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Introduction: Human aortic tissues in vitro are tools to clarify the pathophysiological mechanisms of the cardiovascular system, cell culture, and transplants. Therefore, this study aims to analyze and compare the preservation of human aneurysmatic aortic tissues in three different solutions., Methods: Six human abdominal aortic aneurysms were obtained from patients after surgical ablation. The aorta samples were incubated in different solutions - 0.9% normal physiological saline solution, Ringer's lactate solution, and histidine-tryptophan-ketoglutarate solution (Custodiol®). Segments were collected at 0, 6, 24, and 48 hours. Creatine kinase and nitrate/nitrite were quantified for each incubation time. The tissue's alpha-smooth muscle actin was analyzed by immunofluorescence., Results: There was a significant increase in creatine kinase formation in the normal saline group at 0 and 48 hours and in the Ringer's lactate group at 0 and 48 hours (P=0.018 and P=0.028). The lower levels of creatine kinase and nitrate/nitrite and the aortic tissues' morphological integrity show that histidine-tryptophan-ketoglutarate has better tissue protection. These data suggest that histidine-tryptophan-ketoglutarate induces a protective effect on smooth muscle cells, with less tissue depletion in the aortic aneurysm., Conclusion: This study compared three preservation solutions with the potential for human abdominal aortic aneurysm tissue preservation. The histidine-tryptophan-ketoglutarate solution reduced tissue injury and improved tissue preservation in human abdominal aortic aneurysm tissue samples.

Purpose: Methylene blue (MB) has been used to increase blood pressure in patients with septic shock by acting on guanylate cyclase and nitric oxide synthase., Objective: To determine whether the administration of MB to patients in the initial phase of septic shock leads to a reduction in the use of vasopressors compared to the Control group., Methods: This was a 1:1 randomized clinical trial of two groups (MB and Control). Forty-two patients were included in the present study; 23 patients were allocated to the Control group, and 19 were randomized to the MB group. Both groups had access to standard treatment, consisting of fluid replacement, vasopressors, and antibiotic therapy. Patients received a loading dose of MB (3 mg/kg) and maintenance (0.5 mg/kg/h) for 48 h. Vasopressor doses, laboratory test results, inflammatory and anti-inflammatory cytokine levels, and hemodynamic monitoring were recorded before the infusion of MB (T1) and after 20 min (T2), 2 h (T3), 24 h (T4), 48 h after the infusion started (T5) and 24 h after weaning (T6)., Results: MB therapy was started together with the indication of vasopressin (VAS) as a second vasopressor. The MB group showed an immediate reduction in NOR dosage, an earlier reduction in VAS dosage, and higher IL-10 levels compared to the Control group., Conclusion: Early administration of MB in combination with standard treatment for septic shock might be reduce vasopressors dose. Continuous infusion of MB for 48 h was considered safe and there was no adverse events. These results highlight the potential of MB as a safe adjuvant therapeutic option in the treatment of septic shock., Clinical Trial Registration: https://clinicaltrials.gov/, identifier RBR-96584w4., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Luis-Silva, Menegueti, Peres, Sepeda, Jordani, Mestriner, Petroski-Moraes, Brito-de-Sousa, Costa-Rocha, Cruz, Donadel, de Souza, Reis, Bellissimo-Rodrigues, Basile-Filho, Becari, Evora, Martins-Filho and Auxiliadora-Martins.)

