1. Medicine

Metabolic response of blood vessels to TNFα

  1. Abidemi Junaid
  2. Johannes Schoeman
  3. Wei Yang
  4. Wendy Stam
  5. Alireza Mashaghi
  6. Anton Jan van Zonneveld
  7. Thomas Hankemeier  Is a corresponding author
  1. Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, Netherlands
  2. Department of Internal Medicine (Nephrology), Leiden University Medical Center, Netherlands
  3. Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Netherlands
https://doi.org/10.7554/eLife.54754
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Cite this article
  1. Abidemi Junaid
  2. Johannes Schoeman
  3. Wei Yang
  4. Wendy Stam
  5. Alireza Mashaghi
  6. Anton Jan van Zonneveld
  7. Thomas Hankemeier
(2020)
Metabolic response of blood vessels to TNFα
eLife 9:e54754.
https://doi.org/10.7554/eLife.54754
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5 figures, 5 tables and 4 additional files

Figures

Figure 1
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Metabolomics workflow.

(a) Schematic diagram of the OrganoPlate 2-lane design and 3D reconstruction of the microvessels-on-a-chip formed by cultured HUVECs (blue: Hoechst, red: F-actin and green: VE-cadherin). All dimensions are in µm. (b) Collection of culture media after perfusion. The medium of four microvessels were pooled to form one sample. (c) Identification and quantification of prostaglandins, isoprostanes, lysophosphatidic acid (LPA) classes, sphingolipids and platelet activating factor (PAF) in microvessels-on-a-chip by UPLC-MS/MS using two different solvent gradients. (d) Pathway analysis.

Figure 2
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Inflammatory and oxidative stress markers in microvessels-on-a-chip.

Reconstructed LC-MS/MS ion chromatograms of PGE2, 5-iPF2α IV, iPF2α and 8, 12-iPF2α IV in microvessels treated with 50 ng/ml TNFα for 18 hr.

Figure 3
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TNFα-induced concentration profile changes of the signaling lipids in the microvessels-on-a-chip.

Concentrations of (a) prostaglandins, (b) isoprostanes, (c) lysophosphatidic acid (LPA) classes, (d) sphingolipids and (e) platelet activating factor (PAF) with available standards detected in the microvessels without TNFα exposure (control) and after exposure to 0.4 and 15 ng/ml TNFα for 18 hr. Data represent mean and s.e.m. of three biological replicates per condition; n = 4–6 technical replicates. Significance determined by unpaired Student’s t-test; *p<0.1, **p<0.05, ***p<0.01, ****p<0.001.

Figure 4
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TNFα induces the release of oxidative stress and inflammatory markers in endothelial cells.

Exposure to TNFα, causes TNF signaling in the microvessels to produce ROS from endogenous sources: mitochondria, xanthine oxidase, NADPH oxidase and uncoupled eNOS. Sphingosine-1-phosphate (S-1-P) is needed in order for TNF receptor-associated factor 2 (TRAF2) to form a complex with the TNF receptor 1 (TNFR1). These lead to the conversion of arachidonic acid (AA) to isoprostanes and NFκB activation. Moreover, AA is enzymatically converted by cyclooxygenase-2 (COX-2) to prostaglandins (PGs). At the same time, autotaxin (ATX) and phospholipase A2 (PLA2) are upregulated, resulting in the syntheses of lysophosphatidic acid (LPA) classes and platelet activating factor (PAF). Through their receptors, LPAs and PAF further promote the activation of COX-2.

Figure 5
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Expression of platelet endothelial cell adhesion molecule (PECAM-1) and von Willebrand factor (vWF) in isolated human umbilical vein endothelial cells (HUVECs).

Tables

Table 1
The peak area ratio of metabolites in the culture medium (EGM2) normalized with the peak area ratio of metabolites found in the culture medium after perfusion in the microvessels-on-a-chip for 18 hr (EGM2 HUVECs).

The peak area ratio is the peak area of the metabolites divided by the appropriate peak area of the internal standards. Fold changes below the 1 (blue) and above the 1 (red) indicates that low and high concentrations of fatty acids were present in medium before exposure to the microvessels. The data represent one biological replicate; n = 3 technical replicates.

Bioactive lipid*EGM2/EGM2 HUVECsBioactive lipid*EGM2/EGM2 HUVECs
PGF2α0.1LPA C22:518.2
PGF3α2.1LPA C16:021.3
8-iso-13, 14-dihydro-PGF2α0.0LPA C18:148.4
8-iso-PGF2α0.2LPA C22:45.9
5-iPF2α0.4cLPA C20:478.6
8, 12-iPF2α IV0.5LPA C18:00.0
LPA C14:06.2cLPA C18:20.0
LPA C16:125.4cLPA C16:014.8
LPA C22:617.7cLPA C18:125.8
LPA C18:277.2cLPA C18:011.1
LPA C20:431.0S-1-P C18:10.9

  1. * The rest of the metabolites shown in Figure 3 are not displayed, because they were not detected in the EGM2.

