Resistin contributes perivascular adipose tissue dysfunction in a rheumatoid arthritis mouse model
Authors: Fedoce Aline G. , Veras Flavio P. , Rosa Marcos H. , Silva Josiane F. , Paiva Isadora M. , Schneider Ayda H. , Machado Mirele R. , Cunha Fernando Q. , de Cassia Aleixo Tostes Rita 
Published: May 2022

BackgroundPatients with rheumatoid arthritis (RA) experience 50% more risk of mortality attributed to cardiovascular disease (CVD). PVAT dysfunction, which leads to vascular dysfunction, is driven by increased proinflammatory adipokines, and overactivation of immune cells. The proinflammatory adipokine resistin modulates vascular function, and circulating, synovial and serum resistin concentrations are increased in RA patients. We hypothesized that resistin causes PVAT dysfunction, inflammation and macrophage infiltration in a RA experimental model.MethodsAntigen‐induced arthritis (AIA) was induced in 12 weeks‐old C57BL/6 male mice. AIA immunized mice received mBSA (intraarterial injection, 10 µg in10 µl PBS/week) or PBS (10 µl) for 5 weeks. Disease activity was determined based on immune cells profile in lymph nodes, by flow cytometry, and measurement of the mediolateral knee joint diameter. Thoracic aorta, with or without PVAT, were isolated after five weeks of AIA onset for functional, cellular, and molecular assays. Data are represented as mean and standard error, and student´s T test (p<0.05) was used for statistical analysis. All the experiments were approved by the Ethics Committee on Animal Research of the FMRP,USP (protocol nº 15/2020).ResultsInguinal lymph nodes of AIA showed increased CD4+/IL‐17 cells compared to control [(%) AIA: 10.4 ± 1.06 vs. CT 1.8 ± 1.06, n=6] and the mediolateral knee diameter was increased in AIA compared to control mice [(mm) AIA 4.38 ± 0.06 vs. CT 3.50 ± 0.04, n=6]. Aorta from AIA mice had a dysfunctional PVAT, decreased phenylephrine (Pe) maximum responses (Emax) and no changes in Pe logEC50 compared to CT [Emax (mN): CT ‐PVAT 10.6 ± 0.3 vs. CT +PVAT 8.7 ± 0.2; AIA ‐PVAT 6.8 ± 0.3 vs. CT +PVAT 7.0 ± 0.2, n=6‐8]. 40 ng/ml of resistin for 4 hours compromised aortic PVAT function [Emax (mN): WT–PVAT + Resistin = 5,8 ± 0,1 vs. WT+PVAT + Resistin = 6,0 ± 0,1; CT ‐PVAT= 10,6 ± 0,3 vs. CT +PVAT 8,7 ± 0,2, n=7‐8]. Resistin concentrations were increased in the PVAT, plasma, and knee of AIA mice vs. control [(pg/ml) Serum: AIA 899.2 ± 11 vs. CT 837.9 ± 18; PVAT: AIA 217.0 ± 24 vs. CT 121 ± 18; Knee: AIA 28.3 ± 1.7 vs. CT 19.5 ± 1.1, n=4‐8). mRNA genemarkers of type 1 (M1) macrophages, including monocyte chemoattractant protein‐1 (CCL2), interleukin‐1beta (IL‐1b), inducible nitric oxide synthase (iNOS), and tumor necrosis alpha (TNFα) were increased in AIA PVAT [(‐∆∆ct) CCL2: AIA 2.6 ± 0.3 vs. CT 0.82 ± 0.10; iNOS: AIA 2,5± 0,7 vs. CT 0,6 ± 0,1; IL‐1b: AIA 2.0 ± 0.4 vs. CT 1.0; TNFα: AIA 1,2 ± 0,1 vs. CT 0,5 ± 0,0; n=5‐8, 2]. mRNA gene markers of type 2 macrophages (M2) such as resistin‐like molecule alpha (Retnla), L‐arginase (Arg1), Mannose Receptor C‐Type 1 (Mrc1) was increased in PVAT from AIA mice vs. control [(‐∆∆ct) Arg1: AIA 3,4 ± 0,6 vs. CT 0,8 ± 0,1; Retna AIA 2,0 ± 0,6 vs. CT 1,0 ± 0,2; n=5‐7, 2]. Flow cytometry analysis confirmed increased M1 and M2 markers in the PVAT of AIA mice [(% of cells) M1:F4/80+CD11b+:AIA 11,8 ± 1,6 vs. Sham 6,0 vs. 1,1; M2: CD206+CD11b+ :AIA 20,4 ± 1,7 vs. Sham 11,8 ± 1,2; n=6‐8].ConclusionAIA‐associated PVAT dysfunction, i.e. loss of its anti‐contractile effect, is linked to increased resistin and an inflammation profile involving macrophages‐related cytokines and chemokines.