Levels Of Endothelial Cells Research Articles

Deep transcriptomic profiling reveals the similarity between endothelial cells cultured under static and oscillatory shear stress conditions.

Atherosclerosis is a multifactorial disease that preferentially develops in specific regions in the arterial tree. This characteristic is mainly attributed to the unique pattern of hemodynamic shear stress in vivo. High laminar shear stress (LS) found in straight lumen exerts athero-protective effects. Low or oscillatory shear stress (OS) present in regions of lesser curvature and arterial bifurcations predisposes arterial intima to atherosclerosis. Shear stress-regulated endothelial function plays an important role in the process of atherosclerosis. Most in vitro research studies focusing on the molecular mechanisms of endothelial function are performed in endothelial cells (ECs) under cultured static (ST) condition. Some findings, however, are not recapitulated in subsequent translational studies, mostly likely due to the missing biomechanical milieu. Here, we profiled the whole transcriptome of primary human coronary arterial endothelial cells (HCAECs) under different shear stress conditions with RNA sequencing. Among 16,313 well-expressed genes, we detected 8,177 that were differentially expressed in OS vs. LS conditions and 9,369 in ST vs. LS conditions. Notably, only 1,618 were differentially expressed in OS vs. ST conditions. Hierarchical clustering of ECs demonstrated a strong similarity between ECs under OS and ST conditions at the transcriptome level. Subsequent pairwise heat mapping and principal component analysis gave further weight to the similarity. At the individual gene level, expressional analysis of representative well-known genes as well as novel genes showed a comparable amount at mRNA and protein levels in ECs under ST and OS conditions. In conclusion, the present work compared the whole transcriptome of HCAECs under different shear stress conditions at the transcriptome level as well as at the individual gene level. We found that cultured ECs are significantly different from those under LS conditions. Thus using cells under ST conditions is unlikely to elucidate endothelial physiology. Given the revealed high similarities of the endothelial transcriptome under OS and ST conditions, it may be helpful to understand the underlying mechanisms of OS-induced endothelial dysfunction from static cultured endothelial studies.

40 Sepiapterin, a potential therapeutic approach to restore the metabolic phenotype switch induced by sFlt-1 in preeclampsia

Introduction Preeclampsia (PE) is an often fatal cardiovascular complication related to pregnancy with no current effective way of treatment. Evidence of early increased circulating anti-angiogenic factors (AAFs) support the onset of a multi-systemic disorder and widespread maternal endothelial dysfunction in PE. However, the molecular mechanisms are still not well understood. Objectives To determine the potential early metabolic perturbations and mitochondrial bioenergetics effects of sFlt-1 in bovine aortic endothelial cells (ECs) and first trimester extravillous trophoblasts (HTR-8/SVneo). Also, to evaluate the potential use of sepiapterin (SE), a precursor of eNOS’s cofactor tetrahydrobiopterin (BH4), in abrogating the effects of sFlt-1 in cells. Methods Metabolic perturbations and mitochondrial bioenergetics were assessed in ECs and HTR-8/SVneo in an in-vitro model of preeclampsia using exogenous sFlt-1 and serum from preeclamptic women. Mitochondrial bioenergetics was assessed using an XFe24 Extracellular Flux Analyzer. Nitric oxide (NO) was determined by chemiluminescence. Mitochondrial function and metabolism in sFlt-1-treated cells was evaluated also in galactose media. Mitochondrial membrane potential and superoxide was evaluated by JC-1 and Mito-Sox, respectively, by flow cytometry. Results We found that treatment with sFlt-1 affected the mitochondrial maximal respiration and spare respiratory capacity in ECs in a dose dependent manner leading to a metabolic phenotype switch to glycolysis. In contrast, HTR-8/SVneo, displayed an unexpected strong glycolytic metabolism. sFlt-1 was found not to disturb the trophoblast mitochondrial metabolic and bioenergetics profile even at relatively high doses. In addition, we found that sFlt-1 treatment caused concentration dependent decreases in mitochondrial membrane potential and diminished NO levels in ECs. Moreover, treatment of ECs with sFlt-1 in galactose strongly impaired cell viability suggesting the role of sFlt-1 as a mitochondrial disruptor. SE, protected ECs and HTR8/SVneo cells from sFlt-1-induced superoxide formation and restored the NO levels and metabolic phenotype switch induced by sFlt-1 and maternal PE serum in ECs. Conclusions sFlt-1 disrupts mitochondria bioenergetics and metabolism in both, ECs and HTR-8/SVneo. This evidence could explain the potential detrimental effects of AAFs in the maternal endothelium and the hallmark of hypertension in PE. Hence, we demonstrate that SE, by enhancing BH4/NO bioavailability and diminish mitochondrial superoxide formation, restored the metabolic phenotype switch induced by sFlt-1 and PE serum. Based on this evidence, we postulate the use of SE as a potential therapeutic approach to prevent or treat PE.

Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells.

Thrombomodulin is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, antifibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of thrombomodulin expression in ECs remains largely unknown. In this study, we characterized nuclear receptor 4A family as a novel regulator of thrombomodulin expression in vascular ECs. We demonstrated that both nuclear receptors 4A, Nur77 and Nor1, robustly increase thrombomodulin mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and vascular endothelial growth factor165-stimulated thrombomodulin expression. Mechanistically, we found that Nur77 and Nor1 increase thrombomodulin expression by acting through 2 different mechanisms. We showed that Nur77 barely affects thrombomodulin promoter activity, but significantly increases thrombomodulin mRNA stability, whereas Nor1 enhances thrombomodulin expression mainly through induction of Kruppel-like factors 2 and 4 in vascular ECs. Furthermore, we demonstrated that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor α-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, whereas enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. Our results identified nuclear receptors 4A as novel regulators of thrombomodulin expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders.

Sphingosine-1-phosphate receptor subtype 2 signaling in endothelial senescence-associated functional impairments and inflammation.

Endothelial inflammation is an important risk factor in the initiation and development of vascular disease. Therefore, signaling cascades and patho-physiological outcomes of endothelial inflammation are important questions in vascular biology. Recent studies suggest that sphingosine-1-phosphate receptor subtype 2 (S1PR2) signaling in endothelial cells (ECs) play a critical role in endothelial inflammation. For example, ECs present in atherosclerotic plaques exhibit senescence phenotype. Levels of S1PR2 are markedly increased in cultured senescent ECs and in lesion regions of atherosclerotic endothelium. Also, inflammatory cytokines and mechanical flow stress profoundly increase S1PR2 levels in ECs. Inhibition of endothelial S1PR2 signaling diminishes endothelial senescence-associated functional impairments and atherogenic stimuli-induced endothelial activation. In contrast, activation of endothelial S1PR2 stimulates the production of pro-inflammatory chemokines/cytokines and lipid mediators in ECs. In this article, we will review signaling and functions of sphingosine-1-phosphate (S1P) receptors in endothelial biology, with particular focus on endothelial S1PR2 signaling-mediated endothelial inflammation.

Transient receptor potential vanilloid type 1 is vital for (-)-epigallocatechin-3-gallate mediated activation of endothelial nitric oxide synthase.

Epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, has beneficial effects on physiological functions of endothelial cells (ECs), yet the detailed mechanisms are not fully understood. In this study, we investigated the role of transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated nonselective calcium channel, in EGCG-mediated endothelial nitric oxide (NO) synthase (eNOS) activation and angiogenesis. In ECs, treatment with EGCG time-dependently increased the intracellular level of Ca(2+) . Removal of extracellular calcium (Ca(2+) ) by EGTA or EDTA or inhibition of TRPV1 by capsazepine or SB366791 abrogated EGCG-increased intracellular Ca(2+) level in ECs or TRPV1-transfected HEK293 cells. Additionally, EGCG increased the phsophorylation of eNOS at Ser635 and Ser1179, Akt at Ser473, calmodulin-dependent protein kinase II (CaMKII) at Thr286 and AMP-activated protein kinase (AMPK) at Thr172, all abolished by the TRPV1 antagonist capsazepine. EGCG-induced NO production was diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Moreover, blocking TRPV1 activation prevented EGCG-induced EC proliferation, migration, and tube formation, as well as angiogenesis in Matrigel plugs in mice. EGCG may trigger activation of TRPV1-Ca(2+) signaling, which leads to phosphorylation of Akt, AMPK, and CaMKII; eNOS activation; NO production; and, ultimately, angiogenesis in ECs.

