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. 2017 Aug 14:8:925.
doi: 10.3389/fimmu.2017.00925. eCollection 2017.

DT-13 Ameliorates TNF-α-Induced Vascular Endothelial Hyperpermeability via Non-Muscle Myosin IIA and the Src/PI3K/Akt Signaling Pathway

Yuanyuan Zhang  1 , Yuwei Han  1 , Yazheng Zhao  1 , Yanni Lv  1 , Yang Hu  1 , Yisha Tan  1 , Xueyuan Bi  1 , Boyang Yu  1 , Junping Kou  1
Affiliations

DT-13 Ameliorates TNF-α-Induced Vascular Endothelial Hyperpermeability via Non-Muscle Myosin IIA and the Src/PI3K/Akt Signaling Pathway

Yuanyuan Zhang et al. Front Immunol. .

Abstract

DT-13(25(R,S)-ruscogenin-1-O-[β-d-glucopyranosyl-(1→2)][β-d-xylopyranosyl-(1→3)]-β-d-fucopyranoside) has been identified as an important factor in TNF-α-induced vascular inflammation. However, the effect of DT-13 on TNF-α-induced endothelial permeability and the potential molecular mechanisms remain unclear. Hence, this study was undertaken to elucidate the protective effect of DT-13 on TNF-α-induced endothelial permeability and the underlying mechanisms in vivo and in vitro. The in vivo results showed that DT-13 could ameliorate endothelial permeability in mustard oil-induced plasma leakage in the skin and modulate ZO-1 organization. In addition, the in vitro results showed that pretreatment with DT-13 could increase the transendothelial electrical resistance value and decrease the sodium fluorescein permeability coefficient. Moreover, DT-13 altered the mRNA and protein levels of ZO-1 as determined by real-time PCR, Western blotting, and immunofluorescence analyses. DT-13 treatment decreased the phosphorylations of Src, PI3K, and Akt in TNF-α-treated human umbilical vein endothelial cells (HUVECs). Further analyses with PP2 (10 µM, inhibitor of Src) indicated that DT-13 modulated endothelial permeability in TNF-α-induced HUVECs in an Src-dependent manner. LY294002 (10 µM, PI3K inhibitor) also had the same effect on DT-13 but did not affect phosphorylation of Src. Following decreased expression of non-muscle myosin IIA (NMIIA), the effect of DT-13 on the phosphorylations of Src, PI3K, and Akt was abolished. This study provides pharmacological evidence showing that DT-13 significantly ameliorated the TNF-α-induced vascular endothelial hyperpermeability through modulation of the Src/PI3K/Akt pathway and NMIIA, which play an important role in this process.

Keywords: DT-13; Src/PI3K/Akt; non-muscle myosin IIA; tight junctions; vascular endothelial hyperpermeability.

