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. 2020 Feb 13;21(4):1240.
doi: 10.3390/ijms21041240.

Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge

Bisera Stepanovska  1 , Antonia I Lange  2   3 , Stephanie Schwalm  4 , Josef Pfeilschifter  4 , Sina M Coldewey  2   3   5 , Andrea Huwiler  1
Affiliations

Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge

Bisera Stepanovska et al. Int J Mol Sci. .

Abstract

Sphingosine 1-phosphate (S1P) is a key bioactive lipid that regulates a myriad of physiological and pathophysiological processes, including endothelial barrier function, vascular tone, vascular inflammation, and angiogenesis. Various S1P receptor subtypes have been suggested to be involved in the regulation of these processes, whereas the contribution of intracellular S1P (iS1P) through intracellular targets is little explored. In this study, we used the human cerebral microvascular endothelial cell line HCMEC/D3 to stably downregulate the S1P lyase (SPL-kd) and evaluate the consequences on endothelial barrier function and on the molecular factors that regulate barrier tightness under normal and inflammatory conditions. The results show that in SPL-kd cells, transendothelial electrical resistance, as a measure of barrier integrity, was regulated in a dual manner. SPL-kd cells had a delayed barrier build up, a shorter interval of a stable barrier, and, thereafter, a continuous breakdown. Contrariwise, a protection was seen from the rapid proinflammatory cytokine-mediated barrier breakdown. On the molecular level, SPL-kd caused an increased basal protein expression of the adherens junction molecules PECAM-1, VE-cadherin, and β-catenin, increased activity of the signaling kinases protein kinase C, AMP-dependent kinase, and p38-MAPK, but reduced protein expression of the transcription factor c-Jun. However, the only factors that were significantly reduced in TNFα/SPL-kd compared to TNFα/control cells, which could explain the observed protection, were VCAM-1, IL-6, MCP-1, and c-Jun. Furthermore, lipid profiling revealed that dihydro-S1P and S1P were strongly enhanced in TNFα-treated SPL-kd cells. In summary, our data suggest that SPL inhibition is a valid approach to dampenan inflammatory response and augmente barrier integrity during an inflammatory challenge.

