Abstract C5: T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells.

Abstract Prostaglandin E2 (PGE2) plays a crucial role in angiogenesis as well as in ischemic and inflammatory disorders of the brain associated with breakdown of the blood-brain barrier. However, the effects of PGE2 on brain endothelial cell migration, a key process in the angiogenic response and blood-brain barrier stability, are not well defined. Exposure of human brain endothelial cells (HBECs) to PGE2 elicited a chemotactic response in a time- and dose-dependent manner. The maximum migratory response was detected following 8 hours exposure of HBECs to PGE2 (100 nM). Migration of HBECs in response to PGE2 was accompanied by profound changes in the reorganization of actin filaments. Fluorescence microscopy examination of NBD-phallacidin-labeled endothelial cells showed increased formation of stress fibers, lamellipodia and podosomes after treatment with PGE2 (100 nM) compared to control. Based on these results, we hypothesized that Rho-kinase (ROCK), an enzyme involved in regulation of actin dynamics and cell migration, mediated the effects of PGE2 on HBEC migration. Western blot analyses revealed that ROCK II (type alpha), but not ROCK I (type beta), was expressed in HBECs. To examine ROCK II activation, we performed immunocomplex kinase assays using myosin light chain (MLC) as a substrate. PGE2 (100 nM) induced a 2-fold increase of 32P-incorporation into MLC indicating activation of ROCK II. Pretreatment of HBECs with the selective ROCK inhibitor, Y27632 (150 nM), blunted HBEC migration in response to PGE2 but had no effect on migration induced by fetal bovine serum (10%). Knockdown of ROCK II by siRNA also abrogated the migratory response of HBECs to PGE2. In contrast, similar treatment had no effect of HBEC migration stimulated by hepatocyte growth factor. Taken together, these results are consistent with the hypothesis that stimulation of HBECs with PGE2 leads to activation of ROCK II, reorganization of the actin cytoskeleton and ultimately migration. A better characterization of the molecular events that regulate migration of brain endothelial cells is critical for the development of novel strategies to treat cerebrovascular diseases associated with deregulated angiogenesis.

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Autocrine Effects of Brain Endothelial Cell-Produced Human Apolipoprotein E on Metabolism and Inflammation in vitro

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.668296 ◽

2021 ◽

Vol 9 ◽

Author(s):

Felecia M. Marottoli ◽

Troy N. Trevino ◽

Xue Geng ◽

Zarema Arbieva ◽

Pinal Kanabar ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Endothelial Cells ◽

Endothelial Cell ◽

Apolipoprotein E ◽

Paracellular Permeability ◽

Mitochondrial Activity ◽

Oxygen Species ◽

Brain Endothelial Cell ◽

Brain Endothelial Cells ◽

Lps Treatment

Reports of APOE4-associated neurovascular dysfunction during aging and in neurodegenerative disorders has led to ongoing research to identify underlying mechanisms. In this study, we focused on whether the APOE genotype of brain endothelial cells modulates their own phenotype. We utilized a modified primary mouse brain endothelial cell isolation protocol that enabled us to perform experiments without subculture. Through initial characterization we found, that compared to APOE3, APOE4 brain endothelial cells produce less apolipoprotein E (apoE) and have altered metabolic and inflammatory gene expression profiles. Further analysis revealed APOE4 brain endothelial cultures have higher preference for oxidative phosphorylation over glycolysis and, accordingly, higher markers of mitochondrial activity. Mitochondrial activity generates reactive oxygen species, and, with APOE4, there were higher mitochondrial superoxide levels, lower levels of antioxidants related to heme and glutathione and higher markers/outcomes of oxidative damage to proteins and lipids. In parallel, or resulting from reactive oxygen species, there was greater inflammation in APOE4 brain endothelial cells including higher chemokine levels and immune cell adhesion under basal conditions and after low-dose lipopolysaccharide (LPS) treatment. In addition, paracellular permeability was higher in APOE4 brain endothelial cells in basal conditions and after high-dose LPS treatment. Finally, we found that a nuclear receptor Rev-Erb agonist, SR9009, improved functional metabolic markers, lowered inflammation and modulated paracellular permeability at baseline and following LPS treatment in APOE4 brain endothelial cells. Together, our data suggest that autocrine signaling of apoE in brain endothelial cells represents a novel cellular mechanism for how APOE regulates neurovascular function.

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Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

10.1101/2021.10.29.466491 ◽

2021 ◽

Author(s):

Kandarp Dave ◽

Michael John Reynolds ◽

Donna B Stolz ◽

Riyan Babidhan ◽

Duncan X Dobbins ◽

...

