Faculty Opinions recommendation of Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling.

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O2.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2.−, pulmonary microvascular endothelial cells were preloaded with the O2.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2.−resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O2.−triggered Ca2+release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2.−production and cellular apoptosis. These “signaling” effects of O2.−were prevented by DIDS treatment, by depletion of intracellular Ca2+stores with thapsigargin and by chelation of intracellular Ca2+. This study demonstrates that O2.−flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2+release, which activates mitochondrial O2.−generation.

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Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.275.2.l203 ◽

1998 ◽

Vol 275 (2) ◽

pp. L203-L222 ◽

Cited By ~ 52

Author(s):

Timothy M. Moore ◽

Paul M. Chetham ◽

John J. Kelly ◽

Troy Stevens

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Intracellular Signaling ◽

Endothelial Permeability ◽

Cell Permeability ◽

Gap Formation ◽

Pulmonary Endothelium ◽

Intracellular Signals ◽

Endothelial Cell Permeability ◽

Cell Cell

Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]ipromotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]idecreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]iand cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]ipromotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]iand cAMP in mediating site-specific alterations in endothelial permeability.

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Orchestration of Primary Hemostasis by Platelet and Endothelial Lysosome-Related Organelles

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314245 ◽

2020 ◽

Vol 40 (6) ◽

pp. 1441-1453 ◽

Cited By ~ 4

Author(s):

Ellie Karampini ◽

Ruben Bierings ◽

Jan Voorberg

Keyword(s):

Endothelial Cells ◽

Intracellular Signaling ◽

Protein Complexes ◽

Biological Properties ◽

Bleeding Disorders ◽

Granule Formation ◽

Primary Hemostasis ◽

Granule Maturation ◽

Lysosome Related Organelles ◽

Von Willebrand

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.

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Abstract TP275: “Touch-and-Go”: Touching of Endothelial Cells by Endothelial Progenitor Cells Increases Activation of Pro-survival ERK1/2 Signaling

Stroke ◽

10.1161/str.48.suppl_1.tp275 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Emiri T Mandeville ◽

Su Jing Chen ◽

Kazuhide Hayakawa ◽

Ken Arai ◽

Eng H Lo

Keyword(s):

Endothelial Cells ◽

Rat Brain ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Short Duration ◽

Intracellular Signaling ◽

Infarct Volume ◽

Dependent Manner ◽

Brain Endothelial Cells ◽

Endothelial Progenitor

Background: Cell-based therapies can potentially promote neurological repair for CNS diseases including stroke. Pre-clinical data showed improved infarct volume and neurological scores following injection of Endothelial progenitor cells (EPCs). However relatively few EPCs were found in infarct areas, and mechanisms by which injected EPCs enhance neovascularization are largely unknown. In this study, we hypothesized that circulating EPCs would positively impact intracellular signaling cascades in rat brain endothelial cells (RBECs) even by short-duration contact due to activation of pro-survival ERK1/2 cascades. Methods: Primary RBECs and EPCs were isolated from rat brain and spleen, respectively. These cells were cultured separately, and 10 days later, cultured EPCs were transferred to plates of cultured RBECs. After 1 or 10 min incubation with cell-culture plate shaking, EPCs were washed from the plates and RBECs were subjected to western blot analysis to assess ERK1/2 phosphorylation. As a negative control for EPCs, we prepared neutrophils from different rats. Results: We confirmed that our RBECs and EPCs were viable in vitro by LDH assay and these cells were positive for their cell-type markers assessed by immunostaining. Ten min incubation of EPCs phosphorylated ERK1/2 in RBECs in an EPC-number-dependent manner, whereas identical conditions of neurtrophil incubation did not. Importantly, only 1 min incubation with EPCs significantly upregulated ERK cascades in RBECs. Remaining EPCs on RBEC surfaces may not contribute to ERK1/2 phosphorylation because very few EPCs were observed after washout. In addition, experiments by the same procedure without RBECs did not show ERK phosphorylation. Conclusion: We demonstrated increased activation of pro-survival ERK1/2 signaling in RBECs following short-duration incubation of EPCs. Results suggest that circulating EPCs may not need to be integrated into existing blood vessels to promote neovascularization. Rather, short-duration interactions between EPCs and RBECs may provide a “Touch-and-Go” stimulus that supports brain endothelial cells to make favorable environments for neovascularization.

