Materials and Methods
6. EXPERIMENTAL METHODS
6.1 Endothelial cell preparation
ECs were obtained from human umbilical cord veins 145. A sterile technique was utilized in all manipulations of the cord. The cord was severed from the placenta soon after birth, placed in a sterile container filled with 100 U/ml penicillin, 100 µg/ml streptomycin in physiologic solution (NaCl 0.9%), and held at 4°C until processing. Storage time averaged about 4 h, and cords were discarded if held more than 12 h. The cord was inspected, and all areas with clamp marks were cut off. The umbilical vein was cannulated with a blunt 14 gauge needle, 2 cm long, and the needle was secured by clamping the cord over the needle. The vein was perfused 2 times with 30 ml of physiologic solution to wash out the blood and allowed to drain. The other end of the umbilical vein was clamped and 10 ml of 0.1% collagenase (specific activity: 316 U/ml, Gibco, Invitrogen) in physiologic solution was infused. After incubation of the cord at 37°C for 10 min, the collagenase solution containing the ECs was flushed from the cord by perfusion with 15 ml of culture medium. The effluent was collected in a sterile 50 ml conical centrifuge tube and sedimented at 280 g for 5 min and washed once with 10 ml of culture medium, and the cell button was resuspended by trituration in 5 ml of fresh culture medium.
6.2 Endothelial cell culture
The cell suspension was plated on 25-cm2 tissue culture flasks previously coated with 1% gelatine and cultured in supplemented culture medium (M199 with 10% heat inactivated foetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2mM L-glutamine, 10mM HEPES pH 7.4, heparin 12 U.I./ml, 1% RDGF, Sigma). The flasks were placed at 37°
in 5% CO2 humidified incubator. The cells were fed three times a week with a complete change of fresh culture medium until 90% confluent.
For subculture, cells were harvested with 0.25% trypsin-0.02% EDTA. After trypsinization, the viability was determinated by trypan blue staining and the cells were identified by their typical cobblestone morphology with phase contrast microscopy. The ECs were characterized by immunofluorescence staining with monoclonal antibodies specific for typical ECs’markers (see below 6.4). After each pharmacological intervention, cell viability was also checked by MTT assay (see below 6.7). Cells up to the fourth passage were used for all experiments. The investigation conformed with the principles outlined in the declaration of Helsinki for use of human tissue.
6.3 Preparation of Retinal Derived Growth Factor (RDGF)
Eyes from recently slaughtered adult cows were obtained from a local abattoir. Retinas were removed from eyes and suspended in phosphate buffered saline (PBS) without Ca2+ and Mg2+ (one retina per ml) at 4°C for 30 min. The suspension was centrifuged at 450 g for 10 min at 4°C. The supernatant was ultracentrifuged at 2200 g for 15 min at 4°C. The RDGF was then sterilized with 0.2 µm filter and used immediately or aliquoted and stored at -20°C in the dark.
6.4 HUVEC characterization performed by Flow Cytometry
Cells were detached with trypsin and 105 cells were labeled for 20 minutes at room temperature with the following primary monoclonal antibodies: anti von Willebrand Factor (vWF) (Immunotech), anti kinase insert domain receptor (KDR) (Sigma) and anti vascular endothelial (VE)-cadherin (Sigma). An isotype-matched IgG1 antibody (Sigma) was used as negative control. The cells were further incubated with a fluorescein
isothiocyanate(FITC)-conjugated goat anti-mouse IgG (H+L) antibody (Beckman Coulter) and analyzed by flow cytometry analysis (FACscan, BD Biosciences).
6.5 Drugs
All reagents were dissolved in the appropriate vehicle and stored at −80 °C for no longer than 6 months. TEL stock solution (10-2M) was obtained dissolving the drug in NaOH 1M and taking it to pH 7.4. LOS was dissolved in water (10-2M). Dexamethasone (DEX) was dissolved in ethanol 50% (2x10-2M). EXP3174 (10-1M) and EXP3179 (10-2M) were dissolved in dimethilformamide. Ciglitazone (CIG: 10-1M) was dissolved in dimethilformamide. Rosiglitazone (RSG) was dissolved in ethanol 50% (10-1M ).
Before every cell treatment all reagents were diluted in serum-free M199 and old medium was replaced with fresh pre-warmed M199 containing 20% FCS.
Internal controls were also performed with all the diluents used to dissolve the inflammatory stimuli and the drugs under investigation. Neither drugs nor vehicles modified cell morphology, cell number, viability and unstimulated CAMs expression.
We repeated the experiments at least three times, that is, with three different HUVEC donors, to ensure the reproducibility of results.
6.6 ELISA
The endothelial inflammation grade and its modulation by the drugs under investigation were assessed by ELISA (Enzyme-Linked ImmunoadSorbent Assay) and surface adhesion molecules expression was measured as absorbance units (AU).
