Macrophage A2aR Alleviates LPS ‐Induced Vascular Endothelial Injury and Inflammation via Inhibiting M1 Polarisation and Oxidative Stress

Figures

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  FIGURE 1. LPS treatment reduces A2aR expression and causes the generation of oxidative stress. (A) Representative western blot (WB) images and quantification of A2aR protein expression levels in hearts of mice injected with LPS (5 mg/kg) or 0.9% normal saline (NS) for 24 h. (n = 5). (B) Quantitative PCR (qRT‐PCR) of A2aR mRNA expression levels (n = 10). (C) A2aR protein expression levels in primary bone marrow‐derived macrophages (BMDMs) after treatment with PBS or LPS (1 μg/mL, 12 h) (n = 3). (D) QRT‐PCR of tumour necrosis factor‐alpha (TNF‐α), IL‐1β and IL‐6 mRNA expression levels in mice hearts injected with LPS (n = 5–6). (E) QRT‐PCR of superoxide dismutase Type 1/2 (SOD1/2), glutathione peroxidase 1 (Gpx1) and heme oxygenase 1 (HO‐1) mRNA expression levels in mice hearts injected with LPS (5 mg/kg) or 0.9% NS for 24 h (n = 6). (F) Measurement of total reactive oxygen species (ROS) from PMA‐differentiated THP‐1 cells challenged with LPS (1 μg/mL, 12 h). ROS values normalised to protein concentration (n = 3). Error bars represent mean ± SEM. *p or #p < 0.05, **p or ##p < 0.01.

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  FIGURE 2. Knockout macrophage A2aR enhanced endotoxemia‐induced oxidative stress, vascular permeability and oedema. (A) Schematic illustration showed that WT and Mac‐A2aR KO mice were challenged with LPS (5 mg/kg, i.p., 24 h) or vehicle. Evans Blue was injected (i.v.) 1 h before the tissue collection. (B) The mRNA expression of SOD1, SOD2, Gpx1 and HO‐1 in lung tissues from the above groups (n = 3–4). (C) WB analysis of SOD1 protein expression levels in the heart (n = 3–4). (D and E) Vascular permeability of heart and liver measured by Evans blue‐conjugated albumin (EBA) extravasation. (D) Representative heart appearance after EBA administration. (E) Quantitative analysis of EBA extravasation in the liver (n = 2–3). (F) Intravascular near‐infrared fluorescence imaging of the heart and liver, with total fluorescence intensity calculation. (G) Dry‐to‐wet (W/D) weight ratio of liver (n = 4–6). (H) Heart histology. Magnification 40 ×, scale bar: 100 μm. Myocardial injury score (n = 3). (I) 24 h survival rates after LPS treatment (n = 8), Mantel–Cox log‐rank test. Error bars represent mean ± SEM. *p or #p < 0.05, **p or ##p < 0.01, ***p or ###p < 0.001, ****p or ####p < 0.0001.

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  FIGURE 3. Knockout macrophage A2aR aggregated endotoxemia‐induced cardiopulmonary vascular endothelial adhesion and injury in vivo. (A) Representative WB images and quantification of intercellular adhesion molecule‐1 (ICAM‐1) and E‐selectin protein expression levels in heart from WT or Mac‐A2aR‐KO mice after LPS (5 mg/kg, i.p.,24 h) challenge (n = 5–6). (B) Immunofluorescence images of E‐selectin and ICAM‐1 stained with platelet endothelial cell adhesion molecule‐1 (PECAM‐1) in lungs by immunofluorescence staining. Scale bar: 50 μm. Quantitative data illustrated the changes in relative mean fluorescent intensity (MFI) of ICAM‐1 and E‐selectin expression. Error bars represent mean ± SEM. *p or #p < 0.05, **p or ##p < 0.01.

