment is available. In this review, the existing perivascular medical devices and perivascular drug delivery systems, such as polymeric gels, meshes, sheaths, wraps, matrices, and metal meshes, are jointly evaluated. The key criteria for the design of an ideal perivascular system are identified. Perivascular treatments should have mechanical specifications that ensure system localization, prolonged retention and adequate vascular constriction. From the data gathered, it appears that a drug is necessary to increase the efficacy of these systems. As such, the release kinetics of pharmacological agents should match the development of the pathology. A successful perivascular system must combine these optimized pharmacological and mechanical properties to be efficient.
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http://dx.doi.org/10.1016/j.biomaterials.2017.02.028DOI Listing
June 2017

Connexins and pannexins: from biology towards clinical targets.

Swiss Med Wkly 2016 5;146:w14365. Epub 2016 Dec 5.

Department of Medicine, Université de Lausanne, Lausanne, Switzerland.

Efficient cell communication is a prerequisite for the coordinated function of tissues and organs. In vertebrates, this communication is mediated by a variety of mechanisms, including the exchange of molecules between cells, and between cells and the extracellular medium, via membrane channels made of connexin and pannexin proteins. These channels are a necessary component of all human tissues. Here, we review the biological essentials of the connexin and pannexin families, and the roles of these proteins in the function of cells which are central to major human diseases. We then discuss how connexins and pannexins participate in human pathology, and the clinical perspectives that this knowledge opens.
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http://dx.doi.org/10.4414/smw.2016.14365DOI Listing
March 2017

Connexins and pannexins: from biology towards clinical targets.

Swiss Med Wkly 2016 Dec 5;146:w14365. Epub 2016 Dec 5.

Department of Medicine, Université de Lausanne, Lausanne, Switzerland.

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http://dx.doi.org/10.4414/smw.2016.14365DOI Listing
December 2016

Perivascular sustained release of atorvastatin from a hydrogel-microparticle delivery system decreases intimal hyperplasia.

J Control Release 2016 06 16;232:93-102. Epub 2016 Apr 16.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ansermet, 30, 1205 Geneva, Switzerland. Electronic address:

Intimal hyperplasia (IH) is the major cause of grafted vessel occlusion and occurs frequently after bypass intervention. No pharmaceutical formulation is currently available to prevent this pathology. Local perivascular delivery of an appropriate active compound released in a time-dependent manner (from day one up to 4weeks) is necessary for an efficient single-administration preventive therapy. To this aim, we propose the combination of gel and microparticles delivery system containing atorvastatin (ATV). The incorporation of ATV in a cross-linked hyaluronic acid gel, provided in vitro a fast release over 3days, while ATV-loaded poly-lactic-co-glycolic acid (PLGA) microparticles dispersed in the gel gave a sustained release over 4weeks. In vivo, ATV formulations were applied perivascularly in mice undergoing carotid artery ligation. IH was significantly reduced (-68%) in presence of ATV incorporated in hyaluronic acid gel and encapsulated in microparticles compared to control. No significant IH alteration was observed when ATV was incorporated only in the gel (fast release) or only in the microparticles (slow release) demonstrating that a biphasic release of ATV is essential to interfere with the development of IH. ATV was detected in adjacent tissues 28days after the intervention, showing the sustained presence of the drug in vivo. After four weeks ATV was not detected in remote tissues, except at a very low concentration (0.044ng/mg) in the liver, suggesting a very low risk of systemic toxicity of locally delivered ATV. Additionally, the ex vivo data showed that ATV in solution permeates through isolated human saphenous veins and thus is a good candidate for perivascular delivery. Our data demonstrate that a local biphasic ATV release on the mice ligated carotid efficiently prevents the development of IH without apparent toxicity.
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http://dx.doi.org/10.1016/j.jconrel.2016.04.023DOI Listing
June 2016

A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

PLoS One 2016 9;11(3):e0150880. Epub 2016 Mar 9.

Department of Cell Physiology and Metabolism, University of Geneva Faculty of Medicine, Geneva, Switzerland.

Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150880PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784816PMC
August 2016

Targeting endothelial connexin40 inhibits tumor growth by reducing angiogenesis and improving vessel perfusion.

Oncotarget 2016 Mar;7(12):14015-28

Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland.

Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40-/-), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40-/- but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40-/- mice. As a result, Cx40-/- mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment.
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http://dx.doi.org/10.18632/oncotarget.7370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924695PMC
March 2016

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells.