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Figures

Figure 1
Figure 1
DT-13 prevented TNF-α-induced vascular hyperpermeability and ZO-1 disassembly in vivo. (A) Photographs of ears after treatment with mustard oil (30 min). (B) OD measurements of extravasated Evans blue in mouse ears 30 min after topical application of mustard oil. (C) The expression of ZO-1 in the mouse aorta was measured by Western blotting. (D) Immunostaining of ZO-1 in the mouse aorta endothelium. Bar = 20 μm. The ZO-1 in green and the DAPI in blue. The data represent the mean ± SD from three experiments. ##P < 0.01 vs. the control group; **P < 0.01 vs. the TNF-α group.
Figure 2
Figure 2
DT-13 ameliorated TNF-α-induced endothelial hyperpermeability. (A) EC permeability was measured using a Millicell-ERS voltohmeter. Human umbilical vein endothelial cells (HUVECs) were pretreated with various concentrations of DT-13 (0.01, 0.1, and 1 µM) or dexamethasone (1 µM) for 1 h followed by TNF-α (10 ng/mL) stimulation for 4 h. (B) The transendothelial permeability was assessed using the paracellular transport marker (sodium fluorescein) permeability coefficient, which was measured using a fluorescence multiwall plate reader [Ex (λ) 485 nm; Em (λ) 530 nm]. (C) Ultrastructural characteristics of tight junctions (TJs) (magnification ×50,000). TJs were intact in the group control; TJs were disrupted in the TNF-α-induced group; TJs were not disrupted in HUVECs pretreated with DT-13 (1 µM). (D) The mRNA level of ZO-1 was determined by real-time PCR. High-dose and moderate-dose DT-13 groups showed increases in the mRNA level of ZO-1 following TNF-α treatment. (E) Western blotting analysis revealed the effects of high-dose DT-13 on the protein expression of ZO-1, whereas the moderate-dose DT-13 group exhibited no significant change. (F) ZO-1 localization within the TJs of endothelial cells is altered by TNF-α, which was inhibited by DT-13. The ZO-1 in green and the DAPI in blue. Bar = 50 μm. The data represent the mean ± SD from three experiments. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the TNF-α group.
Figure 3
Figure 3
DT-13 modulated Src/PI3K/Akt pathway proteins in human umbilical vein endothelial cells (HUVECs). (A) Western blotting analyses of Src, (B) PI3K, and (C) Akt were performed after 4 h of 10 ng/mL TNF-α stimulation with or without various concentrations of DT-13 (0.01, 0.1, 1 µM) pretreatment for 1 h. The blots are representative images from three separate experiments. DT-13 decreased the expression of p-Src, p-PI3K, and p-Akt in HUVECs. The data represent the mean ± SD from three experiments. ##P < 0.01 vs. the control group; **P < 0.01 vs. the TNF-α group.
Figure 4
Figure 4
The Src pathway was related to DT-13 regulation of tight junction function induced by TNF-α. Permeability was assessed by transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na-F) across the human umbilical vein endothelial cell (HUVEC) monolayer. (A) TEER was significantly elevated in HUVECs pretreated with 10 µM PP2. (B) Na-F permeability was significantly reduced in HUVECs pretreated with 10 µM PP2. Western blotting analysis for the effect of DT-13 on (C) ZO-1, (D) phosphorylation of Src, (E) PI3K, and (F) Akt in endothelial cells. The cells were pretreated with 10 µM PP2 for 1 h prior to addition of 1 µM DT-13 for 1 h and were then incubated with TNF-α for another 4 h for Western blot analysis. The data represent the mean ± SD from three experiments. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the TNF-α group.
Figure 5
Figure 5
PI3K regulated TNF-α-induced permeability downstream of Src. Permeability was assessed by transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na-F) across the human umbilical vein endothelial cell (HUVEC) monolayer. (A) TEER was significantly elevated in HUVECs pretreated with 10 µM LY294002. (B) Na-F permeability was significantly reduced in HUVECs pretreated with 10 µM LY294002. Western blotting analysis for the effect of DT-13 on (C) PI3K and (D) Src. Endothelial cells were pretreated with 10 µM LY294002 for 1 h before addition of 1 µM DT-13 for 1 h and then incubated with TNF-α for another 4 h for Western blot analysis. The data represent the mean ± SD from three experiments. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the TNF-α group.
Figure 6
Figure 6
Non-muscle myosin IIA (NMIIA) plays an important role in DT-13-attenuated TNF-α-induced endothelial hyperpermeability and related pathways. (A) The complex of DT-13 and proteins of NMMHC IIA. (B) 5 Å residues of NMMHC IIA around DT-13. Western blotting analysis for the effect of DT-13 on (C) Src, (D) Akt, and (E) PI3K. Endothelial cells were pretreated with NMIIA siRNA for 43 h before addition of 1 µM DT-13 for 1 h and then incubated with TNF-α for another 4 h for Western blot analysis. The data represent the mean ± SD from three experiments. #P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the TNF-α group.
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