Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery

2.1. HUVEC Culture

Cryopreserved human umbilical vein endothelial cells (HUVECs) (ATCC Cat number PCS-100-010) were cultured in endothelial growth medium (Lonza) supplemented with 10% heat-inactivated foetal bovine serum (Sigma-Aldrich). Cultures were maintained at 37°C in a 5% CO₂ atmosphere at 95% humidity. The HUVECs were serially passaged (the replicative senescence model). Cells passaged < 8 times (population doubling (PD) < 20; with PD calculated as [ln{number of cells harvested} − ln{number of cells seeded}/ln2]) were regarded as young endothelial cells, while those passaged 27–35 times (PD > 96) were regarded as senescent [28]. The proliferation rate of the latter cells is remarkably reduced, and more than 70% are positive for senescence-associated β-galactosidase. Prior to use, HUVEC extracts from cells passaged 4–8 (young pool) and from cells passaged 27–35 (senescent pool) were mixed (performed in quadruplicate).

2.2. Isolation and Characterization of Young and Senescent eMV

Young and senescent HUVEC-derived MVs were isolated from their culture medium. Briefly, samples were centrifuged using serial centrifugations (15 min at 3000 rpm, 30 min at 14,000 rpm), and pellets were frozen and stored at −20°C until use. Prior to use, MVs from cells passaged 4–8 (young pool) and from cells passaged 27–35 (senescent pool) were mixed (performed in quadruplicate).

MVs from a medium containing young and senescent HUVEC cells were characterized in terms of size using a Beckman Coulter Cytomics FC 500 flow cytometer running CXP software. MVs were considered to be those events gated with a size between 0.5–1.5 μm; this gate was established from the side scatter versus forward scatter dot plot produced in a standardization experiment using the SPHERO™ Flow Cytometry Nano Fluorescent Size Standard Kit (Spherotech). The latter has size-calibrated fluorescent beads ranging from 0.1–1.9 μm in diameter. Events below 0.2 μm were excluded in order to adequately distinguish true events from the background; events > 1.9 μm were excluded to prevent possible confusion with apoptotic bodies. The absolute number of MVs (events) per μL was determined using Flow Count calibrator beads (Beckman Coulter) according to the manufacturer's recommendations and employing CXP software: (MVs counted × standard beads/L)/(standard beads counted). Data were recorded as the mean of three independent measurements of the same sample. The same number of senescent and young MVs was used in comparative analyses.

2.3. Western Blotting

The total protein content of extracts from young and senescent eMVs (performed using CytoBuster Protein Extraction Reagent lysis buffer (Millipore)), which contains a protease and a phosphatase inhibitor cocktail (Roche), was quantified using a BCA Protein Assay Kit (Pierce), employing BSA as the standard. Briefly, equal amounts of protein (25 μg protein/lane) were diluted with a reducing sample buffer and separated by 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels under reducing conditions. These proteins were then transferred onto nitrocellulose membranes (BioRad), blocked with TBS containing 0.1% Tween 20 and 5% dry nonfat milk for 1 h at room temperature, and incubated in the same buffer with different primary antibodies (anti-6PGL, Santa Cruz, sc-398833, dilution 1 : 500, 28 kDa; anti-GK, Santa Cruz, sc-393555, dilution 1/250, 61 kDa; anti-PSPH, Santa Cruz, sc-271421, dilution 1/250, 25 kDa). After washing with TBST, the membranes were incubated with Novex horseradish peroxidase-conjugated secondary antibodies followed by 2 additional washing steps with TBST. Bands were visualized with Luminata Crescendo Western HRP substrate (Millipore). Ponceau red (Sigma) staining was used as a loading control. Bands were quantified using Image J software (NIH) and normalized to Ponceau red.