Abstract 18933: Regulation of Impaired Angiogenesis in Diabetes by Microrna26a

Wound healing is a physiological reparative response to injury that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix deposition, wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. In recent years, microRNAs (miRNAs) has been implicated in a wide-range of disease states. We have investigated the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to diabetic stimuli (e.g. high glucose, PKC activation) in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~2-fold) four days post wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to 80%), increased granulation tissue thickness (by 2.5 fold) and accelerated wound closure (by 50% after nine days) compared to scrambled anti-miR controls using a db/db mouse model of diabetes. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene, SMAD1, both at the mRNA and protein levels in ECs. In addition, increased SMAD1 levels correlated with increased ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27 nine days post-wounding of diabetic mice. Interestingly, inhibition of miR-26a increased migration and growth of both human microvascular ECs (HMVECs), human umbilical endothelial cells (HUVECs), and fibroblasts under normal glucose conditions. However, in the presence of high glucose, anti-miR-26a functional effects were abolished in fibroblasts, but not in HUVECs or HMVECs, suggesting cell-specific miR-26a regulated effects under diabetic conditions. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy.

Biomimetic modified clinical-grade POSS-PCU nanocomposite polymer for bypass graft applications: a preliminary assessment of endothelial cell adhesion and haemocompatibility.

To date, there are no small internal diameter (<5mm) vascular grafts that are FDA approved for clinical use due to high failure rates from thrombosis and unwanted cell proliferation. The ideal conditions to enhance bioengineered grafts would be the blood contacting lumen of the bypass graft fully covered by endothelial cells (ECs). As a strategy towards this aim, we hypothesized that by immobilising biomolecules on the surface of the polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) nanocomposite polymers, which contain binding sites and ligands for cell surface receptors similar to extracellular matrix (ECM) will positively influence the attachment and proliferation of ECs. Since, the surface of POSS-PCU is inert and not directly suitable for immobilisation of biomolecules, plasma graft polymerisation is a suitable method to modify the surface properties ready for immobilisation and biofunctionalisation. POSS-PCU was activated by plasma treatment in air/O2 to from hydroperoxides (-OH, -OOH), and then carboxylated via plasma polymerisation of a 30% acrylic acid solution (Poly-AA) using a two-step plasma treatment (TSPT) process. Collagen type I, a major component of ECM, was covalently immobilised to mimic the ECM structures to ECs (5mg/ml) using a two-step chemical reaction using EDC chemistry. Successful immobilisation of poly-AA and collagen on to the nanocomposites was confirmed using Toluidine Blue staining and the Bradford assay. Un-treated POSS-PCU served as a simple control. The impact of collagen grafting on the physical, mechanical and biological properties of POSS-PCU was evaluated via contact angle (θ) measurements, scanning electron microscopy (SEM), atomic force microscopy (AFM), dynamic mechanical thermal analysis (DMTA), ECs adhesion and proliferation followed by platelet adhesion and haemolysis ratio (HR) tests. Poly-AA content on each of the plasma treated nanocomposite films increased on Low, Med and High samples due to more carboxylic acid (-COOH) groups at the surface forming amide (-NH2) bonds. The amount of -COOH groups on each of the Low, Med and High nanocomposites correlated with Poly-AA grafting density at 14.7±0.9, 18.9±0.9, and 34.2±2.4 μg/cm(2). Immobilisation of collagen type I on to nanocomposite surface was also found to increase significantly on the Low, Med and High samples from 22±4, 150±15, and 219±17 μg/cm(2), respectively. The level of ECs and their adhesion efficiency were improved with increasing amounts of grafted collagen I. The maximum adhesion of ECs was found on the highest collagen type I coated nanocomposites. Platelet adhesion and activation also increased with increasing collagen density. The obtained HR values for all of the treated samples were well within the acceptable standards for biomaterials (<5% HR). Poly-AA-g-POSS-PCU surfaces offer binding sites for the covalent bonding of collagen type I and other biomolecules such as fibronectin by exposure of RGD cell binding domains and growth factors using EDC cross-linking chemistry. Collagen type I modification can yield accelerated EC growth and enhance the endothelialisation of POSS-PCU nanocomposites, and the amount of immobilised collagen can control the level of platelet adhesion on functionalized POSS-PCU via TSPT and poly acrylic acid (poly-AA) treatment. Such surface modification procedures of polymeric surfaces can improve the patency rate of POSS-PCU nanocomposites as vascular bypass grafts in the preparation of a range of medical devices ready for pre-clinical and in vivo evaluation.

