VLDL Induced Modulation of Nitric Oxide Signalling and Cell Redox Homeostasis in HUVEC.
Atherosclerosis, one of the principal causes of morbidity and mortality in occidental countries, is a disease affecting large and medium-sized muscular arteries, characterised by endothelial dysfunction and vascular inflammation [1-3]. Dyslipidaemia, and in particular, the increased concentration of cholesterol esters and low-density lipoproteins (LDL) at the arterial intima surface, has long been recognised as a major proatherogenic factor  potentially more dangerous for the vessels upon increasing the lipid oxidation level [5-8]. Not only the LDL but also the very-low-density lipoproteins (VLDL), the physiological precursors of the LDL, might share the responsibility of the onset and builds up of the atherosclerotic plaques . The implication in the atherosclerotic lesion of VLDL can be indirect, since the more elevated the VLDL concentration, the higher the LDL accumulation, but also direct due to the straight molecular interactions with the arterial cell components, membranes, and receptors [10, 11].
Evidence has been collected suggesting that, as reported for the LDL , also the oxidation of VLDL triggers a cascade of proatherogenic and proinflammatory cell responses . These include lipoprotein retention in the arterial wall and recruitment of macrophages at the vessel level; under these conditions, foam cells are formed and eventually the atherosclerotic plaque builds up [13, 14].
Interestingly, while the role of LDL and their redox state in the atherogenesis have been both intensively studied [15-18], the experimental evidence supporting the direct involvement of VLDL, native or oxidized, in the onset and development of atherosclerosis is still limited.
Previous reports have shown an increased production of reactive oxygen species (ROS) by mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction associated with atherosclerosis
[19-23]. A growing body of evidence suggests that alterations of the nitric oxide (NO) synthesis may be involved with this disease [18,24,25].
As a gaseous cell messenger, NO regulates several pathways both in prokaryotes and in eukaryotes [18, 26-29], including the modulation of cell energetic through the interaction with electron transport chain proteins [30-32]. Very relevant to cardiovascular diseases, the NO generated by endothelial cells plays a crucial role in the blood pressure control via relaxation of the vessel muscular smooth muscle cells .
Studies on knockout mice demonstrated that NO produced at low (nM) concentration by the constitutive NO synthases (eNOS and nNOS) exerts vasculo-protective effects, whereas higher NO concentration levels ([micro]M), as those synthesized by the inducible NOS (iNOS), are more often associated with oxi/nitrosative stress of vessels and tissues [34, 35].
Moreover, it has been observed that under condition favoring oxidative stress and inflammation, the impairment of the physiological activity of the eNOS may occur, leading to the so called "uncoupling state" characterized by the production of superoxide [O.sup.-*.sub.2] instead of NO [36-38].
The present work was aimed at characterising the response of vascular endothelial cells in culture to a time-persistent (24 h) administration of VLDL as purified from human blood sera (native), or oxidised, focusing on the NO metabolism, the ROS production, and the changes of the primary mitochondrial function . Human umbilical vein endothelial cells (HUVEC) have been taken as a model of vascular endothelium, and within the simplification of a monoculture, it allowed a detailed biochemical characterisation of eNOS biosynthesis and function, pointing to a key role played by this enzyme in the early redox unbalance associated to the onset of atherosclerosis.
2. Materials and Methods
2.1. Cell Culture. Human umbilical vein endothelial cells (HUVEC, ATCC CR[L.sup.-1]730) were cultured on 0.2% (w/v) gelatin-precoated flasks or multiwell plates (Falcon, BD Biosciences, USA) and grown at 37[degrees]C, 5% C[O.sub.2], 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 [micro]g/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 [micro]g protein m[L.sup.-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 xg) 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[TM] 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 .
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. . 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 xg, for 18 h at 15[degrees]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[degrees]C overnight. All VLDL preparations were filtered through a sterile 0.45 f m filter and, after the evaluation of protein concentration by the BCA (~500 [micro]g/mL), were stored at -70[degrees]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 SDSPAGE (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 Universita 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  using [Cu.sup.2+] ions (CuS[O.sub.4]). Briefly, a solution of purified n-VLDL, at the concentration 0.2-0.4 mg m[L.sup.-1] in EDTA-free PBS, was incubated with CuS[O.sub.4], 40 [micro]M 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 [micro]g m[L.sup.-1]), total RNA was extracted using the Nucleo Spin[R] RNA isolation kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. Total RNA, 1 [micro]g, 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 x [10.sup.6]) 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[degrees]C with primary mouse monoclonal antiphospho-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); [alpha]-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[degrees]C, and chemiluminescence was determined (Amersham, GE Healthcare, UK). Densitometric analysis was carried out by the ChemiDoc[TM] 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 xg for 5 min at 20[degrees]C. Cell pellets were resuspended in Hank's buffer at the density 1 x [10.sup.5] cells m[L.sup.-1] and plated in 24-well (black) plates.
Immediately after the addition of 10 [micro]M DCFDA, the relative fluorescence emission was followed kinetically at 520 nm for 60 min (VICTOR[TM] 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 x [10.sup.5] cell/mL) with n-VLDL or ox-VLDL, the culture supernatant was centrifuged at 4[degrees]C, 1000 xg 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 [micro]g/mL), HUVEC were trypsinized, pelleted at 500 xg for 10 min, and washed twice using PBS buffer. Then, cells (~1 x [10.sup.6]) were suspended in sample extraction buffer and incubated on ice for 20 min. After centrifugation at 12,000 xg 4[degrees]C for 20 min, the 3-NT levels were determined colorimetrically according to the manufacturer's instructions.
2.9. [O.sub.2] Consumption Measurements. HUVEC incubated with n-VLDL or ox-VLDL (140 f g/mL) were harvested and resuspended in the oxygraph medium consisting of 3 mM Mg[Cl.sub.2] x 6 [H.sub.2]O, 10 mM K[H.sub.2]P[O.sub.4], 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 x [10.sup.6]) 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 [micro]g/mL, and the incubation time, 10 minutes, were fixed according to .
The contribution to cell respiration of any respiratory complex was evaluated according to Kuznetsov et al. with minor modifications . 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 [micro]M, to specifically inhibit complex I; succinate, 10 mM, was added to induce complex II-supported respiration, and antimycin, 5 [micro]M, 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 x [10.sup.6]), lysed, and centrifuged at 13,000 xg for 10 min. Cell lysates were assayed for citrate synthase activity  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 [micro]mol/min/1 x [10.sup.6] cellsnot shown).
2.11. Statistical Analysis. Data are the mean [+ or -] 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.
The transcriptional activity of HUVEC exposed to 140 [micro]g/mL n-VLDL or ox-VLDL was investigated by targeting genes related to NO metabolism and to oxidative stress. The genes included in the screening have been grouped in functional classes (Table 1), and the mRNA production of each gene has been reported as the relative to that of untreated cells (see Materials and Methods).
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 [micro]gm[L.sup.-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 [micro]g m[L.sup.-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 [10.sup.6] 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 [micro]g/mL and 140 [micro]g/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 [O.sub.2] 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 [micro]g m[L.sup.-1], were allowed to consume [O.sub.2] in the presence and absence of selective activators or inhibitors of specific respiratory chain complexes [49, 50]. The basal rates of [O.sub.2] 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 [O.sub.2] consumption, from ~85 pmoles [O.sub.2] [s.sup.-1] to ~67 pmoles [O.sub.2] [s.sup.-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 [O.sub.2] 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)).
