VLDL Induced Modulation of Nitric Oxide Signalling and Cell Redox Homeostasis in HUVEC

2.1. Cell Culture

Human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) were cultured on 0.2% (w/v) gelatin-precoated flasks or multiwell plates (Falcon, BD Biosciences, USA) and grown at 37°C, 5% CO₂, 95% air in F-12K medium containing 1.26 g/L glucose, 0.1 mg/mL heparin, 0.03 mg/mL endothelial cell growth supplement (ECGS), 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1.5 g/L sodium bicarbonate.

The day before the experiments, cells were depleted of serum for 4 h and thereafter incubated in serum-free medium for 24 h with native VLDL (n-VLDL) or oxidised VLDL (ox-VLDL), at the concentration of 75 or 140 μg protein mL⁻¹, the former assayed only in a subset of experiments.

Controls were carried out in the absence of VLDL, under otherwise identical conditions. When necessary, HUVEC were harvested by trypsinization and centrifugation (900 ×g) and carefully suspended in the working medium, at suitable density (see text). None of the treatments affected cellular viability of HUVEC, as determined by cell counts, morphology, and trypan blue exclusion analysis.

Cell lysis was performed by CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich, USA) in the presence of the Protease Inhibitor Cocktail (Sigma-Aldrich, USA); protein content was determined according to bicinchoninic acid (BCA) assay (Sigma-Aldrich, USA), and citrate synthase activity, considered as representative of the mitochondrial mass, was measured as described [40].

2.2. VLDL Isolation and Ethics Statement

n-VLDL (density: 0.95–1.006 kg/L) were isolated from human sera by preparative ultracentrifugation following the procedure described by Redgrave et al. [41]. Serum samples were collected from typically 40–50 healthy volunteers of age comprised between 20 and 40 years (gender equally represented) after 12 h fasting, in order to minimize the biological variability among the preparations. The presence of chylomicrons in fresh sera was eliminated centrifuging for 30 min at 15,000 rpm. Then, n-VLDL were obtained by ultracentrifugation at 105,000 ×g, for 18 h at 15°C in a 1.006 kg/L density solution. After ultracentrifugation, the supernatant from the top of each tube was carefully aspirated and pooled. n-VLDL were dialysed against 0.25 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS), using a membrane (molecular weight cut-off: 6–8 kDa) at 4°C overnight. All VLDL preparations were filtered through a sterile 0.45 μm filter and, after the evaluation of protein concentration by the BCA (~500 μg/mL), were stored at −70°C until used. The purity of isolated VLDL was checked on agarose gel and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), staining with a lipid-specific dye (Sudan Black B) and a protein-specific dye (Coomassie Blue R 250), respectively.

n-VLDL with high degree of purity were obtained as outcome by its apolipoprotein composition determined by SDS-PAGE (Supplementary Figure 1(a) available online at https://doi.org/10.1155/2017/2697364) and characteristic mobility (Supplementary Figure 1(b)) [42, 43]. Human sera were collected anonymously by the Immunohematology Laboratory, Policlinico Umberto I, Sapienza Università di Roma. The procedure described has been approved by the Ethics Committee of Sapienza University (Prot. number 3610-2015).

2.3. VLDL Oxidation

VLDL oxidation was carried out according to Guha and Gursky [44] using Cu²⁺ ions (CuSO₄). Briefly, a solution of purified n-VLDL, at the concentration 0.2–0.4 mg mL⁻¹ in EDTA-free PBS, was incubated with CuSO₄, 40 μM for 6 h; the reaction was then stopped using 1 mM EDTA. Conjugated dienes and thiobarbituric acid-reactive substances (TBARS) were quantified in order to monitor lipid peroxidation [45, 46] (Supplementary Figure 1(c) and (d), resp.).