HUVECs grown to 80% confluence in 96-well plates (Gibco, Invitrogen) previously coated with gelatine 1% were treated with the indicated concentrations of drugs added to each well in absence or presence of the inflammatory stimulus. After the incubation period, HUVECs were washed three times in PBS containing 1% BSA (PBSA) and fixed with 2% (wt/vol)
paraformaldehyde for 15 min at room temperature and incubated with 3% (wt/vol) BSA in PBS at 37°C for 1h to block specific binding sites. ELISA was performed by incubating mono-layers at 37 °C for 2 h with primary monoclonal antibodies (Ancell) or appropriate control isotype (Gibco, Invitrogen), (5 µg/ml in PBSA for both), then adding for 1 h the secondary goat antimouse IgG-peroxidase coniugate antibody (Immunotech), diluted 1/10.000 in PBSA. The cells were washed three times with PBSA between each incubation step, and the integrity of the monolayer was monitored by phase-contrast microscopy. As a final step, the peroxidase substrate O-phenylenediamine dihydrochloride (Gibco, Invitrogen) was added and colour development was stopped by adding HCl (3 M). ICAM-1 and VCAM-1 expression was quantified by absorbance determination (Titertek Instruments Inc.) at 490 nm wavelength. The absorbance of the control isotype was subtracted from the readings. Within- and between-assay variability was 3.5 and 5.5%, respectively.
6.7 MTT assay
Cell viability was evaluated by 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), an assay used to determine cell vitality by measuring the activity of one of the oxidative enzymes. The dye, positively charged tetrazolium salts, is reduced to an insoluble violet formazan product by mitochondria succinic dehydrogenase 146. Formazan production is decreased in presence of impaired mitochondrial redox function and enhanced free radical production and may, therefore, be used as an index of cell viability responses to experimental interventions. Briefly, after removing treated plates from the incubator, fresh medium containing a fixed volume of reconstituted MTT (final concentration: 0.5 mg/ml) was added to the wells; the plates were kept at 37 °C in a 5% CO2 environment for 2 h. Subsequently, formazan crystals were dissolved in dimethyl sulfoxide(DMSO) and absorbance was measured by spectrophotometry at 570 nm wavelength. The within- and between-assay variability was 3 and 5%, respectively.
6.8 Comet assay
DNA damage in response to exogenous H2O2 was assessed by modified alkaline single cell gel electrophoresis (Comet), an assay based on the free moving capacity of broken DNA loops towards the anode during high pH-electrophoresis, resulting in “comet-like” structures whose tail-to-head relative intensity at fluorescence microscopy is proportional to DNA damage. As described previously 147, slides were spread with 1% normal melting agarose (Sigma) in Ca2+ and Mg2+-free PBS (Sigma–Aldrich) and left to solidify until subsequent use. After H2O2 exposure and pharmacological treatment, HUVECs were centrifuged, then resuspended in 0.5% low melting agarose (Agarose wide range, Sigma) and incubated at 37 °C. Cell suspension was rapidly dropped on top of the first agarose layer, covered with a coverslip and allowed to solidify at 4 °C. After coverslip removal, a final layer of 0.5% low melting agarose was added to the slide. The slides were then lysed by immersion into a coupling jar containing freshly prepared cold lysing buffer (2.5M NaCl, 100mM Na2EDTA, 10mM Tris, 10% DMSO, and 1% Triton X-100, pH 10.0) for 1 h at 4 °C. After the lysis step, the slides were placed in a horizontal gel electrophoresis chamber filled with freshly prepared cold alkaline buffer (300mM NaOH, 1mM Na2EDTA, pH > 13). Slides were left in this solution for 20 min to allow DNA unwinding; the DNA fragments were then separated by electrophoresis for 20 min at 25V (300 mA). After electrophoresis the slides were washed with neutralizing buffer (0.4M Tris, pH 7.5) and stained with ethidium bromide (20 µg/ml). Coded slides were viewed using a fluorescence microscope (Nikon Eclipse E800) with 200× magnification. For each experimental phase, 100 randomly selected cells (50 cells from each replicate slide) were scored and analyzed by the Comet Image Analysis System, version 5.5 (Kinetic Imaging Ltd.).
