Apolipoprotein (a)/Lipoprotein(a)‐Induced Oxidative‐Inflammatory α7‐nAChR/p38 MAPK/IL‐6/RhoA‐GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation‐Associated Development of Coronary Artery Spasm

Figures

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  Figure 1. Lp(a) levels positively correlate with patient monocytic α7-nAChR levels and are implicated in inflammation-associated CAS. (a) Box and whisker plot of the differential serum Lp(a) levels in control subjects and patients with CAS. Spearman dots and regression line plots showing the correlation between serum Lp(a) levels and (b) hs-CRP levels, (c) IL-6 expression, or (d) monocytic α7-nAChR expression, in patients with CAS. Spearman dots and regression line plots showing the correlation between serum Lp(a) levels and (e) IL-6 expression or (f) monocytic α7-nAChR expression, in the controls. R2: coefficient of determination; MFI: median fluorescence intensity; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  Figure 2. The apolipoprotein (a) component of Lp(a) interacts with and induces α7-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 α7-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 α7-nAChR in the patient monocyte-derived macrophages. (e) Methyllycaconitine (MLA) dose dependently inhibited the Lp(a)-induced activation of α7-nAChR. (f) Visualization of the direct molecular interaction between Lp(a) and α7-nAChR using the PyMoL molecular docking and visualization software. Complex formation criteria are indicated. (g) Immunofluorescence demonstrated induced expression of α7-nAChR after Lp(a) treatment and fluorescent-protein tagging showed a high correlation for protein localization with α-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 α7-nAChR.

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  Figure 3. Lp(a) preferentially induces patient monocyte-derived macrophage M1 polarization. Dots and line plot showing the expression profile of (a) CHRNA7, (b) CD163, (c) CD206, or (d) CD80 in the macrophages, resting monocytes, stem cells, stimulated monocytes, and T cells of patients with CAD using the GSE9820/GPL6255/GDS3690 dataset, n = 153. Flow cytometry cell count polygons (upper panel) and histograms (lower panel) depicting the effect of treatment with 500 nM LDL or Lp(a) on the (e) CD80 median fluorescence intensity or (f) CD206 median fluorescence intensity of CAS monocyte-derived macrophages. Histogram colors are green for control antibody and purple for target antibody. The macrophages were exposed to either Lp(a) or LDL for 24 hours. LDL: low-density lipoprotein; PE: phycoerythrin. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  Figure 4. Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α7-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 α7-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, α7-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…

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  Figure 5. The human monoclonal antibody, tocilizumab, disrupts Lp(a)-induced α7-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 α7-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.

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  Figure 6. Graphical abstract depicting how Lp(a)-triggered inflammation drives CAS through macrophage M1 polarization, activation of coronary VSMC, and α7-nAChR/p38 MAPK signal induction. Tocilizumab disrupts Lp(a)-induced α7-nAChR/p38 signaling by attenuating the inflammation in coronary VSMCs and patient monocyte-derived macrophages.