The endothelium of vessels exposed to oxidative stress displays an altered availability of reactive oxygen and nitrogen species (RONS) that may result in disturbance of mitochondrial function and unbalancing of cell physiological signalling, aspects in which NO is known to play a crucial role [31, 51-54].
Bioavailability of NO at cell level depends on the activity of the NOS isoforms, so that physiological pathways ascribable to eNOS activity are featured by a limited (nM) amount of NO, whereas higher NO concentrations ([micro]M) resulting from the activation of iNOS are responsible for structural modifications such as membrane lipid peroxydation and protein nitrosation. These modifications are associated to the onset and maintenance of severe inflammatory states, including atherosclerosis [34, 55, 56].
In this study, human umbilical vein endothelial cells (HUVEC) have been used as a model system to investigate the endothelium response to a persistent VLDL exposure.
The incubation time and the VLDL concentrations were chosen so to set a mild though clear dyslipidemic condition of pathophysiological relevance. Within the experimental limits of cells in culture mimicking endothelium microenvironment, cells were allowed to face for 24 h about twice as much the physiological blood concentration of VLDL (140 [micro]g/mL), as prepared (native) or oxidized. The experimental design was such to investigate the role of VLDL in inducing nitro-oxidation of endothelium-like cells.
The analysis of NOS expression suggested the activation of a regulative antagonistic cross talk among the two NOS isoforms (eNOS and iNOS), so that the upregulation of the iNOS mRNA was accompanied, under comparable conditions, by a significant downregulation of the eNOS.
The iNOS activation is fully consistent with the increased production of NOx independently detected in the culture medium, suggesting a pathway of NO production which escapes the eNOS control. Furthermore, the increased production of ROS and 3-nitrotyrosine herein reported is consistent with the evidence of peroxynitrite formation compatible with a cogent inflammatory state of the cells .
The whole framework is also coherent with the upregulation of IL8 and VEGFA, genes involved in NO production and cell inflammatory response  and with the increased expression of NCF2 and NQO1 factors related to the NADPH oxidase function and biosynthesis [58, 59].
Consistently with the setting of an inflammatory state, the lowered eNOS synthesis induced by VLDL in HUVEC may imply an alteration of the physiological vasodilatory function relying on nanomolar NO fluxes. It is worth to notice that the eNOS is not only downregulated, according to both the mRNA and protein expression changes: based on the detected eNOS phosphorylation pattern (by targeting phospho-Ser1177 and phospho-Thr495), the enzyme appears also largely uncoupled hence supporting the production of superoxide ion [60-62].
The relevance of the proposed involvement of VLDL in the biosynthetic and functional regulation of eNOS was supported by changes found, over the same time scale, on the regulation of genes involved to NOS stability and function [63, 64]. The decreased expression of DDAH2, scavenger of an eNOS inhibitor (ADMA) , as well as the upregulation of ARG2, responsible for the depletion of arginine, were all elements pointing to the impairment of the physiological production of NO.
Interestingly, and somehow discordant with the above findings, some differences in the expression of other genes from the same array, as the modest upregulation of HSP90, involved in maintaining the dimeric structure of eNOS  and of GLA, responsible for degradation of the eNOS uncoupler Gb3 , seemed to point out the activation of compensatory pathways possibly aimed to the preservation of the eNOS function. Furthermore, the upregulation of the enzyme GCH1, involved in BH4 biosynthesis, combined to the downregulation of its feedback regulator GCHFR, suggests that under conditions in which BH4 oxidation is feasible, such as under oxidising conditions, signals enhancing BH4 bioavailability may be activated by endothelial cells, aimed at preventing or possibly reverting eNOS uncoupling [38, 68].
It is interesting to underline that upon VLDL treatment, the observed increase in ROS concentration follows a trend fully consistent with the progress of eNOS uncoupling, detected by Western blot. A peak in ROS concentration was found after 140 [micro]g/mL ox-VLDL administration, a condition matching that responsible for the ultimate eNOS uncoupling (lowest phospho-Ser1177, highest phospho-Thr495).
It may be also interesting to point out that the increased production of superoxide goes along with a mild upregulation of SOD1 and SOD2 mRNAs (Figure 1(d)), thus suggesting a cell attempt to tempering ROS rise.
Beside NO, ROS are also involved in the maintenance of physiological vascular homeostasis, through a tight control exerted by healthy endothelial cells.
The setting of prooxidative conditions was shown to result in increased ROS production, associated with the activation of signals promoting oxidative damage [22, 69].
Hence, the VLDL-induced cell redox changes are characterised by the concurrent synthesis of NO by the iNOS and of [O.sup.-*.sub.2] by the uncoupled eNOS. These two evidences are fully compatible with the observed production of the highly reactive species peroxynitrite, probed by the increased tyrosine nitration.
Ox-VLDL treatment appears to be the condition necessary and sufficient to determine peroxynitrite formation, as the increase of 3-NT was found significant already at 75 [micro]g/mL, compared to control cells; this may suggest a specific interaction of ox-VLDL with the scavenger receptors. As already reported for ox-LDL, this class of cell surface receptors may specifically recognise oxidised lipoproteins, thus activating typical inflammatory cell response [70, 71].
The whole picture appears consistent with the onset and/or maintenance of a nitrosative stress in endothelial cells undergoing VLDL treatments; within this frame, the determination of the functional state of mitochondria is relevant to define the level of cell dysfunction.
VLDL induced a functional decrease (mild if native, more evident if oxidised) of mitochondrial respiration. The mitochondrial respiratory control ratio (RCR) also followed a similar trend. The respiratory chain activity, evaluated at the level of the specific mitochondrial complexes, showed a decrease of the electron transfer efficiency (~20%), detected almost uniformly along the chain, apparently just slightly more at the level of complex II (~50% inhibition by ox-VLDL).
As the observed impairment on mitochondrial respiration may not be unequivocally attributed to a specific respiratory complex, it appears feasible to envisage that the oxi/ nitrosative stress induced by VLDL may determine relevant modification of mitochondrial proteins, such as protein nitrosation, causing an interference with the electron transfer process. The persistency of these conditions would bring to supercomplex disaggregation and alteration of the mitochondrial proteolipid arrangement.
From these experiments, we can propose that 24 h incubation of HUVEC with native and oxidised VLDL triggers a signalling leading to iNOS activation and eNOS uncoupling.
These events, resulting in an unbalance of NO and ROS metabolism, are more clearly produced by excess of ox-VLDL leading to production of harmful proxynitrite and triggering a cell inflammatory state. Consistently, cells display a lower mitochondrial [O.sub.2] consumption and RCR (Figure 5).
It is tempting to speculate on the biomedical relevance of these results: an altered lipid metabolism arising from both a genetic or epigenetic background could represent a condition in which an inadequate lifestyle (smoking abuse, highly processed food consumption, and pollution) provides an additional detrimental contribution, perceived by the endothelium of vessel as a prooxidant insult.