6.9 TOSC assay
The total oxyradical scavenging capacity (TOSC)assay allows to discriminate the relative antioxidant capacity of a test compound by measuring the amount of ethylene produced during the oxidation of α-keto-γ-methiolbutyric acid (KMBA, Sigma, 0.2 mM) by hydroxyl, peroxyl radicals and peroxynitrite 148. As described in more detail 149, hydroxyl radicals were generated at 35 °C by the iron plus ascorbate-driven Fenton reaction (1.8 µM Fe3+, 3.6 µM EDTA, and 180 µM ascorbic acid in 100 mM potassium phosphate buffer, pH 7.4). Peroxyl radicals derived from thermal homolysis of 20 mM 2,2’-azo-bis-amidinopropane (ABAP, Sigma) at 35 °C in 100 mM potassium phosphate buffer, pH 7.4. Peroxynitrite was generated from the decomposition of N-ethylcarbamide (SIN-1, Sigma) in the presence of 0.2 mM KMBA, 100 mM potassium phosphate buffer, pH 7.4, and 0.1 mM DTPA, at 35 °C. The concentration of SIN-1 was varied to achieve an ethylene yield equivalent to the iron– ascorbate and ABAP systems. Reactions with KMBA were carried out in 10 ml vials sealed with gas-tight Mininert® valves (Supelco) in a final volume of 1 ml. Ethylene production was measured by gas-chromatographic analysis of 200 µl aliquots taken from the head space of vials at timed intervals during the course of the reaction. Analyses were performed with a Hewlett-Packard gas chromatograph (HP 6890 Series) equipped with a Supelco SPB-1 capillary column and a flame ionization detector (FID). The oven, injection and FID temperatures were, respectively, 35, 160 and 220 °C. Helium was the carrier gas (at a flow rate of 1 ml/min) at a split ratio of 20:1. Data were expressed as area under the kinetic curve (trapezoid method). The within- and between-assay variability was 2 and 4%, respectively.
7. EXPERIMENTAL DESIGN
7.1 TNF-α induced CAM expression
HUVECs were incubated with TNF-α (10 ng/ml) (Peprotech; specific activity 2×107 U/mg) either per se or in presence of TEL (10−7–10−4 M), LOS (10−7–10−4 M) or PDTC (10−6–10−4 M, Sigma) in separate HUVEC wells. HUVECs exposed at that concentration of TNF-α showed no evidence of cytotoxicity according to MTT test or trypan blue staining. The two LOS active metabolites (EXP-3174, EXP-3179) and the steroid DEX were used at a single 10−4M concentration. Incubations lasted 48 h, a time interval allowing maximum VCAM-1 and ICAM-1 expression as validated in pilot studies.
7.2 Pharmacological PPAR-γ manipulation
To evaluate the effect of PPAR-γ stimulation on TNF-α-inducible VCAM-1 expression, HUVECs were separately exposed to TEL (10−4M), CIG (10−7–10−4 M ×48 h, Sigma) and RSG (10−7–10−4 M ×48 h), two PPAR-γ receptor agonists 150. The contribution of partial PPAR-γ agonism by TEL 141 was assessed by evaluating its effect per se (10−4 M) or in presence of either T0070907 (10−9–10−6 M, Cayman) or GW9662 (10−9–10−6, Alexis), two specific PPAR-γ receptor antagonists 150,151. Since the results did not differ throughout the explored concentration range, only the highest one was reported in the Results.
7.3 AII-mediated cell damage and effect of AT1R blockade
In order to test whether AII-mediated responses could be evoked in our experimental conditions, AII (10-8-10-5M) was added for 150 min by measuring cell viability (MTT test). To analyse AT1R antagonism, TEL and LOS (10−7–10−5 M for both) were added 30 min prior to AII at a concentration (10−6M×150 min) that reduced cell viability by 50%. Experiments were carried out in 24 h serum starved HUVECs cultured in serum-free medium during AII stimulation.
7.4 H2O2-induced cell damage
The effects of TEL, LOS and DEX (10−4M for all) on cell viability (MTT assay) and DNA damage (Comet assay) was evaluated in HUVECs exposed to H2O2 (3×10−4M×90 min), an experimental condition that reduced cell viability by 50% in preliminary experiments. Each drug was added 30 min before adding H2O2 to the wells.
7.5 Effect of TEL on in vitro TOSC
In vitro TOSC was evaluated in presence of vehicle or increasing concentrations (10−7–10−4
8. DATA PROCESSING AND STATISTICS
Adhesion molecule data (each single value representing the mean of four replicate wells) were reported as absolute values or percent changes from TNF-α-stimulated values.
Cell viability (MTT assay, each single value representing the mean of at least four replicate wells) was expressed as percent changes from control conditions.
Comet assay results were reported as tail DNA percentage (TDNA%, each value representing the mean of four replicates).
TOSC was expressed as the percent change in the area under the kinetic curves in presence of vehicle or TEL. Accordingly, TOSC may potentially range between 0 and 100%, corresponding to no (i.e. unchanged ethylene formation) and total (completely blocked ethylene formation) scavenging capacity, respectively.
Data were analysed by non parametric statistics for paired (Wilcoxon’s test) comparisons. Strength of association was tested by non parametric Spearman’s correlation coefficients (rho). P-values <0.05 were considered statistically significant. The results were expressed as the mean±S.D.