2.4. Mass Spectrometry for NADPH Analysis

The presence of NADP⁺ and NADPH in eMVs was analyzed using a HPLC system Agilent 1100 coupled in-line to a TSQ Quantum triple quadrupole mass spectrometer (Thermo Scientific) equipped with an ESI source. Samples were prepared according to the “hot water/buffer extraction” protocol from Ortmayr [29]. Briefly, eMVs (50 × 10⁶) were added to 250 μL of 5 mM ammonium acetate buffer at pH 8.0. The supernatant was collected after sample incubation for 3 min at 85°C, cooling on dry ice, and centrifugation for 10 min at 4000 ×g. Equipment settings were also obtained from Ortmayr [29] (see Table 1). Mass spectra were recorded in negative mode for NADP⁺ and NADPH. The column used was ACE Excel 3 C18 -PFP 150 mm × 3.0 mm + 3 μm. Mass spectra of the column eluates were recorded in MS/MS mode using methanol and H₂O, adding 5 mM ammonium acetate. Nitrogen was used as the ion source gas. The sheath gas pressure was set at 40 (arbitrary units), the auxiliary gas pressure was set at 2 (arbitrary units), and the spray voltage was set at 3000 V. The capillary temperature was set at 350°C. Argon was used as the collision gas for collision-induced dissociation at a pressure of 1.5 mTorr (Q2). Data were acquired using Xcalibur Control Software.

2.5. Proteomic Analysis

Proteomic analysis involved in-gel protein digestion followed by HPLC and mass spectrometry (MS). In order to obtain sufficiently large samples, the four HUVEC “young pool” extracts (see Section 2.2) were mixed, as were the four HUVEC “senescent pool” extracts, and the corresponding pools for the MVs. These four samples were dissolved in lysis buffer (8 M urea, 2 M thiourea, 5% CHAPS, 2 mM TCEP-HCl, and protease inhibitors). MVs were lysed by ultrasonication (10 strokes, low amplitude) on ice. The lysates were then centrifuged at 20,000 ×g for 10 min at 4°C, and the supernatant containing the solubilized proteins was used for LC-MS/MS experiments. Total protein concentration was determined using the Pierce 660 nm protein assay (Thermo). An aliquot of each sample was diluted with loading sample buffer and then loaded onto 1.2 cm wide wells of a conventional SDS-PAGE gel (1 mm thick, 4% stacking gel, and 12% resolving gel). The run was stopped as soon as the front entered 1 cm into the resolving gel, so that the whole proteome became concentrated at the stacking/resolving gel interface. The separated protein bands were visualized by Coomassie staining, excised, cut into cubes (cross section 1 mm²), deposited in 96-well plates, and processed automatically in a Proteineer DP (Bruker Daltonics). The digestion protocol used was based on Shevchenko et al. [30] with minor variations: gel plugs were washed firstly with 50 mM ammonium bicarbonate and secondly with acetonitrile prior to reduction with 10 mM dithiothreitol in 25 mM ammonium bicarbonate solution; alkylation was performed with 55 mM indoleacetic acid in 50 mM ammonium bicarbonate solution. The gel pieces were then rinsed, firstly with 50 mM ammonium bicarbonate and secondly with acetonitrile, and subsequently dried under a stream of nitrogen. Proteomics grade trypsin (Sigma-Aldrich) at a final concentration of 16 ng/μL in 25% acetonitrile/50 mM ammonium bicarbonate solution was added and digestion allowed to proceed at 37°C for 4 h. The reaction was stopped by adding 50% acetonitrile/0.5% trifluoroacetic acid for peptide extraction. The tryptic-eluted peptides were dried by speed-vacuum centrifugation and then desalted on StageTip C18 Pipette tips (Thermo Scientific) until analysis by mass spectrometry.

A 1 μg aliquot of each sample was subjected to 1D-nano LC ESI-MSMS analysis using an Eksigent Technologies nanoLC Ultra 1D plus nanoliquid chromatography system coupled to a high-speed Triple TOF 5600 mass spectrometer (SCIEX) with a Nanospray III source. The analytical column used was a silica-based reversed phase Acquity UPLC M-Class Peptide BEH C18 Column (75 μm × 150 mm, 1.7 μm particle size, and 130 Å pore size) (Waters). The trap column was a C18 Acclaim PepMap™ 100 (Thermo Scientific) (100 μm × 2 cm, 5 μm particle diameter, and 100 Å pore size), switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 μL/min. The nanopump provided a flow-rate of 250 nL/min and was operated under gradient elution conditions. Peptides were separated using a 2–90% mobile phase B gradient (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid) for 250 min. The injection volume was 5 μL.

