Fig 1: Schematic diagram of the signalling pathways regulated by NIPA1-SO. Our study has uncovered an athero-protective role of the lncRNA NIPA1-SO, which, by interacting with a single transcription factor, is capable of inhibiting monocyte adhesion and foam cell formation, two fundamental processes in atherosclerosis. The transcription factor FUBP1 negatively regulates NIPA1 expression; this inhibitory effect is increased by the interaction of NIPA1-SO with FUBP1, resulting in lower transcription of NIPA1. A reduction in NIPA1 protein results in increased BMPR2 protein due to a reduction in NIPA1-mediated BMPR2 endocytosis and degradation, leading to higher levels of Smad1/5/8 phosphorylation (pSmad1/5/8), which, through complexation with Smad4, inhibit transcription of the adhesion molecules VCAM1 and ICAM1, reducing monocyte adhesion to endothelial cells. Further, the pSmad1/5/8:Smad4 complex promotes ABCA1 and ABCG1 transcription, both of which promote cholesterol efflux via high-density lipoprotein (HDL) particles, thereby inhibiting foam cell formation.
Fig 2: NIPA1-SO and NIPA1 influence atherosclerotic plaque formation in mice in vivo. (A) En face analysis of aortas from LDLR-/-/NIPA1+/+ mice injected with lentivirus to overexpress NIPA1-SO or a control lentivirus (LV-Mock) was performed to demonstrate if NIPA1-SO is involved in atherogenesis in vivo (n = 5). Tissue samples were stained with Oil Red O and the lesion area quantified as a percentage of total en face area. Immunohistochemistry analysis of lesions in LDLR-/-/NIPA1+/+ mice overexpressing NIPA1-SO was performed to determine if the composition of atherosclerotic lesions in such mice differs from control mice. Data shown is the % of lesion area positive for Oil Red O (lipid) staining or Masson staining. (B) Aortic ring monocyte adhesion assays were performed on LDLR-/-/NIPA1+/+ mice injected with NIPA1-SO lentiviral particles or a control lentivirus as described in C (n = 5). (C) En-face dissection and subsequent Oil Red O lipid staining and immunohistochemical analysis of LDLR-/-/NIPA1+/+ and LDLR-/-/NIPA1-/- mice fed an atherogenic Western diet for 12 weeks was performed (n = 5). En face aorta samples were stained with Oil Red O (red) to visualize atherosclerotic plaques. Lesion areas, as a percentage of total en face area, are shown. Further analysis of atherosclerotic lesions from the two mouse strains was also performed using Oil Red O staining (quantifying the level of lipid as percentage of the lesion area). Hematoxylin (blue) and eosin (pink) (H&E) staining was also performed to visualize the nuclei and cytoplasm/extracellular matrix (respectively). H&E staining was quantified as a percentage of total lesion area. Immunohistochemistry was used to stain for monocytes (using the marker LGALS3) and smooth muscle cells (using a-smooth muscle actin) as indicated by brown staining. The abundance of these cell types as a percentage of total lesion area is shown. Staining with Masson’s trichrome stain was used to determine the quantity of collagen, as a percentage of total lesion area. (D) Immunohistochemistry of LDLR-/-/NIPA1+/+ and LDLR-/-/NIPA1-/- mice was performed for BMPR2, pSmad1, ICAM1, VCAM1, ABCA1 and ABCG1, with brown staining indicating the presence of the protein of interest. For all immunohistochemistry experiments, the level of each protein, relative to the control (LDLR-/-/NIPA1+/+) mice, is presented (n = 5). (E) An aortic ring monocyte adhesion assay was performed using LDLR-/-/NIPA1+/+ and LDLR-/-/NIPA1-/- mouse aorta samples to determine if NIPA1 knock-out alters monocyte adhesion to endothelial cells (n = 5). The aortic rings were labeled with calcein-AM and incubated with Dil–labeled mouse peritoneal monocytes. Data presented are the total number of monocytes adhered to the aortic ring for the two groups. Data are presented as the total number of cells adhering to the aortic endothelium. *P < 0.05 by unpaired 2-tailed Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig 3: NIPA1-SO, via BMPR2, increases ABCA1 and ABCG1 levels in THP-1 cells. (A and B) Western blotting of the cholesterol efflux-mediators ABCA1 and ABCG1 (A) and intracellular cholesterol concentration (B), in human monocytes (THP-1 cells) in response to lentiviral overexpression of NIPA1-SO and BMPR2 siRNA knockdown (n = 3). (C and D) Western blotting of ABCA1 and ABCG1 (C) and intracellular cholesterol concentration (D), in human THP-1 cells with NIPA1-SO shRNA knockdown and BMPR2 siRNA transfection (n = 3). Data in (A) and (C) are presented following normalization to ß-actin and untreated control cells. *P < 0.05 by one-way ANOVA with Tukey’s post hoc tests (A and C) or unpaired 2-tailed Student’s t-test (B and D).
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