Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

Porcine Aortic Endothelial Cells were isolated as previously described [4]. The cells were cultured in Human Endothelial Basal Growth Medium (Gibco-Invitrogen, Paisley, UK) supplemented with 5% Foetal Bovine Serum (FBS, Gibco-Invitrogen) and 1% antibiotic/antimicotic (Gibco-Invitrogen). Cell number and viability (95%) were determined using a Thoma chamber under a phase-contrast microscope after vital staining with trypan blue dye. The cells were placed in T-25 tissue culture flasks (approximately 3x10⁵ cells/flask) (T25 Falcon Beckton-Dickinson, Franklin Lakes, NJ, USA) in a 5% CO₂ atmosphere at 38.5°C. The cells were maintained in a logarithmic growth phase by routine passages every 2–3 days at a 1:3 split ratio.

All experiments were performed with cells from the third to the eighth passage.

Previous observations indicated that the time of culture could influence gene expression in a primary culture of pAECs; therefore, we decided to utilise a relative time control point for each time of treatment. The pAECs were grown until confluence in a flat bottom 24-well assay plate (approximately 4x10⁴ cells/well) (353813 Falcon Beckton-Dickinson) or, for immunohistochemical study, in 8-well slide chambers (approximately 4x10⁴ cells/well) (354631 Beckton-Dickinson).

L-NAME 5 mM or 10 mM (N5751, Sigma) was added to the culture with or without LPS (10 μg/ml) and incubated for 15 h. At the end of treatment the total levels of NO and the apoptosis rate were evaluated.

Total ribonucleic acid (RNA) from pAECs was isolated using an RNeasy Mini Kit 50 (Qiagen Sciences Inc, MD, USA) and treated with RNase-free DNase kit (Qiagen) according to the manufacturer’s instructions.

The RNA concentration was spectrophotometrically quantified (A₂₆₀ nm) and 1 μg of total RNA was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit (Bio-RAD Laboratories Inc., California, USA) at a final volume of 20 μl. Swine primers (eNOS, iNOS and Hypoxanthine-guanine phosphoribosiyltransferase -HPRT) were designed using Beacon Designer 2.07 Software (premier Biosoft International, Palo Alto, Ca, USA). Their sequences, expected PCR product length and accession number in the EMBL database are shown in Table 1. Real-time quantitative polymerase chain reaction (PCR) was carried out in an iCycler Thermal Cycler (Bio-RAD) using SYBR green I detection. A master-mix of the following reaction components was prepared to the indicate end-concentrations: forward primer (0.2 μM) and reverse primer (0.2 μM), 1x IQ SYBR Green BioRad Supermix (Bio-RAD Laboratories Inc.). Then, 2.5 μl of cDNA was added to 22.5 μl of the master mix. All samples were carried out in duplicate. The following real-time PCR protocol was employed: initial denaturation for 3 min at 95°C, 40 cycles of 95°C for 15 sec and 60°C for 30 sec, followed by a melting step with slow heating from 55°C-95°C with a rate of 0.5°C/s. The housekeeping gene HPRT data were used to normalise the amount of RNA.

The expression of iNOS and eNOS mRNA under standard culture conditions was calculated as a threshold cycle (deltaC_T) (HPRT C_T – iNOS/eNOS C_T). The expression of iNOS and eNOS mRNA during LPS treatment was calculated as the fold of increase using the ΔΔCt method (ABI Prism 7700 Sequence detection System User Bulletin #2. Relative quantification of gene expression; 1997 and 2001).

Real-time efficiency for each primer set was acquired by amplification of a standardised cDNA dilution series. The specificity of the amplified PCR products was verified by analysis of the melting curve, which is sequence-specific and by an agarose gel electrophoresis run.

The cells were harvested and lysed in SDS solution (Tris–HCl 50 mM pH 6.8; SDS 2%; glycerol 5%). The protein content of cellular lysates was determined by a Protein Assay Kit (TP0300, Sigma). Aliquots containing 20 μg of proteins were separated on NuPage 4-12% bis-Tris Gel (Gibco-Invitrogen) for 50 min at 200 V. The proteins were then electrophoretically transferred onto a nitrocellulose membrane. The blots were washed in PBS and protein transfer was checked by staining the nitrocellulose membranes with 0.2% Ponceau Red. Non-specific binding on nitrocellulose membranes was blocked with 5% milk powder in PBS-T20 (Phosphate Buffer saline-0.1% Tween-20) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20). After several washings with PBS-T20, the membranes were incubated with the secondary biotin-conjugate antibody and then with a 1:1000 dilution of an anti-biotin horseradish peroxidase (HRP)-linked antibody. The western blots were developed using chemiluminescent substrate (Super Signal West Pico Chemiluminescent Substrate, Pierce Biotechnology, Inc, Rockford, IL, USA) according to the manufacturer’s instructions. The intensity of the luminescent signal of the resultant bands was acquired by Fluor-S^TM Multimager using Quantity One Software (Bio-RAD).

