Oxidized low density lipoprotein

Fasting blood was obtained from all patients and plasma was stored at −20°C until analysis. Blood samples were collected from hemodialysis patients after an overnight fasting of 8 h, immediately before the start of a routine 4 h hemodialysis session, as described in previous studies [17 (link), 30 (link)]. Blood was drawn from all patients into EDTA-containing tubes and into tubes without anticoagulant in order to obtain plasma, whole blood, and serum. Samples for fasting blood glucose levels, HbA1c, total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), high-density lipoprotein cholesterol (HDL-cholesterol), triglycerides, and creatinine were transferred to the laboratory and assayed immediately. For adiponectin and ox-LDL, the samples were centrifuged immediately and plasma was stored at −20°C until analysis.
According to guidelines of American clinical practice (K/DOQI), the five stages of renal insufficiency based on eGFR are as follows: I ≥ 90, II = 60–89, III = 30–59, IV = 15–29, and V < 15 [9 (link), 31 (link)]. The glomerular filtration rate (GFR) was estimated (eGFR) using the chronic kidney disease epidemiology collaboration (CKD-EPI) equation, which is more accurate and less biased than the MDRD Study equation, especially in patients with higher GFR, resulting in reduced misclassification of CKD [32 (link)].
Plasma concentrations of total adiponectin and ox-LDL were quantitated by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (human adiponectin ELISA kit, Buhlmann, Switzerland; human ox-LDL ELISA kit, Mercodia, Sweden). According to the manufacturers, the detection limits for adiponectin and ox-LDL assays were 0.08 ng/mL and 0.3 U/L, respectively. Intra- and interassay coefficients of variation were <15% and <10% for adiponectin and ox-LDL, respectively.

Blood samples were collected via brachial puncture into 5-mL heparin vacuum tubes. The samples were centrifuged at 3000 rpm for 10 min at RT, and sera were separated and stored at −80 °C for further analysis (maximum, 6 months). The plasma levels of oxidized low-density lipoprotein (ox-LDL) were measured by using the LP-CHOLOX test carried out on an automated analyzer (Free Carpe Diem, Diacron International, Grosseto, Italy), using a commercial kit (Diacron International, Grosseto, Italy) according to the manufacturer’s instructions. The LP-CHOLOX test evaluates a class of hydroperoxides derived from the lipid peroxidation, which are mainly represented by oxidized cholesterol. Peroxides are able to promote the oxidation of the ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). The LP-CHOLOX test is based on the spectrophotometric measurement (at 505 nm) of the colored complex developed by the binding between the Fe³⁺ and the thiocyanate. The absorbance values are directly proportional to the lipoperoxides concentrations, and the values are related to specific standard solution (400 μEq/L). Results are expressed in μEq/L, and reference values are: Normal, ≤599 μEq/L; slight alteration, from 600 to 799 μEq/L; moderate alteration, from 800 to 999 μEq/L; strong alteration, ≥1000 μEq/L [54 (link),55 (link)].