Table 1—source data 1

Peak area ratios of the identified metabolites in culture medium (EGM2) and in culture medium after perfusion in the microvessels-on-a-chip (EGM2 HUVECs).

https://cdn.elifesciences.org/articles/54754/elife-54754-table1-data1-v2.xlsx
Table 2
Comparison of the concentration of bioactive lipids between living human blood vessel and human microvessels-on-a- chip.

The concentrations in human blood vessel were obtained from HMDB (Wishart et al., 2018; Wishart et al., 2013; Wishart et al., 2007; Wishart et al., 2009).

Human blood vesselMicrovessels-on-a-chip
Bioactive lipidHealthyDiseasedHealthyDiseased
PGF1α∼0.0317–0.376 nM-∼0.350 nM∼0.527–1.412 nM
PGF2α∼0.144–0.371 nM∼0.4–1.6 nM∼3.96 nM∼5.36–12.5 nM
PGE2*∼0.13–0.172 nM-∼0.175 nM∼0.194–0.281 nM
PGE1<0.1 nM-∼0.0225 nM∼0.0246–0.0308 nM
PGD2∼0.065–0.2 nM-∼0.254 nM∼0.257–0.336 nM
PGA2∼0.0448–0.496 nM-∼0.006 nM∼0.0048–0.0058 nM
8-iso-PGF2α∼0.057–0.57 nM-∼0.103 nM∼0.122–0.216 nM
S-1-P C18:1∼0.5–3.0 nM-∼2.12 nM∼1.47–2.11 nM
Sph C18:1∼1.3–50 nM-∼44.2 nM∼45.2–49.8 nM
Spha C18:0∼1.3–50 nM-∼6.0 nM∼6.2–6.7 nM
Table 3
Heatmap of prostaglandins, isoprostanes, lysophosphatidic acid (LPA) classes, sphingolipids and platelet activating factor (PAF) detected in the microvessels-on-a-chip.

The fold changes were measured with respect to the controls and log2 transformed. The controls are microvessels unexposed to TNFα and PMA. The metabolites are characterized by their inflammatory action (anti- or pro-inflammatory), platelet activation (anti- or pro-platelet activation), vascular tone (constriction or dilation) and angiogenic action (anti- or pro-angiogenic). The data were obtained from the experiments done in Figure 3 with three biological replicates per condition; n = 4–6 technical replicates.

Fold change of concentration
Bioactive lipid15 ng/ml50 ng/ml20 ng/mlInflammatoryPlateletVascularAngiogenic
TNFTNFPMAactionactivationtoneaction
ProstaglandinsPGF1α2.01.85.0antinocon
PGF2α1.71.55.0pronoconpro
PGF3α1.51.14.4anti
PGE2*0.70.7.7proantidilpro
PGE10.50.42.7antiantidilpro
PGD20.43.43.5antianticonanti
13, 14-dihydro-PGF2α0.70.52.3pro
PGA2−0.30.02.6antino
Isoprostanes8-iso-13, 14-dihydro-PGF2α1.41.34.6anti
8-iso-PGF2α*1.10.94.2proanticonanti
8-iso-PGE20.10.02.1proanticonanti
8-iso-PGE10.10.00.7anticonanti
5-iPF2α0.00.00.0
8, 12-iPF2α IV−0.10.00.2
Lysophosphatidic acidsLPA C14:0−0.2−0.2−0.4proproconpro
LPA C16:1−0.4−0.3−0.6proproconpro
LPA C22:6*0.40.50.2proproconpro
LPA C18:20.10.0−0.1proproconpro
LPA C20:40.30.40.3proproconpro
LPA C22:5*0.50.60.3proproconpro
LPA C16:0−0.2−0.3−0.3proproconpro
LPA C18:10.10.2−0.1proproconpro
cLPA C20:4−0.1−0.2−0.1antiantino
LPA C18:00.10.0−0.2proproconpro
cLPA C16:0−0.20.00.0antiantino
cLPA C18:0−0.2−0.1−0.2antiantino
SphingolipidsS-1-P C18:1−0.5−0.6−0.9antianticonpro
Sph C18:10.20.10.0antianticonpro
Spha C18:00.20.0−0.1
PAF C16:0−0.2−0.2−0.4proproconpro

  1. * Validated markers of oxidative stress.

Table 3—source data 1

Concentrations of the identified metabolites in the microvessels-on-a-chip.

https://cdn.elifesciences.org/articles/54754/elife-54754-table3-data1-v2.xlsx
Table 4
Heatmap of pro- and anti- inflammatory and oxidative stress markers measured in 3D microvessels-on-a-chip and 2D endothelial cell monolayers.