According to our results, the early dysfunction of the eNOS associated to iNOS activation driven by VLDL can be relevant to set conditions compatible with the development of atherosclerosis.
ADMA: Asymmetric dimethylarginine ADP: Adenosine diphosphate ALOX12: Arachidonate 12-lipoxygenase, 12S type ARG2: Arginase, type II BSA: Bovine serum albumin cDNA: Complementary DNA DDAH2: Dimethylarginine dimethylaminohydrolase 2 EC: Endothelial cells eNOS: Endothelial nitric oxide synthase EDTA: Ethylenediaminetetraacetic acid Gb3: Globotriaosylceramide GCH1: GTP cyclohydrolase 1 GCHFR: GTP cyclohydrolase I feedback regulator GLA: Galactosidase alpha HSP90AB1 Heat shock protein 90 kDa?, class B member 1 HUVEC: Human umbilical vein endothelial cells IL8: Interleukin 8 iNOS: Inducible NOS LDL: Low-density lipoproteins NCF2: Neutrophil cytosolic factor 2 NO: Nitric oxide nNOS: Neuronal NOS NOx: Nitrites and nitrates NQO1: NAD(P)H dehydrogenase, quinone 1 n-VLDL: Native VLDL ox-VLDL: Oxidised VLDL PBS: Phosphate-buffered saline PCR: Polymerase chain reaction P-Ser1177: Phosphorylated serine 1177 P-Thr495: Phosphorylated threonine 495 RNS: Reactive nitrogen species ROS: Reactive oxygen species RONS: Reactive oxygen and nitrogen species Thr: Threonine RCR: Respiratory control ratio Ser: Serine SOD1: Superoxide dismutase 1 SOD2: Superoxide dismutase 2 VEGFA: Vascular endothelial growth factor A VLDL: Very-low-density lipoproteins U-eNOS: Uncoupled eNOS 3-NT: 3-Nitrotyrosine.
Conflicts of Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
Maria Chiara Magnifico and Roxana Elena Oberkersch have contributed equally to this work.
This work was supported by the Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR) of Italy (PRIN20107Z8XBW 005) to Paolo Sarti and Regione Lazio of Italy (FILAS-RU-2014-1020) to Paolo Sarti. The Erasmus Mundus Programme Action 2 Arcoiris Partnership (UID ARCO1100237) and ERASMUS + Programme (KA103) are acknowledged for supporting Roxana Elena Oberkersch and Yasmine Grooten. The authors gratefully acknowledged the staff from the "Immunohematology and Transfusion Medicine" UOC of Policlinico Umberto I and particularly Dr. Rosato V. and Dr. Malanga G. for the selection of blood samples from healthy volunteers used in this study. The authors are grateful to Dr. Alessio Paone for supporting them in the statistical analysis of the results.
 Y. Nakashima, T. N. Wight, and K. Sueishi, "Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans," Cardiovascular Research, vol. 79, no. 1, pp. 14-23, 2008.
 I. Tabas, G. Garcia-Cardena, and G. K. Owens, "Recent insights into the cellular biology of atherosclerosis," The Journal of Cell Biology, vol. 209, no. 1, pp. 13-22, 2015.
 H. A. Jensen and J. L. Mehta, "Endothelial cell dysfunction as a novel therapeutic target in atherosclerosis," Expert Review of Cardiovascular Therapy, vol. 14, no. 9, pp. 1021-1033, 2016.
 M. S. Rahman, A. J. Murphy, and K. J. Woollard, "Effects of dyslipidaemia on monocyte production and function in cardiovascular disease," Nature Reviews Cardiology, vol. 14, no. 7, pp. 387-400, 2017.
 A. Negre-Salvayre, N. Auge, C. Camare, T. Bacchetti, G. Ferretti, and R. Salvayre, "Dual signaling evoked by oxidized LDLs in vascular cells," Free Radical Biology & Medicine, vol. 106, pp. 118-133, 2017.
 V. Lubrano and S. Balzan, "LOX-1 and ROS, inseparable factors in the process of endothelial damage," Free Radical Research, vol. 48, no. 8, pp. 841-848, 2014.
 V. N. Sukhorukov, V. P. Karagodin, and A. N. Orekhov, "Atherogenic modification of low-density lipoproteins," Biomeditsinskaya Khimiya, vol. 62, no. 4, pp. 391-402, 2016.
 K. E. Petersen, J. Lykkesfeldt, K. Raun, and G. Rakipovski, "Brief communication: plasma lipid oxidation predicts atherosclerotic status better than cholesterol in diabetic apolipoprotein E deficient mice," Experimental Biology and Medicine, vol. 242, no. 1, pp. 88-91, 2017.
 G. Ferretti, T. Bacchetti, T. P. Johnston, M. Banach, M. Pirro, and A. Sahebkar, "Lipoprotein(a): a missing culprit in the management of athero-thrombosis?," Journal of Cellular Physiology, 2017.
 S. Takahashi, J. Sakai, T. Fujino et al., "The very low-density lipoprotein (VLDL) receptor: characterization and functions as a peripheral lipoprotein receptor," Journal of Atherosclerosis and Thrombosis, vol. 11, no. 4, pp. 200-208, 2004.
 P. Xie, Y. Duan, X. Guo, L. Hu, and M. Yu, "SalA attenuates hypoxia-induced endothelial endoplasmic reticulum stress and apoptosis via down-regulation of VLDL receptor expression," Cellular Physiology and Biochemistry, vol. 35, no. 1, pp. 17-28, 2015.
 G. D. Norata, A. Pirillo, E. Callegari, A. Hamsten, A. L. Catapano, and P. Eriksson, "Gene expression and intracellular pathways involved in endothelial dysfunction induced by VLDL and oxidised VLDL," Cardiovascular Research, vol. 59, no. 1, pp. 169-180, 2003.
 M. C. Jong, W. L. Hendriks, L. C. van Vark, V. E. Dahlmans, J. E. Groener, and L. M. Havekes, "Oxidized VLDL induces less triglyceride accumulation in J774 macrophages than native VLDL due to an impaired extracellular lipolysis," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 20, no. 1, pp. 144-151, 2000.
 L. Wang, R. Gill, T. L. Pedersen, L. J. Higgins, J. W. Newman, and J. C. Rutledge, "Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation," Journal of Lipid Research, vol. 50, no. 2, pp. 204-213, 2009.
 T. Kita, N. Kume, M. Minami et al., "Role of oxidized LDL in atherosclerosis," Annals of the New York Academy of Sciences, vol. 947, pp. 199-205, 2001.
 V. Mollace, M. Gliozzi, V. Musolino et al., "Oxidized LDL attenuates protective autophagy and induces apoptotic cell death of endothelial cells: role of oxidative stress and LOX-1 receptor expression," International Journal of Cardiology, vol. 184, pp. 152-158, 2015.
 D. P. Hajjar and A. M. Gotto Jr., "Biological relevance of inflammation and oxidative stress in the pathogenesis of arterial diseases," The American Journal of Pathology, vol. 182, no. 5, pp. 1474-1481, 2013.
 S. H. Chan, C. H. Hung, J. Y. Shih et al., "Baicalein is an available anti-atherosclerotic compound through modulation of nitric oxide-related mechanism under oxLDL exposure," Oncotarget, vol. 7, no. 28, pp. 42881-42891, 2016.
