ERK5 is required for VEGF-mediated survival and tubular morphogenesis of primary human microvascular endothelial cells

Extracellular signal-regulated kinase 5 (ERK5) is activated in response to environmental stress and growth factors. Gene ablation of Erk5 in mice is embryonically lethal as a result of disruption of cardiovascular development and vascular integrity. We investigated vascular endothelial growth factor (VEGF)-mediated ERK5 activation in primary human dermal microvascular endothelial cells (HDMECs) undergoing proliferation on a gelatin matrix, and tubular morphogenesis within a collagen gel matrix. VEGF induced sustained ERK5 activation on both matrices. However, manipulation of ERK5 activity by siRNA-mediated gene silencing disrupted tubular morphogenesis without impacting proliferation. Overexpression of constitutively active MEK5 and ERK5 stimulated tubular morphogenesis in the absence of VEGF. Analysis of intracellular signalling revealed that ERK5 regulated AKT phosphorylation. On a collagen gel, ERK5 regulated VEGF-mediated phosphorylation of the pro-apoptotic protein BAD and increased expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and apoptosis suppression. Our findings suggest that ERK5 is required for AKT phosphorylation and cell survival and is crucial for endothelial cell differentiation in response to VEGF.

Specific Increase in MDR1 Mediated Drug-Efflux in Human Brain Endothelial Cells following Co-Exposure to HIV-1 and Saquinavir

Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

Bisphenol A alters transcript levels of biomarker genes for major depressive disorder in vascular endothelial cells and colon cancer cells

Bisphenol A (BPA) is capable of mimicking endogenous hormones with potential consequences for human health and BPA exposure has been associated with several human diseases including neuropsychiatric disorders. Here, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results show that BPA at low concentrations (10 ng/mL and 1 μg/mL) induces differential transcript levels of four biomarker genes for Major Depressive Disorder (MDD) in HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). These results substantiate increasing concerns of BPA exposure in levels currently detected in humans.

Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells

The production of reactive oxygen species (ROS) within endothelial cells may have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies indicate that a phagocyte-type NAD(P)H oxidase is a major source of endothelial ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specificity (NADH.NADPH). In the present study, we (1) cloned and characterized the cDNA and predicted amino acid structures of the 2 major subunits of rat coronary microvascular endothelial cell NAD(P)H oxidase, gp91-phox and p22-phox; (2) undertook a detailed comparison with phagocytic NADPH oxidase sequences; and (3) studied the subcellular location of these subunits in endothelial cells. Although these studies revealed an overall high degree of homology (.90%) between the endothelial and phagocytic oxidase subunits, the endothelial gp91-phox sequence has potentially important differences in a putative NADPH-binding domain and in putative glycosylation sites. In addition, the subcellular location of the endothelial gp91-phox and p22-phox subunits is significantly different from that reported for the neutrophil oxidase, in that they are predominantly intracellular and collocated in the vicinity of the endoplasmic reticulum. This first detailed characterization of gp91-phox and p22-phox structure and location in endothelial cells provides new data that may account, in part, for the differences in function between the phagocytic and endothelial NAD(P)H oxidases.

High glucose mediates prooxidant and antioxidant enzyme activities in coronary endothelial cells

Objective: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O2-)are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMEC) exposed to hyperglycaemia. Methods: CMEC were cultured under normal (5.5 mmol/L) or high glucose (22 mmol/L)concentrations for 7 days. The activity and expression (protein level) of eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses,respectively while the effects of hyperglycaemia on nitrite and O2 - generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Results: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p<0.05) and increased the protein expressions and/or activities of p22-phox, a membrane-bound component of pro-oxidant NAD(P)H oxidase and antioxidant enzymes (p<0.05). Free radical-scavengers, namely, Tiron and MPG (0.1-1 mol/L) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p<0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced prooxidant enzyme activity and diminished generation of 3 antioxidant glutathione. By increasing the antioxidant enzyme capacity CMEC may protect themselves against free radical-induced cell damage in diabetic conditions. The definitive version is available at http://www.blackwell-synergy.com

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells

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The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