J Biol Chem 2015 Dec 22;290(51):30530-9. Epub 2015 Oct 22.

the Department of Medicine, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland, and.

Store-operated Ca(2+) channels (SOCs) are voltage-independent Ca(2+) channels activated upon depletion of the endoplasmic reticulum Ca(2+) stores. Early studies suggest the contribution of such channels to Ca(2+) homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca(2+) depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca(2+) imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca(2+) entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.
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http://dx.doi.org/10.1074/jbc.M115.682583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683273PMC
December 2015

Role of Connexins and Pannexins in the Pancreas.

Pancreas 2015 Nov;44(8):1234-44

From the *Department of Genetic Medicine, University of Geneva, Geneva; and †Department of Medicine, University of Lausanne, Lausanne, Switzerland; ‡Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and §Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.

The pancreas produces enzymes with a digestive function and hormones with a metabolic function, which are produced by distinct cell types of acini and islets, respectively. Within these units, secretory cells coordinate their functioning by exchanging information via signals that flow in the intercellular spaces and are generated either at distance (several neural and hormonal inputs) or nearby the pancreatic cells themselves (inputs mediated by membrane ionic-specific channels and by ionic- and metabolite-permeant pannexin channels and connexin "hemichannels"). Pancreatic secretory cells further interact via the extracellular matrix of the pancreas (inputs mediated by integrins) and directly with neighboring cells, by mechanisms that do not require extracellular mediators (inputs mediated by gap and tight junction channels). Here, we review the expression and function of the connexins and pannexins that are expressed by the main secretory cells of the exocrine and endocrine pancreatic cells. Available data show that the patterns of expression of these proteins differ in acini and islets, supporting distinct functions in the physiological secretion of pancreatic enzymes and hormones. Circumstantial evidence further suggests that alterations in the signaling provided by these proteins are involved in pancreatic diseases.
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http://dx.doi.org/10.1097/MPA.0000000000000378DOI Listing
November 2015

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

PLoS One 2015 23;10(9):e0138847. Epub 2015 Sep 23.

Department of Vascular Surgery, Centre Hospitalier Universitaire Vaudois, Laboratory of Experimental Medicine, Lausanne, Switzerland.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138847PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580578PMC
May 2016

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

Cell Commun Signal 2015 Jul 22;13:34. Epub 2015 Jul 22.

Department of Medicine, University Hospital, CHUV, Lausanne, Switzerland.

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.
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http://dx.doi.org/10.1186/s12964-015-0110-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510910PMC
July 2015

REST represses a subset of the pancreatic endocrine differentiation program.

Dev Biol 2015 Sep 5;405(2):316-27. Epub 2015 Jul 5.

Swiss Institute for Experimental Cancer Research, Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland; DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen N, Denmark. Electronic address:

To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program.
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http://dx.doi.org/10.1016/j.ydbio.2015.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732702PMC
September 2015

Interplay between connexin40 and nitric oxide signaling during hypertension.

Hypertension 2015 Apr 23;65(4):910-5. Epub 2015 Feb 23.

From the Departments of Medicine (L.L.G., F.A., J.-A.H.) and Angiology (L.M.), Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland; and Department of Cell Physiology and Metabolism, University of Geneva, Medical Center, Geneva, Switzerland (P.M.).

Connexins (Cxs) and endothelial nitric oxide synthase (eNOS) contribute to the adaptation of endothelial and smooth muscle cells to hemodynamic changes. To decipher the in vivo interplay between these proteins, we studied Cx40-null mice, a model of renin-dependent hypertension which displays an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels. These mice, which were either untreated or subjected to the 1-kidney, 1-clip (1K1C) procedure, a model of volume-dependent hypertension, were compared with control mice submitted to either the 1K1C or the 2-kidney, 1-clip (2K1C) procedure, a model of renin-dependent hypertension. All operated mice became hypertensive and featured hypertrophy and altered Cx expression of the aorta. The combination of volume- and renin-dependent hypertension in Cx40-/- 1K1C mice raised blood pressure and cardiac weight index. Under these conditions, all aortas showed increased levels of Cx40 in endothelial cells and of both Cx37 and Cx45 in smooth muscle cells. In the wild-type 1K1C mice, the interactions between Cx40 and Cx37 with eNOS were enhanced, resulting in increased NO release. The Cx40-eNOS interaction could not be observed in mice lacking Cx40, which also featured decreased levels of eNOS. In these animals, the volume overload caused by the 1K1C procedure resulted in increased phosphorylation of eNOS and in a higher NO release. The findings provide evidence that Cx40 and Cx37 play an in vivo role in the regulation of eNOS.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.114.04775DOI Listing
April 2015

Procedure for human saphenous veins ex vivo perfusion and external reinforcement.

