Cigarette smoke total particulate matter generation
Particulate matter from cigarette smoke was generated using 3R4F reference cigarettes (University of Kentucky). Cigarettes were conditioned for a minimum of 48 h at 22°C and 60% humidity (ISO 3402:1999) before smoking on a RM20S smoking machine (Borgwaldt KC, Hamburg, Germany) under ISO standard conditions (35 ml puff taken over 2 sec. every minute; ISO 3308:2000). Smoke particulate matter (PM) was collected on a Cambridge filter pad at a minimum of 250 mg tar per pad. These are non-ISO standard conditions designed to provide a higher yield of particulate matter than conventionally achieved. PM was eluted with dimethylsulphoxide (DMSO) at 24 mg/ml and stored protected from light in single use aliquots at -20°C for no longer than 1 month.
Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs; Lifeline Cell Technology, California, U.S.A.) were maintained at 37°C in a 5% CO2 humidified atmosphere. Cells were cultured in VascuLife® VEGF media (Lifeline Cell Technology) supplemented with VEGF (5 ng/ml), EGF (5 ng/ml); basic FGF (5 ng/ml), IGF-1 (15 ng/ml), ascorbic acid (50 μg/ml), L-glutamine (10 mM), hydrocortisone hemisuccinate (1.0 μg/ml), heparin sulphate (0.75 units/ml) and foetal bovine serum (2%). Cells were seeded at 1×104 cells/ml for all assays with the exception of PCR experiments where cells were seeded at 2x104 cells/ml. Cells were treated for 24 h either with PM or with 1% DMSO as a diluent control. To examine the contribution of oxidative stress in regulating osteopontin levels, cells were incubated with the anti-oxidant ascorbate (200 μM) for 5 h prior to PM exposure.
Assessment of cell viability
Cell viability was measured by neutral red uptake assays. In brief, HUVECs were incubated in 150 μl neutral red solution diluted 1:65 in VascuLife® VEGF media. Cells were incubated for 3 h at 37°C to allow active uptake of the dye into viable cells. Plates were washed twice in PBS before the addition of 150 μl de-stain solution (50% ethanol, 49% distilled water and 1% glacial acetic acid). After 20 min. of agitation, the optical density was determined at 540 nm with a reference filter of 630 nm using a microplate spectrophotometer (Thermo Labsystems, Waltham, MA, U.S.A.). Background readings taken from blank wells were subtracted from wells containing both untreated and treated cells. Neutral red uptake was expressed as a percentage of the level in untreated cells.
Quantification of osteopontin and MMP-3 gene expression
Gene expression was assessed with the RT2 profiler™ PCR array system (SABiosciences) using an atherosclerosis-specific panel. Cells were lysed using TRIzol® reagent and centrifuged with chloroform. The supernatant was added to an equal volume of isopropanol and incubated at -20°C to precipitate RNA. Samples were centrifuged to pellet mRNA followed by an ethanol wash. Genomic DNA was eliminated using the supplied First Strand Kit and mRNA was diluted to 1 μg total mRNA. The components of the RT cocktail were combined in a microfuge tube and added in equal volume to RNA. The resultant mixture was incubated at 42°C for 15 min., after which the reaction was inactivated at 95°C for 5 min. cDNA was diluted in dH20 and combined with the PCR mastermix and 25 μl of solution was added to each well of the array plate. The following thermal cycler protocol was used: 10 min. at 95°C, 40 cycles of 15 sec. at 95°C and 1 min. at 60°C.
Quantification of osteopontin, IL-6 and MMP-3 protein levels
Osteopontin and MMP-3 levels were evaluated by electrochemiluminescence using the MSD® platform (Meso Scale Discovery, MD, U.S.A.), in supernatants collected from HUVEC cultures or sera from smokers and abstainers. 25 μl of conditioned media or serum and calibrators (0-100 ng/ml osteopontin, 0-2,500 pg/ml IL-6 and 0-100 ng/ml MMP-3) were incubated with agitation on the assay plate (osteopontin singleplex, ultrasensitive pro-inflammatory-4 II (IL-6) MULTI-SPOT® and MMP-3-plex MULTI-SPOT® 96-well small spot plates) for 1.5 h. After this, SULFO-TAG anti-human osteopontin, anti-human IL-6 or anti-human MMP-3 antibodies were added to each well at 1 μg/ml. After a further 1.5-hour incubation period, plates were washed in 0.05% (v/v) PBS-Tween-20 solution followed by the addition of 150 μl Read Buffer. Plates were read using a MSD® SECTOR 2400 imager and protein concentration assessed against a standard curve, using MSD Workbench® software.
