Increased Rho-kinase expression and activity and pulmonary endothelial dysfunction in smokers with normal lung function

Subjects and tissue preparations

Patients who underwent lung resection for carcinoma were enrolled in the present study after giving informed consent, as approved by the local ethics committee (Comité de protection des personnes Ile de France, Paris, France). They were divided into two groups: nonsmokers and current smokers. All but two nonsmokers had never smoked. The two remaining subjects were ex-smokers (5 and 10 pack-yrs) but had quit smoking for 20 and 25 yrs, respectively. All patients had normal lung function, as defined by slow and forced vital capacity (FVC), total lung capacity and forced expiratory volume in 1 s (FEV₁) ≥80% of predicted values. Absence of airflow obstruction was defined as a ratio of FEV₁/FVC ≥0.7 15.

Peripheral lung tissues and proximal pulmonary arteries were carefully dissected from lobectomy tissue free from macroscopic signs of lung tumour. Rings of 2–3 mm external diameter and 4–6 mm of length were cut and immediately placed in cold Krebs-Henseleit solution (4°C) for vasoreactivity study within the following 30 min. The other specimens of pulmonary arterial and lung parenchyma were stored at -80°C for Western blotting, or they were fixed overnight in 4% formalin and embedded in paraffin for immunohistochemistry assessment.

Chemical products

eNOS antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA). Primary antibodies for ROCK-1, ROCK-2 and RhoA were provided by Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Pierce Biotechnology, Inc (Rockford, IL, USA). Rho activation kit for pull-down assay was purchased from Stressgen Bioreagent Corp. (Victoria, BC, Canada). Rho-kinase assay kit was supplied by CycLex Co., Ltd (Tera-Sawaoka Ina, Negano, Japan). Nitric oxide synthase assay kit was purchased from Cayman Chemical Europe, (Massy, France). Cyclic guanosine monophosphate (cGMP) enzyme immunoassay Biotrak (EIA) system was provided by GE Healthcare Life Sciences, (Little Chalfont, UK). All other chemical products were purchased from Sigma-Aldrich (Steinheim, Germany).

Measurement of isometric tension of pulmonary arterial vascular rings

Each pulmonary arterial ring was mounted between two parallel stainless steel wires for measurement of isometric tension in organ chambers (Emka Technologies, Paris, France). Individual organ baths were filled with 20 mL of Krebs-Henseleit solution (glucose 10 mM, pyruvate 2 mM, HEPES 10 mM, EDTA 0.03 mM, NaCl 118 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, KH₂PO₄ 1.2 mM, MgSO₄ 1.2 mM, NaHCO₃ 15 mM), heated at 37°C and continuously aerated with 95% O₂ and 5% CO₂. The isometric force generated by the ring segment was measured and recorded by Macintosh Performa 630-Software (Apple). Pulmonary arterial rings were initially stabilised for 30 min between 1–1.5 g, at a resting tension that has been previously determined as corresponding to the optimal length for tension development.

The arterial rings were precontracted with increasing concentrations of phenylephrine (10⁻⁸–10⁻⁵ mol·L⁻¹) as previously described 16. For each ring, the effective concentration of phenylephrine needed to achieve optimal contraction was calculated. Indomethacin (10⁻⁵ mol·L⁻¹) was added to the organ baths 30 min prior to phenylephrine to exclude the influence of cyclooxygenase pathway products 17. After the phenylephrine contraction series, the baths were washed three times with fresh Krebs solution and the rings were allowed to stabilise over a resting tension for 1 h.

Endothelium-dependent relaxation was assessed by exposing pulmonary arterial rings to increasing concentrations of acetylcholine (Ach; 10⁻⁹–10⁻⁴ mol·L⁻¹). At least four rings from each patient were studied for obtained results.

Immunohistochemistry assessment for eNOS, ROCK-1 and ROCK-2

For eNOS immunohistochemical staining, sections were prepared as previously described to block nonspecific binding of primary and secondary antibodies and nonspecific reactions 18. The sections were incubated with the specific eNOS antibody (1 in 100 titre). All bound antibody sections were incubated with HRP-conjugated secondary antibody. The sections were incubated with metal enhanced 3,3′-diaminobenzidine peroxidase substrate until the desired staining was obtained. Tissues were counterstained with haematoxylin and examined using light microscopy. Negative control sections were incubated in blocking buffer alone without primary antibody. eNOS immunostaining expression was measured in endothelial cells of pulmonary arterial rings. The staining expression was measured by using intensity score. At least ten counted images were performed for each patient.

For ROCK-1 and ROCK-2 immunostaining, sections were stained with ImmunoCruz™ staining system. Primary antibody (goat polyclonal immunoglobulin (Ig)G) was diluted to 1 in 50, as determined by titration in serum block. After 2 h of incubation with the primary antibody, sections were washed in PBS and incubated with biotinylated secondary antibody and HRP-streptavidin complex for 30 min each. The desired stain was obtained in 60 s after addition of HRP substrate mixture to the sections. The last steps with counterstained, dehydrated and mounted slides were then performed. The expression of ROCK-1 and ROCK-2 peroxidase staining was measured in SMCs of pulmonary arteries. Ten magnified visual fields were performed for each subject.

