Plasma Pattern of Extracellular Vesicles Isolated from Hepatitis C Virus Patients and Their Effects on Human Vascular Endothelial Cells

Abstract

Hepatitis C virus (HCV) patients are at increased risk of cardiovascular disease (CVD). In this study, we aimed to evaluate the role of extracellular vesicles (EVs) as pathogenic factors for the onset of HCV-related endothelial dysfunction. Sixty-five patients with various stages of HCV-related chronic liver disease were enrolled in this case series. Plasma EVs were characterized and used to stimulate human vascular endothelial cells (HUVEC), which were examined for cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) release. The results showed that EVs from HCV patients were mainly of endothelial and lymphocyte origin. Moreover, EVs were able to reduce cell viability and mitochondrial membrane potential of HUVEC, while increasing ROS release. Those harmful effects were reduced by the pretreatment of HUVEC with the NLR family pyrin domain containing 3 (NLRP3)/AMP-activated protein kinase and protein kinase B blockers. In conclusion, in HCV patients, we could highlight a circulating pattern of EVs capable of inducing damage to the endothelium. These data represent a novel possible pathogenic mechanism underlying the reported increase of CVD occurrence in HCV infection and could be of clinical relevance also in relation to the widespread use of antiviral drugs.

Plasma Redox Balance in Advanced-Maternal-Age Pregnant Women and Effects of Plasma on Umbilical Cord Mesenchymal Stem Cells

“…Plasma NO was quantified by the Griess assay (Promega Italia Srl, Milan, Italy) [23,25]. To achieve this, 5 mL plasma sample was deproteinated by adding 10 mL sulfosalicylic acid.…”

“…Cell viability of hUMSCs was investigated by using 1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT assay; Cayman Chemical), as previously per-formed [25,56]. Briefly, after the 10% plasma stimulation of hUMSCs (50,000 cells/well in 24-well plates), the media were removed and 200 µL of the MTT solution diluted in Dulbecco's Modified Eagle Medium high glucose w/o phenol red, supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin, was added to each well.…”

“…ROS release by hUMSCs was analyzed through the DCFDA assay, which evaluated the oxidation of 2,7-dichlorodihydrofluorescein diacetate into 2,7-dichlorodihydrofluorescein (Abcam, Cambridge, UK) [25,56]. To achieve this, 50,000 hUMSCs/well were plated in 24-Transwells plates in complete medium, as executed for MTT assay.…”

“…NO release by hUMSCs was quantified through the Griess assay (Promega) [25,56]. To achieve this, 50,000 hUMSCs were plated in 24-Transwells plates in complete medium and then the same experimental protocol followed for MTT and ROS assays was used.…”

“…Considering what has been reported above regarding the role of lipid peroxides, GSH and NO in the physiopathology of AMA pregnancy and the lack of detailed information, here, our primary endpoint was to examine the plasma levels of lipid markers of peroxidation, GSH and NO at the first trimester (T0), at the second trimester during the morphologic ultrasound (T1) and at 48-72 h after the delivery (T2) in pregnant women over 40 years (median age 41) compared with pregnant women younger than 40 years (median age 30). In particular, we used the thiobarbituric acid reactive substances (TBARS) assay for measurement of plasma lipid peroxides such as malonyldialdeide (MDA) and specific assays for GSH and NO, as previously conducted [23][24][25]. Secondly, we wanted to analyze the effects of plasma taken from AMA pregnant women at different time points on hUMSCs in order to highlight the role of circulating factors in the modulation of their viability and oxidant release.…”