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Primary cell cultures are essential tools for elucidating the physiopathological mechanisms of the cardiovascular system. Therefore, a primary culture growth protocol of cardiovascular smooth muscle cells (VSMCs) obtained from human abdominal aortas was standardized. Ten abdominal aorta samples were obtained from patients diagnosed with brain death who were organ and tissue donors with family consent. After surgical ablation to capture the aorta, the aortic tissue was removed, immersed in a Custodiol® solution, and kept between 2 and 8 °C. In the laboratory, in a sterile environment, the tissue was fragmented and incubated in culture plates containing an enriched culture medium (DMEM/G/10% fetal bovine serum, L-glutamine, antibiotics and antifungals) and kept in an oven at 37 °C and 5% CO 2 . The aorta was removed after 24 h of incubation, and the culture medium was changed every six days for twenty days. Cell growth was confirmed through morphological analysis using an inverted optical microscope (Nikon®) and immunofluorescence for smooth muscle alpha-actin and nuclei. The development of the VSMCs was observed, and from the twelfth day, differentiation, long cytoplasmic projections, and adjacent cell connections occurred. On the twentieth day, the morphology of the VSMCs was confirmed by actin fiber immunofluorescence, which is a typical characteristic of VSMCs. The standardization allowed VSMC growth and the replicability of the in vitro test, providing a protocol that mimics natural physiological environments for a better understanding of the cardiovascular system. Its use is intended for investigation, tissue bioengineering, and pharmacological treatments., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Rationale: Methylene blue (MB) has been used to increase blood pressure in septic shock, acting on the activity of guanylate cyclase and nitric oxide synthase., Patience Concerns: The aim of this study is to demonstrate the benefit of MB in early phase of septic shock.Diagnoses: We report 6 cases of patients with septic shock with up to 72 hours of evolution., Interventions: We used MB after fluid replacement, use of norepinephrine and vasopressin. Patients received a loading dose of MB and maintenance for 48 hours., Outcomes: All patients presented a reduction in the dose of vasopressors and lactate levels soon after the administration of the loading dose of MB, an effect that was maintained with the maintenance dose for 48 hours. Interleukin 6 and interleukin 8 were elevated at the beginning of the septic condition, with a progressive and marked reduction after the beginning of MB infusion, demonstrating a role of MB in reducing the inflammatory activity., Lessons: This case series suggests that MB used early in the treatment of septic shock may be useful in reducing vasopressor dose and lactate levels. Further studies are still required to further validate these findings., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

In the present study, we hypothesized that endothelin (ET) receptors (ET A and ET B ) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3 -/- and caspase -/- mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ET A receptor antagonist reversed the effect. The ET B receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1β levels in a concentration-dependent manner (100 nM-10 μM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ET A - and ET B -mediated activation of NLRP3 in mouse CC via Ca 2+ -dependent ROS generation., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Respiratory symptoms are one of COVID-19 manifestations, and the metalloproteinases (MMPs) have essential roles in the lung physiology. We sought to characterize the plasmatic levels of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9) in patients with severe COVID-19 and to investigate an association between plasma MMP-2 and MMP-9 levels and clinical outcomes and mortality. MMP-2 and MMP-9 levels in plasma from patients with COVID-19 treated in the ICU (COVID-19 group) and Control patients were measured with the zymography. The study groups were matched for age, sex, hypertension, diabetes, BMI, and obesity profile. MMP-2 levels were lower and MMP-9 levels were higher in a COVID-19 group (p < 0.0001) compared to Controls. MMP-9 levels in COVID-19 patients were not affected by comorbidity such as hypertension or obesity. MMP-2 levels were affected by hypertension (p < 0.05), but unaffected by obesity status. Notably, hypertensive COVID-19 patients had higher MMP-2 levels compared to the non-hypertensive COVID-19 group, albeit still lower than Controls (p < 0.05). No association between MMP-2 and MMP-9 plasmatic levels and corticosteroid treatment or acute kidney injury was found in COVID-19 patients. The survival analysis showed that COVID-19 mortality was associated with increased MMP-2 and MMP-9 levels. Age, hypertension, BMI, and MMP-2 and MMP-9 were better predictors of mortality during hospitalization than SAPS3 and SOFA scores at hospital admission. In conclusion, a significant association between MMP-2 and MMP-9 levels and COVID-19 was found. Notably, MMP-2 and MMP-9 levels predicted the risk of in-hospital death suggesting possible pathophysiologic and prognostic roles., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Angiotensin-II (Ang-II), a major target for treatment of cardiovascular disease, promotes cardiovascular dysfunction by directly modulating structure and function of vascular cells. Inflammasome components are expressed in the vasculature and are activated by specific stimuli. However, whether Ang-II activates the inflammasome in vascular cells or inflammasome activation contributes to Ang-II-induced vascular damage is still not fully elucidated. We tested the hypothesis that Ang-II induces endothelial dysfunction, vascular remodeling, and high blood pressure via inflammasome activation. C57BL6/J wild type (WT) and Caspase-1 knockout (Casp1-/-) mice were infused with vehicle or Ang-II for two weeks (490 ng/Kg/day) to determine whether the inflammasome contributes to vascular damage induced by Ang-II. Rat Aortic Vascular Smooth Muscle cells (RASMC) were used to determine if the interaction between Ang-II and inflammasomes causes migration and proliferation of vascular smooth muscle cells. Ex vivo studies revealed that Ang-II infusion induced vascular oxidative stress, endothelial dysfunction and vascular remodeling in WT mice. Casp1-/- mice were protected against Ang-II-induced vascular injury. In vitro experiments, Ang-II activated the NLRP3 inflammasome in RASMC, i.e. Ang-II increased Caspase-1 (Casp1) activity and cleavage of pro-interleukin (IL)-1β. MCC950 (NLRP3 receptor antagonist) prevented Ang-II-induced vascular migration and proliferation, but failed to reduce reactive oxygen species production. In conclusion, Ang-II leads to inflammasome activation in the vasculature contributing to endothelial dysfunction and vascular remodeling. Taken together, we place inflammasomes as a possible therapeutic target in conditions associated with increased Ang-II levels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