Detection of osteopontin by immunocytochemistry
After PM treatment, HUVECs were washed in PBS and fixed in ice-cold methanol for 15 min. Cells were then blocked in PBS containing 2% BSA for 45 min at room temperature. After two PBS washes, the cells were incubated with 4 μg/ml rabbit anti-osteopontin polyclonal antibody (Abcam, Cambridge, U.K.) for 1 h. The cells were washed twice in PBS and incubated with 5 μg/ml Alexa Fluor® 568-labelled goat anti-rabbit antibody (Invitrogen, Paisley, U.K.) for 1 h. Cells were further washed with PBS and counterstained with DAPI (10 μg/ml in PBS). Coverslips were mounted onto slides using fluorescence mounting media (Dako, Denmark), viewed with a TE2000 inverted fluorescence microscope (Nikon, Surrey, U.K.) and images captured with an Orca 12-bit camera (Hamamatsu, Japan). Images were taken of the DAPI (blue) and osteopontin (red) channels using IPLabTM software. Image data were quantified using MetaMorph® software. Regions of interest were created using a 70 × 70 pixel circle around the DAPI-stained nucleus by examining only the blue channel. The red fluorescence of interest was then examined within this region and the average pixel intensity of fluorescence in each region of interest assessed. 10 nuclei per treatment were evaluated.
Detection of osteopontin by western blotting
Whole-cell lysates were prepared by scraping cells off in 1 ml ice cold PBS and pelleting by centrifugation at 1500 g for 5 min. The resultant cell pellet was resuspended in 200 μl lysis buffer. Cell extracts were incubated for 20 min at 65°C with reducing agent and sample buffer. Equal amounts of proteins (25 μg per lane) were resolved on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked using 5% non-fat dried milk solution in PBS containing 0.1% (v/v) Tween-20 (PBST). Membranes were probed for 1 h with either 1 μg/ml rabbit polyclonal osteopontin (Abcam, Cambridge, U.K.) or 0.5 μg/ml mouse monoclonal β-actin (Sigma, Poole, U.K.) antibodies. Membranes were washed in PBST and then incubated with secondary 4 μg/ml goat anti-rabbit (Abcam, Cambridge, U.K.) or 1:1000 goat anti-mouse (R&D systems, Minneapolis, U.S.A.) HRP-conjugated antibodies with 1:3000 dilution Strep-Tactin® HRP conjugate (Bio-Rad, California, U.S.A) for a further hour with agitation. Following two washes in 5% milk-PBST and one in PBST, membranes were incubated in Immun-StarTM WesternCTM chemiluminescence substrate (Bio-Rad) for 5 min before detection using an XRS Gel DocTM system (Bio-Rad). Membrane images were evaluated using Quantity One® software. The molecular weights of bands were calculated against the protein standard lanes and quantified by average band density.
Obtaining sera from human smokers
A single-centre, randomized, controlled, open-label, parallel group clinical study (approved by an Institutional Review Board) was conducted targeting enrolment of at least 120 smokers smoking their usual brand of cigarettes. U.S. adult smokers between 21 and 65 years old were enrolled in the study. Subject inclusion/exclusion criteria and baseline demographics can be found in Additional file 1: Table S1. The study was conducted in compliance with the principles of the Declaration of Helsinki. The study protocol and informed consent forms were reviewed and approved by the IntegReview Ethical Review Board (Austin, Texas, USA; http://www.integreview.com). Written, informed consent was obtained from all study subjects before participating in the study. This study was conducted according to the applicable principles of the US Code of Federal Regulations (CFR) governing the protection of human subjects (21 CFR 50), financial disclosure by clinical investigators (21 CFR 54), and IRBs (21 CFR 56).
Pre-study assessment (e.g. medical history, physical exam, hematology, serology, virology, urinalysis) showed that all subjects were generally healthy. Subjects were confirmed to smoke between 19 and 25 cigarettes per day and had been smoking for at least 3 years. Subjects resided in the clinic for approximately 8 days. Smoking status was verified during screening and daily in-clinic by measuring exhaled carbon monoxide.
On Days −2 and −1 in clinic, all subjects acclimated to smoking 20 cigarettes per day (20 CPD). The intervention started on Day 1 and consisted of maintaining or reducing to zero the number of CPD. On Day 1, subjects were randomized to 1 of 4 groups (20 CPD, 10 CPD, 5 CPD, and 0 CPD). From Day 1 until the end of the study on Day 5, subjects smoked up to the maximum allocated number of CPD/group.
Blood was collected from subjects in the 20 CPD and 0 CPD groups on Day −1 and Day +5 and serum was separated using BD diagnostics vacutainer serum separator tubes. After blood samples were drawn, tubes were inverted 5–6 times and placed upright for ≤2 h. Tubes were then centrifuged at 1300-2000 x g. Using disposable pipettes, serum was transferred into 2 ml cryovials and stored frozen at ≤ -80°C until analysis. Levels of osteopontin in sera were determined using the MSD® platform as described.
Data analysis and statistics
Data are expressed as means ± standard deviation (S.D.). For both the in vitro studies and the osteopontin/IL-6 measurements in human sera, differences between treatment means were examined using Student’s unpaired t-tests. In the western blotting studies, data are expressed as a ratio of osteopontin to β-actin band density. Data were then normalised by dividing the band density of the treatment group by that obtained in control (DMSO-treated) cells.