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Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of pro-inflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Pro-inflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of severe COVID-19 patients might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Introduction Testosterone (Testo) modulates vascular tone and cardiac performance. Athletes who use Testo at supraphysiological doses exhibit increased blood pressure, higher inflammatory marker levels, vascular dysfunction, and cardiac hypertrophy. NLRP3 inflammasome activation, as part of the innate immune system response, contributes to proinflammatory cytokines production, leading e.g to cardiac dysfunction. Hypothesis We hypothesized that supraphysiological levels of Testo promotes cardiac dysfunction via generation of mitochondrial reactive oxygen species (mROS) and activation of the NLRP3 inflammasome. Methods Male, 12 week‐old wild type (WT) and NLRP3 knockout (NLRP3‐/‐) mice were used. Mice were treated with testosterone propionate [Testo‐P (10 mg/kg)] or vehicle for 30 days. Cardiac function was evaluated using echocardiography. After in vivo experiments, western blot and ELISA were performed to evaluate NLRP3 inflammasome components. ROS generation was evaluated by lucigenin. Bone marrow‐derived macrophages (BMDMs) were isolated, primed with lipopolysaccharide (LPS [500 ng/mL 4 h]) and stimulated with Testo [10‐7 M], for 4, 6, 12 and 24 h. ROS generation was evaluated by DHE fluorescence and MitoSOX. Results Echocardiography showed cardiac dysfunction in WT mice treated with Testo‐P, characterized by reduced ejection fraction, shortening fraction, cardiac output and systolic volume. Testo‐P also increased interventricular septum, left ventricle posterior wall and decreased left ventricle internal diameter. All these effects were observed in NLRP3‐/‐mice. Furthermore, WT mice treated with Testo‐P showed increased cardiac expression of NLRP3 receptor, active Caspase‐1 and IL‐1β levels. These effects were prevented in NLRP3‐/‐. In addition, increased ROS generation was observed in the left ventricle of WT mice, but not NLRP3‐/‐ mice, treated with TP. Testo‐P‐treated WT mice showed increased macrophage infiltrate in the left ventricle, and this effect was not seen in the WT vehicle mice. In in vitro experiments, BMDMs primed with LPS and stimulated with Testo showed increased expression of the NLRP3 receptor and IL‐1β levels after 12 and 24 h. In addition, Testo‐stimulated BMDMs exhibited increased mROS generation. Conclusion Supraphysiological levels of testosterone induce cardiac dysfunction via mROS generation and NLRP3 inflammasome activation. This study was approved by the Ethics Committee on Animal Experimentation of the Ribeirao Preto Medical School (020/2021).

Background Patients 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. Methods Antigen‐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). Results Inguinal 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 gene markers 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...