Withaferin A promotes proliferation and migration of brain endothelial cells

Purpose: To investigate the effect of withaferin A (WFA) on the proliferation and migration of brain endothelial cells. Methods: BALB-5023 mouse microvascular cells were treated with a range of withaferin A (WFA) concentrations from 10 to 100 ng/mL. Dojindo’s CCK-8 cell proliferation kit was used for the analysis of cell proliferation. Transwell cell culture inserts were used to determine the migration potential of WFAtreated endothelial cells. Absorbance was measured at 450 nm on an enzyme-linked immunosorbent (ELISA) reader. Results: The results revealed a significant increase in the proliferation and migration of endothelial cells following treatment with a low concentration (30 ng/mL) of WFA compared with the higher concentration (> 10 ng/mL). The effect was further enhanced when WFA was used in combination with soluble Fas ligand (sFasL). Autocrine signaling of vascular endothelial growth factor (VEGF) by endothelial cells was significantly increased following treatment with WFA or in combination with sFasL. WFA increased the expression of Fas on endothelial cells, suggesting the involvement of sFasL in the proliferation and migration of brain endothelial cells. Conclusion: Thus, WFA promotes the proliferation and migration of endothelial cells through increase in the expression of Fas and secretion of VEGF.

Transthyretin Proteins Regulate Angiogenesis by Conferring Different Molecular Identities to Endothelial Cells

Familial amyloidotic polyneuropathy (FAP) has a high prevalence in Portugal, and the most common form of hereditary amyloidosis is caused by an amyloidogenic variant of transthyretin (TTR) with a substitution of methionine for valine at position 30 (V30M). Until now, the available efficient therapy is liver transplantation, when performed in an early phase of the onset of the disease symptoms. However, transplanted FAP patients have a significantly higher incidence of early hepatic artery thrombosis compared with non-FAP transplanted patients. Because FAP was described as an independent risk factor for early hepatic artery thrombosis, more studies to understand the underlying mechanisms involved in this outcome are of the utmost importance. Knowing that the liver is the major site for TTR production, we investigated the biological effects of TTR proteins in the vasculature and on angiogenesis. In this study, we identified genes differentially expressed in endothelial cells exposed to the WT or V30M tetramer. We found that endothelial cells may acquire different molecular identities when exposed to these proteins, and consequently TTR could regulate angiogenesis. Moreover, we show that V30M decreases endothelial survival by inducing apoptosis, and it inhibits migration. These findings provide new knowledge that may have critical implications in the prevention of early hepatic artery thrombosis in FAP patients after liver transplantation.

Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis

Introduction: Chronic insufficiency alters homeostasis, in part due to endothelial inflammation. Plasminogen activator inhibitor-1 (PAI-1) is increased in renal disease, contributing to vascular damage. We assessed PAI-1 activity and PAI-1 4G/5G polymorphism in hemodialysis (HD) subjects and any association between thrombotic vascular access (VA) events and PAI-1 polymorphism. Methods: Prospective, observational study in 36 HD patients: mean age: 66.6 +/- 12.5 yr, males n=26 (72%), time on HD: 28.71 +/- 22.45 months. Vascular accesses: 10 polytetrafluoroethylene grafts (PTFEG), 22 arteriovenous fistulae (AVF), four dual lumen catheters (CAT). Control group (CG): 40 subjects; mean age: 60.0 +/- 15 yrs, males n=30 (75%). Group A (GA): thrombotic events (n=12), and group B (GB): No events (n=24). Groups were no different according to age (69.2 +/- 9.12 vs. 65.3 +/- 14.5 yrs), gender (males: 7; 58.3% vs. 18; 81.8%), time on HD (26.1 +/- 14.7 vs. 30.1 +/- 38.7 months), causes of renal failure. Time to follow-up, for access thrombosis: 12 months. Results: PAI-1 levels in HD: 7.21 +/- 2.13 vs. CG: 0.42 +/- 0.27 U/ml (p < 0.000 1). PAI-1 4G/5G polymorphic variant distribution in HD: 5G/5G: 6 (17%),4G/5G: 23 (64%); 4G/4G: 7 (19%) and in CG: 5G/5G: 14 (35%); 4G/5G: 18 (45%); 4G/4G: 8 (20%). C-reactive protein (CRP) in HD: 24.5 +/- 15.2 mg/L vs. in CG 2.3 +/- 0.2 mg/L (p < 0.0001). PAI-1 4G/5G variants: GA: 5G/5G: 3; 4G/5G: 8; 4G/4G: 1; GB: 5G/5G: 3; 4G/5G: 15; 4G/4G: 6. Thrombosis occurred in 8/10 patients (80%) with PTFEG, 3/22 (9%) in AVF, and 1/4 (25%) in CAT. Among the eight PTFEG patients with thrombosis, seven were PAI 4G/5G. Conclusions: PAI-1 levels were elevated in HD patients, independent of their polymorphic variants, 4G/5G being the most prevalent variant. Our data suggest that in patients with PTFEG the 4G/5G variant might be associated with an increased thrombosis risk.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.