Attenuation of Glomerular Endothelial Cells from High Glucose-Induced Injury by Blockade of MAD2B

Background/Aims: To assess the role of mitotic arrest-deficient 2-like protein 2 (MAD2B) in high glucose-induced injury in mouse glomerular endothelial cells (GEnCs). Methods: GEnCs were cultured in vitro, and MAD2B protein levels were measured by Western blot in cells stimulated with high glucose (30 mM) for various periods of time. MAD2B and scrambled shRNA were introduced into GEnCs by liposomal transfection. Cell proliferation, apoptosis, nitric oxide (NO) production, and monolayer permeability were then measured in cells grown in the following conditions: control, high glucose treatment, MAD2B shRNA transfection with high glucose treatment, and scrambled shRNA transfection with high glucose treatment. Results: High glucose increased the protein levels of MAD2B in GEnCs. Compared with control cells, apoptosis was increased by high glucose treatment, which was attenuated by transfection with MAD2B shRNA transfection. Cells treated with high glucose produced less NO than control cells, whereas MAD2B shRNA transfection increased NO production. Cell monolayer permeability was enhanced in high glucose treated cells, but MAD2B shRNA transfection reduced permeability. Conclusion: High glucose levels induced the expression of MAD2B in GEnCs, whereas suppressing its expression reduced high glucose-induced endothelial cell apoptosis and high permeability, and promoted cell proliferation and NO production.

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Inhibition of Cx43 gap junction uncoupling prevents high glucose-induced apoptosis and reduces excess cell monolayer permeability in retinal vascular endothelial cells

Experimental Eye Research ◽

10.1016/j.exer.2018.05.003 ◽

2018 ◽

Vol 173 ◽

pp. 85-90 ◽

Cited By ~ 11

Author(s):

Dongjoon Kim ◽

Ulrik Mouritzen ◽

Bjarne Due Larsen ◽

Sayon Roy

Keyword(s):

Endothelial Cells ◽

Gap Junction ◽

High Glucose ◽

Vascular Endothelial Cells ◽

Cell Monolayer ◽

Vascular Endothelial ◽

Monolayer Permeability ◽

Induced Apoptosis

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Hypoxic upregulation of arginase II in human lung endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00068.2010 ◽

2010 ◽

Vol 299 (6) ◽

pp. C1541-C1548 ◽

Cited By ~ 49

Author(s):

Karina Krotova ◽

Jawaharlal M. Patel ◽

Edward R. Block ◽

Sergey Zharikov

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Immune Cell ◽

Human Lung ◽

Pulmonary Artery Hypertension ◽

Arginase Activity ◽

No Production ◽

Protein Levels ◽

Lung Endothelial Cells ◽

Arginase Ii

Activated arginase has been implicated in many diseases including cancer, immune cell dysfunction, infections, and vascular disease. Enhanced arginase activity has been reported in lungs of patients with pulmonary artery hypertension. We used hypoxia as a model for pulmonary hypertension and studied the effect of exposure to hypoxia on arginase activity in human lung microvascular endothelial cells (HMVEC). Hypoxia induces upregulation of arginase activity as well as mRNA and protein levels of arginase II (Arg II), the only arginase isoform we were able to identify in HMVEC. In endothelial cells, arginase shares and competes for the substrate l-arginine with nitric oxide (NO) synthase (NOS). Through regulation of substrate availability for NOS, arginase is able to modulate NO production. To evaluate the role of Arg II in regulation of NO production under hypoxia, we compared NO output (RFL-6 reporter assay) in cells with normal and silenced Arg II. Exposure to hypoxia led to an increase in NO levels produced by HMVEC. Inhibition of Arg II by specific small interfering RNA or by the pharmacological inhibitor BEC additionally enhanced the levels of NO. Another possible role for activated arginase is involvement in regulation of cell proliferation. However, we showed that hypoxia decreased cell proliferation and upregulated Arg II did not have an effect on cell proliferation. Since hypoxia-inducible factors (HIF) are a family of transcriptional factors activated by hypoxia, we tested the possibility of involvement of HIF-1 and HIF-2 in regulation of Arg II under hypoxia. The silencing of HIF-2 but not HIF-1 prevented the activation of Arg II by hypoxia.

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The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

Journal of Cell Science ◽

10.1242/jcs.112.10.1599 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1599-1609 ◽

Cited By ~ 2

Author(s):

B.M. Kraling ◽

D.G. Wiederschain ◽

T. Boehm ◽

M. Rehn ◽

J.B. Mulliken ◽

...