J Vis Exp 2014 Oct 1(92):e52079. Epub 2014 Oct 1.

Department of Thoracic and Vascular Surgery, CHUV University Hospital.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672965PMC
http://dx.doi.org/10.3791/52079DOI Listing
October 2014

Restoration of connexin 40 (Cx40) in Renin-producing cells reduces the hypertension of Cx40 null mice.

Hypertension 2014 Jun 10;63(6):1198-204. Epub 2014 Mar 10.

Department of Medicine, Laboratory of Experimental Medicine, c/o Department of Physiology, Bugnon 7a, 1005 Lausanne, Switzerland.

Connexin 40 (Cx40) is expressed by the renin-producing cells (RSCs) of the kidneys and the endothelial cells of blood vessels. Cx40 null mice (Cx40(-/-)) feature a much increased renin synthesis and secretion, which results in chronic hypertension, and also display an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels and nitric oxide production. To discriminate the effect of Cx40 in renin secretion and vascular signaling, we targeted Cx40 to either the RSCs or the endothelial cells of Cx40 null mice. When compared with Cx40(-/-) controls, the animals expressing Cx40 in RSCs were less hypertensive and featured reduced renin levels, still numerous RSCs outside the wall of the afferent arterioles. In contrast, mice expressing Cx40 in the endothelial cells were as hypertensive as Cx40(-/-) mice, in spite of control levels of Cx37 and eNOS. Our data show that blood pressure is improved by restoration of Cx40 expression in RSCs but not in endothelial cells, stressing the prominent role of renin in the mouse hypertension linked to loss of Cx40.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.113.02976DOI Listing
June 2014

The use of external mesh reinforcement to reduce intimal hyperplasia and preserve the structure of human saphenous veins.

Biomaterials 2014 Mar 13;35(9):2588-99. Epub 2014 Jan 13.

Department of Thoracic and Vascular Surgery, University Hospital, Laboratory of Experimental Medicine, Bugnon 21, 1011 Lausanne, Switzerland. Electronic address:

The saphenous vein is the conduit of choice in bypass graft procedures. Haemodynamic factors play a major role in the development of intimal hyperplasia (IH), and subsequent bypass failure. To evaluate the potential protective effect of external reinforcement on such a failure, we developed an ex vivo model for the perfusion of segments of human saphenous veins under arterial shear stress. In veins submitted to pulsatile high pressure (mean pressure at 100 mmHg) for 3 or 7 days, the use of an external macroporous polyester mesh 1) prevented the dilatation of the vessel, 2) decreased the development of IH, 3) reduced the apoptosis of smooth muscle cells, and the subsequent fibrosis of the media layer, 4) prevented the remodelling of extracellular matrix through the up-regulation of matrix metalloproteinases (MMP-2, MMP-9) and plasminogen activator type I. The data show that, in an experimental ex vivo setting, an external scaffold decreases IH and maintains the integrity of veins exposed to arterial pressure, via increase in shear stress and decrease wall tension, that likely contribute to trigger selective molecular and cellular changes.
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http://dx.doi.org/10.1016/j.biomaterials.2013.12.041DOI Listing
March 2014

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

J Pharmacol Exp Ther 2013 Dec 26;347(3):574-81. Epub 2013 Sep 26.

Department of Thoracic and Vascular Surgery, University Hospital, Laboratory of Experimental Medicine, Lausanne, Switzerland (C.D., L.M., F.A., S.D., J.-M.C., F.S., J.-A.H.); School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Geneva, Switzerland (L.L., I.M., F.D., O.J.); and Department of Cell Physiology and Metabolism, University of Geneva, Medical Center, Geneva, Switzerland (P.M.).

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.
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http://dx.doi.org/10.1124/jpet.113.208769DOI Listing
December 2013

Intravaginal and subcutaneous immunization induced vaccine specific CD8 T cells and tumor regression in the bladder.

J Urol 2014 Mar 13;191(3):814-22. Epub 2013 Aug 13.

