HUVEC re-established their hurdle integrity within hours, as the observed increased level of resistance in EA

HUVEC re-established their hurdle integrity within hours, as the observed increased level of resistance in EA.hy926 after incubation with conditioned moderate from HUVEC subsequently reduced further and fell below the worthiness Cyclosporin A of HUVEC (Figure 2E). Open in another window Figure 2 Evaluation of S1P-signaling in EA and HUVEC.hy926. elevated serum albumin leakage in bronchoalveolar lavage liquid (BALF). Lung ECs isolated from Spns2 lacking mice revealed elevated leakage of fluorescein isothiocyanate (FITC) tagged dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2. 0.05, ** 0.01, and *** 0.001. 3. Results 3.1. EC Barrier Stabilizing Function of S1P and S1PR1 To investigate the role of S1P in EC barrier function, the human endothelial cell line EA.hy926 and primary HUVEC Cyclosporin A were used. EA.hy926 represents a somatic cell hybrid of HUVEC and the lung epithelial carcinoma cell line A549. Quantitative PCR exhibited that both, HUVEC and EA. hy926 expressed mainly followed by = 3. (B) Flow Cytometric analysis cell surface expression of S1PR1 on EC before and after treatment with 1 M FTY720 overnight. means SEM, = 3. (C) Intracellular calcium responses in EA.hy926 and HUVEC upon stimulation with 100 nM S1P. Data were normalized to the response of 10 M ATP. Means SEM, = 3. (D) Resistance following treatment with 1 M S1P, normalized resistance values were taken at the time of the established maximum resistance after S1P treatment divided by resistance of carrier-treated control cells at the same time and are means SEM, = 3, ** 0.01, determined by two-sided SPERT Students t-test. Line plots represent one experiment out of three with black arrows indicating the addition of S1P or vehicle at the corresponding time. (E) Difference in initial non-stimulated resistance of EA.hy926 and HUVEC in ECIS measurements 60 h after seeding, means SEM, = 3, * 0.05, determined by a two-sided Students t-test. Line plot represents one experiment out of three. 3.2. Endogenous Differences in S1P Signaling between HUVEC and EA. hy926 To explore the reason for the different behavior of HUVEC and EA.hy926 in ECIS measurements, both cells were treated with 3 M of the S1PR1 antagonist W146. While EA.hy926 resistance was not affected by W146 treatment, HUVEC monolayers showed significantly reduced resistance by 60% in ECIS measurements, suggesting involvement of S1PR1 in constitutive basal EC barrier maintenance in HUVEC, but not in EA.hy926 (Determine 2A). A similar observation was recorded in ECIS measurements after treatment with the anti-S1P antibody Sphingomab. Sphingomab (120 g/mL) reduced the basal resistance of the HUVEC monolayer by 30%, while EA.hy926 did not respond at all (Physique 2B). Determination of S1P in the supernatant of both cell types revealed three fold greater S1P level in HUVEC medium than EA.hy926 medium Cyclosporin A (Figure 2C). Conditioned HUVEC medium consequently provided a four-fold enhanced calcium signal in S1PR1, overexpressing rat hepatoma HTC4 cells compared to EA.hy926 conditioned medium (Determine 2D). Conditioned medium from HUVEC induced a significant 20% increase of the measured resistance in ECIS experiments when added to EA.hy926, while conditioned medium from EA.hy926 in contrast reduced the corresponding resistance by 20% of a HUVEC monolayer (Physique 2E). HUVEC re-established their barrier integrity within hours, while the observed increased resistance in EA.hy926 after incubation with conditioned medium from HUVEC subsequently decreased further and fell below the value of HUVEC (Figure 2E). Open in a separate windows Physique 2 Comparison of S1P-signaling in HUVEC and EA.hy926. (A) Resistance following treatment with 3 M of the S1PR1 antagonist W146. Normalized resistance values were taken at the time of the established maximal change of resistance after W146 treatment divided by resistance of carrier-treated control cells at the same time and are means SEM, = 3, ** 0.001, determined by two-sided Students t-test. Line plots represent one experiment out of three with black arrows indicating the addition of W146 or vehicle at the corresponding time. (B) Resistance following treatment with 120 g/mL of the anti-S1P antibody Sphingomab. The difference in resistance is the difference between S1P-antibody treatment and isotype control antibody treatment taken at the time of maximal change of resistance after treatment. Shown are means SEM, = 3, *** 0.001, determined by a two-sided Students t-test. Line plot represents one experiment out of three with a black arrow indicating the addition of Sphingomab (S1P Ab) or isotype control antibody (Isotype Ctrl) at the corresponding time. (C) LC-MS/MS quantification.