Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor ...(TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells.
Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography.
SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities.
SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.
Display omitted
•Serum of COVID-19 patients present high levels of mtDNA•SARS-CoV-2 infects endothelial cells, which express ACE2 and TMPRSS2 proteins•SARS-CoV-2 promotes mitochondrial dysfunction, mtDNA release, TLR9 activation, and cytokines release•mtDNA induces vascular dysfunction in wild-type, but not TLR9 KO mice•TLR9 antagonism reduces IL-6 production in endothelial cells infected by SARS-CoV-2
Display omitted
Cardiovascular disease (CVD) is the leading cause of death in rheumatoid arthritis (RA). Resistin is an adipokine that induces adipose tissue inflammation and activation of ...monocytes/macrophages via adenylate cyclase-associated protein-1 (CAP1). Resistin levels are increased in RA and might cause perivascular adipose tissue (PVAT) dysfunction, leading to vascular damage and CVD. This study aimed to investigate the role of resistin in promoting PVAT dysfunction by increasing local macrophage and inflammatory cytokines content in antigen-induced arthritis (AIA). Resistin pharmacological effects were assessed by using C57Bl/6J wild-type (WT) mice, humanized resistin mice expressing human resistin in monocytes-macrophages (hRTN+/-/-), and resistin knockout mice (RTN-/-) with AIA and respective controls. We investigated AIA disease activity and functional, cellular, and molecular parameters of the PVAT. Resistin did not contribute to AIA disease activity and its concentrations were augmented in the PVAT and plasma of WT AIA and hRTN+/-/- AIA animals. In vitro exposure of murine arteries to resistin impaired vascular function by decreasing the anti-contractile effect of PVAT. WT AIA mice and hRTN+/-/- AIA mice exhibited PVAT dysfunction and knockdown of resistin prevented it. Macrophage-derived cytokines, markers of types 1 and 2 macrophages, and CAP1 expression were increased in the PVAT of resistin humanized mice with AIA, but not in knockout mice for resistin. This study reveals that macrophage-derived resistin promotes PVAT inflammation and dysfunction regardless of AIA disease activity. Resistin might represent a translational target to reduce RA-driven vascular dysfunction and CVD.
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 20,4 ± 1,7 vs. Sham 11,8 ± 1,2; n=6‐8.
Conclusion
AIA‐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.
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).
The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target-organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target ...for cytokines. Considering that cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum from individuals with severe COVID-19 decreases endothelial cells' main antioxidant defense, i.e., the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from patients with severe COVID-19 at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from individuals with COVID-19 increased oxidant species, as indicated by higher DHE (dihydroethydine) oxidation, increased protein carbonylation, and induced mitochondrial reactive oxygen species (ROS) generation and dysfunction. Serum from patients with COVID-19, but not serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were decreased in endothelial cells exposed to serum from individuals with COVID-19. In addition, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-CoV-2 infection is linked to decreased endothelial antioxidant defense via IL-6-dependent mechanisms. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.
We demonstrate that endothelial cell dysfunction in SARS-CoV-2-infected individuals is linked to decreased activity of the major antioxidant system regulator, the Nrf2 transcription factor. We provide evidence that this phenomenon relies on IL-6, an important cytokine involved in the pathophysiology of COVID-19. Our data support the view that Nrf2 activation is a potential therapeutical strategy to prevent oxidative stress and vascular inflammation in severe cases of COVID-19.
Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of ...the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 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.
This study aimed to evaluate the skeletal muscle proteome of calves that received an intramuscular application of vitamin A at birth and its impacts on meat quality. Forty male crossbred calves were ...used from birth to feedlot finishing. On the day of birth, 20 calves were injected intramuscularly with a single dose of 300,000 IU of vitamin A and the other 20 animals received placebo (control). After weaning at 210 days, the animals of the two treatments were feedlot finished for 180 days. Longissimus muscle samples were collected for proteome analysis and meat quality assessment. There was a trend (P = 0.07) towards more tender meat in animals that received vitamin A compared to control (WBSF = 3.92 ± 0.16 kg vs. 4.23 ± 0.15). Meat color, pH and cooking loss were not affected (P > 0.05). Vitamin A treatment resulted in a more than 50% increase in intramuscular fat (IMF) content (P < 0.05) compared to control (4.10 ± 0.35% vs. 2.57 ± 0.27%). The intramuscular injection of vitamin A affected the post-mortem muscle proteome and the protein-protein interactions. A greater abundance of proteins involved in three main pathways were observed these being energy metabolism (GAPDH, ENO3, TPI1, CKM, and COX5A), muscle contraction (ACTB, ACTC1, ACTG1, ACTG2, ACTA1, ACTA2, ACTN1, ACTN2, ACTN3, TPM1, TPM2, TPM3, MYH1, PDLIM3, and TNNT3), protein binding, transport, and signaling (TUBA4A, VIM, TBA1B, and EEF1A2). A greater abundance of oxidative stress and cellular defense proteins (HSPA8 and DNAJC18) was specifically observed in the control treatment. Vitamin A upregulated key biological processes related to energy metabolism, which favored IMF accumulation during the finishing phase.
PDF
