Afamin Levels and Their Correlation with Oxidative and Lipid Parameters in Non-diabetic, Obese Patients

Background: Afamin is a liver-produced bioactive protein and features α- and γ-tocopherol binding sites. Afamin levels are elevated in metabolic syndrome and obesity and correlate well with components of metabolic syndrome. Afamin concentrations, correlations between afamin and vitamin E, afamin and lipoprotein subfractions in non-diabetic, obese patients have not been fully examined. Methods: Fifty non-diabetic, morbidly obese patients and thirty-two healthy, normal-weight individuals were involved in our study. The afamin concentrations were measured by ELISA. Lipoprotein subfractions were determined with gel electrophoresis. Gas chromatography–mass spectrometry was used to measure α- and γ tocopherol levels. Results: Afamin concentrations were significantly higher in the obese patients compared to the healthy control (70.4 ± 12.8 vs. 47.6 ± 8.5 μg/mL, p < 0.001). Positive correlations were found between afamin and fasting glucose, HbA1c, hsCRP, triglyceride, and oxidized LDL level, as well as the amount and ratio of small HDL subfractions. Negative correlations were observed between afamin and mean LDL size, as well as the amount and ratio of large HDL subfractions. After multiple regression analysis, HbA1c levels and small HDL turned out to be independent predictors of afamin. Conclusions: Afamin may be involved in the development of obesity-related oxidative stress via the development of insulin resistance and not by affecting α- and γ-tocopherol levels.

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors—classical LDLR and alternative LOX-1—in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Apolipoprotein B and oxLDL levels in plasma of patients with diabetes, cardiovascular disease, and COVID-19

Elevated levels of low-density lipoprotein (LDL-X) cholesterol, apolipoprotein B (ApoB), and especially oxidized LDL in plasma are associated with an increased risk of cardiovascular disease (CVD). The aim of the study was to determine the levels of ApoB and oxLDL in the blood of patients with diabetes mellitus (DM), CVD and COVID-19. ApoB and oxLDL were determined using enzyme-linked immunosorbent assay kits (Elabscience, USA). The measurements were performed at an optical wavelength of 450 nm. It was found that ApoB and oxLDL levels in the blood of patients with diabetes and, especially, with COVID-19 are substantially higher than in the blood of healthy people. Blood levels of ApoB and oxLDL are higher in patients with both COVID-19 and diabetes or CVD as com pared to patients with COVID-19 without comorbidities. Thus, the levels of ApoB and oxidized LDL may be the promising markers of severe COVID-19.

An Untargeted Lipidomic Analysis Reveals Depletion of Several Phospholipid Classes in Patients with Familial Hypercholesterolemia on Treatment with Evolocumab

Rationale: Familial hypercholesterolemia (FH) is caused by mutations in genes involved in low-density lipoprotein cholesterol (LDL-C) metabolism, including those for pro-protein convertase subtilisin/kexin type 9 (PCSK-9). The effect of PCSK-9 inhibition on the plasma lipidome has been poorly explored. Objective: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, the plasma lipidome of FH subjects before and at different time intervals during treatment with the PCSK-9 inhibitor Evolocumab was explored. Methods and Results: In 25 FH subjects, heterozygotes or compound heterozygotes for different LDL receptor mutations, untargeted lipidomic revealed significant reductions in 26 lipid classes belonging to phosphatidylcholine (PC), sphingomyelin (SM), ceramide (CER), cholesteryl ester (CE), triacylglycerol (TG) and phosphatidylinositol (PI). Lipid changes were graded between baseline and 4- and 12-week treatment. At 12-week treatment, five polyunsaturated diacyl PC, accounting for 38.6 to 49.2% of total PC at baseline; two ether/vinyl ether forms; seven SM; five CER and glucosyl/galactosyl-ceramide (HEX-CER) were reduced, as was the unsaturation index of HEX-CER and lactosyl—CER (LAC-CER). Although non quantitative modifications were observed in phosphatidylethanolamine (PE) during treatment with Evolocumab, shorter and more saturated fatty acyl chains were documented. Conclusions: Depletion of several phospholipid classes occurs in plasma of FH patients during treatment with the PCSK-9 inhibitor Evolocumab. The mechanism underlying these changes likely involves the de novo synthesis of SM and CER through the activation of the key enzyme sphingomyelin synthase by oxidized LDL and argues for a multifaceted system leading to vascular improvement in users of PCSK-9 inhibitors.