In order to normalise the NOS data on the housekeeping protein, membranes were stripped (briefly: the membranes were washed 5 min in water, then 5 min in 0.2 M NaOH and then washed again in water) and re-probed for housekeeping β-tubulin (1:500 sc-5274 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

The relative protein content (NOS/ β-tubulin) was expressed as arbitrary units (AUs).

The cells were fixed with ethanol:acetic acid (2:1) for 10 min at −20°C, and permeabilised with 0.1% Triton X-100. Thereafter, the cells were washed with PBS three times and incubated for 1 h at RT with 10% FBS in DPBS (Dulbecco Phosphate Buffer Solution, Cambrex Bio-Science INC, Wakersville, MA, USA). Primary antibodies: 1:100 anti-iNOS (BD Transduction) and 1:100 anti eNOS (Biomol) were added, and the slides were incubated in a humidified chamber at 4°C overnight. After several washes with PBS, a 1:800 dilution of fluorescein isothiocyanate (FITC)-conjugated secondary antibodies was added. The cells were then counterstained with propidium iodide (PI) and examined under an epifluorescence microscope (Eclipse e600 Nikon, Japan) equipped with appropriate filters and a Nikon DXM 1200 digital camera with ACT-2U software for DMX 1200.

The NO concentrations in culture media were determined by the “Total Nitric Oxide Assay” (DE 1600 R&D Systems, Minneapolis, MN 55413, USA) using Appliskan 100-240 V (Thermo Electron Corporation, Vantaa Finland). This assay determines total nitric oxide concentration based on the enzymatic conversion of nitrate to nitrites by nitrate reductase. The reaction is followed by a colorimetric detection of nitrite as an azo dye product of the Griess Reaction. The sensitivity of the assay was less than 1.35 μmol/L, and the intra- and interassay coefficients of variation were less than 5% and 7% respectively. The basal level of NO present in the fresh endothelial medium was subtracted from all samples (CTR or treated cells).

Under standard culture conditions, the iNOS mRNA expression level was notably lower than eNOS (Figure 2).

Lipopolysaccharide induced a significant increase of iNOS mRNA levels starting after 2 h of continuous treatment, remaining stable until 7 h and declining at 15 and 24 h, without reaching the iNOS mRNA level in the pAECs cultured under standard conditions (Figure 3A).

Conversely, eNOS mRNA levels were decreased by LPS treatment starting after 5 h of continuous treatment until the end of the experiment (Figure 3B).

The iNOS protein level detected in the pAECs cultured under standard culture conditions was very low while the LPS treatment induced a significant increase of iNOS at 7 h; the protein content then decreased without reaching the basal level of the control cells (Figure 4A).

The eNOS protein is largely expressed in pAECs cultured under standard conditions while LPS treatment induced a significant decrease of eNOS protein expression (Figure 5A).

The iNOS protein was detected in the cytoplasm of the pAECs cultured under standard culture conditions (Figure 6A-C) while, in LPS treated cells, in addition to a cytoplasmatic signal, nuclear staining was detected (Figure 6D-F).

Under standard culture conditions, the eNOS protein is diffusely distributed in the cytoplasm of pAECs (Figure 7A, B); LPS treatment resulted in a modification of the signal with more intense staining in the perinuclear region (Figure 7C-F).

Lipopolysaccharide treatment did not induce any significant change in the total level of nitric oxide (Figure 8). L-NAME (5-10 mM) induced a dose dependent decrease of total NO, both under standard control conditions and with LPS treatment (Figure 9).

Lipopolysaccharide induced a significant increase of the apoptosis rate in pAECs while L-NAME treatment (5 mM) partially protected cells against LPS-induced apoptosis (Figure 10). L-NAME 10 mM produced a slight but significant increase of the apoptosis rate in pAECs cultured under standard culture conditions and was unable to reduce LPS-induced apoptosis.