METHODS

The cell culture work reported in this chapter was performed by Dr Allan Lawrie and Dr Clauida Paiva. I specifically contributed to performing PASMC proliferation assays and only contributed data to Figure 3B.

3.3.1 Isolation and culture of pulmonary artery smooth muscle cells (PA-SMCs) from

human lung tissue

Ribonucleic acid (RNA) from PASMCs isolated from human lung tissue (with prior ethical approval) was provided by Prof. Nick Morrell, University of Cambridge, Cambridge U.K. The isolation and culture of PASMCs as well as the subsequent extraction of RNA from these cells had been performed by the Morrell group, as previously described (Morrell, Upton et al. 1999; Morrell, Yang et al. 2001).

Briefly PASMCs grown in culture had been isolated from the proximal and peripheral segmental pulmonary arteries (<1-2mm) of explanted lungs from female patients who had undergone lung or heart/lung transplantation for advanced PAH (n=3 with 1 case harboring a mutation in BMPR-2) and control patients with emphysema or cancer undergoing lung surgery (n=3). The PASMC phenotype was confirmed by immunofluorescence with antibodies to alpha- smooth muscle actin and smooth muscle myosin (hsm-v). Total RNA was extracted from growth arrested PASMCs using Trizol reagent (Invitrogen).

3.3.2 Quantification of TRAIL gene expression in PA-SMCs

Real time PCR was used to determine the gene expression of TRAIL in PASMCs. Briefly, total RNA isolated (as above) from PASMCs was reverse transcribed using Superscript III reverse transcriptase as per manufacturer’s instructions (Invitrogen, Paisley, UK). Gene expression was performed using Taqman PCR with commercial gene master mix and gene expression assays (Applied Biosystems U.K) with primers for TRAIL (Hs00234356_m1), TRAIL-R1 (Hs00269492_m1), TRAIL-R2 (Hs00366278_m1), TRAIL-R3 (Hs00182570_m1), and TRAIL-R4 (Hs04187520_m1). Relative gene expression was normalised to the control housekeeping gene ribosomal 18s RNA using the comparative delta/delta CT quantification method.

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3.3.3 Culture of primary human PA-SMCs in-vitro.

Commercially available human pulmonary arterial smooth muscle cells (hPASMCs) (Cascade biologics, Invitrogen, UK) were used in proliferation and migration experiments. Each supplied vial contained at least 5x105 viable cells that had been cryopreserved after the tertiary stage of culture in a medium containing 10% DMSO by the manufacturer. The subsequent storage, initiation of culture from cryopreserved cells and subculture were performed according to the manufacturer’s instruction). A haemocytometer was used to determine the concentration of cells and viability was assessed with Tryptan blue.

Unless indicated otherwise, PASMCs were grown in Media 231 containing smooth muscle growth supplement (SMGS) with added gentamicin/amphotericin (all from Cascade biologicsTM, InvitrogenTM, UK). hPASMCs were allowed to grow to 70-80% confluence in a monolayer before use in all experiments below. Cells were not used beyond passage 10. All tissue culture work was performed in class II laminar flow hoods using aseptic precautions. Sterile (autoclaved) pipettes, tips and flasks were used. Sterile cell culture media and reagents were pre-warmed at 37oC in a water bath that was regularly inspected and cleaned. Cells were cultured at 37oC in 5% CO2/95% air in a humidified cell culture incubator that underwent

regular inspection and cleaning. Unless stated otherwise, wells were washed with sterile PBS three times (500µl/well) with aspiration between each wash step.

3.3.4 In vitro assay of human PA-SMC proliferation.

PASMC proliferation experiments were performed in 24 well polystyrene plates (Costar3524, Corning, N.Y, U.S). To facilitate cell adhesion, all wells were pre coated with 500µl of 0.2% (w/v) gelatin, for 15mins at room temperature. Wells were washed with PBS and hPASMCs were seeded at a concentration of 2.5x104/ml (500µl/well) and incubated overnight at 37oC in 5% CO2/95% air.

Subsequently, cells were washed in PBS and then growth arrested for 48 hours by exchange of media to Dulbecco’s Modified Eagle medium (500µl/well; DMEM, Biowhittaker®, Lonza, UK) supplemented with 0.2% (v/v) fetal calf serum (FCS) 100IU/ml penicillin, 100µg/ml Streptomycin and 0.25µg/ml Actinomycin B (all Gibco, Invitrogen, UK). After quiescence, cells

84 were washed in PBS and were stimulated for 72 hours, before being counted as described below.

All recombinant proteins and antibodies (purchased from R&D systems, Abingdon, UK unless otherwise indicated; # indicates catalogue number) were reconstituted and stored as indicated by the manufacturer. Antibodies, recombinant proteins, inhibitors and controls (unstimulated) were all diluted in supplemented DMEM with 0.2% (v/v) FCS as described (250µl/well).