Coiled-coil domain containing 3 (CCDC3) represses tumor necrosis factor-α/nuclear factor κB-induced endothelial inflammation

Coiled-coil domain containing 3 (CCDC3) is a newly identified secretory protein that is expressed in vascular endothelial cells (ECs) and adipose tissues. Here, we investigate the role of CCDC3 in tumor necrosis factor (TNF)-α-induced inflammatory response in ECs. Our results show that stable overexpression of CCDC3 decreases, while stable knockdown of the endogenous CCDC3 increases TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) at the mRNA and protein level in ECs. The IκB kinase inhibitor Bay 11–7082 completely blocks TNF-α-induced expression of VCAM-1, confirming that TNF-α-induced expression of VCAM-1 in ECs is nuclear factor κB (NF-κB) dependent. Stable overexpression of CCDC3 decreases TNF-α-induced p65 and p50 nuclear translocation and NF-κB transcriptional activity, suggesting that CCDC3 inhibits TNF-α-induced NF-κB activation in ECs. Similar to CCDC3 overexpression, both CCDC3-containing conditioned medium (CM) and purified CCDC3 decrease TNF-α-induced expression of VCAM-1 in receiving ECs, suggesting a paracrine/autocrine function of CCDC3. Interestingly, CCDC3 in CM can enter the receiving ECs. Taken together, our work demonstrates that CCDC3 represses TNF-α/NF-κB-induced pro-inflammatory response in ECs, providing an insight into the functional role of CCDC3.

Paris Saponin II suppresses the growth of human ovarian cancer xenografts via modulating VEGF-mediated angiogenesis and tumor cell migration

Paris Saponin II (PSII) is an active component of Rhizoma Paridis-an essential ingredient in traditional Chinese herbal medicines. PSII can induce cytotoxic effects in cancer cells and inhibit ovarian cancer growth. Since pathological angiogenesis (henceforth, angiogenesis) is often associated with gynecological cancers, here, we investigated whether PSII renders effects on angiogenesis and examined possible molecular mechanisms underlying the effects of PSII. The effects of PSII on the biofunctions of endothelial cells (EC), the crucial components of blood vessels, were examined by standardized angiogenesis in vitro and ex vivo assays, Western blot analysis, ELISA, and kinase assay. Angiogenesis in a xenograft mouse model of ovarian cancer was evaluated by color Doppler ultrasound and immunohistochemistry. PSII exerted marked inhibitory effect on the growth of VEGF-stimulated human umbilical vein endothelial cells in a dose-time-dependent manner, inhibited cell's motility, and interfered with tubulogenesis. PSII also blocked microvessel outgrowth in a rat aortic ring assay and compromised angiogenesis in a mouse model of ovarian carcinoma using either SKOV3 or HOC-7 cell lines. VEGF levels in PSII-treated EC and tumor cells were reduced. In EC, PSII blocked the activation of VEGFR2 in dose-dependent manner leading to the reduction of VEGF-induced phosphorylation on several intracellular pro-angiogenic kinase, including the extracellular signal-related kinase, Src family kinase, focal adhesion kinase, and AKT kinase. The results provided the first insight into the anti-angiogenesis properties of Saponin family in solid tumors and suggested a promising therapeutic potential of PSII in the ovarian cancer treatment.