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Endothelial Cell ◽

Tight Junction ◽

Human Brain ◽

Blood Brain Barrier ◽

Extracellular Vesicles ◽

Brain Barrier ◽

Brain Endothelial Cell ◽

Brain Endothelial Cells

Ischemic stroke causes brain endothelial cell death and damages tight junction integrity of the blood-brain barrier (BBB). We engineered endothelial cell-derived extracellular vesicles (EVs) for the delivery of exogenous heat shock protein 27 (HSP27) and harnessed the innate EV mitochondrial load as a one, two-punch strategy to increase brain endothelial cell survival (via mitochondrial delivery) and preserve their tight junction integrity (via HSP27 delivery). We demonstrated that endothelial microvesicles but not exosomes transferred their mitochondrial load that subsequently underwent fusion with the mitochondrial network of the recipient primary human brain endothelial cells. This mitochondrial transfer increased the relative ATP levels and mitochondrial function in the recipient endothelial cells. EV-mediated HSP27 delivery to primary human brain endothelial cells decreased the paracellular permeability of small and large molecular mass fluorescent tracers in an in vitro model of ischemia/reperfusion injury. This one, two-punch approach to increase the metabolic function and structural integrity of brain endothelial cells is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

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Angiopoietin-1 promotes endothelial cell proliferation and migration through AP-1–dependent autocrine production of interleukin-8

Blood ◽

10.1182/blood-2007-08-110338 ◽

2008 ◽

Vol 111 (8) ◽

pp. 4145-4154 ◽

Cited By ~ 54

Author(s):

Nelly A. Abdel-Malak ◽

Coimbatore B. Srikant ◽

Arnold S. Kristof ◽

Sheldon A. Magder ◽

John A. Di Battista ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Interleukin 8 ◽

Endothelial Cell Migration ◽

Endothelial Cell Proliferation ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration ◽

Angiopoietin 1

Abstract Angiopoietin-1 (Ang-1), ligand for the endothelial cell–specific Tie-2 receptors, promotes migration and proliferation of endothelial cells, however, whether these effects are promoted through the release of a secondary mediator remains unclear. In this study, we assessed whether Ang-1 promotes endothelial cell migration and proliferation through the release of interleukin-8 (IL-8). Ang-1 elicited in human umbilical vein endothelial cells (HUVECs) a dose- and time-dependent increase in IL-8 production as a result of induction of mRNA and enhanced mRNA stability of IL-8 transcripts. IL-8 production is also elevated in HUVECs transduced with retroviruses expressing Ang-1. Neutralization of IL-8 in these cells with a specific antibody significantly attenuated proliferation and migration and induced caspase-3 activation. Exposure to Ang-1 triggered a significant increase in DNA binding of activator protein-1 (AP-1) to a relatively short fragment of IL-8 promoter. Upstream from the AP-1 complex, up-regulation of IL-8 transcription by Ang-1 was mediated through the Erk1/2, SAPK/JNK, and PI-3 kinase pathways, which triggered c-Jun phosphorylation on Ser63 and Ser73. These results suggest that promotion of endothelial migration and proliferation by Ang-1 is mediated, in part, through the production of IL-8, which acts in an autocrine fashion to suppress apoptosis and facilitate cell proliferation and migration.

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Estrogen-Receptor-Mediated Protection of Cerebral Endothelial Cell Viability and Mitochondrial Function after Ischemic Insult in vitro

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.226 ◽

2009 ◽

Vol 30 (3) ◽

pp. 545-554 ◽

Cited By ~ 59

Author(s):

Jiabin Guo ◽

Diana N Krause ◽

James Horne ◽

John H Weiss ◽

Xuejun Li ◽

...

Keyword(s):

Endothelial Cells ◽

Estrogen Receptor ◽

Endothelial Cell ◽

Cell Viability ◽

Mitochondrial Function ◽

Protective Effects ◽

Brain Endothelial Cells ◽

Ischemic Insult ◽

Mitochondrial Superoxide

Protective effects of estrogen against experimental stroke and neuronal ischemic insult are well-documented, but it is not known whether estrogen prevents ischemic injury to brain endothelium, a key component of the neurovascular unit. Increasing evidence indicates that estrogen exerts protective effects through mitochondrial mechanisms. We previously found 17β-estradiol (E2) to improve mitochondrial efficiency and reduce mitochondrial superoxide in brain blood vessels and endothelial cells. Thus we hypothesized E2 will preserve mitochondrial function and protect brain endothelial cells against ischemic damage. To test this, an in vitro ischemic model, oxygen-glucose deprivation (OGD)/reperfusion, was applied to immortalized mouse brain endothelial cells (bEnd.3). OGD/reperfusion-induced cell death was prevented by long-term (24, 48 h), but not short-term (0.5, 12 h), pretreatment with 10 nmol/L E2. Protective effects of E2 on endothelial cell viability were mimicked by an estrogen-receptor (ER) agonist selective for ERα (PPT), but not by one selective for ERβ (DPN). In addition, E2 significantly decreased mitochondrial superoxide and preserved mitochondrial membrane potential and ATP levels in early stages of OGD/reperfusion. All of the E2 effects were blocked by the ER antagonist, ICI-182,780. These findings indicate that E2 can preserve endothelial mitochondrial function and provide protection against ischemic injury through ER-mediated mechanisms.

