Article Figures & Data
Figures
- Fig. 1.
Protective effect of selepressin (SEL) against thrombin-induced endothelial barrier dysfunction. HLMVECs were pretreated for 48 hours with either vehicle (VEH; 0.01% DMSO) or 1 nM (A), 10 nM (B), 100 nM (C) selepressin and were exposed to either vehicle (PBS) or thrombin (1 U/ml). A gradual increase in endothelial permeability (reduced TEER) was observed in the thrombin-treated cells, which was reduced in selepressin-treated cells. Similarly, HLMVECs were pretreated for 72 hours with either vehicle (0.01% DMSO) or 1 nM (D), 10 nM (E), 100 nM (F) selepressin and were exposed to either vehicle (PBS) or thrombin (1 U/ml). A gradual increase in endothelial permeability (reduced TEER) was again observed in thrombin-treated cells, which was significantly prevented in 10 and 100 nM selepressin-treated cells. In all the cases, N = 4 per group. Means ± S.E. Two-way ANOVA for repeated measures followed by Bonferroni post-hoc test. Text color reflects the group that is being compared. Arrow indicates the time of addition of either thrombin or vehicle.
- Fig. 2.
Protective effect of selepressin against VEGF- and angiopoietin 2–induced endothelial barrier dysfunction. HLMVECs were pretreated for 72 hours with either vehicle (VEH; 0.01% DMSO) or 1 nM (Fig. 2A), 10 nM (B), or 100 nM (C) selepressin (SEL) and were exposed to either vehicle (PBS) or VEGF (100 ng/ml). A gradual increase in endothelial permeability (reduced TEER) was observed in the VEGF-treated cells, which was concentration dependently reduced in selepressin-treated cells. Similarly, HLMVECs were pretreated for 72 hours with either vehicle (0.01% DMSO) or 100 nM selepressin (D) and were exposed to either vehicle (PBS) or Ang2 (400 ng/ml). Selepressin significantly suppressed Ang2-induced hyperpermeability. N = 4 per group. Means ± S.E. Two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Text color reflects the group that is being compared. Arrow indicates the time of addition of VEGF, Ang2, or vehicle.
- Fig. 3.
Protective effect of selepressin against LPS-induced endothelial barrier dysfunction. HLMVECs were pretreated for 72 hours with either vehicle (VEH; 0.01% DMSO) or 1 nM (A), 10 nM (B), and 100 nM (C) selepressin (SEL) and were exposed to either vehicle (PBS) or LPS (1 EU/ml). A gradual increase in endothelial permeability (reduced TEER) was observed in LPS-treated cells, which was significantly suppressed in all selepressin-treated cells. N = 4 per group. Means ± S.E. Two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Text color reflects the group that is being compared. Arrow indicates the time of addition of either LPS or vehicle.
- Fig. 4.
Efficiency of selepressin blockade of increased permeability with edemogenic agents. Based on data in Figs. 1–3, the percent efficiency of blockade of increased permeability was calculated for each concentration of selepressin and thrombin, VEGF, or LPS, either with 48 or 72 hours pretreatment with selepressin.
- Fig. 5.
Vasopressin receptor inhibition abolishes the protective effects of selepressin toward LPS-induced endothelial hyperpermeability. (A) HLMVECs were pretreated with selepressin (SEL; 100 nM) and the V1AR antagonist atosiban (1 μM) or 0.01% DMSO [vehicle (VEH)] prior to vehicle (PBS) or LPS (1 EU/ml) exposure. Atosiban abolished the protective effects of selepressin against LPS, seen in Fig. 3. (B) HLMVECs were transfected with an irrelevant siRNA (siCTR) or siRNA against the V1AR (siV1AR) and then pretreated for 72 hours with either selepressin or vehicle (0.01% DMSO) prior to vehicle (PBS) or LPS (1 EU/ml) exposure. Treatment with siV1AR abolished the protective effects of selepressin against LPS, seen in Fig. 3. N = 4 per group. Means ± S.E. Two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Text color reflects the group that is being compared. Arrow indicates the time of addition of either LPS or vehicle. NTC, non-targeting control.
- Fig. 6.
Selepressin (SEL) protects HLMVECs against LPS-induced VE-cadherin and actin reorganization. HLMVECs grown on glass coverslips were treated with PBS (vehicle) or LPS (1 EU/ml) after 72 hours of pretreatment with either 0.01% DMSO (vehicle) or selepressin (1, 10, 100, 1000 nM). The cells were then fixed and double-stained for VE-cadherin and actin. Quantification of cortical VE-cadherin staining and of the cortical-to-total-actin distribution is shown in panels (B) and (C), respectively. #P < 0.05 vs. LPS, *P < 0.05 vs. vehicle. Means ± S.E.M. Three slides per group, 10 observations per slide. One-way ANOVA for independent samples followed by Bonferroni post hoc test.
- Fig. 7.
Silencing of vasopressin receptor 1a does not prevent the protective effects of selepressin against LPS–VE-cadherin reorganization but prevents selepressin (SEL) antagonism of LPS-induced gap formation. HLMVECs grown on glass coverslips were treated with irrelevant, control siRNA, or siRNA against the V1AR for 72 hours. After the transfection, the cells were exposed to either vehicle (0.01% DMSO) or selepressin (100 nM) prior to vehicle (PBS) or LPS (1 EU/ml) exposure. Cells were then fixed and double-stained for VE-cadherin and actin. Quantification of cortical VE-cadherin staining and endothelial gaps (expressed as percent coverage of slide surface by cells) is shown in (B). #P < 0.05 vs. negative control (Neg. Cont.) siRNA + LPS, *P < 0.05 vs. AVPR1A siRNA + SEL. Means ± S.E.M. Three slides per group, 10 observations per slide. One-way ANOVA for independent samples followed by Bonferroni post hoc test. AVPR1A, arginine vasopressin receptor 1A.
- Fig. 8.
Signaling pathways involved in the protective effects of selepressin (SEL) against LPS-induced endothelial barrier dysfunction. (A) Quantification of Western blot analysis of p53 expression after treatment of HLMVECs with either vehicle (VEH; 0.01% DMSO) or selepressin (1, 10, 100, and 10,000 nM) for 24 or 48 hours. p53 band signal intensity was analyzed by densitometry. Protein levels were normalized to β-actin. *P < 0.05 vs. vehicle. Means ± S.E.M. (B) Quantification of Western blot analysis of total and active Rac1 after treatment of HLMVECs with selepressin (100 nM) or vehicle (0.01% DMSO) prior to exposure to LPS (1 EU/ml for 1 hour) or vehicle (PBS). Signal intensity was analyzed by densitometry. Protein levels were normalized to total Rac1. *P < 0.05 vs. vehicle. Means ± S.E.M. (C) Western blot analysis of active RhoA and total RhoA levels after treatment of HLMVECs with selepressin (100 nm) or vehicle (0.01% DMSO) for 48 hours and post-treated with LPS (1 EU/ml) or vehicle (PBS) for 1 hour. The blots shown are representative of three independent experiments. Protein levels were normalized to RhoA. ***P < 0.001 vs. vehicle; $$P < 0.01 vs. LPS. (D) Quantification of Western blot analysis of phospho-MLC2 after treatment of HLMVECs with selepressin (100 nM) or vehicle (0.01% DMSO) for 48 hours prior to exposure to LPS (1 EU/ml for 1 hour) or vehicle (PBS). Protein levels were normalized to total MLC2. *P < 0.05 vs. vehicle. Means ± S.E.M. of three independent experiments. One-way ANOVA for independent samples followed by Bonferroni post hoc test.