MiRNA-155 targets myosin light chain kinase and modulates actin cytoskeleton organization in endothelial cells

Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3'UTR was verified by luciferase-MYLK 3'UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.

Abstract 9623: Stimulatory Effects of Cardiotrophin-1 on Atherosclerosis

Cardiotrophin-1 (CT-1), a cytokine belonging to the interleukin-6 superfamily, was recently shown to be expressed in the intima of early atherosclerotic lesions in the human carotid artery. CT-1 stimulates pro-atherogenic molecule expression in human vascular endothelial cells (EC) and monocyte migration. However, it has not been reported whether CT-1 accelerates atherosclerosis. This study was performed to examine the stimulatory effects of CT-1 on human macrophage foam cell formation, human EC proliferation, and the migration and proliferation of human vascular smooth muscle cells (VSMCs) in vitro, and on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice in vivo. CT-1 was expressed at high levels in ECs and macrophages in both humans and ApoE-/- mice. CT-1 significantly enhanced oxidized low-density lipoprotein-induced foam cell formation associated with increased levels of CD36 and acyl-CoA:cholesterol acyltransferase-1 (ACAT1) expression in human monocyte-derived macrophages. CT-1 also significantly stimulated the migration and proliferation of human aortic VSMCs, but not EC proliferation. Four-week-infusion of CT-1 into ApoE-/- mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration and VSMC content in the aortic wall. Activation of inflammasome, such as apoptosis-associated speck-like protein containing a caspase recruitment domain, nuclear factor-κB, and cyclooxygenase-2, was observed in exudate peritoneal macrophages from ApoE-/- mice infused with CT-1. Infusion of anti-CT-1 neutralizing antibody alone into ApoE-/- mice significantly suppressed monocyte/macrophage infiltration in atherosclerotic lesions. These results indicate that CT-1 accelerates the development of atherosclerotic lesions by stimulating the inflammasome and foam cell formation associated with CD36 and ACAT1 up-regulation in macrophages and VSMC migration and proliferation, contributing to the occurrence of atherosclerotic vascular diseases. Blockade of cardiotrophin-1 is expected to emerge as a new line of therapy against atherosclerosis and its related diseases.

Abstract 117: MicroRNA-138 regulates S100A1 in Endothelial Cells

Rationale: The EF-hand Ca2+ sensor S100A1 is essential for proper endothelial nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in endothelial cells (ECs) subjected to hypoxia, rendering them dysfunctional. Objective: To determine if the 3’UTR mediates the rapid hypoxia-induced downregulation of S100A1 in ECs. Methods and Results: ECs transfected with a S100A1 - 3’ untranslated region (UTR) luciferase reporter construct displayed significantly reduced gene expression when subjected to gas or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (miR-138) could target the 3’UTR of S100A1. Hypoxia greatly increased miR-138 levels in ECs, but not in skeletal muscle C2C12 myotubes. Consistent with this finding, patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased miR-138 levels. Transfection of a miR-138 mimic into ECs reduced S100A1 - 3 ‘UTR reporter gene expression, while transfection of an anti miR-138 (antagomir) prevented the hypoxia-induced downregulation of the reporter gene. The increased levels of miR-138 are dependent on Hif1-α activation as treatment with siRNA against Hif1-α prevented S100A1 reporter gene downregulation after hypoxia. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression. Finally, ECs transfected with miR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. Conclusions: Our study shows that miR-138 is an essential mediator of EC dysfunction via its ability to target the 3’UTR of S100A1 in a hypoxia-induced manner. MiR-138 might thus be an attractive target for the treatment of pathologies that are linked to endothelial dysfunction.