Keyword(s):

Cell Culture ◽

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Protein Levels ◽

Matrix Deposition ◽

Capillary Endothelial Cells ◽

Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

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High Glucose Increases Monolayer Permeability to Macromolecules in Retinal Endothelial Cells

Frontiers in Diabetes - Molecular and Cell Biology of Type 2 Diabetes and Its Complications ◽

10.1159/000060885 ◽

1998 ◽

pp. 217-220

Author(s):

F. Pricci ◽

G. Pugliese ◽

G. Romeo ◽

G. Leto ◽

G. Galli ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Retinal Endothelial Cells ◽

Monolayer Permeability

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Activation of general control nonderepressible 2 kinase protects human glomerular endothelial cells from harmful high-glucose-induced molecular pathways

International Urology and Nephrology ◽

10.1007/s11255-016-1377-x ◽

2016 ◽

Vol 48 (10) ◽

pp. 1731-1739 ◽

Cited By ~ 10

Author(s):

Theodoros Eleftheriadis ◽

Konstantina Tsogka ◽

Georgios Pissas ◽

Georgia Antoniadi ◽

Vassilios Liakopoulos ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Molecular Pathways ◽

General Control ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells

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miR-450a-5p eliminats MGO-induced insulin resistance via targeting CREB

10.21203/rs.2.12119/v1 ◽

2019 ◽

Author(s):

Cuifeng Wei ◽

Li Meng ◽

Yuting Zhang

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Cell Invasion ◽

High Glucose ◽

Target Gene ◽

Cell Activity ◽

Luciferase Reporter ◽

Akt Pathway ◽

Potential Target ◽

In Cells

Abstract Background miR-450a-5p was involved in fat formation, but its role in insulin resistance remains unclear. This study further investigated the effects of miR-450a-5p in endothelial cells, with the aim of finding a potential target for diabetes mellitus. Methods Human umbilical vein endothelial cells (HUVECs) were severally treated with low-glucose, high-glucose, methylglyoxal (MGO), and insulin only or plus MGO. miR-450a-5p was up-regulated or down-regulated in treated HUVECs. miR-450a-5p expression in cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity was determined through MTT experiments. Transwell assay and oil red O staining were used for the detection of cell invasion and fat formation. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by double-luciferase reporter analysis, and its influence in eNOS/AKT pathway was estimated. Results miR-450a-5p decreased obviously in endothelial cells with high-glucose and MGO. Through in vitro cell experiments, we knew that MGO could not only intensify the activity of endothelial cells, but also accelerate cell invasion and fat accumulation, which could be reversed by up-regulated miR-450a-5p. Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin, which were eliminated by up-regulated miR-450a-5p. Furthermore, CREB was the target gene of miR-450a-5p that had an activation effect on the eNOS/AKT pathway. Conclusions Up-regulated miR-450a-5p eliminated MGO-induced insulin resistance via targeting CREB, which might be a potential target to improve insulin resistance and benefit patients with related diseases.

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Shiga Toxin 2 Reduces Complement Inhibitor CD59 Expression on Human Renal Tubular Epithelial and Glomerular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.01079-12 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2678-2685 ◽

Cited By ~ 23

Author(s):

Silvia Ehrlenbach ◽

Alejandra Rosales ◽

Wilfried Posch ◽

Doris Wilflingseder ◽

Martin Hermann ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Shiga Toxin ◽

Human Kidney ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Protein Levels ◽

Glomerular Endothelial Cells ◽

Shiga Toxin 2 ◽

Renal Tubular

ABSTRACTInfections with enterohemorrhagicEscherichia coli(EHEC) are a primary cause of hemolytic-uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55, and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells derived from human kidney cells that are involved in HUS. Incubation with Stx2 did not influence the amount of CD46 or CD55 on the surface of HK-2 and GEnC cells, as determined by fluorescence-activated cell sorter analysis. In contrast, CD59 was significantly reduced by half on GEnC cells, but the reduction on HK-2 cells was less pronounced. With increasing amounts of Stx2, reduction of CD59 also reached significance in HK-2 cells. Enzyme-linked immunosorbent assay analyses showed that CD59 was not present in the supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, reverse transcription-quantitative PCR analyses showed downregulation of CD59 mRNA as the likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as a possible consequence of CD59 downregulation. In summary, Stx2 downregulates CD59 mRNA and protein levels on tubular epithelial and glomerular endothelial cells, and this downregulation likely contributes to complement activation and kidney destruction in EHEC-associated HUS.

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Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

Circulation Research ◽

10.1161/circresaha.121.318982 ◽

2021 ◽

Author(s):

Sarah Basehore ◽

Samantha Bohlman ◽

Callie Weber ◽

Swathi Swaminathan ◽

Yuji Zhang ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Laminar Flow ◽

No Production ◽

Disturbed Flow ◽

Enos Activity ◽

Enos Phosphorylation ◽

Steady Laminar ◽

In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

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Abstract 546: L-NAME Attenuates the Cardiotonic Steroids-induced Monolayer Permeability in Lymphatic Endothelial Cells

Hypertension ◽

10.1161/hyp.62.suppl_1.a546 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Juan Castor ◽

Darijana Horvat ◽

Walter E Cromer ◽

Thomas J Kuehl ◽

David C Zawieja ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Tight Junctions ◽

Cell Monolayer ◽

Hypertensive Disorder ◽

Lymphatic Endothelial Cells ◽

Vascular Leak Syndrome ◽

Vascular Leak ◽

Monolayer Permeability

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na + /K + ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture. Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of β-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure β-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The β-catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not. Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the β-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.

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