2.4. Real-Time Polymerase Chain Reaction (PCR)

After HUVEC incubation, for 24 h, with n-VLDL or ox-VLDL (140 μg mL⁻¹), total RNA was extracted using the Nucleo Spin® RNA isolation kit (Macherey-Nagel, Germany), according to the manufacturer’s instructions. Total RNA, 1 μg, was used for the reverse transcription reaction using RT² First Strand Kit (Qiagen, Germany). The cDNA obtained was hybridised to the Human Nitric Oxide Signalling Pathway RT² Profiler PCR Array (Qiagen, Germany).

This array contains oligonucleotide primers matching 84 genes whose expression is controlled by or involved in the signalling of NO, superoxide metabolism, and response to oxidative stress (see Table 1). The array includes also genes that are known to be induced or repressed by NO and whose finding therefore can be used as indicator for the activation of NO cell pathways.

2.5. Western Blot

After HUVEC treatments, cells (1 × 10⁶) were lysed with CelLytic MT Reagent (Sigma-Aldrich, USA) in the presence of Protease Inhibitor Cocktail (Sigma-Aldrich, USA). The proteins were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on nitrocellulose membranes (Whatman, GE Healthcare, UK), 1 h at 100 mA. After 2 h blocking (PBS with 0.1% Tween 20 and 3% BSA), the membranes were incubated overnight at 4°C with primary mouse monoclonal anti-phospho-Ser1177 and anti-phospho-Thr495 antibodies (BD Transduction Laboratories, USA). Thereafter, the membranes were stripped and reprobed for monoclonal purified mouse anti-eNOS antibodies (BD Transduction Laboratories, USA); α-tubulin was used as the reference. The enhanced chemiluminescence (ECL) Horseradish Peroxidase (HRP) anti-mouse secondary antibody (Jackson, Baltimore, PA, USA) was incubated 1 h at 25°C, and chemiluminescence was determined (Amersham, GE Healthcare, UK). Densitometric analysis was carried out by the ChemiDoc™ MP Image Analysis Software (Bio-Rad, USA).

2.6. ROS Quantification

Cell ROS generation was assessed by using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Sigma-Aldrich, USA), with modifications respect to the protocol generally adopted for adherent cells.

Briefly, HUVEC (70–75% confluent) were incubated with VLDL for 24 h as already described, then trypsinized, and pelleted at 900 ×g for 5 min at 20°C. Cell pellets were resuspended in Hank’s buffer at the density 1 × 10⁵ cells mL⁻¹ and plated in 24-well (black) plates.

Immediately after the addition of 10 μM DCFDA, the relative fluorescence emission was followed kinetically at 520 nm for 60 min (VICTOR™ Multilabel Counter, PerkinElmer, USA). The value of ROS production is taken at 30 min.

2.7. Nitrate/Nitrite Determination

The accumulation of nitrate/nitrite (NOx) in the culture medium was assessed by using the fluorescent probe 2,3-diaminonaphthalene (DAN) (Fluorometric Assay Kit, Cayman Chemical, USA).

After 24 h incubation of HUVEC (~2.5 × 10⁵ cell/mL) with n-VLDL or ox-VLDL, the culture supernatant was centrifuged at 4°C, 1000 ×g for 10 min and the NOx content was measured by adding DAN according to the manufacturer’s instructions and using a Fluorescence Plate Reader VICTOR Multilabel Counter (PerkinElmer, USA).

2.8. 3-Nitrotyrosine Quantification

The level of 3-nitrotyrosine- (3-NT-) modified proteins was used as marker of protein damage induced in HUVEC by peroxynitrite and quantified using nitrotyrosine ELISA kit (Abcam ab113848, UK).

After treatments with n-VLDL or ox-VLDL at different concentration (75, 140 μg/mL), HUVEC were trypsinized, pelleted at 500 ×g for 10 min, and washed twice using PBS buffer. Then, cells (~1 × 10⁶) were suspended in sample extraction buffer and incubated on ice for 20 min. After centrifugation at 12,000 ×g 4°C for 20 min, the 3-NT levels were determined colorimetrically according to the manufacturer’s instructions.