 A. Almeida and J. P. Bolanos, "A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons," Journal of Neurochemistry, vol. 77, no. 2, pp. 676-690, 2001.
 P. Dromparis and E. D. Michelakis, "Mitochondria in vascular health and disease," Annual Review of Physiology, vol. 75, pp. 95-126, 2013.
 X. Tang, Y. X. Luo, H. Z. Chen, and D. P. Liu, "Mitochondria, endothelial cell function, and vascular diseases," Frontiers in Physiology, vol. 5, p. 175, 2014.
 N. R. Madamanchi and M. S. Runge, "Mitochondrial dysfunction in atherosclerosis," Circulation Research, vol. 100, no. 4, pp. 460-473, 2007.
 I. A. Sobenin, M. A. Sazonova, A. Y. Postnov, Y. V. Bobryshev, and A. N. Orekhov, "Changes of mitochondria in atherosclerosis: possible determinant in the pathogenesis of the disease," Atherosclerosis, vol. 227, no. 2, pp. 283-288, 2013.
 J. D. Erusalimsky and S. Moncada, "Nitric oxide and mitochondrial signaling: from physiology to pathophysiology," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 12, pp. 2524-2531, 2007.
 R. P. Patel, D. Moellering, J. Murphy-Ullrich, H. Jo, J. S. Beckman, and V. M. Darley-Usmar, "Cell signaling by reactive nitrogen and oxygen species in atherosclerosis," Free Radical Biology & Medicine, vol. 28, no. 12, pp. 1780-1794, 2000.
 A. Giuffre, V. B. Borisov, M. Arese, P. Sarti, and E. Forte, "Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress," Biochimica et Biophysica Acta (BBA)--Bioenergetics, vol. 1837, no. 7, pp. 1178-1187, 2014.
 J. B. Vicente, F. Malagrino, M. Arese, E. Forte, P. Sarti, and A. Giuffre, "Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine [beta]-synthase," Biochimica et Biophysica Acta (BBA)--Bioenergetics, vol. 1857, no. 8, pp. 1127-1138, 2016.
 N. Zhang, Y. Diao, R. Hua et al., "Nitric oxide-mediated pathways and its role in the degenerative diseases," Frontiers in Bioscience (Landmark Edition), vol. 22, pp. 824-834, 2017.
 C. Bogdan, "Nitric oxide synthase in innate and adaptive immunity: an update," Trends in Immunology, vol. 36, no. 3, pp. 161-178, 2015.
 A. Galkin, A. Y. Abramov, N. Frakich, M. R. Duchen, and S. Moncada, "Lack of oxygen deactivates mitochondrial complex I: implications for ischemic injury?," The Journal of Biological Chemistry, vol. 284, no. 52, pp. 36055-36061, 2009.
 P. Sarti, E. Forte, A. Giuffre, D. Mastronicola, M. C. Magnifico, and M. Arese, "The chemical interplay between nitric oxide and mitochondrial cytochrome c oxidase: reactions, effectors and pathophysiology," International Journal of Cell Biology, vol. 2012, Article ID 571067, 11 pages, 2012.
 P. Sarti, E. Lendaro, R. Ippoliti, A. Bellelli, P. A. Benedetti, and M. Brunori, "Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy," The FASEB Journal, vol. 13, no. 1, pp. 191-197, 1999.
 S. Moncada and E. A. Higgs, "The discovery of nitric oxide and its role in vascular biology," British Journal of Pharmacology, vol. 147, Supplement 1, pp. S193-S201, 2006.
 P. Pacher, J. S. Beckman, and L. Liaudet, "Nitric oxide and peroxynitrite in health and disease," Physiological Reviews, vol. 87, no. 1, pp. 315-424, 2007.
 A. Chatterjee, S. M. Black, and J. D. Catravas, "Endothelial nitric oxide (NO) and its pathophysiologic regulation," Vascular Pharmacology, vol. 49, no. 4-6, pp. 134-140, 2008.
 S. Karbach, P. Wenzel, A. Waisman, T. Munzel, and A. Daiber, "eNOS uncoupling in cardiovascular diseases-the role of oxidative stress and inflammation," Current Pharmaceutical Design, vol. 20, no. 22, pp. 3579-3594, 2014.
 U. Forstermann and W. C. Sessa, "Nitric oxide synthases: regulation and function," European Heart Journal, vol. 33, no. 7, pp. 829-837, 2012, 837a-837d.
 H. Li and U. Forstermann, "Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease," Current Opinion in Pharmacology, vol. 13, no. 2, pp. 161-167, 2013.
 P. Sarti, M. Arese, E. Forte, A. Giuffre, and D. Mastronicola, "Mitochondria and nitric oxide: chemistry and pathophysiology," Advances in Experimental Medicine and Biology, vol. 942, pp. 75-92, 2012.
 P. A. Srere, "Citrate synthase," Methods in Enzymology, vol. 13, pp. 3-11, 1969.
 T. G. Redgrave, D. C. Roberts, and C. E. West, "Separation of plasma lipoproteins by density-gradient ultracentrifugation," Analytical Biochemistry, vol. 65, no. 1-2, pp. 42-49, 1975.
 A. Cwiklinska, B. Kortas-Stempak, A. Gliwinska, A. Pacanis, A. Kuchta, and M. Wroblewska, "Interaction between VLDL and phosphatidylcholine liposomes generates new [gamma]-LpE-like particles," Lipids, vol. 49, no. 2, pp. 143-153, 2014.
 M. Guha, C. England, H. Herscovitz, and O. Gursky, "Thermal transitions in human very-low-density lipoprotein: fusion, rupture, and dissociation of HDL-like particles," Biochemistry, vol. 46, no. 20, pp. 6043-6049, 2007.
 M. Guha and O. Gursky, "Effects of oxidation on structural stability and remodeling of human very low density lipoprotein," Biochemistry, vol. 49, no. 44, pp. 9584-9593, 2010.
 U. P. Steinbrecher, S. Parthasarathy, D. S. Leake, J. L. Witztum, and D. Steinberg, "Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids," Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 12, pp. 3883-3887, 1984.
 K. Yagi, "A simple fluorometric assay for lipoperoxide in blood plasma," Biochemical Medicine, vol. 15, no. 2, pp. 212-216, 1976.
 S. Robin, M. Arese, E. Forte, P. Sarti, A. Giuffre, and T. Soulimane, "A sulfite respiration pathway from Thermus thermophilus and the key role of newly identified cytochrome c550," Journal of Bacteriology, vol. 193, no. 15, pp. 3988-3997, 2011.
 A. Masci, D. Mastronicola, M. Arese et al., "Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells," Biochimica et Biophysica Acta (BBA)--Bioenergetics, vol. 1777, no. 1, pp. 66-73, 2008.
 E. Gnaiger, A. V. Kuznetsov, B. Lassnig et al., "High-resolution respirometry--optimum permeabilization of the cell membrane by digitonin," BioThermoKinetics in the Post Genomic era, C. Larsson, I. Pahlman and L. Gustafsson, Eds., p. 7, 1998.
 A. V. Kuznetsov, V. Veksler, F. N. Gellerich, V. Saks, R. Margreiter, and W. S. Kunz, "Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells," Nature Protocols, vol. 3, no. 6, pp. 965-976, 2008.