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Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis

Cells ◽

10.3390/cells9010038 ◽

2019 ◽

Vol 9 (1) ◽

pp. 38 ◽

Cited By ~ 6

Author(s):

Nicolas Ricard ◽

Jiasheng Zhang ◽

Zhen W. Zhuang ◽

Michael Simons

Keyword(s):

Endothelial Cells ◽

Intracellular Signaling ◽

Clinical Importance ◽

Macrophage Infiltration ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Ischemic Event ◽

Intracellular Signaling Pathway ◽

First Time

Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of each ERK isoform in arteriogenesis, we used mice with a global Erk1 knockout as well as Erk1 and Erk2 floxed mice to delete Erk1 or Erk2 in endothelial cells, macrophages, and smooth muscle cells. We found that ERK1 controls macrophage infiltration following an ischemic event. Loss of ERK1 in endothelial cells and macrophages induced an excessive macrophage infiltration leading to an increased but poorly functional arteriogenesis. Loss of ERK2 in endothelial cells leads to a decreased arteriogenesis due to decreased endothelial cell proliferation and a reduced eNOS expression. These findings show for the first time that isoform-specific roles of ERK1 and ERK2 in the control of arteriogenesis.

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Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-02-0080 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3553-3564 ◽

Cited By ~ 48

Author(s):

Naoko Kogata ◽

Michitaka Masuda ◽

Yuji Kamioka ◽

Akiko Yamagishi ◽

Akira Endo ◽

...

Keyword(s):

Endothelial Cells ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Adhesion Molecule ◽

Intracellular Signaling ◽

Vascular Endothelial Cells ◽

Expression Cloning ◽

Vascular Endothelial ◽

Cell Contacts ◽

Cell Cell

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

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A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2008.04.090 ◽

2008 ◽

Vol 371 (3) ◽

pp. 437-440 ◽

Cited By ~ 9

Author(s):

Zhi-yong Ma ◽

Wei Zhang ◽

Liang Chen ◽

Rong Wang ◽

Xiao-hong Kan ◽

...

Keyword(s):

Endothelial Cells ◽

Chloride Channel ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

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Endothelial Cells Induce Multiple Myeloma Cell Proliferation Protect Against Conventional and Novel Therapies.

Blood ◽

10.1182/blood.v104.11.2354.2354 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2354-2354

Author(s):

Shaji Kumar ◽

Noopur Raje ◽

Teru Hideshima ◽

Klaus Podar ◽

Kenji Ishitsuka ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Endothelial Cells ◽

Tumor Cell ◽

Blood Vessels ◽

Intracellular Signaling ◽

De Novo ◽

Cell Contact ◽

Protective Effects ◽

Tumor Cell Proliferation

Abstract Angiogenesis or formation of new blood vessels from existing blood vessels, in contrast to vasculogenesis or de novo formation of new vessels, plays an important role in the progression and spread of most cancers. Multiple myeloma (MM) is characterized by increased microvessel density (MVD), a quantitative estimate of angiogenesis, which correlates with stage of disease. MVD increases with progression from MGUS to smoldering MM to newly diagnosed MM and relapsed MM. It is a powerful prognostic factor, predicting for overall survival. To further elucidate the biological basis for the prognostic value of increased angiogenesis in MM, we studied the interactions of MM cells with endothelial cells using HUVECS as a model system. Co-culture of MM cells (MM1.S, OPM2, U266) with HUVECS induced tumor cell proliferation. Enhanced tumor cell proliferation correlated with the number of HUVECs and was greater than that triggered by co-culture with patient bone marrow stromal cells. When HUVECs were fixed prior to co-culture there was a significant decrease in the tumor cell proliferation. Addition of HUVEC conditioned media to the MM cell lines also induced proliferation. Importantly, HUVECS protected against anti-MM agents including conventional agents (Dexamethasone, Doxorubicin, Melphalan) and novel drugs (Revlimid™). The protective effect afforded by co-culture was lost on HUVEC fixation. Intracellular signaling events following MM cell-endothelial cell contact were studied to understand the mechanisms of the proliferative and protective effects. Western blotting demonstrated activation of the JAK/STAT, PI3K/Akt and the MAPK pathways, mediating proliferation and survival. Ongoing studies focused on understanding cytokine as well as adhesion-mediated interactions between the endothelial cells and the MM cells will identify targets for new therapeutic approaches in MM.

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LFA1 Mediated Attachment of Primary Human Monocytes to Activated Brain Microvascular Endothelial Cells Is Regulated by SDF-1α through the SRC Family Kinase Lyn; Potential Involvement of This Signaling Pathway in HIV-Associated Dementia.