Department of Urology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Electronic address:

Purpose: Vaccines targeting tumor associated antigens are in development for bladder cancer. Most of these cancers are nonmuscle invasive at diagnosis and confined in the mucosa and submucosa. However, to our knowledge how vaccination may induce the regression of tumors at such mucosal sites has not been examined previously. We compared different immunization routes for the ability to induce vaccine specific antitumor CD8 T cells in the bladder and bladder tumor regression in mice.

Materials And Methods: In the absence of a murine bladder tumor model expressing a tumor antigen relevant for human use we established an orthotopic model expressing the HPV-16 tumor antigen E7 as a model. We used an adjuvant E7 polypeptide to induce CD8 T cell mediated tumor regression.

Results: Subcutaneous and intravaginal but not intranasal vaccination induced a high number of TetE7(+)CD8(+) T cells in the bladder as well as bladder tumor regression. The entry of vaccine specific T cells in the bladder was not the only key since persistent regression of established bladder tumors by intravaginal or subcutaneous immunization was associated with tumor infiltration of total CD4 and CD8 T cells. This resulted in an increase in TetE7(+)CD8(+) T cells and a decrease in T regulatory cells, leading to an increased number of effector interferon-γ secreting vaccine specific CD8 T cells in the regressing bladder tumor.

Conclusions: These data show that immunization routes should be tailored to each mucosal tumor site. Subcutaneous or intravaginal vaccination may be of additional value to treat patients with bladder cancer.
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http://dx.doi.org/10.1016/j.juro.2013.08.009DOI Listing
March 2014

Hyperglycemia downregulates Connexin36 in pancreatic islets via the upregulation of ICER-1/ICER-1γ.

J Mol Endocrinol 2013 17;51(1):49-58. Epub 2013 May 17.

Service of Internal Medicine, Department of Physiology, University Hospital Lausanne, Lausanne, Switzerland.

Channels formed by the gap junction protein Connexin36 (CX36) contribute to the proper control of insulin secretion. We previously demonstrated that chronic exposure to glucose decreases Cx36 levels in insulin-secreting cells in vitro. Here, we investigated whether hyperglycemia also regulates Cx36 in vivo. Using a model of continuous glucose infusion in adult rats, we showed that prolonged (24-48 h) hyperglycemia reduced the Cx36 gene Gjd2 mRNA levels in pancreatic islets. Accordingly, prolonged exposure to high glucose concentrations also reduced the expression and function of Cx36 in the rat insulin-producing INS-1E cell line. The glucose effect was blocked after inhibition of the cAMP/PKA pathway and was associated with an overexpression of the inducible cAMP early repressor ICER-1/ICER-1γ, which binds to a functional cAMP-response element in the promoter of the Cx36 gene Gjd2. The involvement of this repressor was further demonstrated using an antisense strategy of ICER-1 inhibition, which prevented glucose-induced downregulation of Cx36. The data indicate that chronic exposure to glucose alters the in vivo expression of Cx36 by the insulin-producing β-cells through ICER-1/ICER-1γ overexpression. This mechanism may contribute to the reduced glucose sensitivity and altered insulin secretion, which contribute to the pathophysiology of diabetes.
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http://dx.doi.org/10.1530/JME-13-0054DOI Listing
January 2014

Connexins and M3 muscarinic receptors contribute to heterogeneous Ca(2+) signaling in mouse aortic endothelium.

Cell Physiol Biochem 2013 1;31(1):166-78. Epub 2013 Feb 1.

Department of Zoology and Animal Biology, Laboratory of Vascular Cell Physiology, University of Geneva, Geneva 4, Switzerland.

Background/aims: Smooth muscle tone is controlled by Ca(2+) signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40) and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca(2+) signaling of the mouse aorta.

Methods: Ca(2+) imaging was performed on intact aortic endothelium from both wild type (Cx40+/+) and Connexin40-deficient (Cx40 -/-) mice.

Results: Acetylcholine (ACh) induced early fast and high amplitude Ca(2+) transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca(2+) transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca(2+) waves, indicating that Cx40 contributes to the spreading of Ca(2+) signals. The propagation of those Ca(2+) responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca(2+) waves.

Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca(2+) signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.
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http://dx.doi.org/10.1159/000343358DOI Listing
September 2013

Reduction of connexin36 content by ICER-1 contributes to insulin-secreting cells apoptosis induced by oxidized LDL particles.

PLoS One 2013 30;8(1):e55198. Epub 2013 Jan 30.