 J. Gutierrez, S. W. Ballinger, V. M. Darley-Usmar, and A. Landar, "Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascular cells," Circulation Research, vol. 99, no. 9, pp. 924-932, 2006.
 M. A. Kluge, J. L. Fetterman, and J. A. Vita, "Mitochondria and endothelial function," Circulation Research, vol. 112, no. 8, pp. 1171-1188, 2013.
 G. C. Brown, "Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase," FEBS Letters, vol. 369, no. 2-3, pp. 136-139, 1995.
 U. Forstermann, "Nitric oxide and oxidative stress in vascular disease," Pflugers Archiv, vol. 459, no. 6, pp. 923-939, 2010.
 M. Falabella, E. Forte, M. C. Magnifico et al., "Evidence for detrimental cross interactions between reactive oxygen and nitrogen species in Leber's hereditary optic neuropathy cells," Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 3187560, 9 pages, 2016.
 M. Arese, M. C. Magnifico, D. Mastronicola et al., "Nanomolar melatonin enhances nNOS expression and controls HaCaTcells bioenergetics," IUBMB Life, vol. 64, no. 3, pp. 251-258, 2012.
 Y. I. Wang, J. Schulze, N. Raymond et al., "Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal," American Journal of Physiology Heart and Circulatory Physiology, vol. 300, no. 3, pp. H784-H791, 2011.
 K. Bedard and K. H. Krause, "The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology," Physiological Reviews, vol. 87, no. 1, pp. 245-313, 2007.
 K. Itoh, P. Ye, T. Matsumiya, K. Tanji, and T. Ozaki, "Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria," Journal of Clinical Biochemistry and Nutrition, vol. 56, no. 2, pp. 91-97, 2015.
 B. J. Michell, Z. Chen, T. Tiganis et al., "Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase," The Journal of Biological Chemistry, vol. 276, no. 21, pp. 17625-17628, 2001.
 L. A. Biwer, E. P. Taddeo, B. M. Kenwood, K. L. Hoehn, A. C. Straub, and B. E. Isakson, "Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition," Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1861, no. 7, pp. 671-679,2016.
 F. Chen, S. Kumar, Y. Yu et al., "PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+-toxins," PLoS One, vol. 9, no. 7, article e99823, 2014.
 Q. Li, J. Y. Youn, and H. Cai, "Mechanisms and consequences of endothelial nitric oxide synthase dysfunction in hypertension," Journal of Hypertension, vol. 33, no. 6, pp. 1128-1136, 2015.
 G. H. Oliveira-Paula, R. Lacchini, and J. E. Tanus-Santos, "Endothelial nitric oxide synthase: from biochemistry and gene structure to clinical implications of NOS3 polymorphisms," Gene, vol. 575, no. 2, Part 3, pp. 584-599, 2015.
 S. Lambden, P. Kelly, B. Ahmetaj-Shala et al., "Dimethylarginine dimethylaminohydrolase 2 regulates nitric oxide synthesis and hemodynamics and determines outcome in polymicrobial sepsis," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 35, no. 6, pp. 1382-1392, 2015.
 W. Chen, H. Xiao, A. N. Rizzo, W. Zhang, Y. Mai, and M. Ye, "Endothelial nitric oxide synthase dimerization is regulated by heat shock protein 90 rather than by phosphorylation," PLoS One, vol. 9, no. 8, article e105479, 2013.
 J. L. Park, S. E. Whitesall, L. G. D'Alecy, L. Shu, and J. A. Shayman, "Vascular dysfunction in the alpha-galactosidase A-knockout mouse is an endothelial cell-, plasma membrane-based defect," Clinical and Experimental Pharmacology & Physiology, vol. 35, no. 10, pp. 1156-1163, 2008.
 K. Zschiebsch, C. Fischer, G. Pickert et al., "Tetrahydrobiopterin attenuates DSS-evoked colitis in mice by rebalancing redox and lipid signalling," Journal of Crohn's & Colitis, vol. 10, no. 8, pp. 965-978, 2016.
 M. E. Widlansky and D. D. Gutterman, "Regulation of endothelial function by mitochondrial reactive oxygen species," Antioxidants & Redox Signaling, vol. 15, no. 6, pp. 1517-1530, 2011.
 I. A. Zani, S. L. Stephen, N. A. Mughal et al., "Scavenger receptor structure and function in health and disease," Cell, vol. 4, no. 2, pp. 178-201, 2015.
 A. Pirillo, G. D. Norata, and A. L. Catapano, "LOX-1, OxLDL, and atherosclerosis," Mediators of Inflammation, vol. 2013, Article ID 152786, 12 pages, 2013.
Maria Chiara Magnifico, (1) Roxana Elena Oberkersch, (2) Azzurra Mollo, (1) Luca Giambelli, (3) Yasmine Grooten, (4) Paolo Sarti, (1) Graciela Cristina Calabrese, (2) and Marzia Arese (1)
(1) Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy
(2) Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Ciencias Biologicas, Catedra de Biologia Celular y Molecular, Buenos Aires, Argentina
(3) Blood Transfusion Service and Hematology, Umberto I Hospital, Rome, Italy
(4) Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Vrije Universiteit Brussel, Brussels, Belgium
Correspondence should be addressed to Graciela Cristina Calabrese; email@example.com and Marzia Arese; firstname.lastname@example.org
Received 21 April 2017; Revised 31 July 2017; Accepted 15 August 2017; Published 20 September 2017
Academic Editor: Vicenta L. Cortes
Caption: Figure 1: Gene expression changes induced by VLDL. HUVEC were incubated 24 h with n-VLDL (grey) orox-VLDL (black), both 140 [micro]g/mL. cDNA were assayed by RT-PCR (Human Nitric Oxide Signalling Pathway [RT.sup.2] Profiler PCR Array). The relative expression of the genes of interest is shown. (a) Endothelial nitric oxide synthase (eNOS). (b) Inducible nitric oxide synthase (iNOS). (c) Genes involved in the regulation of eNOS activity. (i) HSP90AB1: heat shock protein 90kDa [alpha] (cytosolic), class B member 1; (ii) GLA: galactosidase a; (iii) GCH1: GTP cyclohydrolase 1; (iv) GCHFR: GTP cyclohydrolase I feedback regulator; (v) DDAH2: dimethylarginine dimethylaminohydrolase 2; (vi) ARG2: arginase, type II. (d) Genes induced by nitric oxide and involved in superoxide metabolism/oxidative stress response. (i) IL8: interleukin 8; (ii) VEGFA: vascular endothelial growth factor A; (iii) SOD1: superoxide dismutase 1, soluble; (iv) SOD2: superoxide dismutase 2, mitochondrial; (v) NCF2: neutrophil cytosolic factor 2; (vi) NQO1: NAD(P)H dehydrogenase, quinone 1; (vii) ALOX12: arachidonate 12-lipoxygenase, 12S type. Relative expression calculated after [beta]-actin normalisation versus control cells. Data [+ or -] SD; n. of biological experiments = 3. P values were considered statistically significant by ANOVA.
