Patients and treatments
All patients provided written consent to the study and received careful clinical and neurosurgical assessments before being enrolled into the trial, as we described previously [16, 20]. The inclusion and exclusion criteria are listed in the Additional files section. Briefly, patients with mild or moderate CSDH who had a minimal risk of cerebral hernia and did not need immediate surgery were included in the trial. They were randomized to receive ATO monotherapy (20 mg daily) or a combined regimen of ATO 20 mg daily and DEX for five weeks. DEX was given at 2.25 mg daily for 2 weeks followed by 1.5 mg daily for 2 weeks and then 0.75 mg daily for 1 week [20]. Patients were switched to surgery to remove haematomas when their neurological deficits deteriorated or when CT or MRI scan found haematoma enlargement and/or a midline shift of more than 1 cm.
During the trial, peripheral venous blood samples were collected at the baseline before treatment, 7 days and 5 weeks after the treatment started. Haematoma fluids were collected during surgery from patients who underwent surgery without the drug treatment and those who were switched to surgery after unsuccessful drug treatment. Blood samples were collected from these patients immediately before and 7 days after surgery. Venous blood samples were drawn into a serum separator tube and left to stand at room temperature for 30 min to ensure full coagulation to collect serum. CSDH fluids were collected into vacuum tubes and centrifuged at 2000g for 20 min at 4 °C to collect the supernatants. Both haematoma supernatants and serum samples were aliquoted and stored at −80 °C until analyses [21]. Cells in haematoma freshly collected from CSDH were immediately analyzed using flow cytometry.
Cell culture and drug treatments
It has been shown that haematoma fluid contains high levels of angiogenic factors than in blood, including vascular endothelial growth factor (VEGFA), IL-1β, IL-6 and IL-8. These factors are primarily produced by monocytes/macrophages and contribute to inflammation-induced formation and aberrant angiogenesis of the CSDH membrane [4, 22, 23]. In particular, VEGFA produced by infiltrating macrophages in the haematoma capsule is thought to play a major role in inducing ongoing rebleeding [24, 25]. This is because VEGFA is the most important proangiogenic factor involved in excessive microvascular permeability [9, 26, 27]. Macrophage-derived VEGFA or other angiogenic factors were one of major focuses of the study.
Cells from the human acute monocytic leukaemia cell line (THP-1; 3111C0001CCC000057, NICR, China), which a well-established monocytic cell line phenotypically homologous to primary human monocytes, can be differentiated into macrophages by phorbol-12-myristate-13-acetate (PMA) [28]. They were seeded in 6-well plates at a density of 1×106/well and cultured in the RPMI 1640 medium (C11875500BT, Gibco, New Zealand) supplemented with 10% fetal bovine serum (10091-148, Gibco, New Zealand) in a humidified 5% CO2 incubator. After stimulation with 0.1 μM PMA (Sigma-Aldrich, St Louis, MO) for 24 h, the cells were washed and cultured in the regulator growth medium for 24 h at 37 °C with the following treatments: (1) untreated control; (2) 100 μg/L lipopolysaccharide (LPS; Sigma-Aldrich); (3) LPS and 10 μM ATO (Pfizer, USA); (4) LPS and 0.1 μM ATO; (5) LPS and 1 μM DEX (Sigma-Aldrich); (6) LPS and 0.01 μM DEX; and (7) LPS and a combination of 0.1 μM ATO and 0.01 μM DEX. The treated cells and conditioned media were harvested for analyses.
Mass spectrometry (MS)
Serum samples and haematoma fluids were extracted with ethyl acetate, and cell samples were processed with acetonitrile for protein precipitation before LC-MS/MS. Ultra-high-performance liquid chromatography (UPLC) coupled with triple quadrupole MS was performed to detect ATO, its two active metabolites ortho-hydroxy-atorvastatin (o-ATO) and para-hydroxy-atorvastatin (p-ATO), and DEX in haematoma fluids, serum samples, and cultured cells. A Waters ACQUITY UPLC I-Class system (Waters, USA) equipped with an Acquity UPLC BEH C18 column (1.7 μm, 100×2.1 mm, Waters, USA) was coupled online to a Waters Xevo TQD IVD triple quadrupole mass spectrometer (Waters, Ireland) with electrospray ionization and selective reaction monitoring in positive ion mode. Solvent A (acetonitrile with 0.01% formic acid) and solvent B (0.01% aqueous formic acid) were used with a flow rate of 0.4 mL/min and the following solvent (time [min], vol% solvent B): 0.0, 80%; 1.0, 60%; 4, 10%; 4.5, 90%; 4.51, 80%; and 5, 80%. The injection volume was 10 μL.