Data acquisition was performed with a Triple TOF 5600 System (SCIEX) (ionspray voltage floating 2300 V, curtain gas 35 psi, interface heater temperature 150°C, ion source gas 1 25 psi, and declustering potential 100 V). All data were acquired using information-dependent acquisition (IDA) mode with Analyst® TF 1.7 software (SCIEX). The following IDA parameters were chosen: a 0.25 s MS survey scan in the mass range 350–1250 Da, followed by 35 MS/MS scans of 100 ms in the mass range 100–1800 (total cycle time: 4 s). Switching criteria were set to ions greater than a mass to charge ratio (m/z) of 350 and smaller than 1250, with a charge state of 2–5 and an abundance threshold of more than 90 counts/s (cps). Former target ions were excluded for 15 s. The IDA rolling collision energy (CE) parameters script was used for automatically controlling the CE.

MS and MS/MS data obtained from individual samples were processed using Analyst TF 1.7 software. Raw data file conversion tools were used to generate mgf files which were then compared (using Mascot Server v.2.5.1 software; Matrix Science) to those in the UniProt Homo sapiens protein database. The latter contains 40,530 coding genes and their corresponding reversed entries. The search parameters were set as follows: carbamidomethyl (C) as the fixed modification and acetyl (protein N-term) and oxidation (M) as the variable modifications. Peptide mass tolerance was set to 25 ppm and 0.05 Da for fragment mass. Two missed cleavages were allowed. False discovery rates (≤1% at the spectral level) for peptide identification were calculated manually.

2.6. Functional Experiments

Two functional experiments were carried out in order to (1) test the ability of MVs to use blood metabolites to feed the NAD(P)H synthesis machinery, and (2) visually demonstrate that these metabolites induce NAD(P)H formation.

In the first experiment, five different blood metabolites were tested separately on young and senescent eMVs: lactate, pyruvate, glucose, branched-chain amino acids (BCAA; Val, Leu, Ileu), and glycerol. The concentration used was higher than the plasma value found in the bibliography (included in Figure 1): 7 mM, 0.5 mM, 15 mM, 0.3 mM, and 0.3 mM, respectively. In each tube, the test sample was formed by adding: 2 μL of blood metabolite, 5 μL of lysate including 10⁶ eMVs, and 5 μL of 3 mM NADPH (Santa Cruz Biotechnology, reference number 202725A). No metabolites were included in the control samples. NADPH was used as the internal control; in fact, this experimental design allowed us to analyze the metabolite-induced changes in the NAD(P)⁺/NAD(P)H ratio. For example, for a certain metabolite, a lower ratio indicates that this metabolite increases the reduced form. After a 10 min incubation, 1 μL of the sample was analyzed in a microvolume spectrophotometer (DeNovis DS-11 spectrophotometer), and the NAD(P)⁺/NAD(P)H ratio was determined by absorbance measurement at 260 nm/340 nm, respectively. The experiment was repeated three times using duplicated samples; twelve measures were performed in each case.

To visualize NAD(P)H synthesis, the eMVs (young and senescent) were incubated for 10 min with the blood metabolites previously described. Subsequently, 10 μL of each sample including 40,000 eMVs was dropped into the center of a small water-repellent circle made on a slide with a PAP pen. The drop and the circle were wrapped up with a coverslip and observed in a Zeiss LSM 780 multiphoton confocal microscope with a Mai-Tai DS (690–1040 nm tunable) laser. The excitation wavelength was 735 nm, more than twice that of NAD(P)H (340 nm), and the laser intensity was set at 5.5%. The detection range was 396–502 nm.

2.7. Statistical Analysis

Data were analyzed using R software. The two-tailed Student t-test was used to analyze differences in Western blotting results. Data of our first functional experiment (ability of MVs to use blood metabolites to feed the NAD(P)H synthesis machinery) were analyzed as follows. Kruskal-Wallis test (with post hoc Dunn's test) was performed for the comparison of the effect of metabolites in young eMVs and senescent eMVs. Mann–Whitney–Wilcoxon rank-sum test was used to compare the effect of every molecule between young and senescent: glucose in young versus glucose in senescent and so on. Significance was set at p < 0.05.

3.1. Proteomic Analysis

The pentose phosphate pathway (PPP) is the main biochemical route to synthesize NADPH. Hence, our first objective was to search for the presence of enzymes that participate in this pathway using mass spectrometry (MS) analysis of young and senescent eMVs. The majority of PPP enzymes were detected in this MS analysis, as shown in Table 2, except for 6PGL, GK, and PSPH, which were detected by Western blot (WB) analysis (Figure 2).

