Exposure to Concentrated Ambient Fine Particulate Matter Induces Vascular Endothelial Dysfunction via miR-21

Figures

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  Figure 1. Aortic arch permeability alteration in SD rats exposed to PM2.5. PM2.5 or normal saline was injected PM2.5 intratracheally at 4 mg/kg body weight in each SD rat once every 3 days for 4 weeks. (A) 2% Evans Blue (solution in normal saline) was injected into the caudal vein of SD rats, and the aortic arch was lavaged using normal saline and photographed after 90 min, after which the Evans Blue was extracted with formamide at 60 ˚C for 24 h and quantified at 620 nm. (B) Hematoxylin and eosin staining analysis of the aortic arch from SD rats treated or untreated with PM2.5. Labels indicate areas of endothelial injury. **P< 0.01. Bar = 50 μm.

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  Figure 2. miR-21 is involved in the PM2.5-induced increase in vascular endothelial cell permeability. Total RNAs were extracted from NS- or PM2.5-treated HUVECs and aortic arches of SD rats. (A) Affymetrix GeneChip miRNA 1.0 Array analysis of the differential expression miRNAs in NS or PM2.5 treated HUVECs. Hierarchical clustering was performed using 84 miRNAs selected from among 2,536 miRNAs. Each row corresponds to one miRNA, and the two columns correspond to the NC and PM2.5 groups. (B) Volcano plot of miRNA analysis. Plots of log P value versus log2fold change for HUVECs treated and untreated with PM2.5. The two vertical lines in each panel mark twofold changes. Red spots indicate miRNAs with highly significant fold changes. Labels indicate miR-21. (C) miR-21 expression was verified using qRT-PCR from HUVECs treated or untreated with PM2.5 (80 µg/mL) for 24 h. (D) The expression of miR-21 in the aortic arches of rats treated with NS or PM2.5 was measured using qRT-PCR. (E) Effect of PM2.5 on HUVEC monolayer permeability. Cells were cultured until confluent in transwell inserts and incubated with or without the miR-21 inhibitor for 24 h and then stimulated with PM2.5 (80 µg/mL) or left…

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  Figure 3. TIMP3/MMP9 is the direct target of miR-21 and is involved in miR-21-induced increases in vascular endothelial cell permeability underexposure to PM2.5. (A) The formation and regulation of miR-21 in inhibiting TIMP3 translation, and the putative binding site and mutation site of miR-21 in the TIMP3 3'UTR predicted by RNA22. (B) Luciferase assays indicated that miR-21 directly down-regulated the expression of TIMP3. psi-check2-TIMP3 3'UTR vector and the mutation vector were built, and then relative luciferase values were normalized to the group cotransfected with the NC. Renilla luciferase activity was normalized to firefly luciferase expression. (C) Western blotting shows the expression of TIMP3 and MMP9 in miR-21-overexpressing HUVECs. Western blotting (D) and IHC staining (E) show the expression of TIMP3 and MMP9 in NS- and PM2.5-treated HUVECs and aortic arches of SD rats. (F) Western blotting shows that the expression of TIMP3 was inhibited and MMP9 was up-regulated by PM2.5, and the effect was abolished by restraint of miR-21 in HUVECs. β-actin served as an internal control.*P< 0.05,**P< 0.01, ***P< 0.001. Bar = 50 μm.

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  Figure 4. VE-cadherin is regulated by miR-21 and is involved in PM2.5-induced increases in vascular endothelial cell permeability. Western blotting (A) and IHC staining (B) show the expression of VE-cadherin was inhibited in PM2.5-treated HUVECs and aortic arches of SD rats. (C) Western blotting shows that the expression of VE-cadherin was inhibited by PM2.5, and the effect was abolished by restraint of miR-21 in HUVECs. β-actin served as an internal control.*P< 0.05,**P< 0.01, ***P< 0.001. Bar = 50 μm.

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  Figure 5. p-STAT3 is involved in the promotion of transcriptional regulation of miR-21 in PM2.5-treated HUVECs. (A) Schematic representation of predicted binding sites of STAT3 in the human miR-21 gene and information on the STAT3 binding site mutations in the miR-21 promoter region. (B) Western blotting shows that p-STAT3 was up-regulated by PM2.5, but there was no differential expression of STAT3 in NS- and PM2.5-treated HUVECs or aortic arches of SD rats. β-Actin served as an internal control. (C) Luciferase assays show the effects of STAT3 on the reporter constructs containing the wild-type and mutant miR-21 promoter. pGL3-miR-21 promoter and the mutation vectors were built, and then the constructs were co-transfected into HEK-293T cells with vectors expressing STAT3 as well as the pRL-TK vector as a transfection control. A vector expressing red fluorescence protein (dsRed) serves as a negative control for STAT3. The relative luciferase activities are the ratio of Firefly/Renilla luciferase normalized to the negative control. The data of the luciferase assays are presented as the mean ± SD from three separate experiments. (D) Chromatin immunoprecipitation assays show the in vivo…

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  Figure 6. A schematic illustration of how PM2.5 might increase endothelial permeability.