Isolation and primary culture of human abdominal aorta smooth muscle cells from brain-dead donors: an experimental model for vascular diseases.

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% CO2. 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.

Assessment of physicians' knowledge about brain death and organ donation and associated factors.

Precocity and assertiveness when diagnosing brain death are essential for identifying potential donors. To assess the knowledge of physicians about brain death and organ donation, cross-sectional web-based survey was carried out with physicians from different specialties. The knowledge about brain death and organ donation was assessed by a questionnaire with 12 multiple-choice or multiple-answer questions (possible range from 0 to 12). The nonparametric Mann-Whitney and Kruskal-Wallis tests were performed to verify the association between the physicians' knowledge and others variables. The project was approved by the Research Ethics Committee of the Hospital das Clínicas, Faculty of Medicine of Ribeirão Preto, University of São Paulo, under number 4.022.657, and all patients agreed to participate and provided free prior-informed consent. Three hundred sixty physicians were included in this study, most of them have postgraduate (55%) and 59.2% were intensive care physicians. The median of responses was 5 (obtained range from 0 to 10). The participants were classified in 2 groups: with satisfactory knowledge (scores above 5) or without satisfactory knowledge (scores equal/below 5). There was better performance among participants who: completed graduation between 6 and 10 years (P < .012); were intensive care physicians (P < .002); had participated in training courses (P < .001); and those who had worked in intensive care unit (ICU) from 6 to 10 years (P < .023); had performed over 10 brain death protocols (P < .001), and felt safe to talk to family members about brain death (P < .001). The results showed that the participants had low knowledge about diagnosis of brain death and organ donation protocols despite the majority working in ICUs. Be an intensive care physician, had large time experience in ICU, and had performed brain death protocols were associated with unsatisfactory knowledge concerning the subject.

MMP-2 and MMP-9 levels in plasma are altered and associated with mortality in COVID-19 patients.

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.