2.9. O₂ Consumption Measurements

HUVEC incubated with n-VLDL or ox-VLDL (140 μg/mL) were harvested and resuspended in the oxygraph medium consisting of 3 mM MgCl₂ × 6 H₂O, 10 mM KH₂PO₄, 20 mM HEPES, 1 g/L BSA, 110 mM mannitol, 0.5 mM EGTA, and pH 7.1. Cell density and viability were determined by trypan blue exclusion test. Respiration was assayed as previously described [47, 48] using a high-resolution respirometer (Oxygraph-2k; Oroboros Instruments) equipped with two 1.5 mL chambers with thermostats; data were collected and analysed using the built in software DatLab 4.

Cells (1 × 10⁶) were added to the oxygraph chamber containing the medium, and the system let equilibrate for 5 min. Cell plasma membrane was permeabilised to reducing substrates and effectors with digitonin (see text). The optimal digitonin concentration, 2.7 μg/mL, and the incubation time, 10 minutes, were fixed according to [49].

The contribution to cell respiration of any respiratory complex was evaluated according to Kuznetsov et al. with minor modifications [50]. Briefly, 10 min after the addition of digitonin, 8.8 mM pyruvate and 4.4 mM malate were added and the resting complex I-supported respiration was recorded (state 4); ADP, 2 mM, was then added to induce the maximal mitochondrial respiration (state 3), followed by rotenone, 0.5 μM, to specifically inhibit complex I; succinate, 10 mM, was added to induce complex II-supported respiration, and antimycin, 5 μM, was added to inhibit complex III; finally, complex IV-dependent respiration was activated by 2 mM ascorbate and 0.5 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD).

2.10. Citrate Synthase

HUVEC were harvested (1 × 10⁶), lysed, and centrifuged at 13,000 ×g for 10 min. Cell lysates were assayed for citrate synthase activity [40] and for total protein content. HUVEC, used as controls or after incubation with n-VLDL or ox-VLDL, displayed the same protein content (~0.2 mg/mL) and citrate synthase activity (~0.18 μmol/min/1 × 10⁶ cells) (not shown).

2.11. Statistical Analysis

Data are the mean ± standard deviation (SD) of at least three independent biological experiments (as specified in the figure legends), each repeated in three technical replicates. For statistical analysis, one-way analysis of variance (ANOVA), followed by Bonferroni-Holm post hoc test, was used for multiple comparisons. P values indicated in figures were considered statistically significant by ANOVA. In Table 1, the P values for all genes are shown; when followed by  ^∗, P values were considered significant by ANOVA.

3.1. eNOS and iNOS Genes

As shown in Figures 1(a) and 1(b) following cell incubation with n-VLDL, the eNOS mRNA expression is slightly downregulated by ~0.25-fold, whereas the iNOS is upregulated by ~2.5-fold. When cells were exposed to ox-VLDL, the eNOS mRNA expression was more clearly downregulated (~0.4-fold) while the iNOS increased largely by approximately 15-fold.

3.2. Other NO-Related Genes

This group includes genes involved in the stability and functional regulation of the NOS enzyme. Following cell incubation with ox-VLDL, the HSP90AB1, GLA, GCH1, and ARG2 genes are upregulated, whereas the DDAH2 and the GCHFR genes are downregulated (see Figure 1(c)).

Changes of the expression levels of this selection of genes are very small when induced by n-VLDL, varying by a larger extent if VLDL were preliminarily oxidised (see Table 1).

3.3. Inflammation-Related Genes

Along with the iNOS mRNA, also the VEGFA-, IL8-, and ALOX12-encoding genes are upregulated by ox-VLDL, pointing to the activation of inflammation pathways (Figure 1(d)). The expression of endothelial genes involved in the NADPH oxidase biosynthesis and function, namely, the NCF2 and NQO1, also appears upregulated together with the SOD1 and SOD2 mRNA, the latter increased by ~1.8-fold (see Table 1 and Figure 1(d)). In this frame, the 24 h cell incubation with n-VLDL is clearly less effective.