We next carried out a more detailed analysis of the enzymes detected in our MS results in order to connect the PPP with other biochemical routes. Table 2 includes the enzymes of the PPP and those of related metabolic routes proposed according to our proteomic analysis. To clarify these routes, a diagram of all the metabolic pathways proposed is included in Figure 1.

The organization of the different enzymes found in our proteomic analysis led us not only to establish the hypothetical biochemical routes that may be operative in the eMVs but also to point out the metabolites that feed these biochemical routes (Figure 1). All in all, it seems that both young and especially senescent eMVs have a very well-developed enzymatic organization designed to optimize NADPH production, and this is mainly due to the bidirectionality of most of these enzymatic activities. Besides the glycolytic pathways, which use glucose, lactate, and pyruvate, it is very important to point out the possibility that BCAA and glycerol can also be used to direct metabolites to the PPP. Mitochondria are also involved in this strategic metabolic organization: they can act as an NADPH biosynthetic organelle (note the high content of GLUD1 (number 22)) and supply AKG to the serine and 3PHP pathway. Enzymes involved in serine and glycerate metabolism are also included in these biochemical pathways using 3PHP and 3PG as metabolic intermediates.

One of the most interesting results was the higher content of most of these enzymes in senescent eMVs when compared with young eMVs; in fact, their enzymatic machinery seems to be designed to direct metabolites to the PPP or to obtain NADPH from new routes not included in young eMVs. The bold numbers in Table 2 indicate an increase > 25% when young and senescent eMVs were compared. A route specially used in senescent eMVs is the serine and PHP pathway, as suggested by the high content of the enzymes involved (numbers from 23 to 27) in senescent eMVs. The use of glutamine to form F6P using GFPT2 (number 28) is also especially elevated in senescent eMVs. Moreover, at the bottom of Table 2, we have included five enzymes that synthesize NADPH but are not included in Figure 1. Note that IDH1 and IDH2 are decreased in senescent eMVs, whereas the last three enzymes are increased.

3.2. Functional Analysis

We carried out two types of functional analysis; one to test the capacity of the proposed blood metabolites to feed the enzymatic machinery involved in reducing power synthesis and the other one to visualize this reducing power in eMVs. Unfortunately, NADH and NADPH are closely related molecules whose absorption and emission spectra are almost equal and, thereby, it has been impossible for us to separate NADH from NADPH in these functional analyses.

In order to study the effect of blood metabolites on NAD(P)H synthesis, eMVs were incubated 30 min with the proposed metabolites (lactate, pyruvate, glucose, glycerol, and BCCA). These metabolites were incubated separately at a concentration that doubled the maximal value indicated in Figure 1. The absorbance ratio 260 nm/340 nm is a measure of the relative proportion of NAD(P)⁺/NAD(P)H. In young eMVs, lactate and pyruvate diminished significantly this ratio, which indicates that both metabolites induced the formation of NAD(P)H. However, in senescent eMVs, all the metabolites tested induced a decrease in this ratio, glycerol and pyruvate being statistically significant. Statistically significant differences were also observed for glucose, glycerol, and BCAA when the effect of metabolites was compared in young and senescent eMVs (Figure 3).

An experiment using multiphoton confocal microscopy was performed to visualize the production of NAD(P)H by eMVs after incubation with the previously used metabolites. In order to detect only the reduced forms, eMVs were excited at 340 nm (670 nm in the multiphoton confocal microscope), and emission was detected at 460 nm. Only after incubation with the proposed blood metabolites could eMVs be clearly visualized, demonstrating that eMVs have the capacity to synthesize reducing power (NAD(P)H) (Figure 4), although not all vesicles were able to produce fluorescence. Only lactate and pyruvate induced fluorescence in young eMVs, and senescent eMVs seemed to be more efficient in producing fluorescence.

3.3. NADP⁺ and NADPH in eMVs

An MS analysis was carried out to accurately detect the presence of NADP⁺ and NADPH in eMVs. As can be seen in Figure 5, the presence of both cofactors was demonstrated in eMVs, although the content of NADP⁺ was greater than that of the reduced form. Differences in the NADP⁺/NADPH ratio were not observed either following incubation with the blood metabolites or between young and senescent eMVs.