Oxidized LDL Mediated Upregulation of ADAMTS-4 in Monocytes/Macrophages Involves The ROS-NFκB Pathway

ADAMTS-4 is a protease enzyme which involves in vascular remodeling and atherosclerosis. It was found to be upregulated in macrophages seen in atherosclerotic lesions. The aim of this study was to investigate the expression and regulation of ADAMTS-4 in oxLDL induced human monocytes/macrophages system. PBMCs isolated from human blood(hPBMCs), treated with oxLDL (50μg/ml) were used as the model system for the study. mRNA and protein expressions were studied by qRT-PCR, ELISA, and western blot analysis. ROS production and cell viability were determined by fluorescence imaging and MTT assay respectively. In the presence of oxLDL, monocytes get differentiated into macrophages, which were confirmed by the increased expression of CD-36, b- D glucuronidase activity and by the morphological changes. OxLDL increased the mRNA and protein expression of ADAMTS-4 and TIMP-3 in monocytes/ macrophages. A significant increase in the mRNA and protein expression of TNF-α was also observed in oxLDL treated cells compared to untreated control. In the presence of NAC, the ROS scavenger, the production of NFκB and ADAMTS-4 was decreased significantly. Our study suggests that oxLDL significantly upregulated the expression of ADAMTS-4 in the monocyte/macrophage system. OxLDL mediated upregulation of ADAMTS-4 in hPBMCs involves TNF-α and ROS- NFκB pathway.

Synergistic Effect of 3′,4′-Dihidroxifenilglicol and Hydroxytyrosol on Oxidative and Nitrosative Stress and Some Cardiovascular Biomarkers in an Experimental Model of Type 1 Diabetes Mellitus

The objective of this study was to assess a possible synergistic effect of two extra-virgin olive oil polyphenols, 3,4,-dyhydroxyphenylglycol (DHPG) and hydroxytyrosol (HT), in an experimental model of type 1 diabetes. Seven groups of animals were studied: (1) Nondiabetic rats (NDR), (2) 2-month-old diabetic rats (DR), (3) DR treated with 5 mg/kg/day p.o. HT, (4) DR treated with 0.5 mg/kg/day p.o. DHPG, (5) DR treated with 1 mg/kg/day p.o. DHPG, (6) DR treated with HT + DHPG (0.5), (7) DR treated with HT + DHPG (1). Oxidative stress variables (lipid peroxidation, glutathione, total antioxidant activity, 8-isoprostanes, 8-hydroxy-2-deoxyguanosine, and oxidized LDL), nitrosative stress (3-nitrotyrosine), and some cardiovascular biomarkers (platelet aggregation, thromboxane B2, prostacyclin, myeloperoxidase, and vascular cell adhesion protein 1 (VCAM-1)) were analyzed. The diabetic animals showed an imbalance in all of the analyzed variables. HT exerted an antioxidant and downregulatory effect on prothrombotic biomarkers while reducing the fall of prostacyclin. DHPG presented a similar, but quantitatively lower, profile. HT plus DHPG showed a synergistic effect in the reduction of oxidative and nitrosative stress, platelet aggregation, production of prostacyclin, myeloperoxidase, and VCAM-1. This synergism could be important for the development of functional oils enriched in these two polyphenols in the proportion used in this study.

Accumulation of LDL/ox-LDL in the necrotic region participates in osteonecrosis of the femoral head: a pathological and in vitro study

Background Osteonecrosis of the femoral head (ONFH) is a common but intractable disease that appears to involve lipid metabolic disorders. Although numerous studies have demonstrated that high blood levels of low-density lipoprotein (LDL) are closely associated with ONFH, there is limited evidence to explain the pathological role of LDL. Pathological and in vitro studies were performed to investigate the role of disordered metabolism of LDL and oxidized LDL (ox-LDL) in the femoral head in the pathology of ONFH. Methods Nineteen femoral head specimens from patients with ONFH were obtained for immunohistochemistry analysis. Murine long-bone osteocyte Y4 cells were used to study the effects of LDL/ox-LDL on cell viability, apoptosis, and metabolism process of LDL/ox-LDL in osteocytes in normoxic and hypoxic environments. Results In the pathological specimens, marked accumulation of LDL/ox-LDL was observed in osteocytes/lacunae of necrotic regions compared with healthy regions. In vitro studies showed that ox-LDL, rather than LDL, reduced the viability and enhanced apoptosis of osteocytes. Pathological sections indicated that the accumulation of ox-LDL was significantly associated with impaired blood supply. Exposure to a hypoxic environment appeared to be a key factor leading to LDL/ox-LDL accumulation by enhancing internalisation and oxidation of LDL in osteocytes. Conclusions The accumulation of LDL/ox-LDL in the necrotic region may contribute to the pathology of ONFH. These findings could provide new insights into the prevention and treatment of ONFH.

Neutrophil extracellular traps modulate inflammatory markers and uptake of oxidized LDL by human and murine macrophages

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1β, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages’ phenotype are altered under hypoxia.