Animals and Smoke Exposure Protocols
All studies were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals
as adopted and promulgated by the National Institutes of Health (http://oacu.od.nih.gov/regs/guide/guide.pdf
). Eight pregnant Rhesus macaque monkeys were obtained from the California National Primate Research Center breeding colony. Estimated gestational age for each dam was established by sonography performed before gestational day 40. Animals were selected based on a history of successful vaginal delivery and previous infant rearing experience with estimated delivery dates separated by approximately 1 week per animal to facilitate experimental procedures.Two inhalation chambers were used, each with an air capacity of 3.5 m3
, and each housed two dams. Aged and diluted sidestream smoke was used as a surrogate control for ETS. Standardized 1R4F research cigarettes were smoked simultaneously with a single puff volume of 35 ml per cigarette and a duration of 2 s, once per min. ETS was generated by a smoke exposure system (Teague Enterprises, Davis, CA) using IR4F conditioned cigarettes from the Tobacco and Health Research Institute of the University of Kentucky. Sidestream smoke from the smoldering end of each cigarette was collected into a conditioning chamber where it was aged over a period of approximately 2 to 3 min while being diluted with filtered air and then further diluted as it passed into the exposure chambers to produce total suspended particulate concentrations of 1.0 mg/m3
, 4–5 ppm carbon monoxide and 200–300 μg/m3
nicotine. This level of exposure is highly reminiscent of ETS concentrations found in homes or the workplace where smoking is permitted. The exposure chambers were stainless steel and glass Hinners type and 4.2 m3
in size. Each chamber has an air capacity of 3.5 m3
. Airflow through the system was set for 15 changes per hour.Exposure to ETS (1 mg/m3
) occurred for 6 h/day, 5 days/week, beginning on gestational day 100. All dams were allowed to give birth spontaneously (five male and three female infants born), and then ETS or sidestream control exposure continued for 70–80 days postnatally for the four experimental and four control animals, respectively with the chamber containing both the mother and infant. Infants were then sacrificed (mean age, 72 days) and bilateral carotid arteries dissected and cleansed of adventitial fat. Carotid arteries from four control and four ETS-treated newborns (mean weight of animals at sacrifice, 0.84 kg) were then analyzed for mRNA by gene macroarray and for protein by Western blotting. Tissues were flash-frozen and stored at −80°C until assayed.
RNA Isolation All procedures were performed under RNAse-free conditions. RNA was isolated from the carotid artery samples from the neonatal monkeys using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) following tissue disruption with a mortar and pestle and subsequent use of a tissue hand-held glass homogenizer (Kontes). Total RNA was extracted using chloroform or 1-Bromo-3-Chloropropane (BCP) Phase Separation Reagent (Molecular Research). Each carotid (25–33 mg tissue) yielded approximately 2.0–3.3 mg of total RNA. Following extraction, RNA was treated with DNAse using Clontech DNAse-1 per the manufacturer’s instructions (MessageClean DNAse-1; GenHunter Corporation, Nashville, TN). Total RNA from the control and ETS-exposed aortas were used to prepare two radioactive probes for array hybridization.
Probe Preparation and Array Hybridization
The Atlas Human Cardiovascular cDNA Expression Array Kit and reagents (Clontech, Palo Alto, CA) were used for these studies. The array contains duplicate copies of 588 cardiovascular genes and multiple control and housekeeping genes (http://www.clontech.com
). The cardiovascular genes consist of six major categories as follows: (1) cell cycle regulators/intermediate filament markers, (2) apoptosis/oncogenes/tumor suppressors, (3) DNA damage/repair and cell development, (4) cell adhesion/mobility and angiogenesis, (5) cell–cell interactions, and (6) growth factors/cytokines.The 33
P-labeled cDNA probes were synthesized by reverse transcription of total RNA from filtered air (control) and environmental tobacco smoke-exposed monkey carotids using a gene-specific primer mix (1
μl, 0.2 μM of each primer) and annealed to 2.13 μg of total RNA. The mixture was heated for 2 min at 70°C in a thermal cycler (Perkin Elmer, Waltham, MA), then incubated for 2 min at 50°C. First strand cDNA synthesis was initiated by mixing the annealed primer with the RNA and master mix [containing 4 μl of 5X reaction buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 2 μl of dNTP mix (0.5 mM of each dGTP, dCTP, and dTTP), 5 μl of [α
P]dATP (3,000 Ci/mmol), (10 mCi/ml; NEN, Boston, MA.), 1 μl DTT (100 mM), and 2 μl of MMLV reverse transcriptase (50 U/μl)]. The reaction was incubated in a thermal cycler (Perkin Elmer) at 50°C for 25 min and stopped by addition of 1 μl termination mix [0.1 M EDTA (pH 8.0), 1 mg/ml glycogen]. The labeled cDNA probe was purified from unincorporated 33
P-labeled nucleotides by gel filtration chromatography using a 1-ml NucleoSpin extraction spin column (Clontech) per the manufacturer’s instructions. The purified-labeled cDNA probes (approximately 1X106
cpm for control and ETS-exposed monkey carotid) were utilized for macroarray hybridization.
Macroarray Hybridization The probes were prepared for hybridization by adding 10X denaturing solution (1 M NaOH, 10 mM EDTA) to the entire pool of labeled probe and incubated at 68°C for 20 min. Cot-1 DNA and 2X neutralizing solution (1 M NaH2PO4, pH 7.0) was added to the denatured probe and incubated at 68°C for 10 min. The labeled probe solution was mixed together with preheated ExpressHyb hybridization solution. Each of the two duplicate Atlas Human Cardiovascular Array membranes were pre-wet with de-ionized H2O and prehybridized for 30 min at 68°C. Under continuous agitation, the array membranes were hybridized overnight at 68°C, washed three times with 2X SSC, 1% sodium dodecyl sulfate (SDS) at 68°C, washed twice with 0.1X SSC, 0.5% SDS for 30 min at 68°C, and then washed for 5 min with 2X SSC at room temperature (RT).
Signal Detection and Image Analysis The washed membranes were sealed in plastic wrap and exposed directly to a storage phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 8 days. The screen was scanned using Storm 840 in storage phosphor mode. Signal intensity was quantified using an ImageQuant software for identification of differentially expressed genes. In ImageQuant, grid objects were created and used for simultaneous quantitation of all duplicate spots on each array. Grids of seven columns by 14 rows were used to analyze the six main gene groupings on the macroarrays. Separate grids were created for housekeeping gene spots and for determining background (unspotted, unhybridized areas of the membrane), which was subtracted from volume densitometry values for the grid cells. Twenty-three kilo Daltons highly basic protein and 60S ribosomal protein L13A (aka RPL 13A) housekeeping gene values were used for normalization based on the equality of signal strength between both arrays. This normalization scheme was further validated by the equality in normalized expression of many of the common genes on both arrays.
Real-Time RT-PCR-Based Confirmation of Differential Gene Expression
To further confirm differential mRNA expression of the genes identified to be regulated by ETS in the macroarray analysis, we performed real-time polymerase chain reaction (PCR) utilizing two additional ETS-exposed and control monkey aortas. Thus, for these experiments, a total of six aortas were studied for ETS-exposed and control neonatal monkeys, respectively. The aortas were from the same experimental animal cohort as described earlier in the “Materials and Methods”
section. Briefly, total RNA from the ETS and control aortas was treated with DNAse (MessageClean DNAse-I; GenHunter) and cDNA generated by reverse transcriptase (RT)-PCR. A mixture of oligo (dT) oligodeoxynucleotide primers (T12–18) and random hexamer primers were used for these studies following the protocol for SuperScript RNAse H-Reverse Transcriptase (GibcoBRL, Rockville, MD). Real-time detection of PCR was performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The same gene-specific primers used for semiquantitative PCR analysis were used for real-time PCR detection of the genes of interest. Equal amounts of duplicate or triplicate sample cDNA were amplified with the SYBR Green I Master Mix (Applied Biosystems). The thermal cycling parameters were thermal activation for 10 min at 95°C followed by 40 cycles of PCR (melting for 15 s at 95°C and annealing extension for 1 min at 60°C). A standard curve was constructed using a template dilution series (1:10, 1:20, 1:40, 1:80, and 1:160) of total RNA from monkey aorta. A “no template” negative control was included with each PCR. Amplification efficiency for each of the genes of interest was then validated and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In order to compare the relative expression of the various genes, data was expressed as the normalized densitometry ratio of ETS to control aortas.
Protein was prepared from monkey carotid sections from filtered air (control) and ETS-exposed monkeys using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) or NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL), and mammalian Protease Inhibitor Cocktail (Sigma, Saint Louis, MO) per the manufacturer’s protocol. Protein quantity was assayed by detergent-compatible DC protein assay (BioRad, Hercules, CA). Ready gels for SDS-PAGE electrophoresis (BioRad) were used with the appropriate gel concentration for the size of the protein of interest (loading ~44 mg of protein per lane). Kaleidoscope molecular weight markers (BioRad) were loaded in one or two lanes of the gels for size markers. The gels were electrophoresed at 60 V for 15–30 min, followed by 60–90 min at 120 V depending on the speed of migration. After electrophoresis, electroblotting proceeded for 1 h at 100 V on ice.The blot was blocked in 10% w
non-fat dry milk Tris-Buffered Saline Tween-20 (TBST) solution overnight at 4°C. The nitrocellulose membrane was washed three times with TBS, 0.05% Tween. The primary antibody was diluted in TBST to concentrations previously determined or according to manufacturer’s recommendations and incubated for ~1 h at RT. The secondary antibody was diluted in TBST and incubated for ~1 h at RT. The nitrocellulose was washed two times with TBST. Electrochemiluminescence (ECL) Detection Reagents (Amersham Pharmacia Biotech, Buckinghamshire, England) horseradish peroxidase (HRP)-based detection was used. Blots were wrapped in saran and exposed to autoradiograph film (Kodak Biomax, Rochester, NY). Densitometry was performed on a Kodak 1D gel imaging system. Some blots were stripped with Restore Western Blot Stripping Buffer (Pierce) and re-probed with mouse anti-actin or a different primary antibody than that stripped, followed by a secondary antibody and ECL detection as described. Details of the antibodies used and protein products of the genes studied (see “Results”
) are as follows:RANTES (8 kDa protein)—primary antibody goat polyclonal anti-RANTES (Santa Cruz Biotech, Santa Cruz, CA; 1:1000 dilution) and secondary antibody bovine anti-goat IgG HRP (Santa Cruz Biotech; 1:10,000–1:30,000 dilution); VEGFR2 [flk-1] (130 kDa protein)—primary antibody rabbit polyclonal anti-neuropilin-1 (Oncogene Research Products, La Jolla, CA; 1:100–1:200 dilution) and secondary antibody goat anti-rabbit IgG HRP (BioRad; 1:15,000 dilution), as well as primary antibody goat polyclonal anti-neuropilin-2 (Santa Cruz Biotech; 1:100 dilution) and secondary antibody bovine anti-goat Ig G HRP (Santa Cruz Biotech; 1:10,000–1:30,000 dilution); LFA-1 [integrin alpha L] (180 kDa protein)—primary antibody goal polyclonal anti-LFA1 (Santa Cruz Biotech; 1:100 dilution) and secondary antibody bovine anti-goat IgG HRP (Santa Cruz Biotech; 1:10,000–1:30,000 dilution); actin control (41 kDa protein)—primary antibody mouse monoclonal anti-actin (Sigma; 1:1,000 dilution) and secondary antibody goat anti-mouse IgG HRP (BioRad; 1:15,000 dilution).
Densitometry values were obtained for each array cDNA from ETS-treated and control samples, and quantification of spots on arrays from ETS-treated and control (sidestream air) aortas was performed. In order to allow the array products to be quantified comparatively, arrays were also spotted with two housekeeping genes. This permitted the spot densitometry values of the ETS-treated and control array signals to be normalized to the housekeeping signals and to calculate normalized densitometry ratios. Background signal was subtracted from the control and ETS spots prior to analysis. Normalized ratio data from the arrays were then evaluated for differential expression (i.e., greater or less than a 2-fold difference in expression in the ETS-treated versus the control samples). All arrays were spotted in duplicate. Four aortas each were used for the ETS-treated and the control arrays.Quantification of PCR products was performed in a similar fashion. PCR amplifications were conducted with GAPDH in order to permit normalization of signals to GAPDH and to account for any minor variations in DNA loading in the PCR product signals. Normalized ratio data from PCR reactions were then evaluated for differential expression (i.e., greater or less than a 2-fold difference in expression in the ETS-treated versus the control samples). All PCR reactions were performed in duplicate. Six aortas each were used for the ETS-treated and the control PCRs. Normalized data from arrays and PCR were expressed as relative fold change in expression for ETS compared to control-treated aortas.Western blot experiments were performed in triplicate or greater, and data expressed as mean +/− standard error of the mean
(SEM) for net densitometry values for the four control and the four ETS-treated aortas. Means +/− SEM and mean fold change in protein expression in the ETS-treated and control protein products was then calculated. Statistical significance was at the p
0.05 level.For the array, PCR, and Western assays, the calculated fold-change differences between the ETS-treated and control samples were based on the mean values of the four subjects in each of the control and study groups.