The microvascular endothelial redoxome established through tandem mass tag mass spectrometry

Abstract Redox signalling plays an important role in endothelial cell (EC) physiology and pathophysiology. Proteins sense redox signals via cysteine thiol groups. A common oxidative post-translational modification (oxPTM) on cysteine thiols is S-glutathionylation which is reversed to a free thiol state by glutaredoxin (Glrx). OxPTMs alter protein function, location and stability. Identifying which proteins undergo modification will help determine the role of redox signalling in EC function. A proteome-wide screen of human cardiac microvascular endothelial cells identified redox-sensitive proteins involved in vascular signalling mechanisms. Methods Human microvascular endothelial cells (HCMVEC, Lonza) were exposed to VEGF (50ng/ml, 24h) or hypoxia (0.5% O2, 24h) with adenoviral (ad) Glrx (Vectorlabs) overexpression or adLacZ (control). A tandem mass tag mass spectrometry system (TMT) coupled with a thiol-switch technique was used to quantify changes in redox sensitive thiol modifications. Protein lysates were treated with MMTS to alkylate unmodified thiols. Iodo-TMT six-plex probes were tagged to redox-sensitive sites after reversal of oxPTMs by DTT. Samples were pooled and processed by nLC-MS/MS. The abundance of each peptide in different conditions was compared with either adGlrx or adLacZ (control) expression to provide a ratio of changes in redox modifications. Results Iodo-TMT analysis revealed 113 unique thiol modifications identified on 78 different proteins using a ±1.5-fold threshold in a given treatment. Additionally, 44 modifications in 33 proteins were present in at least 2 different conditions, namely Glrx under VEGF and hypoxic conditions. A STRING interaction network identified clusters of 10 proteins involved in organonitrogen synthesis and 6 proteins in angiogenesis. Jagged-1 involved in the regulation of angiogenic sprouting through the Notch pathway was established as a target of redox signalling. Identified redox sensitive cysteines were found in extracellular EFG1 and the calcium binding EGF12 domains. Seven different In Silico programs (including MutationTaster, PolyPhen-2 and PANTHER) predicting the impact of substitution mutations indicated a functional affect for these redox sensitive sites, demonstrating the importance of these residues. Conclusion A non-biased proteomics approach identified novel thiol modifications on proteins involved in microvascular function. Future work will demonstrate the impact of these redox-sensitive thiol modifications on microvascular function to provide a better understanding of redox signalling in protein function and disease. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): MRC-DTP,European Union's Horizon 2020 research and innovation programme.

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The Impaired Bioenergetics of Diabetic Cardiac Microvascular Endothelial Cells

Frontiers in Endocrinology ◽

10.3389/fendo.2021.642857 ◽

2021 ◽

Vol 12 ◽

Author(s):

Haitao Zhang ◽

Yan Shen ◽

Il-man Kim ◽

Neal L. Weintraub ◽

Yaoliang Tang

Keyword(s):