Service of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

Connexin36 (Cx36), a trans-membrane protein that forms gap junctions between insulin-secreting beta-cells in the Langerhans islets, contributes to the proper control of insulin secretion and beta-cell survival. Hypercholesterolemia and pro-atherogenic low density lipoproteins (LDL) contribute to beta-cell dysfunction and apoptosis in the context of Type 2 diabetes. We investigated the impact of LDL-cholesterol on Cx36 levels in beta-cells. As compared to WT mice, the Cx36 content was reduced in islets from hypercholesterolemic ApoE-/- mice. Prolonged exposure to human native (nLDL) or oxidized LDL (oxLDL) particles decreased the expression of Cx36 in insulin secreting cell-lines and isolated rodent islets. Cx36 down-regulation was associated with overexpression of the inducible cAMP early repressor (ICER-1) and the selective disruption of ICER-1 prevented the effects of oxLDL on Cx36 expression. Oil red O staining and Plin1 expression levels suggested that oxLDL were less stored as neutral lipid droplets than nLDL in INS-1E cells. The lipid beta-oxidation inhibitor etomoxir enhanced oxLDL-induced apoptosis whereas the ceramide synthesis inhibitor myriocin partially protected INS-1E cells, suggesting that oxLDL toxicity was due to impaired metabolism of the lipids. ICER-1 and Cx36 expressions were closely correlated with oxLDL toxicity. Cx36 knock-down in INS-1E cells or knock-out in primary islets sensitized beta-cells to oxLDL-induced apoptosis. In contrast, overexpression of Cx36 partially protected INS-1E cells against apoptosis. These data demonstrate that the reduction of Cx36 content in beta-cells by oxLDL particles is mediated by ICER-1 and contributes to oxLDL-induced beta-cell apoptosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055198PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559396PMC
July 2013

Role of hemodynamic forces in the ex vivo arterialization of human saphenous veins.

J Vasc Surg 2013 May 23;57(5):1371-82. Epub 2013 Jan 23.

Department of Vascular Surgery, Pellegrin Hospital, University of Bordeaux, Bordeaux, France.

Background: Human saphenous vein grafts are one of the salvage bypass conduits when endovascular procedures are not feasible or fail. Understanding the remodeling process that venous grafts undergo during exposure to arterial conditions is crucial to improve their patency, which is often compromised by intimal hyperplasia. The precise role of hemodynamic forces such as shear stress and arterial pressure in this remodeling is not fully characterized. The aim of this study was to determine the involvement of arterial shear stress and pressure on vein wall remodeling and to unravel the underlying molecular mechanisms.

Methods: An ex vivo vein support system was modified for chronic (up to 1 week), pulsatile perfusion of human saphenous veins under controlled conditions that permitted the separate control of arterial shear stress and different arterial pressure (7 mm Hg or 70 mm Hg).

Results: Veins perfused for 7 days under high pressure (70 mm Hg) underwent significant development of a neointima compared with veins exposed to low pressure (7 mm Hg). These structural changes were associated with altered expression of several molecular markers. Exposure to an arterial shear stress under low pressure increased the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 at the transcript, protein, and activity levels. This increase was enhanced by high pressure, which also increased TIMP-2 protein expression despite decreased levels of the cognate transcript. In contrast, the expression of plasminogen activator inhibitor-1 increased with shear stress but was not modified by pressure. Levels of the venous marker Eph-B4 were decreased under arterial shear stress, and levels of the arterial marker Ephrin-B2 were downregulated under high-pressure conditions.

Conclusions: This model is a valuable tool to identify the role of hemodynamic forces and to decipher the molecular mechanisms leading to failure of human saphenous vein grafts. Under ex vivo conditions, arterial perfusion is sufficient to activate the remodeling of human veins, a change that is associated with the loss of specific vein markers. Elevation of pressure generates intimal hyperplasia, even though veins do not acquire arterial markers.

Clinical Relevance: The pathological remodeling of the venous wall, which leads to stenosis and ultimately graft failure, is the main limiting factor of human saphenous vein graft bypass. This remodeling is due to the hemodynamic adaptation of the vein to the arterial environment and cannot be prevented by conventional therapy. To develop a more targeted therapy, a better understanding of the molecular mechanisms involved in intimal hyperplasia is essential, which requires the development of ex vivo models of chronic perfusion of human veins.
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http://dx.doi.org/10.1016/j.jvs.2012.09.041DOI Listing
May 2013