Primary mouse brain endothelial cell isolation was performed using 6-month-old C57BL/6 wild type and transgenic mice based on methods from our group [12 (link)–14 (link)]. Animals were sacrificed with cervical dislocation. Mouse forebrains were collected in ice-cold sterile phosphate buffered saline (PBS). Meninges were removed using sterile filter paper and the tissue was cut into 1 mm³ using a scalpel and digested with enzymes (1 mg/ml collagenase type II and 15 µg/ml deoxyribonuclease type I, Roche, Switzerland) in Dulbecco’s modified Eagle medium (DMEM/F12, Gibco, Life Technologies, USA) at 37°C for 50 min. Microvessels were separated from myelin by centrifugation on a 20% bovine serum albumin (BSA)-DMEM gradient (1,000×g, 20 min, 3 times). The collected vessels were further digested by enzymes (1 mg/ml collagenase–dispase and deoxyribonuclease type I in DMEM, both from Roche) for 35 min. Digested cerebral vessels were washed three times in cell culture medium then seeded onto Petri dishes (60 mm; Orange Scientific, Belgium) coated with collagen type IV and fibronectin. Mouse brain endothelial cells were cultured in DMEM/F12 supplemented with plasma-derived bovine serum (15%; First Link, UK), heparin (100 µg/ml), basic fibroblast growth factor (1 ng/ml; Roche), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), and gentamycin (50 µg/ml). Cells were grown under selective culture conditions for the first 4 days. Culture medium contained puromycin (3 µg/ml) to eliminate P-glycoprotein negative contaminating cell types [15 (link)]. When endothelial cells reached 90% confluency on the 4th–5th day after seeding to the Petri dish, cells were subcultivated into 96-well plates (Corning, USA; ACEA, USA), cover slips (VWR, USA) and hanging cell culture inserts for different experiments, respectively. All surfaces were coated with collagen type IV and fibronectin. Endothelial cells were passaged at a cell number of 5 × 10³ cells/well for 96-well plates and 3.5 × 10⁴ cells/cover slips. Since primary brain endothelial cell isolation yield from mice is low compared to other species, 24-well format culture inserts were used for permeability studies (ThinCert, 24-well format, polyethylene terephthalate membrane, 0.33 cm² surface, 3 µm pore size, Greiner Bio-one, Germany). These culture inserts were found to be appropriate for BBB permeability studies [16 (link)]. For barrier integrity tests endothelial cells were seeded to the luminal side of culture inserts at a cell number of 2.5 × 10⁴ cells/insert. To enhance BBB characteristics, primary mouse endothelial cells were co-cultured with primary mouse cerebral glial cells and pericytes [17 (link)].
Primary cultures of mouse pericytes were prepared by the same protocol as primary mouse brain endothelial cells, except for a shorter time of the second enzymatic digestion (15 min) and the omission of the puromycin treatment. At the end of the isolation process cerebral microvessels containing pericytes beside endothelial cells were seeded into collagen type IV coated 60 mm culture dishes (Orange Scientific, Belgium). After 4 days of culture in DMEM, 10% fetal bovine serum (FBS), and gentamycin (50 µg/ml) attached cells reached 70% of confluency. Pericytes were passaged into bigger, uncoated dishes (Orange Scientific, Belgium). Mouse pericyte cultures were used at second passage and seeded at a cell number of 5 × 10³ cells/well for 96-well plates, 3 × 10⁴ cells/cover slips and 5 × 10³ cells/culture inserts. Primary mouse pericytes stain positive for α-smooth muscle actin but not for von Willebrand factor or glial fibrillary acidic protein (GFAP) [17 (link), 18 (link)].
Primary mouse glial cells were obtained from 1 or 2-day-old wild type or ApoB-100 transgenic mice [12 (link), 13 (link)]. Meninges were removed by fine forceps from brains. Little pieces of cortices were minced and mechanically dissociated by pressing the tissue through a nylon mesh (40 µm, Millipore, USA). Cell clusters were seeded onto uncoated 75 cm² flasks (TPP, Germany) and cultured in DMEM containing FBS (10%; Lonza, Switzerland) and gentamycin (50 µg/ml) until 90% confluency. Glial cells were passaged at a cell number of 5 × 10³ cells/well for 96-well plates, 3 × 10⁴ cells/cover slips and 10⁵ cells/wells for 24-well plates (Greiner Bio-One, Germany). For the BBB co-culture model mixed glial cells were cultured for 2 weeks before use. Confluent glia cultures contained 90% of astroglia (positive for GFAP), and 10% microglia (positive for CD11b).
To establish the triple co-culture BBB model culture inserts were put into 24-well plates containing confluent glia cultures. Mouse pericytes were subcultured to the under side of the culture membranes and mouse brain endothelial cells were passaged to the upper side of the coated inserts. Both compartments received endothelial culture medium [17 (link)]. After 2 days of co-culture, cells were kept in culture medium containing 550 nM hydrocortisone and 10 mM lithium chloride [19 (link), 20 (link)].

Blood samples were obtained using the brachial puncture method and placed into heparin vacuum tubes with a 5-mL capacity. After being centrifuged at 3000 rpm for 10 min at room temperature, the sera were isolated and stored at a temperature of − 80 °C for subsequent analysis within a maximum period of 6 months. The measurement of plasma levels of oxidized low-density lipoprotein (ox-LDL) was conducted through the employment of the LP-CHOLOX test on an automated analyzer known as Free Carpe Diem, provided by Diacron International, Grosseto, Italy. This test employed a commercial kit from the same manufacturer and was executed following the provided instructions. The LP-CHOLOX test assesses a category of hydroperoxides originating from lipid peroxidation, primarily comprising oxidized cholesterol. These hydroperoxides have the capability to induce the conversion of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺), thereby promoting oxidation. The LP-CHOLOX test is reliant on a spectrophotometric assessment conducted at a wavelength of 505 nm, measuring the development of a colored complex formed by the interaction of Fe3 + and thiocyanate. The resulting absorbance values exhibit a direct correlation to the concentration of lipoperoxides present, and these values are standardized against a specific solution (400 μEq/L). For all the analyzed samples, the calculated intra- and inter-assay CV% values were below t2.8% indicating the high reliability and precision of the used method. The outcomes are expressed in μEq/L units, with reference ranges established as follows: normal (≤ 599 μEq/L), minor alteration (600 to 799 μEq/L), moderate alteration (800 to 999 μEq/L), and significant alteration (≥ 1000 μEq/L) [19 (link)].