LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling
Viorel Simion, … , Peter Libby, Mark W. Feinberg
Viorel Simion, … , Peter Libby, Mark W. Feinberg
Published October 6, 2020
Citation Information: JCI Insight. 2020;5(21):e140627. https://doi.org/10.1172/jci.insight.140627.
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Research Article Vascular biology

LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling

Abstract

Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor–deficient (LDLR–/–) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1β, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow–derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.

Authors

Viorel Simion, Haoyang Zhou, Jacob B. Pierce, Dafeng Yang, Stefan Haemmig, Yevgenia Tesmenitsky, Galina Sukhova, Peter H. Stone, Peter Libby, Mark W. Feinberg

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Figure 1

Identification of the lncRNA VINAS in lesional intima.

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Identification of the lncRNA VINAS in lesional intima. (A) RNA derived f...
(A) RNA derived from aortic intima of LDLR–/– mice (n = 3; each sample represents RNA pooled from 2 mice) that were placed on a high-cholesterol diet (HCD) for 0 (group 1), 2 (group 2), 12 (group 3), and 18 weeks after 6 weeks of resumption of a normal chow diet (group 4). (B) Venn diagram displays significantly dysregulated lncRNAs in genome-wide RNA-Seq profiling using EdgeR and no-overlapping reads (NOR) showing intersecting hits (n = 11), uniquely identified in EdgeR (n = 14) or NOR (n = 39), (log2 fold change [1.5]; FDR < 0.05). (C) Heatmap for 11 lncRNAs that were dynamically regulated with progression and regression of atherosclerosis (n = 3). (D) RNA-Seq results for VINAS across groups 1–4 obtained by RNA-Seq analysis and verified by RT-qPCR (n = 3). (E) RT-qPCR expression analysis for VINAS in different cell types (n = 3). (F) VINAS expression in body organs and PBMCs of 24-week-old C57BL/6 mice (n = 4). (G) To test the coding potential, VINAS sequence was cloned upstream of 3xFlag-Tag cassette, transfected in HEK293T cells, and immunoblotted for Flag antibody. Positive control was provided with the kit (representative of 3 experiments). (H) RNA from mouse extracellular cells (ECs) was isolated for polyA+ and polyA– enriched RNA and analyzed by real-time quantitative PCR (RT-qPCR) (n = 3). (I) RT-qPCR analysis for RNA derived from mouse ECs separated into cytoplasmic and nuclear fractions and normalized to the cytoplasmic fraction (n = 3). (J) RNA in situ hybridization for negative control and VINAS probes on paraformaldehyde-fixed mouse ECs. Scale bar: 5 μm. Data represent the mean ± SD. Statistical differences were calculated using unpaired 2-tailed Student’s t test except for multiple comparisons (E and F) in which 1-way ANOVA with Bonferroni’s correction was used. *P < 0.05, **P < 0.01, ***P < 0.001.