Caption: Figure 2: eNOS protein expression and evaluation of uncoupling in response to VLDL by Western blot. HUVEC were incubated 24 h with (75 and 140 [micro]g/mL) n-VLDL or ox-VLDL and then assayed by Western blot with anti-eNOS antibodies (see Materials and Methods). (a) Levels of eNOS protein after n-VLDL or ox-VLDL treatment. Densitometric values are shown as fold change versus the eNOS protein expressed by control cells. Data [+ or -] SD, n. of biological experiments = 8. P values were considered statistically significant by ANOVA. Inset: Western blot pattern of eNOS after 24 h incubation with n-VLDL or ox- VLDL; [alpha]-tubulin as reference ([alpha]-tub). (b) Phosphorylation of eNOS at Ser-1177 and Thr-495 in response to n-VLDL or ox-VLDL (see Materials and Methods). Data [+ or -] SD, n. of biological experiments = 4. P values were considered statistically significant by ANOVA. Inset: Western blot pattern of eNOS (P- S1177) and eNOS (P-T495) after 24 h incubation with n-VLDL or ox-VLDL.
Caption: Figure 3: Oxidative and nitrosative stress driven in HUVEC by VLDL. Assays were carried out following 24 h incubation of HUVEC with n-VLDL or ox-VLDL (75 and 140 [micro]g/mL). (a) Nitrite-Nitrate (NOx) accumulation. NOx accumulation (24 h) was quantified in the cell supernatant downstream treatments by 2,3-diaminonaphthalene (DAN) as described in the Materials and Methods section. Data [+ or -] SD; n. of biological experiments = 5. P values were considered statistically significant by ANOVA. (b) Reactive oxygen species (ROS) production. Intracellular ROS levels were measured in VLDL-treated and VLDL-untreated EC in the presence of 2,7-dichlorodihydrofluorescein diacetate (DCFDA see Materials and Methods). The DCFDA fluorescence was followed kinetically and the values taken at 30 min. Data are shown as percentage of ROS amount detected in untreated cells and normalised for protein content. Data are the means [+ or -] SD; n. of biological experiments = 6. P values were considered statistically significant by ANOVA. (c) 3-Nitrotyrosine (3-NT) determination. The 3-NT content was measured by nitrotyrosine competitive ELISA (see Materials and Methods) in VLDL-treated and VLDL-untreated cells (lysate). Data values are the means [+ or -] SD; n. of biological experiments = 5. P values were considered statistically significant by ANOVA.
Caption: Figure 4: Evaluation of mitochondrial OXPHOS in response to VLDL treatment by [O.sub.2] consumption measurements. (a) [O.sub.2] consumption profiles. The oxygen consumption was monitored in 1 x 106 HUVEC after 24 h incubation under the conditions indicated; thin line: control cells (1); grey line: 140 [micro]g m[L.sup.-1] n-VLDL (2); bold line: 140 [micro]g m[L.sup.-1] ox-VLDL (3). Digitonin (Dig) was used to permeabilise cells. Substrates/inhibitors added along the trace: pyruvate and malate (Pyr/Mal); adenosine diphosphate (ADP); rotenone (Rot); succinate (Suc); antimycin A (Ant A); ascorbate and N,N,N',N'-tetrametil-p-fenilendiammina (Asc/TMPD). (b) [O.sub.2] consumption rates. Respiration rate values measured in VLDL-treated HUVEC after each addition step (of substrate/inhibitor). Data values are the means [+ or -] SD; n. of biological experiments = 3. P values were considered statistically significant by ANOVA. (c) Respiratory control ratio (RCR). The values of RCR were obtained as the ratio between the [O.sub.2] consumption rate measured in the presence of saturating [ADP] (state 3) and in its absence (state 4). Data values are the means [+ or -] SD; n. of biological experiments =3. P values were considered statistically significant by ANOVA.
Caption: Figure 5: ox-VLDL-dependent EC dysfunction: schematic representation of the eNOS and iNOS involvement. The activity of eNOS (yellow) and iNOS (red) dimers are sketched. The coupled (native) eNOS is featured by the utilisation of substrates such as L-Arg and [O.sub.2] and production of NO, in the presence of the cofactor calmodulin (CAM) and under condition of Ser1177 phosphorylation, promoting activity (white cell background). ox-VLDL triggers eNOS phosphorylation at residue Thr495, with dephosphorylation of Ser1177: under these conditions, the eNOS is uncoupled (U-eNOS) and results in the generation of [O.sup.-*.sub.2]. In parallel, high level of iNOS biosynthesis occurs, leading to increased ROS and RNS production and resulting in the down- modulation of mitochondrial function (gray background).
