Fig 1.
(A) IL-1β Suppressed VE-cadherin Expression in a Time-dependent Manner. (B) VE-cadherin expression was suppressed by VEGF and pro-inflammatory cytokines at 24 hours. (C) and (D) 5-MTP inhibited IL-1β- (C) or TNF-α-induced (D) downregulation of VE-cadherin in a concentration-dependent manner. (E) and (F) 5-MTP protected VE-cadherin from VEGF and pro-inflammatory cytokines. Upper panels show representative Western blots and lower panels the densitometry. * indicates P < .05 compared to control and # indicates P < .05 compared to IL-1β or TNF-α group.
Fig 2.
5-MTP protects barrier function in vitro and in vivo.
(A) Analysis of 5-MTP (100 μmol/L) effect on endothelial permeability by an in vitro permeability assay with FITC-labeled dextran. Each bar denotes mean ±SD of three independent experiments. * indicates P < .05. (B) Mice were injected with L-5-MTP (23.4 mg/kg) for 30 minutes prior to challenge with LPS (60 mg/kg) for 24 hours. Evans blue dye was used to analyze pulmonary microvascular permeability as described in Methods. The error bars denote mean ± SD (n = 8 mice/group). * indicates P < .05 compared to control and # indicates P < .05 compared to LPS group. (C) Mice were injected with L-5-MTP (23.4 mg/kg) for 30 minutes prior to challenge with LPS (60 mg/kg) for 24 hours. BALF was collected by lavage. Total number of leukocytes was determined by cell counting. Total protein and albumin were analyzed. The error bars denote mean ± SD (n = 5 mice/group). * indicates P < .05 compared to control and # indicates P < .05 compared to LPS group.
Fig 3.
Endothelial cells release 5-MTP.
(A) Comparison of single mass spectra of HUVEC-CM vs. control medium and pure 5-MTP by UPLC-QTof MS. (B) Comparison of the chemical structure of 5-MTP with that of major 5-HTP metabolites. The dashed circles depict the active groups of 5-MTP. Melatonin (N-acetyl 5-methoxytryptamine) possesses 5-methoxy but not the carboxyl group due to decarboxylation. It has weak anti-COX2 action. 5-methoxytryptamine, a major catabolic product of melatonin, is inactive. Serotonin (5-hydroxytryptamine) is also inactive. (C) Analysis of daughters of the m/z 235.1 peak in HUVEC-CM vs. that of pure 5-MTP by LC-MS-MS. (D) Measurement of 5-MTP in HUVEC-CM by ELISA. “cont” denotes control medium. * indicates P < .05.
Fig 4.
HUVEC-CM prevents VE-cadherin reduction and endothelial hyperpermeability induced by pro-inflammatory mediators.
(A) and (B) HUVEC-CM protected VE-cadherin from VEGF and pro-inflammatory cytokines. The upper panel shows a representative Western blot and the lower panel the densitometry. * indicates P < .05. (C) Analysis of HUVEC-CM effect on endothelial permeability by an in vitro permeability assay with FITC-labeled dextran. Each bar denotes mean ±SD of three independent experiments. * indicates P < .05.
Fig 5.
5-MTP blocks p38 MAPK activation.
(A) and (B) Activation of p38, Src and ERK½ was determined in HUVECs treated with VEGF or pro-inflammatory cytokines. Upper panels show representative blots and lower panels show the densitometry of p-p38 MAPK. Error bars denote mean ± SD (n = 3). * indicates P < .05.
Fig 6.
SB202190 protects HUVEC barrier function.
(A) Pretreatment with SB202190 (10 μmol/L), a p38 inhibitor, attenuated VEGF- or pro-inflammatory cytokines-induced HUVECs permeability. Error bars denote mean ± SD (n = 3). * indicates P < .05 compared to control. (B) and (C) SB202190 protected VE-cadherin from VEGF and pro-inflammatory cytokines. Upper panels show representative blots and the lower panel, densitometry. Error bars denote mean ± SD (n = 3). * indicates P < .05.
Fig 7.
5-MTP suppresses pro-inflammatory cytokines-induced ICAM-1 and VCAM-1 expression.
Analysis of ICAM-1 and VCAM-1 expression in HUVECs treated with IL-1β or TNF-α for 24 hours. Pretreatment with 5-MTP (100 μmol/L) attenuated pro-inflammatory cytokines-induced ICAM-1 and VCAM-1 expression. The upper panel shows representative blots and the lower panels, densitometry. Error bars denote mean ± SD (n = 3). * indicates P < .05 compared to control and # indicates P < .05 compared to cytokine alone group.
Fig 8.
Pro-inflammatory cytokines downregulate 5-MTP production.
(A) Measurement of 5-MTP in HUVEC-CM by ELISA. HUVECs were treated with VEGF or pro-inflammatory cytokines for 24 hours. H2O denotes deionized water, Medium, control medium and cont, vehicle control. * indicates P < .05 compared to control. (B) Cell vitality is not affected by vascular permeability factors. Cell survival was analyzed by MTT assay. (C) Analysis of TPH-1 and TPH-2 transcripts in HUVECs treated with VEGF or pro-inflammatory cytokines for 24 hours. TPH-1 and TPH-2 transcripts were measured by RT-PCR. Upper panel shows a representative gel and the lower panel, densitometry of three experiments. Y79 retinoblast cells were included as reference. Cont indicates vehicle control. Error bars denote mean ± SD of three experiments. * indicates P < .05 compared to control.