Expression levels of purinergic receptors in the endothelium of human and murine cornea

AbstractPurpose Since loss of corneal endothelial cells (EC) due to cell death is the major problem in numerous corneal conditions as well as in tissue storage for corneal transplantation, finding ways to improve cell survival is of great importance. Purinergic receptors P2RY [G protein‐coupled receptors] and P2RX [ligand‐gated ion channels] are both known to be implicated in inflammation and apoptosis pathways, therefore modulation of these receptors may provide an efficient way to increase EC survival. The aim of this study was to study expression levels of P2RY2 and P2RX7 receptors (P2R) in murine and human corneal endothelium, as well as the association to the purinergic receptor ligand ATP.Methods Expression levels of P2R were determined by qRealTime‐PCR relying on untreated and Interleukin‐1b‐treated murine and human EC. EC apoptosis was also studied in response to ATP in a “cornea‐in‐a‐cup” assay.Results In murine EC P2RX7 were significantly predominant compared to P2RY1 and P2RY2, while in human EC P2RY2 were significantly prevailing compared to P2RX7. Exposure to ATP in increasing concentrations resulted in a steady increase of apoptosis. As indicators of EC stress, changes in cell size (polymegathism) and cell shape (loss of EC hexagonality, polymorphism) could be demonstrated.Conclusion Our data describe distinct differences in P2R expression levels in murine versus human EC showing the limitations of using the mouse model in P2R EC research. As P2R seem to be evenly distributed over the entire concave surface of the EC monolayer, inhibiting P2R interaction with their ligands (e.g. extracellular ATP) may present an effective way to prevent EC apoptosis.

173 INTERLEUKIN-1 ALPHA ACTIVITY IN NECROTIC ENDOTHELIAL CELLS IS DYNAMICALLY CONTROLLED BY INTRACELLULAR INTERLEUKIN-1 RECEPTOR 2

Inflammation is a key driver of both atherosclerosis and graft rejection. Interleukin-1 alpha (IL-1α), a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from damaged endothelial cells (ECs) during vessel transplant drives allogeneic graft rejection. Therefore it is important to understand how IL-1α activity is controlled after EC necrosis, and how this affects atherosclerosis and graft rejection. We investigated IL-1α activity in control and TNFα or IL-1β stimulated ECs. Necrotic ECs have much lower IL-1α activity than VSMCs, but show comparable levels to other cell-types, even though they contain a tenth of the IL-1α. Following TNFα or IL-1β stimulation IL-1α activity in necrotic ECs is increased up to 8-fold without alteration to IL-1α protein level. Together, these data imply that IL-1α activity is controlled independently of protein level in necrotic ECs. Immunofluorescence and proximity ligation assays show a cytosolic association between IL-1α and IL-1R2, which is known to inhibit IL-1αactivity. Following TNFα or IL-1β stimulation necrotic EC lysates contain more calpain cleaved IL-1α, which shows increased activity. In addition, IL-1R2 in stimulated lysates is less able to protect exogenously added pro-IL-1α,suggesting TNFα or IL-1β dissociates IL-1α from IL-1R2, subsequently allowing calpain to cleave and increase IL-1α activity upon necrosis. We conclude that necrotic EC-derived IL-1α is regulated by binding to cytosolic IL-1R2, and that TNFα and IL-1 modulate this protective mechanism to license IL-1α after necrosis. These and previous data suggest that necrotic ECs may play an important role in vessel wall inflammation during graft rejection, and may also drive atherosclerosis.

Different Redox Response Elicited by Naturally Occurring Antioxidants in Human Endothelial Cells

Evidences that higher natural antioxidant (NA) intake provides protection against cardiovascular disease (CVD) are contradictory. Oxidative-induced endothelial cells (ECs) injury is the key step in the onset and progression of CVD and for this reason the cellular responses resulting from NA interaction with ECs are actively investigated. This study was designed to investigate the direct impact of different naturally occurring antioxidants on the intracellular ROS levels in cultured human ECs. NA-induced redox changes, in terms of modulation of the intracellular ROS levels, were assessed by using the ROS fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). While caffeic and caftaric acid exerted an anti-oxidant effect, both coumaric acid and resveratrol were pro-oxidant. Anti- and pro-oxidant effects of the tested compounds were concentration dependent, showing the induction or the tendency to promote a pro-oxidant outcome with increasing concentrations. Interestingly, the anti- and pro-oxidant behavior of chlorogenic and ferulic acid was dependent on the basal intracellular redox state. Our data indicate that naturally occurring antioxidants are able to induce a rapid modification of the intracellular ROS levels in human ECs, which is dependent on both the applied concentration and the intracellular redox state.

