Fig 1: Acute exposure to inhaled e-cig aerosols containing PG with or without nicotine leads to increased levels of nicotinic acetylcholine receptors in mouse lungs. The protein abundance of nicotinic acetylcholine receptors was measured in lung homogenates using western blotting. ß-actin was used as an endogenous control. Representative blots for nAChR a3 and nAChR a7 levels in air, PG and PG with nicotine exposed male (A) and female (B) mice are shown. (C) The band intensity was measured by densitometry and data are shown as fold change relative to air group control; ß-actin was used as a loading control. Data are shown as mean ± SEM (n = 6/group; equal number of male and female mice were used; n = 3 for male and female groups respectively; *P < 0.05 significant compared with air-exposed control)
Fig 2: Lifelong choline supplementation alters the expression of the alpha7 nicotinic acetylcholine receptor (α7nAchR) within microglia. (a) Photomicrographs depicting the Cornus Ammonis 1 (CA1) of the hippocampus from APP/PS1 and NonTg mice fluorescently stained for α7nAchR and Iba1. Images taken at 40X; scale bar = 25 µm (n = 6 mice/group). (b) Quantitative analysis reveals a significant main effect of genotype, where the APP/PS1 mice have a significantly higher intensity of yellow pixels of α7nAchR/Iba1 colocalization than the NonTg mice (p < .01). Additionally, we find a significant main effect of diet, where the Ch+ groups show a significant reduction in α7nAchR/Iba1 colocalization than the CTL groups (p < .05). A genotype by diet interaction was found, where the APP/PS1 Ch+ mice show a significant reduction of α7nR/Iba1 colocalization compared to the CTL counterparts (p < .001). The center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. **p < .01, ***p < .001
Fig 3: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing a7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 µM–2 µM Lp(a) for 60 min (upper panel) or with 1 µM Lp(a) at 15, 30, and 60 min time points (lower panel), on a7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 µM–2 µM Lp(a) for 60 min (upper panel) or with 1 µM Lp(a) at 15, 30, and 60 min time points (lower panel), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 µM–2 µM Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, a7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 µM Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 µM Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 µM) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: a7-nAChR-targeting short hairpin RNA. *p < 0.05, **p < 0.01, and ***p < 0.001; GAPDH served as loading control.
Fig 4: The human monoclonal antibody, tocilizumab, disrupts Lp(a)-induced a7-nAChR/p38 MAPK signaling by attenuating inflammation in patient monocyte-derived macrophages and HCASMCs. (a) 3D chemical structures of tocilizumab with molecular formula C6428H9976N1720O2018S42 and molar mass 144987.06 g/mol. (b) Graphical representation of the effect of 1.25 µM–10 µM tocilizumab on the viability of HCASMCs or PMDMs. Representative western blot photo images and histograms showing how treatment with 1 µM Lp(a) and/or 2.5 µM–10 µM tocilizumab affects the expression of a7-nAChR, p-p38, and p38 proteins in (c) patient monocyte-derived macrophages or in (d) HCASMCs. *p < 0.05, **p < 0.01, and ***p < 0.001; GAPDH served as loading control. PMDM: patient monocyte-derived macrophage.
Fig 5: The apolipoprotein (a) component of Lp(a) interacts with and induces a7-nAChR expression in the monocyte-derived macrophages of patients with CAS. Graphical representation of the effect of 500 nM LDL or Lp(a) on the (a) relative expression of CHRNA7 mRNA or (b) relative luciferase reporter activity of a7-nAChR in the patient monocyte-derived macrophages. Histograms showing the effect of 100 nM–1000 nM Lp(a) on the (c) relative expression of CHRNA7 mRNA or (d) relative luciferase reporter activity of a7-nAChR in the patient monocyte-derived macrophages. (e) Methyllycaconitine (MLA) dose dependently inhibited the Lp(a)-induced activation of a7-nAChR. (f) Visualization of the direct molecular interaction between Lp(a) and a7-nAChR using the PyMoL molecular docking and visualization software. Complex formation criteria are indicated. (g) Immunofluorescence demonstrated induced expression of a7-nAChR after Lp(a) treatment and fluorescent-protein tagging showed a high correlation for protein localization with a-BTX. Original magnification ×200. DAPI (blue) served as a nuclear marker. *p < 0.05, **p < 0.01, and ***p < 0.001. Lp(a): lipoprotein(a); CHRNA7: gene encoding a7-nAChR.
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