Drugs in cells, including those binding to the cell membrane, were extracted in a lysis buffer. Some cells were treated with 0.25% trypsin-EDTA (25200-056, Gibco, New Zealand) to remove the surface-bound drugs before being lysed.
Flow cytometry
Cells were trypsinized and collected by centrifugation at 800 rpm for 5 min and washed with ice-cold PBS. They were double stained for 30 min at 4 °C with either fluorochrome-tagged antibodies against CD11b-421 (1: 1000, BioLegend, San Diego, CA, USA) and CD86-PE (1:1000, BioLegend) to identify the proinflammatory macrophages (M1) or the CD11b-421 antibody and CD163-PE/Cy7 (1:1000, BioLegend) to identify the anti-inflammatory macrophages (M2). After washing with PBS to remove excess antibodies, cells were analyzed using flow cytometry (LSRFortessaTM Cell Analyzer, Cat No. 647780P3 BD) and FlowJo version 10 (BD, San Jose, USA). Macrophage apoptosis was determined by the binding of APC annexin V (Apoptosis Detection Kit with 7-AAD, BioLegend) according to the manufacturer’s instructions. Briefly, 5×105 cells were incubated in 100 μL annexin V binding buffer containing 5 μL APC annexin V and 5 μL 7-AAD viability staining solution for 15 min at room temperature in the dark.
Cytokine and angiogenesis arrays
Cytokines, chemokines and angiogenesis-related proteins secreted by macrophages into the conditioned media were detected using human cytokine (R&D Systems, Minneapolis, USA) and angiogenesis (R&D Systems, Minneapolis, USA) array kits and quantified by pixel density using Quantity One software version 4.6.6 (Bio-Rad, USA) [21].
Immunoblots
Cultured macrophages were washed in ice-cold phosphate-buffered saline and lysed for 20 min on ice. The cell lysates were centrifuged at 13,000 rpm for 15 min at 4 °C to collect the supernatants. Total protein in the supernatant was quantified using the bicinchoninic acid assay (BCA1, Sigma-Aldrich). The volumes of cell lysates were standardized to an equal amount of protein (20 μg), mixed with 5X SDS sample containing 50 mM dithiothreitol, separated by 10% SDS-PAGE gels, and transferred onto nitrocellulose membranes. The membranes were blocked in 5% dried milk in TBS-T and 0.1% Tween 20, washed, and incubated with anti-IL-8 (ab18672, Abcam, Cambridge, UK, 0.1 μg/mL), anti-IL-1β (12242, CST, USA, 1:1000), anti-TGF-β1 (ab179695, Abcam, Cambridge, UK, 1:1000), anti-CD163 (ab182422, Abcam, Cambridge, UK, 1:1000), anti-CD206 (ab125028, Abcam, Cambridge, UK, 1:2000), anti-Scavenging Receptor SR-BI (ab217318, Abcam, Cambridge, UK, 1:2000), anti-Arginase-1 (9819, CST, USA, 1:1000), anti-OATP1A2 (ab221804, Abcam, Cambridge, UK, 1:1000) and β-actin (Sigma, Germany, 1:1000). After washing to remove excess antibodies, the membranes were incubated with appropriate secondary antibodies followed by chemiluminescence and fluorescence detection agent (Chemi XT4, Syngene, UK). Specific proteins were quantified by densitometry using the ImageJ analysis program (NIH, USA).
ELISA
VEGFA, IL-6 and endothelin-1 (ET-1) were quantified using sandwich ELISA kits (R&D Systems) according to the manufacturer’s instructions. The optical density was measured with a microplate reader (5250040, Varioskan Flash, Thermo, USA) set to 450 nm, with the correction wavelength set to 540 nm.
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
mRNAs for the genes encoding P-glycoprotein, Cytochrome P450 3A4 (CYP3A4) and IL-10 in cultured macrophages were quantitatively amplified using qRT-PCR. RNA expression was normalized to GAPDH. Table S1 lists the primers used in this study.
Statistical analysis
Categorical variables are presented as frequencies and percentages (no [%]) and were compared between groups using the Pearson chi-square test. Continuous variables are expressed as the mean ± standard deviation or median (25th–75th percentile), depending on a normal or skewed distribution of data determined by the Shapiro–Wilk test. Student’s t test, one-way analysis of variance (ANOVA) with Bonferroni correction, the Mann–Whitney U test or the Kruskal-Wallis H test were conducted according to the data distribution. Data were analyzed using SPSS version 22.0 (IBM, Chicago, USA), and a statistically significant level was defined as a P value of <0.05.