Journal: Oxidative Medicine and Cellular Longevity

Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

doi: 10.1155/2022/9964689

Figure Lengend Snippet: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-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 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-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 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M 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 μ M–2 μ M 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, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) 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: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

Article Snippet: The primary HCASMCs (ATCC® PCS-100-021TM, American Type Culture Collection, Manassas, VA, USA) were cultured in smooth muscle cell growth medium 2 (#C-22062, PromoCell GmbH, Heidelberg, Germany), and all patient monocyte-derived macrophage cells were cultured in the RPMI-1640 culture media (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 μ g/mL streptomycin, and 50 U/mL penicillin in 5% CO 2 humidified atmosphere incubator at 37°C to 98%-100% confluence.

Techniques: Activation Assay, Western Blot, Derivative Assay, Expressing, Activity Assay, Fluorescence, shRNA, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

doi: 10.1155/2022/9964689

Figure Lengend Snippet: The human monoclonal antibody, tocilizumab, disrupts Lp(a)-induced α 7-nAChR/p38 MAPK signaling by attenuating inflammation in patient monocyte-derived macrophages and HCASMCs. (a) 3D chemical structures of tocilizumab with molecular formula C 6428 H 9976 N 1720 O 2018 S 42 and molar mass 144987.06 g/mol. (b) Graphical representation of the effect of 1.25 μ M–10 μ M tocilizumab on the viability of HCASMCs or PMDMs. Representative western blot photo images and histograms showing how treatment with 1 μ M Lp(a) and/or 2.5 μ M–10 μ M tocilizumab affects the expression of α 7-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.

Article Snippet: The primary HCASMCs (ATCC® PCS-100-021TM, American Type Culture Collection, Manassas, VA, USA) were cultured in smooth muscle cell growth medium 2 (#C-22062, PromoCell GmbH, Heidelberg, Germany), and all patient monocyte-derived macrophage cells were cultured in the RPMI-1640 culture media (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 μ g/mL streptomycin, and 50 U/mL penicillin in 5% CO 2 humidified atmosphere incubator at 37°C to 98%-100% confluence.

Techniques: Derivative Assay, Western Blot, Expressing, Control

Journal:

Article Title: The orphan nuclear receptor ROR? is a negative regulator of the inflammatory response

doi: 10.1093/embo-reports/kve007

Figure Lengend Snippet: Fig. 1. RORα is expressed in different vascular SMC types. (A and B) RT–PCR (35 cycles) analysis of RORα and GAPDH mRNA. C-PCR and C-RT are negative controls for PCR and RT, respectively; VSMC, smooth muscle cells from saphenous veins; CASMC, human coronary artery smooth muscle cells; HASMC, human aortic smooth muscle cells. (C) RT–PCR (30 cycles) analysis of RORα mRNA in SMC infected with Ad-RORα1 or Ad-GFP for 24 h. (D) Analysis of RORα protein expression in SMC infected for 24 h with or without Ad-RORα1. Immunocytochemistry experiments were performed as previously described (Chinetti et al., 1998) using a rabbit polyclonal RORα antibody raised against aa 163–225.

Article Snippet: Primary human aortic (HA) and coronary artery (CA) SMC (PromoCell, Heidelberg, Germany) and primary SMC from saphenous veins (VSMC: a kind gift of Dr Walsh, Boston, MA) were cultured under standard conditions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Immunocytochemistry