The cells were treated with 15 ng/ml TNFα in the same experiment as Figure 3. The fold changes were measured with respect to the controls and log2 transformed. The controls are microvessels unexposed to TNFα and PMA. The metabolites are characterized by their inflammatory action (anti- or pro-inflammatory), platelet activation (anti- or pro-platelet activation), vascular tone (constriction or dilation) and angiogenic action (anti- or pro-angiogenic). The data represent one biological replicate; n = 2–3 technical replicates.

Fold change of
Concentration
Bioactive lipid2D3DInflammatoryPlateletVascularAngiogenic
TNFTNFactionactivationtoneaction
PGF1α1.93.4antinocon
PGF3α1.06.6anti
PGE12.11.9antiantidilpro
PGD22.36.5antianticonanti
PGA20.70.0antino
cLPA C20:4−0.7−0.5antiantino
cLPA C18:2−0.50.0antiantino
cLPA C16:0−0.6−0.3antiantino
cLPA C18:1−0.9−0.2antiantino
cLPA C18:0−0.6−0.2antiantino
S-1-P C18:1−2.0−0.9antianticonpro
8-iso-PGE11.81.9anticonanti
5-iPF2α0.3−0.1
PGF2α1.92.2pronoconpro
PGE2*2.41.0proantidilpro
13, 14-dihydro-PGF2α0.91.1pro
8-iso-13, 14-dihydro-PGF2α1.91.9anti
8-iso-PGF2α*2.01.3proanticonanti
8-iso-PGE20.6−0.3proanticonanti
LPA C14:00.0−0.8proproconpro
LPA C16:1−1.0−1.0proproconpro
LPA C22:6*0.0−0.3proproconpro
LPA C18:2−1.0−1.2proproconpro
LPA C20:4−0.2−0.4proproconpro
LPA C22:5*1.0−0.1proproconpro
LPA C16:0−0.6−0.6proproconpro
LPA C18:1−0.5−1.0proproconpro
LPA C18:00.1−0.9proproconpro
PAF C16:0−0.5−0.8proproconpro

  1. * Validated markers of oxidative stress.

Table 4—source data 1

Peak area ratios of the identified metabolites in 6-well plates and in the microvessels-on-a-chip.

https://cdn.elifesciences.org/articles/54754/elife-54754-table4-data1-v2.xlsx
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Biological sample (Human)primary human umbilical vein endothelial cellsLeiden University Medical Center (LUMC)freshly isolated from umbilical cord of male newborns
Chemical compound, drugphorbol 12-myristate 13-acetateSigma-AldrichCat#:P8139
Peptide,
recombinant protein		tumor necrosis factor-αSigma-AldrichCat#:H8916
Biological sample (Rat)rat tail collagen type 1TrevigenCat#:3440-005-01
Antibodymouse anti-human CD144BD BiosciencesCat#:555661; RRID:AB_396015IF(1:150)
Antibodysheep anti-human CD31R and D SystemsCat#:AF806; RRID:AB_355617IF(1:150)
Antibodyrabbit anti-human vWFAgilent DakoCat#:A0082; RRID:AB_2315602IF(1:1000)
Antibodyalexa fluor 488-conjugated goat anti-mouseThermoFisherCat#:R37120; RRID:AB_2556548IF(1:250)
Antibodyalexa fluor 488-conjugated donkey anti-sheepThermoFisherCat#:A11015; RRID:AB_141362IF(1:250)
Antibodyalexa fluor 647-conjugated goat anti-rabbitThermoFisherCat#:A27040; RRID:AB_2536101IF(1:250)
Otherrhodamine phalloidinSigma-AldrichCat#:P1951; RRID:AB_2315148IF(1:200)
OtherhoechstInvitrogenCat#:H3569; RRID:AB_2651133IF(1:2000)
Software, algorithmLabSolutionsShimadzuRRID:SCR_018241
Software, algorithmSPSSSPSSRRID:SCR_002865
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798

Additional files

Source data 1

Calibration curve of bioactive lipids.

https://cdn.elifesciences.org/articles/54754/elife-54754-data1-v2.xlsx
Supplementary file 1

References regarding the action of bioactive lipids on inflammation, platelets, vascular tone and angiogenesis.

https://cdn.elifesciences.org/articles/54754/elife-54754-supp1-v2.docx
Supplementary file 2

An overview of the concentrations of the calibration solution.

https://cdn.elifesciences.org/articles/54754/elife-54754-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/54754/elife-54754-transrepform-v2.docx

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  1. Abidemi Junaid
  2. Johannes Schoeman
  3. Wei Yang
  4. Wendy Stam
  5. Alireza Mashaghi
  6. Anton Jan van Zonneveld
  7. Thomas Hankemeier
(2020)
Metabolic response of blood vessels to TNFα
eLife 9:e54754.
https://doi.org/10.7554/eLife.54754