3.4. eNOS Expression and Uncoupling

The protein expression of eNOS was determined by Western blot analysis in HUVEC incubated with n-VLDL or ox-VLDL. For the analysis, primary antibodies against eNOS, or specifically recognising Thr495- or Ser1177-phosphorylated eNOS, were used. Consistent with the data shown in Figure 1, the exposure to n-VLDL leads to a ~20% decrease of the eNOS protein detected regardless to the lipoprotein concentration used, namely, 75 and 140 μg mL⁻¹. The exposure to ox-VLDL, at the same concentrations, induces, respectively, a ~30% and ~40% decrease of protein expression (Figure 2(a)).

The results of Western blot carried out with primary antibodies directed against phosphorylated eNOS indicated differences in phosphorylation at the level of key regulatory sites, Ser1177 and Thr495, upon varying VLDL redox state and concentration. In Figure 2(b), the results were reported as the ratio of phosphorylated eNOS (at Ser1177 or Thr495) over total eNOS (%). Control cells exhibited a ~60% phosphorylation at Ser1177 (P-Ser1177), and incubation with n-VLDL (both 75 and 140 μg mL⁻¹) did not induce significant changes. Nevertheless, the amount of P-Ser1177 decreased significantly to ~30%–40% of the total eNOS, when cells were incubated with ox-VLDL (Figure 2(b)). Somewhat symmetrically, Thr495 (P-Thr495) is ~40% phosphorylated in the controls, reaching ~60% phosphorylation upon incubation with n-VLDL, regardless to their concentration, in the limited range explored. At the highest ox-VLDL concentration, up to ~90% threonin was phosphorylated (Figure 2(b)).

3.5. Nitrite/Nitrate, Reactive Oxygen Species, and 3-Nitrotyrosine

The accumulation of nitrite/nitrate (NOx), the production of ROS, and the concentration level of 3-NT were measured after 24 h incubation with VLDL. As shown in Figure 3(a), ~20 nmoles NOx was produced by 10⁶ control cells in 24 h. Incubation with VLDL resulted in the increase of NOx production, as a function of VLDL concentration and oxidation state. 75 μg/mL and 140 μg/mL ox-VLDL resulted in the highest NOx increase, respectively, by 1.5- and 2-fold with respect to controls. The production of ROS was also measured under similar conditions. As shown in Figure 3(b), the ROS level rises during the 24 h incubation, with increasing the concentration of VLDL, and more markedly if these are oxidised; the larger effect is observed at the highest ox-VLDL concentration. A comparable trend is observed when monitoring the level of 3-nitrotyrosine protein modification; this last finding, particularly, suggests a correlation between the persistence in the HUVEC environment of ox-VLDL and formation of the short-lived detrimental peroxynitrite (Figure 3(c)).

3.6. HUVEC Respiration

The O₂ consumption of HUVEC following treatment with n-VLDL or ox-VLDL was measured oxigraphically. Measurements were carried out under different experimental conditions, such as using intact or digitonin-permeabilised cells in the presence or absence of specific mitochondrial substrates and respiratory chain inhibitors.

Typical traces are reported in Figure 4(a), where HUVEC untreated or incubated with n-VLDL or ox-VLDL, both 140 μg mL⁻¹, were allowed to consume O₂ in the presence and absence of selective activators or inhibitors of specific respiratory chain complexes [49, 50]. The basal rates of O₂ consumption have been measured and reported under all conditions (see Figure 4(b)). As shown in the figure, the incubation with n-VLDL induces a ~21% decrease of the O₂ consumption, from ~85 pmoles O₂ s⁻¹ to ~67 pmoles O₂ s⁻¹. Moreover, the rate of respiration decreases, instead down to ~59% of the initial value, if incubation is carried out with ox-VLDL.

The depression of O₂ consumption appears slightly more evident in the presence of rotenone and succinate, inhibiting complex I and activating complex II, respectively (Figure 4(b)). The basal respiratory control ratio, RCR, was also affected by the VLDL incubation; it decreased from 1.74 to 1.50 and 1.40, upon treatment with n-VLDL and ox-VLDL, respectively (see Figure 4(c)).