NLRP3 plays a role in vascular diseases. Corpora cavernosa (CC) is an extension of the vasculature. We hypothesize that NLRP3 plays a deleterious role in CC relaxation. Male C57BL/6 (WT) and NLRP3 deficient (NLRP3 -/- ) mice were used. Intracavernosal pressure (ICP/MAP) measurement was performed. Functional responses were obtained from CC strips of WT and NLRP3 -/- mice before and after MCC950 (NLRP3 inhibitor) or LPS + ATP (NLRP3 stimulation). NLRP3, caspase-1, IL-1β, eNOS, nNOS, guanylyl cyclase-β1 (GCβ1) and PKG1 protein expressions were determined. ICP/MAP and sodium nitroprusside (SNP)-induced relaxation in CC were decreased in NLRP3 -/- mice. Caspase-1, IL-1β and eNOS activity were increased, but PKG1 was reduced in CC of NLRP3 -/- . MCC950 decreased non-adrenergic non-cholinergic (NANC), acetylcholine (ACh), and SNP-induced relaxation in WT mice. MCC950 did not alter NLRP3, caspase-1 and IL-1β, but reduced GCβ1 expression. Although LPS + ATP decreased ACh- and SNP-, it increased NANC-induced relaxation in CC from WT, but not from NLRP3 -/- mice. LPS + ATP increased NLRP3, caspase-1 and interleukin-1β (IL-1β). Conversely, it reduced eNOS activity and GCβ1 expression. NLRP3 plays a dual role in CC relaxation, with its inhibition leading to impairment of nitric oxide-mediated relaxation, while its activation by LPS + ATP causes decreased CC sensitivity to NO and endothelium-dependent relaxation.

Background: The pathogenesis of myocarditis that occurs in Trypanosoma cruzi-infected mice is still poorly understood. Therefore, it is important to know the mediators that trigger leukocyte migration to the heart as well as the cellular source of these possible mediators. In this study, we investigated (1) NO synthase (NOS) induction, (2) NO synthesis, (3) trypanocidal activity, and (4) chemokine and cytokine mRNA expression by isolated cardiomyocytes infected with T cruzi., Methods and Results: Mouse cardiomyocytes were isolated, infected with T cruzi, and evaluated for induction of inducible NOS (iNOS), nitrite production, trypanocidal activity, and cytokine and chemokine mRNA expression. We found that T cruzi-infected murine embryonic cardiomyocytes produced nitrite and expressed mRNAs for the chemokines chemokine growth-related oncogene, monokine induced by interferon-gamma, macrophage inflammatory protein-2, interferon-gamma-inducible protein, RANTES, and monocyte chemotactic protein, for iNOS, and for the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Separate addition of IL-1beta, interferon-gamma, TNF-alpha or monocyte chemotactic protein, macrophage inflammatory protein-2, and interferon-gamma-inducible protein, to cultured cardiomyocytes resulted in NO production but low trypanocidal activity. However, simultaneous addition of IL-1beta, interferon-gamma, and TNF-alpha or the chemokines to cultures resulted in the induction of iNOS, high levels of nitrite, and a marked trypanocidal activity. The iNOS/L-arginine pathway mediated the latter activity, inasmuch as it was inhibited by treatment with N:(G)-monomethyl-L-arginine., Conclusions: These results indicate that iNOS activation and the proinflammatory cytokines and chemokines produced by cardiomyocytes are likely to control parasite growth and cell influx, thus contributing to the pathogenesis of chagasic cardiomyopathy seen in T cruzi-infected mice.