Patients
Twenty-five JIA patients and twenty-five healthy donors (HDs) age-sex-matched with no history of autoimmune disease were included in this cross-sectional study. The participants were Caucasian and recruited at Rheumatology Department (pediatric unit), Reina Sofia University Hospital, Cordoba, Spain, after approval from the ethics committee and signed the informed consent.
The recruitment participant’s diagnoses were made according to the International League of Associations for Rheumatology (ILAR) criteria [19]. Disease activity was assessed using the Juvenile Arthritis Disease Activity Score-10 (JADAS-10), C reactive protein (CRP) [20] considering states of inactive disease, cutoff values ≤ 1 [21]. Remission state was determined according to Wallace criteria [22]. Regarding treatment regimen, 40% out of patients were in remission with no treatment (n=10) and 60% were under treatments (n=15) (Table 1). Four patients were on minimal doses of prednisone as maintenance treatment (maximum 5mg / day) at the time of the recruitment.
Peripheral blood samples were collected from patients and HDs following fasting for 8h for laboratory tests. Tests were performed in all patients to determine the presence of anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF). Besides, metabolic profile such as, glucose, insulin, hemoglobin 1Ac, total cholesterol (TC), HDL-cholesterol, LDL-cholesterol, triglycerides (TGs), apolipoprotein A (ApoA), apolipoprotein B (ApoB), acute phase reactants such as CRP and erythrocyte sedimentation rate (ESR) and complement factors as complement component 3 (C3) and component 4 (C4) were recorded (Table 1). Additionally, the presence of traditional cardiovascular risk factors including smoking, obesity based on body mass index (BMI > 30 Kg/m2), type 2 diabetes mellitus (T2DM) (fasting blood glucose levels > 126 mg/dL, hemoglobin A1c level > 6.5% or antidiabetic treatment) and hypertension were analyzed. Likewise, the prevalence of Metabolic Syndrome (MetSyn) was evaluated according to the National Cholesterol Education Program (NCEP) adult treatment panel III (ATP III) criteria, where 3 of the 5 following characteristics are met: abdominal obesity (male (>102cm); female (>88cm), TG > 150 mg/dL, HDL cholesterol (male (<40 mg/dL); female (<50 mg/dL); blood pressure > 130/85 mmHg; fasting glucose > 110 mg/dL).
Carotid intima media thickness
All subjects underwent high-resolution B-mode ultrasonography for carotid intima media thickness (CIMT) measurements. All ultrasound scanning was performed by a single experienced vascular sonographer on the left and right common carotid arteries, using carotid duplex equipment (LOGIC E9). IMT was measured at the distal wall of the carotid artery on a 10-mm segment and defined as the distance from the leading edge of the lumen-intima surface to the leading edge of the media–adventitia interface of the far wall.
Atherogenic risk
Atherogenic risk was calculated by atherogenic index (AI) based on the levels of TC (mg/dL) and HDL (mg/dL): AI=TC / HDL. Risk was delimited as > 4.5 in female and > 5 in male [23].
Apolipoprotein B/A risk
Apolipoproteins ratio was used to establish CVD risk due to the levels of apolipoproteins A and B. Relative CVD risk groups were: low CVD risk (female: 0.30-0.59; male: 0.40-0.69), moderate CVD risk (female: 0.60-0.79; male: 0.70-0.89) and high CVD risk (female: 0.8-1.0; male: 0.9-1.1). In this study, to calculate the prevalence of CVD risk by ApoB/ApoA ratio, subjects were separated in two groups: low CVD risk and moderate-high CVD risk [24, 25].
SCORE CVD risk
SCORE model was used to determined CVD risk based on traditional CV risk factors such as sex, age, systolic pressure, smoking, TC and HDL-cholesterol following EULAR recommendations for CVD risk [26].
Insulin resistance
The homeostatic model assessment-insulin resistance (HOMA-IR) index was used to measure IR: [insulin concentration (mU/L) x glucose concentration (mg/dL)]/405 (HOMA-IR values > 2.5 indicated IR) [27].
Serum levels of adipokines, cytokines and adhesion molecules
Serum levels of tumor necrosis factor-alpha (TNF-a), interleukin 1-beta (IL-1b), interleukin 6 (IL-6), intercellular adhesion molecule (ICAM-1), E-selectin and vascular endothelial growth factor A (VEGF-A) were quantified by enzyme-linked immunosorbent assay, following the manufacturer’s instructions (Bionova, Diaclone, Madrid, Spain). Serum levels of leptin, adiponectin (adipoQ), resistin (Bionova, Cusabio, Madrid, Spain) and visfatin (RayBiotech, Norcross, GA (EEUU)) were determined by ELISA following the manufacturer’s instructions.
Peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from JIA patients and HDs through Ficoll density gradient. Total RNA was extracted from PBMCs by using a RNA purification kit following the manufacturer’s instructions (Norgen Biotek Corp., ON, Canada). The RNA purity was verified by optical density (OD) absorption ratio OD260/OD280 between 1.8 and 2.0.
In vitro studies
Mechanistic studies were performed with PBMCs isolated from HDs and cell lines: human umbilical vein endothelial cells (HUVEC) and 3T3-L1 adipocytes (murine).
Isolated PBMCs from HDs were cultured in medium [RPMI 1640 containing 2mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 250 pg/mL fungizone (BioWhittaker/MA Bioproducts, Walkersville, MD, USA) at 37ºC in a humidified 5% carbon dioxide (CO2)] atmosphere and treated with 10% of inactivated serum (incubated at 56% for 30 mins) from JIA patients and HDs for 24 hours. Cells were collected for RNA isolation and applied to RT-PCR analysis.
HUVEC cells were purchased from ATCC (Manasas, VA, USA). Cells were cultured in Endothelial Cell Basal medium (EBM; Lonza, Walkersville, Md) with 10% FBS, 0.1% human epidermal growth factor (hEGF), 0.1% hydrocortisone, 0.1% gentamicin, amphotericin-B (GA-1000), 0.4% bovine brain extract, 100 U/mL penicillin, 100 mg/mL streptomycin, and 250 pg/mL fungizone (BioWhittaker/MA Bioproducts, Walkesvile, Md) at 37ºC in a humified 5% CO2 atmosphere. For in vitro experiments, HUVECs were seeded into 6-well plates (4x105 cells per well) in 1.5 mL of completed medium. After 24 hours, cells were treated with 10% of inactivated serum from JIA patients and HDs for 24 hours. Subsequently, cells were collected for mRNA analyses.
3T3-L1 cells were purchased from ATCC (Manasas, VA, USA). Cells were cultured and differentiated into adipocytes according to the protocol described previously (28). Differentiated cells were used when at least 90% showed an adipocyte phenotype by accumulation of lipid droplets by day 8. On day 8 of differentiation, cells were treated with medium containing 10% of inactivated JIA or HDs serum for 24h. Cells were harvested for total RNA isolation and applied to gene expression studies.
Gene expression analysis
The expression of genes involved in inflammation (TNF-a, IL-1b, IL-6, IL-8, interferon (IFN)-g, monocyte chemoattractant protein-1 (MCP-1), toll like receptor (TLR)-2 and TLR-4), oxidative stress (superoxide dismutase (SOD)-1, SOD-2, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and glutathione peroxidase (GPX)-1, endothelial dysfunction (VEGF-A, ICAM-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin) and adipokines (leptin, adiponectin, visfatin and resistin) was analyzed in PBMCs, endothelial cells and adipocytes through RT-PCR.
Real-time PCR using SYBR green was performed according to the manufacturer’s instructions (Thermo Fisher Scientific, Madrid, Spain). Expression of genes of interest was corrected by the geometrical average of b-actin, glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase using the BestKeeper tool (29).
Statistical analysis
Statistical analysis and graphs were performed by GraphPad Prism 8.0.1. Descriptive data are presented as the mean and standard deviation (SD) for continuous variables and as absolute frequencies and percentages for categorical ones. Kolmogorov-Smirnov test for normal distribution was carried out. To compare two independent groups (HDs vs JIA patients, JIA patients with treatments vs JIA patients with no treatment, cells treated with HD serum vs cells treated with JIA serum), following normality and equal variance tests, Student’s t-test or alternatively a non-parametric test (Mann-Whitney rank sum test) were used. Qualitative data was analyze using Chi-squared test (presence of cardiovascular risk factors). Correlations among the levels of serum adipocytokines, lipid profile and clinical parameters were assessed by Spearman’s rank correlation. p<0.05 was considered statistically significant.
Multiple testing correction with fold discovery rate approach was carried out in all the in vivo measures. In these cases, statistical significance was considered when p value< 0.05 and FDR<0.1.