Endothelial Cells ◽

Oxygen Consumption ◽

Tricarboxylic Acid Cycle ◽

Lower Percentage ◽

Microvascular Endothelial Cells ◽

Tunel Staining ◽

Significant Difference ◽

Microvascular Endothelial ◽

The Impact ◽

Cardiac Microvascular Endothelial Cells

Diabetes causes hyperglycemia, which can create a stressful environment for cardiac microvascular endothelial cells (CMECs). To investigate the impact of diabetes on the cellular metabolism of CMECs, we assessed glycolysis by quantifying the extracellular acidification rate (ECAR), and mitochondrial oxidative phosphorylation (OXPHOS) by measuring cellular oxygen consumption rate (OCR), in isolated CMECs from wild-type (WT) hearts and diabetic hearts (db/db) using an extracellular flux analyzer. Diabetic CMECs exhibited a higher level of intracellular reactive oxygen species (ROS), and significantly reduced glycolytic reserve and non-glycolytic acidification, as compared to WT CMECs. In addition, OCR assay showed that diabetic CMECs had increased maximal respiration, and significantly reduced non-mitochondrial oxygen consumption and proton leak. Quantitative PCR (qPCR) showed no difference in copy number of mitochondrial DNA (mtDNA) between diabetic and WT CMECs. In addition, gene expression profiling analysis showed an overall decrease in the expression of essential genes related to β-oxidation (Sirt1, Acox1, Acox3, Hadha, and Hadhb), tricarboxylic acid cycle (TCA) (Idh-3a and Ogdh), and electron transport chain (ETC) (Sdhd and Uqcrq) in diabetic CMECs compared to WT CMECs. Western blot confirmed that the protein expression of Hadha, Acox1, and Uqcrq was decreased in diabetic CMECs. Although lectin staining demonstrated no significant difference in capillary density between the hearts of WT mice and db/db mice, diabetic CMECs showed a lower percentage of cell proliferation by Ki67 staining, and a higher percentage of cellular apoptosis by TUNEL staining, compared with WT CMECs. In conclusion, excessive ROS caused by hyperglycemia is associated with impaired glycolysis and mitochondrial function in diabetic CMECs, which in turn may reduce proliferation and promote CMEC apoptosis.

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Extrinsic acidosis suppresses glycolysis and migration while increasing network formation in pulmonary microvascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00544.2018 ◽

2019 ◽

Vol 317 (2) ◽

pp. L188-L201 ◽

Cited By ~ 4

Author(s):

Ji Young Lee ◽

Mher Onanyan ◽

Ian Garrison ◽

Roderica White ◽

Maura Crook ◽

...

Keyword(s):

Endothelial Cells ◽

Network Formation ◽

Segment Length ◽

Microvascular Endothelial Cells ◽

Carbonic Anhydrases ◽

Pulmonary Microvascular Endothelial Cells ◽

Ca Ix ◽

Microvascular Endothelial ◽

And Migration ◽

The Impact

Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.

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Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Analytica Chimica Acta ◽

10.1016/j.aca.2019.01.015 ◽

2019 ◽

Vol 1056 ◽

pp. 79-87 ◽

Cited By ~ 5

Author(s):

Sulay H. Patel ◽

Omnia A. Ismaiel ◽

William R. Mylott ◽

Moucun Yuan ◽

Kurt F. Hauser ◽

...

Keyword(s):

Mass Spectrometry ◽

Endothelial Cells ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Microvascular Endothelial Cells ◽

Tandem Mass ◽

Brain Microvascular Endothelial Cells ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

BioMed Research International ◽

10.1155/2015/864169 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Wenying Luo ◽

Jiayu Zhong ◽

Wei Zhao ◽

Jianjun Liu ◽

Renli Zhang ◽

...

Keyword(s):

Mass Spectrometry ◽

Endothelial Cells ◽

Human Brain ◽

Time Of Flight ◽

Enterovirus 71 ◽

Differentially Expressed ◽

Mass Spectrometry Analysis ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Microvascular Endothelial

2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

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Autocrine Activity of Exosomes in Glucocorticoid-Induced Injury of Bone Microvascular Endothelial Cells Identified by Protein Array

10.21203/rs.3.rs-1070782/v1 ◽

2021 ◽

Author(s):

Qingyu Zhang ◽

Tengqi Li ◽

Zirong Li ◽

Jike Lu ◽

Xinjie Wu ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Transforming Growth Factor ◽