Role of VDAC in coronary endothelial cell dysfunction in type 1 diabetic mice

Endothelial dysfunction is a common feature of diabetic vascular complications. Rarefaction of capillaries in the heart, as a result of coronary endothelial dysfunction, represents one of the most critical mechanisms involved in cardiac ischemia. The voltage‐dependent anion channel (VDAC) plays an important role in the regulation of mitochondrial metabolic function and mitochondria‐mediated apoptosis. The objective of this study is to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in the left ventricle of diabetic mice and hyperglycemia induced significant increase in mitochondrial Ca2+ concentration and VDAC protein levels in coronary ECs. The expression of VDAC‐shRNA in diabetic MCECs not only decreased the mitochondrial Ca2+ concentration, but also attenuated mitochondrial superoxide anion (O2−) production and the activity of the mitochondrial permeability transition pore opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC protein expression in diabetic MCECs leads to the activation of pro‐apoptotic cascade and normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.This research was supported by grant HL115578.

CASPASE‐1 ACTIVATION IS CRITICAL FOR ENDOTHELIAL ACTIVATION AND EARLY ATHEROGENESIS

We hypothesized that caspase‐1(casp1)/inflammasome in endothelial cells (ECs) as the innermost cells exposed to circulation can sense hyperlipidemia as a danger signal in early atherosclerosis and initiate EC activation. Our data demonstrated that: 1) early hyperlipidemia induced casp‐1 expression and activation in mouse aorta in apolipoprotein E (ApoE)−/− mice with high fat (HF) diet; 2) proatherogenic oxidized low density lipoprotein(ox‐LDL), two oxLDL active components, such as lysophosphatidylcholine and lysophosphatidic acid, induced casp‐1 activation in human aortic ECs (HAECs); 3) deficiency of casp‐1 attenuated oxLDL‐induced EC activation by inhibiting the up‐regulation of EC adhesion molecules including ICAM‐1, VCAM‐1 and E‐selectin in both mRNA and protein levels in primary mouse aortic ECs; 4) casp‐1 inhibitors attenuated oxLDL‐induced HAEC activation and monocyte adhesion to HAECs. We then generated the ApoE−/−/Casp‐1−/− double knockout mice, and found that the double knockout mice contained significantly less atherosclerotic lesion in mouse aortic arch and less CD11b+/F4/80‐monocyte and CD11b+/F4/80+ monocyte recruitments into mouse aorta. Collectively, our data have demonstrated for the first time that caspase‐1/inflammasome in ECs can sense early hyperlipidemia and become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis.

Distinct potentials in the myoendothelial junction

Myoendothelial junctions (MEJ) bridge the smooth muscle cells (SMCs) and endothelial cells (ECs) in small arteries. In most vessels they project from the EC through the internal elastic lamina and into the SMC, where gap junctions connect the MEJ with the SMC. MEJs are characterised by a long thin neck and on top a “head”, which can be coupled to SMCs with gap junctions. We have used a spatiotemporal model to simulate electrical coupling between the two cell types. The model shows that the MEJ could be electrically isolated from both the EC and the SMC due to the long thin neck and that there could be levels of cations and anions in the MEJ that are different from the EC and SMC bulk levels. Local Ca2+ domains in the MEJ can activate an outward K+ current in the MEJ and we found that activation of less than 10–20 K+ channels (e.g. IK channels) is sufficient to create an electrical potential of 1–5 mV between the SMC and MEJ. This suggests that the MEJ should be considered as a unique structure with properties distinct from those of the smooth muscle cells or of the endothelial cells proper. Supported by The Danish Council for Independent Research ‐ Medical Sciences.

Role of impaired endothelial cell Ca2+signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2

ObjectiveNotch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether...