Animals and cigarette smoke exposure
Thirty-six male Hartley guinea pigs (Harlam Ibérica, Spain), each weighing 300 g, were given a diet of standard chow and water supplemented with vitamin C (1 g/L; Roche Pharma, Madrid, Spain) ad libitum. A group of 19 animals was exposed to the smoke of 7 research cigarettes (1R3F; Kentucky University Research; Lexington, KY, USA) per day, 5 days a week, using a nose-only system  (Protowerx Design Inc; Langley, British Columbia, Canada) for a period of 3 and 6 months (n = 6 and n = 13, respectively). Controls (n = 17) were sham-exposed during the same periods of time (n = 9 for 3 months, n = 8 for 6 months). Animals that died during the study were excluded from the sample size. All procedures involving animals and their care were approved by the Ethics Committee of the University of Barcelona and were conducted following institutional guidelines that comply with national (Generalitat de Catalunya decree 214/1997, DOGC 2450) and international (Guide for the Care and Use of Laboratory Animals, National Institutes of Health, 85-23, 1985) laws and policies.
After 3 or 6 months of CS exposure and 24 h after the last exposure, the animals were anesthetized with ketamine (50 mg/ml; 50 mg/kg. Pfizer Pharmaceuticals, Dun Laoghaire, Ireland) and xylazine (2%; 7 mg/kg. Bayer AG, Leverkusen, Germany) and the cardiopulmonary block was quickly removed to isolate a segment of the aorta and the main pulmonary artery. Arteries were cleaned of fat and connective tissue and cut into rings 3 mm in length. Two rings of the thoracic aorta and the left and right branches of the main pulmonary artery were placed in organ bath chambers (Panlab, Barcelona, Spain) filled with Krebs-Henseleit's buffer (containing (in mM) 118 NaCl, 24 NaHCO3, 11.1 glucose, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2), bubbled with a gas mixture of 21% O2 and 5% CO2 (pH 7.35–7.45) and kept at 37°C by an outer-water bath warmed by a recirculating heater. Ring preparations were attached to an isometric transducer (Panlab, Barcelona, Spain) and equilibrated for 1 h under a resting tension of 1.75 g for pulmonary artery and 2.3 g for aortic rings, as established in preliminary studies. After a period of stabilization, arteries were contracted with KCl (60 mM) to determine their viability and contractile capacity. On the basis of previous experience, arteries with contractions <1 g were considered not viable. All rings were pre-incubated with indomethacin (1 × 10-5 M, Sigma Aldrich. St Louis, USA) 30 min before the experiments in order to inhibit the synthesis of cyclo-oxygenase products. Indomethacin was kept in the solution throughout the experiment. The rings were then contracted with norepinephrine (NE; 1 × 10-7 to 0.2 × 10-6 M, Sigma Aldrich.) to obtain a stable plateau of tension. Endothelial function was evaluated by adding adenosine-5'-diphosphate (ADP, Boehringer GmbH, Mannheim, Germany), an endothelial nitric oxide (NO)-dependent vasodilator, to the organ bath. One of the rings of the aorta and the left branch of the pulmonary artery were tested to cumulative concentrations of ADP (10-9 to 10-5M). Response to cumulative concentrations of the exogenous NO donor, sodium nitroprusside (SNP; 10-10 to 10-5 M, Sigma Aldrich.), was also tested in the other two rings. To corroborate the endothelial function assay performed with ADP, all the procedures were repeated in the presence of N
G-monomethyl-L-arginine (L-NAME; 10-1 M, Sigma Aldrich.), an inhibitor of eNOS. Endothelium-dependent vasodilator responses were assessed by the maximal relaxation induced by ADP, the dose that caused 50% relaxation (EC50), and the area under the curve (AUC)  (Sigmaplot 10.0, Systat Software Inc, San José, CA, USA). Whereas EC50 is a single-point estimated value, the AUC is a summary measure obtained from all experimental points in the dose-response curve, providing a complete profile of vessel responsiveness. Each curve was evaluated by an observer without knowledge of the smoke exposure status.
Explanted lungs were inflated with 4% formaldehyde at a constant pressure of 25 cm H2O during 24 h, and then embedded in paraffin. Histological examination was performed in 4-μm sagital sections stained with hematoxylin-eosin. The presence of emphysema was evaluated by measuring the mean linear intercept of alveolar septa in 20 randomly selected fields per slice using an image analysis system (Leica Qwin, Leica Microsystems Image Solutions Ltd, Cambridge, UK). Pulmonary vessels were analyzed in lung tissue sections stained with orcein. To assess the number of muscularized arterioles, all vessels with an external diameter <50 μm and with double elastic laminas were counted.
After the organ bath studies, all artery rings were fixed in 4% formaldehyde and cryo-embedded in optimal cutting temperature (O.C.T). Morphometric studies were performed in 4-μm slices of aorta and main pulmonary artery sections stained with elastin-Van Gienson. The external and internal elastic laminas were outlined and both total and lumen areas were computed using an image analysis system  (Leica Qwin). The area of the arterial wall was estimated as the difference between the total and luminal areas. Wall thickness was calculated by dividing the arterial wall area by the external perimeter of the artery .
The expression of desmin and α-actin in pulmonary vessels (< 50 μm) was assessed by immunohistochemistry using anti-α-actin and anti-desmin antibodies (Dako, Glostrup, Denmark). An avidin-biotin reaction was performed to amplify the signal. The immunoreactions to α-actin and desmin were quantified as the number of positive vessels per mm2. The intensity and extension of immunoreaction to desmin were also semi-quantitatively evaluated in a scale from 1 to 3 (for intensity, 1: low, 2: medium, 3: high; and for extension, 1: 0–25% of the vessel wall, 2: 26–75%, 3: > 75%).
Total RNA was extracted from lung tissue using TRIzol (Invitrogen, Paisley, Scotland, UK). For reverse transcription, 2.0 mg of total RNA was retrotranscribed using a high-capacity cDNA Archive kit (Applied Biosystems). Quantification of eNOS was done with real-time PCR using SYBR Green I chemistry (SensiMix (dT) DNA Kit, Quantance Ltd, Ballards Lane, London). Normalization of gene expression levels was performed by using β-actin as endogenous housekeeping gene. To generate a standard curve, 7-fold serial dilutions of each purified PCR product were used for templates. Primers were designed based on guinea pig (eNOS) sequence from GeneBank using specific software (Primer Express, Applied Biosystems, Foster City, CA.). Amplification was performed on Chromo4 thermocycler (MJ Research, BioRad, Hercules, CA), and each sample was run in duplicate. The identities of the amplified products were examined using 12% poly-acrylamide gel electrophoresis and melt curve analysis. The primer sequences for eNOS were 3'-AGCCAACGCGGTGAAGATC-5' and 5'-TTAGCCATCACCGTGCCC-3' and for β-actin 3'-ATATCGCTGCGCTCGTTGTC-5' and 5'-AACGATGCCGTGCTCAATG-3'.
To evaluate the effect of CS exposure on endothelial function, a general linear model  was fitted using time, group and time-by-group interaction as independent factors. The estimates of the factors were adjusted by the effect of contraction to NE by including it in the model. The significance of the independent factors was assessed by the common ANOVA F-test using the type-3 sum of squares. The adequacy of the model was checked by examination of the Pearson residuals. All other variables are expressed as mean ± standard deviation (SD) or as median and inter-quartile range depending on whether or not the variables followed a normal distribution. Comparisons between groups were performed by the Student t-test or Mann Whitney test. A p-value lower than 0.05 was considered significant.