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ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells

Frontiers in Physiology ◽

10.3389/fphys.2021.683651 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sharon Harel ◽

Veronica Sanchez ◽

Alaa Moamer ◽

Javier E. Sanchez-Galan ◽

Mohammad N. Abid Hussein ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factors ◽

Endothelial Cell ◽

Binding Sites ◽

Umbilical Vein ◽

Intracellular Localization ◽

Mrna Levels ◽

Mobility Shift ◽

Angiogenic Response ◽

Angiopoietin 1

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

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Vasculotide, an Angiopoietin-1 mimetic, reduces acute skin ionizing radiation damage in a preclinical mouse model

10.32920/14639919 ◽

2021 ◽

Author(s):

Elina Korpela ◽

Darren Yohan ◽

Lee CL Chin ◽

Anthony Kim ◽

Xiaoyong Huang ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Ionizing Radiation ◽

Radiation Damage ◽

Microvascular Endothelial Cell ◽

Skin Damage ◽

Cancer Radiotherapy ◽

Microvascular Endothelial ◽

Angiopoietin 1

Background Most cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Acute skin damage from cancer radiotherapy diminishes patients’ quality of life, yet effective biological interventions for this damage are lacking. Protecting microvascular endothelial cells from irradiation-induced perturbations is emerging as a targeted damage-reduction strategy. Since Angiopoetin-1 signaling through the Tie2 receptor on endothelial cells opposes microvascular perturbations in other disease contexts, we used a preclinical Angiopoietin-1 mimic called Vasculotide to investigate its effect on skin radiation toxicity using a preclinical model. Methods Athymic mice were treated intraperitoneally with saline or Vasculotide and their flank skin was irradiated with a single large dose of ionizing radiation. Acute cutaneous damage and wound healing were evaluated by clinical skin grading, histology and immunostaining. Diffuse reflectance optical spectroscopy, myeloperoxidase-dependent bioluminescence imaging of neutrophils and a serum cytokine array were used to assess inflammation. Microvascular endothelial cell response to radiation was tested with in vitro clonogenic and Matrigel tubule formation assays. Tumour xenograft growth delay experiments were also performed. Appreciable differences between treatment groups were assessed mainly using parametric and non-parametric statistical tests comparing areas under curves, followed by post-hoc comparisons. Results In vivo, different schedules of Vasculotide treatment reduced the size of the irradiation-induced wound. Although skin damage scores remained similar on individual days, Vasculotide administered post irradiation resulted in less skin damage overall. Vasculotide alleviated irradiation-induced inflammation in the form of reduced levels of oxygenated hemoglobin, myeloperoxidase bioluminescence and chemokine MIP-2. Surprisingly, Vasculotide-treated animals also had higher microvascular endothelial cell density in wound granulation tissue. In vitro, Vasculotide enhanced the survival and function of irradiated endothelial cells. Conclusions Vasculotide administration reduces acute skin radiation damage in mice, and may do so by affecting several biological processes. This radiation protection approach may have clinical impact for cancer radiotherapy patients by reducing the severity of their acute skin radiation damage.

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Inhibition of glutathione synthesis in brain endothelial cells lengthens S-phase transit time in the cell cycle: Implications for proliferation in recovery from oxidative stress and endothelial cell damage

Redox Biology ◽

10.1016/j.redox.2013.01.003 ◽

2013 ◽

Vol 1 (1) ◽

pp. 131-139 ◽

Cited By ~ 9

Author(s):

Carmina Buşu ◽

Wei Li ◽

Gloria Caldito ◽

Tak Yee Aw

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Endothelial Cells ◽

Endothelial Cell ◽

Transit Time ◽

Cell Damage ◽

S Phase ◽

Brain Endothelial Cells ◽

Endothelial Cell Damage ◽

Glutathione Synthesis

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Hantaviruses Direct Endothelial Cell Permeability by Sensitizing Cells to the Vascular Permeability Factor VEGF, while Angiopoietin 1 and Sphingosine 1-Phosphate Inhibit Hantavirus-Directed Permeability

Journal of Virology ◽

10.1128/jvi.02397-07 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5797-5806 ◽

Cited By ~ 101

Author(s):

Irina N. Gavrilovskaya ◽

Elena E. Gorbunova ◽

Natalie A. Mackow ◽

Erich R. Mackow

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Permeability ◽

Cell Permeability ◽

Hemorrhagic Disease ◽

Αvβ3 Integrin ◽

Endothelial Cell Permeability ◽

Sphingosine 1 Phosphate ◽

Permeability Assay ◽

Angiopoietin 1

ABSTRACT Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of αvβ3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional β3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of αvβ3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to αvβ3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of β3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering β3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism for vascular leakage after infection by pathogenic hantaviruses and the means to inhibit hantavirus-directed endothelial cell permeability that may be applicable to additional vascular leak syndromes.

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