Table 1: Expression level of genes involved in the NO signalling, superoxide metabolism, and oxidative stress response induced by n-VLDL and ox-VLDL in HUVEC. Functional grouping Gene n-VLDL (rel. exp.)# Nitric oxide metabolism NOS1 (nNOS) -- Nitric oxide biosynthesis NOS2 (iNOS) 3521 [+ or -] 0.684 NOS3 (eNOS) 0.772 [+ or -] 0.031 Nitric oxide biosynthesis DYNLL1 0.996 [+ or -] 0.450 Regulation GLA 0.921 [+ or -] 0.253 HSP90AB1 1157 [+ or -] 0.393 IL8 0.551 [+ or -] 0.507 INS -- NOS1AP 1037 [+ or -] 0.684 Other nitric oxide AKT1 1014 [+ or -] 0.189 Biosynthesis genes ARG2 1196 [+ or -] 0.265 DDAH2 1158 [+ or -] 0.281 EGFR 2750 [+ or -] 2317 GCH1 0.786 [+ or -] 0.119 GCHFR 0.685 [+ or -] 0.281 Nitric oxide induced CDKN1A 1611 [+ or -] 1425 CXCF8 -- JUN 1574 [+ or -] 0.655 VEGFA 1451 [+ or -] 0.620 Nitric oxide suppressed CCNA1 1335 [+ or -] 0.558 MYB -- TROAP 2080 [+ or -] 0.589 Nitric oxide signalling CAMK1 1477 [+ or -] 0.388 DFG4 1505 [+ or -] 0.935 GRIN2D 1461 [+ or -] 0.573 PPP3CA 1003 [+ or -] 0.388 PRKAR1B 1627 [+ or -] 0.620 PRKCA 1083 [+ or -] 0.036 NQOl 3456 [+ or -] 0.606 Superoxide metabolism AFOX12 1425 [+ or -] 0.450 Release DUOX1 1105 [+ or -] 0.317 Oxidoreductases DUOX2 1429 [+ or -] 0.949 Peroxidases NOX5 -- PRG3 -- SOD1 0.897 [+ or -] 0.235 SOD2 0.942 [+ or -] 0.414 SOD3 -- Other superoxide CCS 1296 [+ or -] 0.670 Metabolism genes NCF1 -- NCF2 1250 [+ or -] 0.698 PREX1 0.916 [+ or -] 0.191 Oxidative stress MPO -- Antiapoptotic MTL5 0.756 [+ or -] 0.316 NME5 1049 [+ or -] 0.249 PRDX2 0.999 [+ or -] 0.294 RNF7 1260 [+ or -] 0.655 Antioxidants APOE -- MT3 -- VIMP -- SRXN1 2721 [+ or -] 2766 Glutathione peroxidases GPX1 1355 [+ or -] 0.587 GPX2 1392[+ or -] 1035 GPX3 1639 [+ or -] 0.795 GPX4 1436 [+ or -] 0.894 GPX5 -- GPX6 -- Other oxidoreductases CAT 1079 [+ or -] 0.533 EPX 1132 [+ or -] 0.448 LPO -- MSRA 1298 [+ or -] 0.793 PRDX6 1918[+ or -] 1369 TPO 0.802 [+ or -] 0.688 TXNRD2 1746 [+ or -] 0.944 Other peroxidases CSDE1 1532 [+ or -] 1115 CYGB -- GPR156 -- PRDX2 0.999 [+ or -] 0.294 PRDX5 1412 [+ or -] 0.845 TTN 0.416 [+ or -] 0.095 Regulation FOXM1 1541 [+ or -] 0.369 GLRX2 1006 [+ or -] 0.447 SCRT2 -- SIRT2 1322 [+ or -] 0.502 Other oxidative stress ATOX1 1254 [+ or -] 0.913 Response genes DUSP1 1186[+ or -] 0.189 GSS 1357 [+ or -] 0.684 KRT1 -- MBL2 2039 [+ or -] 1129 NUDT1 1521 [+ or -] 1018 OXR1 0.876 [+ or -] 0.199 PNKP 1382 [+ or -] 0.601 PRNP 2908 [+ or -] 2329 SCARA3 0.762 [+ or -] 0.376 SEPP1 -- SGK2 4371 [+ or -]5854 Functional grouping Gene P value n-VLDL versus 0 VLDL Nitric oxide metabolism NOS1 (nNOS) -- Nitric oxide biosynthesis NOS2 (iNOS) 0.0005 * NOS3 (eNOS) 0.0047 * Nitric oxide biosynthesis DYNLL1 0.9860 Regulation GLA 0.6263 HSP90AB1 0.5550 IL8 0.0794 INS -- NOS1AP 0.1139 Other nitric oxide AKT1 0.5801 Biosynthesis genes ARG2 0.3450 DDAH2 0.4354 EGFR 0.1798 GCH1 0.0579 GCHFR 0.1486 Nitric oxide induced CDKN1A 0.4282 CXCF8 -- JUN 0.2130 VEGFA 0.2582 Nitric oxide suppressed CCNA1 0.4048 MYB -- TROAP 0.0192 * Nitric oxide signalling CAMK1 0.2150 DFG4 0.3199 GRIN2D 0.0832 PPP3CA 0.4840 PRKAR1B 0.1029 PRKCA 0.5305 NQOl 0.0018 * Superoxide metabolism AFOX12 0.0911 Release DUOX1 0.1697 Oxidoreductases DUOX2 0.3574 Peroxidases NOX5 -- PRG3 -- SOD1 0.4863 SOD2 0.8203 SOD3 -- Other superoxide CCS 0.4132 Metabolism genes NCF1 -- NCF2 0.4988 PREX1 0.6021 Oxidative stress MPO -- Antiapoptotic MTL5 0.1880 NME5 0.7349 PRDX2 0.9952 RNF7 0.5334 Antioxidants APOE -- MT3 -- VIMP -- SRXN1 0.2554 Glutathione peroxidases GPX1 0.4132 GPX2 0.4698 GPX3 0.1599 GPX4 0.4714 GPX5 -- GPX6 -- Other oxidoreductases CAT 0.8239 EPX 0.4422 LPO -- MSRA 0.48110 PRDX6 0.3161 TPO 0.5814 TXNRD2 0.1712 Other peroxidases CSDE1 0.4558 CYGB -- GPR156 -- PRDX2 0.9953 PRDX5 0.3940 TTN 0.0008 * Regulation FOXM1 0.0670 GLRX2 0.9657 SCRT2 -- SIRT2 0.3329 Other oxidative stress ATOX1 0.6551 Response genes DUSP1 0.3110 GSS 0.4227 KRT1 -- MBL2 0.1870 NUDT1 0.3459 OXR1 0.3577 PNKP 0.2771 PRNP 0.2226 SCARA3 0.3352 SEPP1 -- SGK2 0.3548 Functional grouping Gene ox-VLDL (rel. exp.)# Nitric oxide metabolism NOS1 (nNOS) -- Nitric oxide biosynthesis NOS2 (iNOS) 15,212 [+ or -] 7287 NOS3 (eNOS) 0.602 [+ or -] 0.173 Nitric oxide biosynthesis DYNLL1 1066 [+ or -] 0.345 Regulation GLA 1438 [+ or -] 0.231 HSP90AB1 1553 [+ or -] 0.343 IL8 6532 [+ or -] 2814 INS -- NOS1AP 2028 [+ or -] 0.395 Other nitric oxide AKT1 1356 [+ or -] 0.513 Biosynthesis genes ARG2 1847 [+ or -] 0.109 DDAH2 0.621 [+ or -] 0.386 EGFR 2639 [+ or -] 1704 GCH1 1354 [+ or -] 0.701 GCHFR 0.327 [+ or -] 0.249 Nitric oxide induced CDKN1A 1533 [+ or -] 0.904 CXCF8 -- JUN 21,111 [+ or -] 0.890 VEGFA 5372 [+ or -] 1224 Nitric oxide suppressed CCNA1 1534 [+ or -] 0.128 MYB -- TROAP 1189 [+ or -] 0.156 Nitric oxide signalling CAMK1 1916 [+ or -] 0.473 DFG4 1178 [+ or -] 0.920 GRIN2D 1012 [+ or -] 0.554 PPP3CA 1386 [+ or -] 0.067 PRKAR1B 1472 [+ or -] 0.137 PRKCA 1909 [+ or -] 0.149 NQOl 2525 [+ or -] 0.835 Superoxide metabolism AFOX12 3253 [+ or -] 0.187 Release DUOX1 0.404 [+ or -] 0.048 Oxidoreductases DUOX2 0.439 [+ or -] 0.069 Peroxidases NOX5 -- PRG3 -- SOD1 1608 [+ or -] 0.492 SOD2 1819 [+ or -] 0.502 SOD3 -- Other superoxide CCS 1173 [+ or -] 0.518 Metabolism genes NCF1 -- NCF2 3637 [+ or -] 1943 PREX1 0.857 [+ or -] 0.255 Oxidative stress MPO -- Antiapoptotic MTL5 0.759 [+ or -] 0.479 NME5 1645 [+ or -] 0.939 PRDX2 1040 [+ or -] 0.029 RNF7 1248 [+ or -] 0.140 Antioxidants APOE -- MT3 -- VIMP -- SRXN1 8175 [+ or -] 3135 Glutathione peroxidases GPX1 1070 [+ or -] 0.387 GPX2 0.890 [+ or -] 0.314 GPX3 1683 [+ or -] 0.682 GPX4 1283 [+ or -] 0.663 GPX5 -- GPX6 -- Other oxidoreductases CAT 1287 [+ or -] 0.542 EPX 2491 [+ or -] 0.655 LPO -- MSRA 1062 [+ or -] 0.458 PRDX6 1526 [+ or -] 0.741 TPO 0.868 [+ or -] 0.095 TXNRD2 0.933 [+ or -] 0.284 Other peroxidases CSDE1 0.868 [+ or -] 0.558 CYGB -- GPR156 -- PRDX2 1040 [+ or -] 0.029 PRDX5 1035 [+ or -] 0.537 TTN 0.350 [+ or -] 0.192 Regulation FOXM1 1695 [+ or -] 0.914 GLRX2 1323 [+ or -] 0.778 SCRT2 -- SIRT2 1339 [+ or -] 0.292 Other oxidative stress ATOX1 1515 [+ or -] 0.757 Response genes DUSP1 1490 [+ or -] 0.797 GSS 1124[+ or -] 0.173 KRT1 -- MBL2 16,376 [+ or -] 10,431 NUDT1 1871 [+ or -] 0.589 OXR1 1044 [+ or -] 0.606 PNKP 1566 [+ or -] 0.907 PRNP 5331 [+ or -] 2944 SCARA3 0.482 [+ or -] 0.277 SEPP1 -- SGK2 8491 [+ or -]5139 Functional grouping Gene P value ox-VLDL versus 0 VLDL Nitric oxide metabolism NOS1 (nNOS) -- Nitric oxide biosynthesis NOS2 (iNOS) 0.0099 * NOS3 (eNOS) 0.0095 * Nitric oxide biosynthesis DYNLL1 0.7615 Regulation GLA 0.0424 HSP90AB1 0.0485 IL8 0.0272 * INS -- NOS1AP 0.0075 * Other nitric oxide AKT1 0.3054 Biosynthesis genes ARG2 0.0006 * DDAH2 0.0616 EGFR 0.1045 GCH1 0.4329 GCHFR 0.0136 * Nitric oxide induced CDKN1A 0.3065 CXCF8 -- JUN 0.0949 VEGFA 0.0134 * Nitric oxide suppressed CCNA1 0.0097 * MYB -- TROAP 0.1658 Nitric oxide signalling CAMK1 0.0161 * DFG4 0.7098 GRIN2D 0.9659 PPP3CA 0.0155 * PRKAR1B 0.0259 * PRKCA 0.0019 * NQOl 0.0312 * Superoxide metabolism AFOX12 0.0001 * Release DUOX1 0.0029 * Oxidoreductases DUOX2 0.0001 * Peroxidases NOX5 -- PRG3 -- SOD1 0.1094 SOD2 0.0539 SOD3 -- Other superoxide CCS 0.5367 Metabolism genes NCF1 -- NCF2 0.0549 PREX1 0.4455 Oxidative stress MPO -- Antiapoptotic MTL5 0.3562 NME5 0.2187 PRDX2 0.7286 RNF7 0.0863 Antioxidants APOE -- MT3 -- VIMP -- SRXN1 0.0075 * Glutathione peroxidases GPX1 0.8239 GPX2 0.5164 GPX3 0.0960 GPX4 0.5417 GPX5 -- GPX6 -- Other oxidoreductases CAT 0.4419 EPX 0.0087 * LPO -- MSRA 0.8025 PRDX6 0.2989 TPO 0.1235 TXNRD2 0.6938 Other peroxidases CSDE1 0.7056 CYGB -- GPR156 -- PRDX2 0.7286 PRDX5 0.9149 TTN 0.0026 * Regulation FOXM1 0.2597 GLRX2 0.5124 SCRT2 -- SIRT2 0.1279 Other oxidative stress ATOX1 0.3056 Response genes DUSP1 0.1381 GSS 0.3452 KRT1 -- MBL2 0.1108 NUDT1 0.0340 OXR1 0.8910 PNKP 0.2675 PRNP 0.0284 SCARA3 0.0318 SEPP1 -- SGK2 0.0698 Functional grouping Gene P value ox-VLDL versus n-VLDL Nitric oxide metabolism NOS1 (nNOS) -- Nitric oxide biosynthesis NOS2 (iNOS) 0.0513 NOS3 (eNOS) 0.1857 Nitric oxide biosynthesis DYNLL1 0.8376 Regulation GLA 0.1046 HSP90AB1 0.2456 IL8 0.0654 INS -- NOS1AP 0.0125 * Other nitric oxide AKT1 0.7300 Biosynthesis genes ARG2 0.0278 DDAH2 0.0439 EGFR 0.9498 GCH1 0.2406 GCHFR 0.1734 Nitric oxide induced CDKN1A 0.9399 CXCF8 -- JUN 0.4297 VEGFA 0.0226 * Nitric oxide suppressed CCNA1 0.8953 MYB -- TROAP 0.1917 Nitric oxide signalling CAMK1 0.5063 DFG4 0.6873 GRIN2D 0.5407 PPP3CA 0.9211 PRKAR1B 0.7621 PRKCA 0.0052 * NQOl 0.1657 Superoxide metabolism AFOX12 0.0017 * Release DUOX1 0.0115 * Oxidoreductases DUOX2 0.1435 Peroxidases NOX5 -- PRG3 -- SOD1 0.1532 SOD2 0.1200 SOD3 -- Other superoxide CCS 0.8134 Metabolism genes NCF1 -- NCF2 0.1460 PREX1 0.7658 Oxidative stress MPO -- Antiapoptotic MTL5 0.9919 NME5 0.3483 PRDX2 0.8198 RNF7 0.9827 Antioxidants APOE -- MT3 -- VIMP -- SRXN1 0.1113 Glutathione peroxidases GPX1 0.5210 GPX2 0.4669 GPX3 0.9458 GPX4 0.8239 GPX5 -- GPX6 -- Other oxidoreductases CAT 0.6603 EPX 0.1634 LPO -- MSRA 0.6788 PRDX6 0.6865 TPO 0.9056 TXNRD2 0.2250 Other peroxidases CSDE1 0.4081 CYGB -- GPR156 -- PRDX2 0.8198 PRDX5 0.5503 TTN 0.7083 Regulation FOXM1 0.8012 GLRX2 0.6323 SCRT2 -- SIRT2 0.9676 Other oxidative stress ATOX1 0.7224 Response genes DUSP1 0.3571 GSS 0.5988 KRT1 -- MBL2 0.1335 NUDT1 0.6342 OXR1 0.6727 PNKP 0.7837 PRNP 0.4218 SCARA3 0.3568 SEPP1 -- SGK2 0.5327 n-VLDL = RT-PCR assay performed after 24 h incubation of HUVEC with native VLDL (140 [micro]g/mL); ox-VLDL = RT-PCR assay performed after 24 h incubation of HUVEC with oxidised VLDL (140 [micro]g/mL). #Relative expression (rel. exp.) versus untreated cells (0 VLDL) grown for an overall comparable time. cDNAs undetectable under the condition assayed were indicated by "--". P values considered statistically significant by ANOVA were marked by "*".
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Research Article; very low density lipoproteins; and human umbilical vein endothelial cells|
|Author:||Magnifico, Maria Chiara; Oberkersch, Roxana Elena; Mollo, Azzurra; Giambelli, Luca; Grooten, Yasmine|
|Publication:||Oxidative Medicine and Cellular Longevity|
|Date:||Jan 1, 2017|
|Previous Article:||Redox Regulating Enzymes and Connected MicroRNA Regulators Have Prognostic Value in Classical Hodgkin Lymphomas.|
|Next Article:||Protective Effect of Icariin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury.|