Blood ◽

10.1182/blood.v110.11.516.516 ◽

2007 ◽

Vol 110 (11) ◽

pp. 516-516

Author(s):

Mobeen Malik ◽

Ying-Yu Chen ◽

Martha F. Kienzle ◽

Ronald G. Collman ◽

Andrzej Ptasznik

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Intracellular Signaling ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Human Monocytes ◽

Src Family Kinase ◽

Down Regulation ◽

Microvascular Endothelial ◽

Monocyte Adherence

Abstract Infiltration of activated monocytes into the brain of HIV-infected patients is a prerequisite for the development of HIV-associated dementia (HAD). The chemokine stromal derived factor-1α (SDF-1α) is expressed at increased levels in the central nervous system (CNS) of HAD patients and elicits chemotaxis and other cellular effects through its receptor CXCR4. In this project, we investigated the intracellular signaling pathway by which SDF-1α mediates the movement and attachment of monocytes to brain microvascular endothelia, and which may contribute to their infiltration into the CNS in HAD. We demonstrated that SDF-1α stimulates migration of primary human monocytes through its receptor CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1. SDF-1α also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) activated with the pro-inflammatory cytokines TNF-α or IL-1β, or with recombinant HIV-1 envelope glycoprotein (gp120), which increase endothelial cells expression of ICAM-1. The decreased monocyte adherence was linked to down regulation of the activation-dependent epitope of the β2 integrin LFA-1 which is a ligand for ICAM-1. We then demonstrated that the Src family kinase Lyn is a central modulator of migration and LFA-1-mediated adhesion of SDF-1α-stimulated primary monocytes. Using siRNA knockdown we achieved 80% down regulation of Lyn kinase in human monocytes. Lyn down regulation decreased SDF-1α-mediated migration and prevented its inhibition of monocyte attachment to ICAM-1 coated surfaces and activated BMVEC. These data indicate that in SDF-1α-stimulated primary human monocytes Lyn is a positive regulator of cell migration, and a negative regulator of cell adhesion to BMVEC by inhibiting the ICAM-1 binding activity of the LFA-1 integrin. Thus, CXCR4-triggered inside-out integrin signaling, through Lyn, inhibits adherence and stimulates movement of monocytes towards SDF-1α gradient on BMVEC monolayers. These results provide new insight into the intracellular signaling cascade that controls primary human monocytes movement and attachment at the blood brain barrier.

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Gas6-Induced Tissue Factor Expression in Endothelial Cells Is Mediated Through Caveolin-1-Enriched Domains

Blood ◽

10.1182/blood.v120.21.3308.3308 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3308-3308

Author(s):

Sandrine Laurance ◽

Catherine A Lemarie ◽

Mark D Blostein

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Tyrosine Kinases ◽

Intracellular Signaling ◽

Receptor Tyrosine Kinases ◽

Tissue Factor Expression ◽

Caveolin 1 ◽

Intracellular Signaling Pathways ◽

Factor Expression ◽

Cell Fractions

Abstract Abstract 3308 Gas6 is the ligand for the TAM family of receptors, which are composed of three members namely Tyro3, Axl and Mer. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Our laboratory has identified important intracellular signaling pathways important in gas6-Axl mediated protection of endothelial cells from apoptosis. However, as both gas6 and Axl null mice are protected from lethal thromboembolism, we sought to explore novel gas6-Axl intracellular signaling pathways regulating thrombosis. Caveolae have been shown to play a crucial role in the activation of signaling cascades following ligand binding to receptor tyrosine kinases. Caveolae are formed from lipid rafts by polymerization of caveolins. Caveolin-1 is the most abundant protein found in caveolae. Caveolin-1-enriched microdomains are well known to play a role as a docking platform for receptor tyrosine kinases and intracellular adaptor signaling proteins. Axl association with these highly specialized domains of the plasma membrane has not been previously elucidated. In the present study, we investigated the role of caveolin-1-enriched microdomains in gas6/Axl signaling in endothelial cells. First, we demonstrated that gas6-induced Akt and Erk1/2 phosphorylation required the presence of a functional Axl receptor as shown by Axl siRNA knockdown experiments in human umbilical vein endothelial cells. Then, caveolin-1 fractions, enriched by a detergent-free cell lysis followed by sucrose gradient ultra centrifugation, were studied by western blot analysis. After 5 and 10 min of gas6 treatment, Axl colocalized with caveolin-1 suggesting Axl recruitment into caveolin-1-enriched cell fractions. We found that c-Src, a signaling molecule known to behave as a transient docking platform in lipid rafts, also moved in caveolin-1-enriched cell fractions after gas6 stimulation. Caveolin-1 siRNA abolished gas6-induced Akt, Erk1/2 and c-Src phosphorylation suggesting that caveolin-1 enriched fractions are required for gas6-Axl signaling. Interestingly, we have shown that gas6-induced Akt phosphorylation required c-Src activation using c-Src siRNA and the pharmacological inhibitor (PP2). However, gas6-induced Erk1/2 phosphorylation was independent of c-Src. Finally, we found that gas6 increased tissue factor expression through the Axl-c-Src-Akt signaling cascade. Taken together, our results demonstrate that caveolin-1-enriched domains are required for gas6-Axl signaling and lead to the upregulation of tissue factor expression by gas6 in endothelial cells. These results highlight new insights of gas6-Axl signaling and function in endothelial cells. Disclosures: No relevant conflicts of interest to declare.

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