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  FIGURE 4. Activation of A2aR attenuates LPS‐induced oxidative stress and vascular endothelial dysfunction in vivo. (A) Schematic illustration showed that CGS21680 (1 mg/kg) or dimethyl sulfoxide (DMSO) was instilled combined with LPS (5 mg/kg) challenge for 24 h. (B) SOD1, SOD2, Gpx1 and HO‐1 mRNA expression levels in the heart (n = 6–7). (C) The kidney W/D ratio was measured as W/D (n = 4). (D) Representative WB images and quantitation of ICAM‐1 protein expression in the aorta (n = 8). (E) Representative WB images and quantitation of ICAM‐1 and E‐selectin protein expression in the heart (n = 6). (F) Representative Immunofluorescence images and quantitation of relative ICAM‐1 MFI in pulmonary vascular endothelial marked by PECAM‐1 (n = 4). Scale bar: 50 μm. (G) Heart histology. Myocardial injury score (n = 3). Magnification 40 ×. (H) WT and Mac‐A2aR‐KO mice were treated with LPS (5 mg/kg) and CGS21680 (1 mg/kg) or PBS for 24 h. Assessment of kidney oedema (n = 3). (I) WB of E‐selectin protein expression levels in the heart (n = 5). (J) QRT‐PCR of ICAM‐1 mRNA expression levels in the lungs. *p or #p < 0.05, **p or ##p < 0.01, ***p or ###p < 0.001.

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  FIGURE 5. Disruption of macrophages A2aR aggregates LPS‐induced enhanced oxidative stress and macrophages M1 polarisation. (A) Schematics of BMDMs obtained from Mac‐A2aR‐KO mice or WT mice stimulated by LPS (1 μg/mL) for 12 h. (B) SOD1 mRNA expression levels (n = 4). (C) Representative images and quantification of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) protein expression levels (n = 4–5). (D) Macrophages sorted from heart and lung from Mac‐A2aR‐KO mice or WT mice after LPS (5 mg/kg, i.p.) or PBS treatment for 24 h. Flow cytometry analysis of macrophages in heart and lung (CD45 + CD11b + F4/80+), M1‐like macrophages (CD45 + CD11b + F4/80 + CD11c+) and M2‐ like macrophages (CD45 + CD11b + F4/80 + CD206+). (E) QRT‐PCR of TNF‐α mRNA expression levels in BMDMs (n = 4–5). *p or #p < 0.05, **p or ##p < 0.01, ***p or ###p < 0.001. ns represents no significant difference.

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  FIGURE 6. Activation of macrophage A2aR attenuates LPS‐induced oxidative stress and M1 polarisation, reducing TNF‐α production. (A) Intracellular total ROS was detected in PMA‐differentiated THP‐1 cells treated with vehicle, CGS21680 (10 nM), LPS (1 μg/mL) or LPS after 30 min CGS21680 pretreatment (n = 3). (B) QRT‐ PCR of SOD1 mRNA expression levels in BMDMs processed as (A) above (n = 3–4). (C and D) PMA‐differentiated THP‐1 cells were treated with vehicle, LPS (1 μg/mL), LPS after 30 min CGS21680 pretreatment (10 nM) or LPS after 30 min N‐acetyl‐L‐cysteine (NAC) pretreatment (20 mM); H2O2 (200 μM), H2O2 after 30 min CGS21680 pretreatment (10 nM) and H2O2 after 30 min NAC pretreatment (20 mM). WB of iNOS and/or SOD1 protein levels expression in cell groups (n = 4). (E) Intracellular total ROS was detected in PMA‐differentiated THP‐1 cells treated with vehicle, LPS (100 μg/mL) and interferon‐gamma (IFN‐γ) (20 ng/mL) with or without 30 min CGS21680 pretreatment before; interleukin (IL)‐4 (20 ng/mL) and IL‐13 (20 ng/mL) with or without 30 min CGS21680 pretreatment before (n = 3). (F) Flow cytometry analysis of macrophages sorted from WT mice heart after LPS (5 mg/kg, i.p.) with CGS21680(1…

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  FIGURE 7. Macrophage A2aR regulates endothelial cell overactivation. (A) QRT‐PCR of E‐selectin mRNA expression levels in human umbilical vein endothelial cells (HUVEC) cultured with conditioned medium (CM) of BMDMs from Mac‐A2aR‐KO mice or WT mice (n = 4–6). (B) WB and quantitative analysis of ICAM‐1 protein expression levels in HUVECs (n = 3). (C) Immunofluorescence staining for ICAM‐1 in HUVECs cultured with CM from RAW264.7 cells. Scale bar: 20 μm. (D) E‐selectin mRNA in HUVECs cultured with CM from PMA‐differentiated THP‐1 cells (n = 3). (E) WB and quantitative analysis of ICAM‐1 protein expression levels in HUVECs cultured with CM from RAW264.7 cells (n = 3). *p or #p < 0.05, **p or ##p < 0.01, ***p or ###p < 0.001 and ****p or ####p < 0.0001.