Genome-wide differential expression profiling of lncRNAs and mRNAs in human induced pluripotent stem cell-derived endothelial cells exposed to e-cigarette extract

Abstract

Background: Electronic-cigarette (e-cig) usage, particularly in the youth population, is a growing concern. It is known that e-cig causes endothelial dysfunction, which is a risk factor for the development of cardiovascular diseases; however, the mechanisms involved remain unclear. We hypothesized that long noncoding RNAs (lncRNAs) may play a role in e-cig-induced endothelial dysfunction.

Methods: Here, we identified lncRNAs that are dysregulated in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following 24 h of e-cig aerosol extract treatment via microarray analysis. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analyses of the dysregulated mRNAs following e-cig exposure and constructed co-expression networks of the top 5 upregulated lncRNAs and the top 5 downregulated lncRNAs and the mRNAs that are correlated with them. Furthermore, the functional effects of knocking down lncRNA lung cancer-associated transcript 1 (LUCAT1) on EC phenotypes were determined as it was one of the significantly upregulated lncRNAs following e-cig exposure based on our profiling.

Results: 183 lncRNAs and 132 mRNAs were found to be upregulated, whereas 297 lncRNAs and 413 mRNAs were found to be downregulated after e-cig exposure. We also observed that e-cig caused dysregulation of endothelial metabolism resulting in increased FAO activity, higher mitochondrial membrane potential, and decreased glucose uptake and glycolysis. These results suggest that e-cig alters EC metabolism by increasing FAO to compensate for energy deficiency in ECs. Finally, the knockdown of LUCAT1 prevented e-cig-induced EC dysfunction by maintaining vascular barrier, reducing reactive oxygen species level, and increasing migration capacity.

Conclusion: This study identifies an expression profile of differentially expressed lncRNAs and several potential regulators and pathways in ECs exposed to e-cig, which provide insights into the regulation of lncRNAs and mRNAs and the role of lncRNA and mRNA networks in ECs associated e-cig exposure.

Keywords: e-cigarettes; endothelial dysfunction; fatty acid oxidation; iPSC-ECs; lncRNAs.

Fig. 1

Assessment of the biological effects of e-cig on iPSC-ECs. A Cell viability, B ROS production, and C caspase 3/7 activity were assessed by exposing cells to various concentrations (2–10 TPE) of menthol-flavored EAE for either 24 or 48 h. D Tube formation ability was also examined by exposing cells to 6.5 TPE EAE for 16 h to form capillary-like structure and then imaged via phase-contrast microscopy (10 x). E Representative images and quantitative data analysis of migration assays preformed on cells treated with 6.5 TPE EAE at 0, 16, and 24 h. F Permeability of ECs was measured by the transwell permeability assay with streptavidin–HRP and TMB following exposure to 6.5 TPE EAE for 24 h. G Flow cytometric data for the Annexin V and PI staining of cells treated with 6.5 TPE EAE. Data were obtained using iPSC-ECs from four healthy donors, and the assays were repeated three times. Data are represented as mean ± SD. * and ** indicate p < 0.05 and p < 0.001, respectively. Scale bars = 500 μm.

Fig. 2

Differentially expressed lncRNAs in iPSC-ECs treated with e-cig. A Volcano plot of differentially expressed lncRNAs is shown, where red and blue dots represent lncRNAs that are up- and downregulated, respectively, with at least a fold change of 2 and p value < 0.05. B Hierarchical clustering analysis showing differentially expressed lncRNAs between non-treated (control) and treated groups with e-cig (6.5 TPE EAE). C PCA of lncRNA expression profiles is shown, with treated and untreated samples connected for each subject. A qPCR validation of D up- and E downregulated lncRNAs following EAE treatment (6.5 TPE). Data were obtained using iPSC-ECs from four healthy donors, and the assays were repeated three times. Data are represented as mean ± SD. * and ** indicate p < 0.05 and p < 0.001, respectively

Fig. 3

Differentially expressed mRNAs in iPSC-ECs treated with e-cig. A Volcano plot of differentially expressed mRNAs is shown, where red and blue dots represent genes that are up- and downregulated, respectively, with at least a fold change of 2 and p value < 0.05. B Hierarchical clustering analysis showing differentially expressed mRNAs between non-treated (control) and e-cig (6.5 TPE)-treated group. C PCA of mRNA expression profiles is shown, with treated and untreated samples connected for each subject

Fig. 4

GO and KEGG pathway analyses of DEGs between e-cig treatment and controls. GO annotation of A up and B downregulated mRNAs with top 10 enrichment score covering domains of biological process (yellow), molecular function (purple), and cellular component (green). KEGG pathway enrichment analysis of C up and D downregulated mRNAs with the top 10 enrichment score

Fig. 5

Correlation between top 5 significantly upregulated and downregulated lncRNAs and mRNA transcripts. LncRNA-mRNA co-expression network was constructed with top 5 up- and downregulated lncRNAs and 353 associated mRNAs. Red nodes, blue nodes, and yellow nodes correspond to upregulated lncRNAs, downregulated lncRNAs, and coding genes/mRNAs. Solid lines signify a positive Pearson’s correlation coefficient (PCC), and dotted lines signify negative PCC. Coding–noncoding pairs are defined as co-expressing pairs if ∣PCC∣  ≥ 0.9 and p ≤ 0.05

Fig. 6

Assessment of metabolic changes in iPSC-ECs following e-cig treatment. A qPCR validation of FAO genes following e-cig treatment (6.5 TPE). B FAO, C glycolysis, and D glucose uptakes of iPSC-ECs following e-cig exposure (6.5 TPE) for 24 h (n = 3). Representative images and corresponding quantitative data of control and e-cig-treated cells taken with a fluorescence microscope (E, F) or flow cytometry (G). Live cells were stained with MitoTracker Red and MitoView Green. H ATP levels in cells treated with vehicle or e-cig (6.5 TPE) and/or 1.5 µM of oligomycin A for 48 h. Data are represented as mean ± SD, and * and ** indicate p < 0.05 and p < 0.001, respectively. Scale bars = 100 μm

Fig. 7

Assessment of the role of LUCAT1 in iPSC-ECs following e-cig treatment. A qPCR validation of LUCAT1 expression following knockdown with 25 µM siRNA for negative control (siNC) and for LUCAT1 (siLUCAT1). B Permeability, C ROS levels, and D, E representative images and corresponding quantitative data of migration assays were assessed in iPSC-ECs treated with either siNC, siLUCAT1, siNC + e-cig (6.5 TPE), or siLUCAT1 + e-cig (n = 3–4). Data are represented as mean ± SD, and * and ** indicate p < 0.05 and p < 0.001, respectively. Scale bars = 500 μm