Quantitative Polymerase Chain Reaction ◽

Protein Array ◽

Inflammatory Factors ◽

Microvascular Endothelial Cells ◽

Total Protein Content ◽

Microvascular Endothelial ◽

The Impact

Abstract Background: Glucocorticoid could induce injury and apoptosis of bone microvascular endothelial cells (BMECs) in the femoral head and the application of icariin showed a protective effect. However, the impact of autocrine exosomes during these processes is still to be confirmed.Methods: Exosomes were extracted from BMECs treated with hydrocortisone or hydrocortisone plus icariin by super-speed centrifugation; exosome-carried proteins were evaluated via BCA assay, Western blotting, protein array assay and Elisa test, while miRNA expression profile was assessed via high-throughput sequencing and confirmed by quantitative polymerase chain reaction (qPCR) to screen candidate molecules responsible for BMEC-Exo function. BMECs were incubated with and without exosomes before glucocorticoid intervention and then the impact of BMECs-derived exosomes on BMECs viability, apoptosis, migration, angiogenesis, and protein expression was further assessed by a series of functional assays. Results: Exosomes secreted by BMECs could ameliorate glucocorticoid-induced endothelial cellular injury, improve cell viability, decrease cell apoptosis, and promote cell migration and angiogenesis compared with the blank control. These effects of secreted exosomes could be reinforced by icariin intervention. Meanwhile, mechanism studies showed that expression level of eNOS, COX-2, and pERK were significantly increased while the cleaved caspase-3 level was decreased in BMECs after coculture with exosomes. Although icariin treatment would not significantly change the size and total protein content of BMECs-derived exosomes, expression of exosome-carried vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) was enhanced and numerous miRNAs involved in cell proliferation and apoptosis were up-regulated (e.g., hsa-miR-1469 and hsa-miR-133a-5p) or down-regulated (e.g., hsa-miR-10b-5p) (p < 0.05). 29 differentially expressed inflammatory factors were detected between the exosomes secreted by the Icariin-treated and the Model groups.Conclusion: To sum up, the present study indicates that autocrine exosomes could significantly improve glucocorticoid-induced injury of BMECs, partially mediated by activation of MAPK/ERK pathway and regulation of several inflammation/apoptosis/proliferation-associated proteins. Icariin intervention could reinforce these effects and may act as a promising drug for improving glucocorticoid-induced injury of BMECs. In vivo or animal studies are still required to better understand the function of BMEC-derived exosomes.

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Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.8955 ◽

2020 ◽

Vol 35 (1) ◽

Author(s):

Zhiyuan Ma ◽

Shuanghui Lu ◽

Hui Zhou ◽

Shirong Zhang ◽

Yuqing Wang ◽

...

Keyword(s):

Mass Spectrometry ◽

Endothelial Cells ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Microvascular Endothelial Cells ◽

Tandem Mass ◽

Brain Microvascular Endothelial Cells ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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The Impact Of Viperistatin And VP12, Snake Venom Derived Inhibitors Of Collagen Receptors ±1²1 And ±2²1 Integrins, On Eosinophils And Lymphocytes Transmigration Through Human Microvascular Endothelial Cells M

10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a4174 ◽

2012 ◽

Author(s):

Stanislawa Bazan-Socha ◽

Joanna Zuk ◽

Cezary Marcinkiewicz ◽

Jacek Musial

Keyword(s):

Endothelial Cells ◽

Snake Venom ◽

Microvascular Endothelial Cells ◽

Collagen Receptors ◽

Microvascular Endothelial ◽

The Impact

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Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo

Cellular Physiology and Biochemistry ◽

10.1159/000470806 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1503-1518 ◽

Cited By ~ 11

Author(s):

Qing Li ◽

He-He Cui ◽

Yue-Jin Yang ◽

Xiang-Dong Li ◽

Gui-Hao Chen ◽

...

Keyword(s):

Endothelial Cells ◽

Reperfusion Injury ◽

Ischemia Reperfusion ◽

Protective Role ◽

Microvascular Endothelial Cells ◽

Heme Oxygenase 1 ◽

Tandem Mass ◽

Altered Expression ◽

Microvascular Endothelial ◽

Cardiac Microvascular Endothelial Cells

Background: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). Methods: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. Results: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. Conclusions: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

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