Keywords: PKC; S1P lyase; blood–brain barrier; endothelial integrity; inflammation; junctional molecules.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Characterization of a stable SPL knockdown in HCMEC/D3 cells. HCMEC/D3 cells, transduced with either an empty lentiviral vector (ctrl) or a lentiviral vector containing SPL shRNA (SPL-kd), were cultured until confluence and were then incubated for 4 h in serum-free DMEM. Proteins and RNA were extracted and taken for either a Western Blot analysis of the SPL protein (A), or a qPCR analysis of SPL mRNA (B). (C) Subconfluent cells grown on gelatin-coated dishes were photographed using a light microscope (Zeiss AxioObserver Z1, Feldbach) with a 200× total magnification and phase contrast setting. (D,E) Control (ctrl) and SPL-kd cells were rendered serum-free for 24 h and were treated for the last 10 min with either vehicle (-) or 1 μM of sphingosine (sph). The lipids were then extracted and processed for LC-MS/MS as described in the Methods section. The results in A and B are expressed as % of the control transduced cells and are depicted as means ± S.D. (n = 3 in A, n = 4 in B, *** p < 0.001). Results in D and E are expressed as pg/1,5 × 106 cells and are means ± S.D. (n = 3; * p < 0.05, ** p < 0.01, **** p < 0.0001 considered statistically significant when compared to the vehicle-treated control; # p < 0.05 compared to the sphingosine-treated control; § p < 0.05, §§ p < 0.01 compared to the vehicle-treated SPL-kd).
Figure 2
Figure 2
Cell number titration of HCMEC/D3 control and SPL-kd using ECISTM. HCMEC/D3 control cells (continuous lines) and SPL-kd cells (dashed lines) were seeded at densities between 20,000 and 50,000 cells/mL. ECISTM measurements were monitored over an observation period of 216 h and were performed as described in detail in the Methods section. Partial medium changes were performed every 24 h until t = 72 h. The data are shown as mean curves of triplicate samples.
Figure 3
Figure 3
Impact of an inflammatory stimulus (lipopolysaccharide (LPS) + Cyt) on the barrier integrity of HCMEC/D3 control and SPL-kd cells. After the development of a stable barrier (t = 0 h), HCMEC/D3 control cells (continuous line) and SPL-kd cells (dashed line) were stimulated with different dilutions of an inflammatory stimulus (LPS + Cyt). Resistance values were analyzed at the two observation time points 24 h and 120 h (indicated by dotted arrows) after LPS + Cyt administration. The data are expressed as a mean ± S.D. (n = 3), * p < 0.05.
Figure 4
Figure 4
Effect of SPL knockdown on the expression of adherens junction molecules in TNFα-stimulated HCMECs. Confluent control (ctrl) and SPL-kd HCMEC/D3 cells were incubated for 4 h in serum-free DMEM before stimulation for 24 h with either vehicle (-) or 1 nM TNFα in DMEM/0.1% FBS. The proteins were extracted, separated by SDS-PAGE, transferred to nitrocellulose membrane and subjected to analysis using antibodies against PECAM-1, VE-cadherin, β-catenin, p120, ZO-1 and α-tubulin. Data show representative blots, out of 3 independent experiments, performed in triplicates. The evaluation of the respective bands is presented in Figure S1.
Figure 5
Figure 5
Effect of TNFα stimulation on the protein and mRNA expression of adhesion molecules in control and SPL-kd HCMEC/D3. Confluent control (ctrl) and SPL-kd HCMEC/D3 cells were rendered serum-free for 4 h prior to stimulation for 24 h with either vehicle (−) or 1 nM TNFα in DMEM/0.1% FBS. Thereafter, cells were taken for either protein extraction and Western Blot analysis of ICAM-1, VCAM-1, and α-tubulin (A), or RNA extraction and qPCR analysis of ICAM-1 and VCAM-1 (B,C). The results are expressed as % of control transduced cells and are means ± S.D. The evaluation of the respective bands (A) is presented in Figure S3. (n = 3 for A, n = 4–6 for B–C; **** p < 0.0001 compared to the vehicle-treated control; # p < 0.05 compared to the TNFα-treated control; § p < 0.05, §§§§ p < 0.0001 compared to the vehicle-treated SPL-kd).
Figure 6
Figure 6
Effect of TNFα treatment on MCP-1, IL-6, and IL-8 expression in control and SPL-kd HCMEC/D3. A human MCP-1 ELISA kit was used to quantify secreted MCP-1 from vehicle and TNFα-stimulated HCMEC/D3 (A). (B–D): cells stimulated for 24 h with either vehicle (−) or 1 nM TNFα in DMEM/0.1% FBS were taken for RNA extraction and qPCR analysis using primers for MCP-1 (B), IL-6 (C), and IL-8 (D). Results in A are expressed as ng/mL MCP-1 in the supernatant and are means ± S.D (n = 3). The results in B–D are expressed as % of vehicle-treated control cells and are means ± S.D. (n = 3); ** p < 0.01, **** p < 0.0001 compared to the vehicle-treated control; ### p < 0.001, #### p < 0.0001 compared to the TNFα-treated control; §§ p < 0.01, §§§§ p < 0.0001 compared to the vehicle-treated SPL-kd.
Figure 7
Figure 7
Effect of SPL-kd on various signaling molecules and transcription factors. Confluent control (ctrl) or SPL-kd HCMEC/D3 cells were rendered serum-free for 4 h prior to stimulation with either vehicle (−), 1 nM TNFα (A) or 50 nM TPA (B) in serum-free DMEM for 10 min. Protein extracts were separated by SDS-PAGE and subjected to Western blot analysis using antibodies against phospho-p38-MAPK, total p38-MAPK, phospho-AMPKα, total AMPKα, phospho-c-Jun, total c-Jun, JunB, phospho-protein kinase C (PKC) substrates, phospho-MARCKS, and α-tubulin. The data show representative blots, out of 3–4 independent experiments, performed in triplicates.
Figure 8
Figure 8
Effect of TNFα on S1P (A) and dihydro-S1P (B) content in HCMEC/D3 cells. Confluent control (ctrl) and SPL-kd cells were rendered serum-free for 24 h prior to stimulation for 24 h with either vehicle (−) or 1 nM TNFα. Lipids were extracted and quantified by LC-MS/MS. The results are depicted as picograms per 1.5 × 106 cells and are means ± S.D. (n = 3; * p < 0.01, ** p < 0.001, **** p < 0.0001 considered statistically significant when compared to the vehicle-treated control; #### p < 0.0001 compared to the TNFα -treated control; §§§§ p < 0.01 compared to the vehicle-treated SPL-kd).
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References

    1. Stamatovic S.M., Keep R.F., Andjelkovic A.V. Brain endothelial cell-cell junctions: How to “open” the blood brain barrier. Curr. Neuropharmacol. 2008;6:179–192. doi: 10.2174/157015908785777210. - DOI - PMC - PubMed
    1. Abbott N.J., Patabendige A.A., Dolman D.E., Yusof S.R., Begley D.J. Structure and function of the blood-brain barrier. Neurobiol. Dis. 2010;37:13–25. doi: 10.1016/j.nbd.2009.07.030. - DOI - PubMed
    1. Weiss N., Miller F., Cazaubon S., Couraud P.O. The blood-brain barrier in brain homeostasis and neurological diseases. Biochim. Biophys. Acta. 2009;1788:842–857. doi: 10.1016/j.bbamem.2008.10.022. - DOI - PubMed
    1. Persidsky Y., Ramirez S.H., Haorah J., Kanmogne G.D. Blood-brain barrier: Structural components and function under physiologic and pathologic conditions. J. Neuroimmune Pharmacol. 2006;1:223–236. doi: 10.1007/s11481-006-9025-3. - DOI - PubMed
    1. Saunders N.R., Ek C.J., Habgood M.D., Dziegielewska K.M. Barriers in the brain: A renaissance? Trends Neurosci. 2008;31:279–286. doi: 10.1016/j.tins.2008.03.003. - DOI - PubMed

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