Protein semi-quantification of eNOS, ROCK-1, ROCK-2, RhoA and p-MYPT-1 by Western blot

Equal amounts of pulmonary arterial homogenates adjusted to protein content were subjected to 7.5% SDS-PAGE (for eNOS, ROCK-1, ROCK-2 and phosphorylated MYPT-1 (p-MYPT-1)) or 12% gels (RhoA) for electrophoresis and then transferred to PVDF membranes (Immobilon-P, Millipore SA, France). Membranes were blocked in 5% non-fat milk, 0.1% Tween-20 in PBS for 1 h at room temperature. The membranes were hybridised overnight with antibodies against eNOS (dilution 1 in 100), ROCKs (dilution 1 in 500), RhoA (dilution 1 in 100) and p-MYPT-1 (dilution 1 in 200), which were then incubated with HRP-linked goat anti-mouse (eNOS, dilution 1 in 5,000) or donkey anti-goat IgG-HRP secondary antibodies for 45 min for ROCKs (dilution 1 in 10,000), RhoA and p-MYPT-1 (dilution 1 in 5,000). The equal sample loadings were confirmed by β-actin. Protein bands were developed on the film by using enhanced chemiluminescence reagent, according to the manufacturer's instructions. The protein densities were quantified by using Image Software System (Genius 2; Syngene, Cambridge, UK) and normalised with β-actin densities.

eNOS activity

The activity of eNOS was measured using a colorimetric NO synthase assay kit (Cayman Chemical Europe) . This kit was used to evaluate the production of NO from l-arginine by the action of eNOS in the presence of nicotinamide adenine dinucleotide phosphate, an essential co-factor for this enzyme. The best index of total NO produced was the sum of both nitrite (NO₂^-) and nitrate (NO₃^-) concentration, which indirectly represented the activity of total eNOS. All steps of the assay were carried out according to the manufacturer’s instructions. The final products of reaction were NO₃^-. The results were read at an absorbance of 540 nm. The concentrations of NO₃^- from the samples were determined from the standard curve of the provided nitrate standard.

cGMP concentration

cGMP concentration in pulmonary arteries was measured by an enzyme immunoassay Biotrak (EIA) system (GE Healthcare Life Sciences) according to the manufacturer’s instructions. It combined the use of a peroxidase-labelled cGMP conjugate, a specific antiserum that can be immobilised on pre-coated microplates, and a one-pot stabilised substrate solution. The assay was based on competition between unlabelled cGMP and a fixed quantity of cGMP labelled with peroxidise, for a limited number of binding sites on a cGMP specific antibody. Results were obtained by colorimetric reading with the absorbance at 450 nm. The concentrations from the sample were quantified by interpolating absorbance readings from a standard curve generated with the calibration of provided cGMP protein standards.

Measurement of RhoA active form by pull-down assay

Rho activation was determined using a Rho activation kit (Stressgen Bioreagent Corp.). It was used to measure the active form of RhoA (GTP-RhoA). This assay used a glutathione S-transferase-fusion protein containing the Rho-binding domain of mouse rhotekin to affinity precipitate active Rho (GTP-Rho) from pulmonary arterial homogenates (500 μg total proteins). After the pull-down assay, 25 μL of eluted samples were separated by 12% SDS-PAGE gels, transferred to PVDF membrane and probed with antibodies against RhoA. The protein bands were detected and quantified as described previously.

Determination of the activity of Rho-kinase by ELISA

Rho-kinase activity in pulmonary arteries was assessed by ELISA, using Rho-kinase assay kit (CycLex Co., Ltd). Plates were pre-coated with a substrate corresponding to the recombinant C-terminus of MBS (myosin-binding subunit of myosin phosphatase or MYPT-1), which contains a threonine residue that was phosphorylated by Rho-kinase. The specific detector antibody was HRP-conjugated anti-phospho-MBS (AF20) that detected only the phosphorylated form of threonine-696 on MBS. The amount of phosphorylated substrate was measured by binding it with a HRP of AF20 which then catalysed the conversion of the chromogenic substrate tetra-methylbenzidine from a colourless solution. It was then quantified by spectrophotometry and reflected the relative amount of Rho-kinase activity on phosphorylation of MYPT in pulmonary arterial SMC.

Statistical analysis

Results are presented as mean±sem. The software SPSS-version 13.0 (SPSS, Chicago, IL, USA) was used for all statistical analysis. Comparisons between groups for characteristics were performed with the unpaired t-test. Comparisons of dose–response curves for relaxation response were analysed by ANOVA for repeated measure. Values of p<0.05 were considered statistically significant.