PASMCs were stimulated with recombinant human TRAIL (rhTRAIL, #375-TL) at doses between of 1-100ng/ml. This preparation of rhTRAIL is an extracellular domain of TRAIL (Thr 95 to Gly 281) and contains a 6x histidine tag at the amino terminal end. In separate experiments TRAIL was cross linked using a monoclonal anti-polyhistidine antibody (#MAB050) and used to assess cell proliferation. Recombinant human PDGF (BB isoform, #220-BB) was used as a positive control and DMEM containing 0.2% (v/v) supplemented FCS (as above) served as control. Where indicated, inhibition of TRAIL receptors by antibodies (all goat IgG) to the extracellular domain of human TRAIL-R1 (#AF347), TRAIL-R3 (#AF630) TRAIL-R4 (#AF633) and the MEK inhibitor PD98059 (MEK inhibits ERK1/2) were all added 30mins prior to the addition of recombinant TRAIL or PDGF-BB.

After 72 hours of stimulation, cell proliferation was assessed by the CoulterTM counting method. Wells were washed with PBS before the addition of Trypsin/EDTA (500µl/well) for 5min. The contents of each well were collected and added to individual small plastic containers containing 9.5ml of a diluent (#8448011; Coulter Isoton II, Beckman Coulter, Bromley UK). Each container was placed consecutively on a loading tray and cells were counted automatically by a Coulter Z1 machine. A numerical result was displayed on the screen and was noted. This number was multiplied by twenty to derive the actual cell count given the prior 1:20 dilution step.

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3.3.5 In-vitro assay of human PA-SMC migration.

The method used to assess cell migration in response to TRAIL was performed using the well established transwell chamber assay, first described by Boyden when investigating leucocyte chemotaxis (Boyden 1962). The principle of this assay is as follows; a chamber divided into two compartments is separated by a micro-porous membrane (of varying pore size). Culture media containing the cell type to be studied is placed in the top chamber and the chemotactic stimulus, is added to the lower chamber. After a period of incubation the membrane separating the two chambers is fixed and removed and the numbers of cells on the undersurface of the membrane are counted to determine the number of cells that have migrated.

As described in section 3.3.3 above, hPASMCs were serum starved for 48 hours in DMEM (0.2%FCS). 24 well cell culture inserts with a special membrane (PET track etched, 8µm pore size, #353097, BD FalconTM) were presoaked in human fibronectin (#F0895, Sigma Aldrich, Poole, UK) for 60min at room temperature so as to coat both inner and outer aspects of the well insert. The cell inserts were washed to remove fibronectin and placed in a 24 well plate with media containing the relevant stimuli. The latter step was done after a 24 well plate had been prepared by adding 750µl/well of starvation media containing, recombinant TRAIL (1- 100ng/ml, #375-TL), PDGF-BB (10ng/ml) or control (DMEM with 0.2%FCS). hPASMCS were seeded at a concentration of 3x10 4 cells/ml, (250µl/well).

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3.3.6 Time course for ERK1/2 expression and western Immunoblotting

To determine a time course for the expression of ERK1/2 in PASMC, recombinant human TRAIL (30ng/ml) was used to stimulate hPASMC for between 0-60minutes in 12-24 well cell culture plates. PASMC lysates were then prepared by washing cells in ice cold PBS. Cells were scraped and collected in ice cold PBS and centrifuged at 200g for 5min at 40C. The supernatant was discarded and the remaining cell pellet was re-suspended in an ice cold lysis buffer and left for 30min on ice. After lysis the tube was spun in a microcentrifuge for 10min at maximum speed. The supernatant containing the lysed cells was collected. The protein concentration was quantified using a DC protein assay as described in the methods chapter. Cell lysates were frozen at -800C.

Western immunoblotting was used to determine the amount of total and phosphorylated levels of p44 and p42 MAP Kinases (ERK1 and 2) in lysates of human PASMC stimulated with TRAIL. The principles and details of western blotting are described in the methods chapter. Briefly a 20µg sample of each PASMC lysates was loaded on to 4-12% Bis-Tris-Nupage Gel and run under reducing conditions in MES running buffer (Invitrogen) before transfer to nitrocellulose membrane (Invitrogen). The membranes were blocked for 1h in 5% non-fat milk at room temperature. The blots were incubated with a phospo-p44/42 MAPK (ERK1/2; Thr202/Tyr204), rabbit monoclonal antibody (1:1000 dilution, #9101, Cell Signalling technology®) or p44/42 MAPK (ERK1/2) mouse monoclonal antibody (1:1000 dilution, #9107, Cell signaling technology®) for 1h at room temperature. A HRP conjugated and species specific antibody was added for 1 hour before performing an enhanced chemoluminescence (ECL, GE healthcare) reaction and exposure to autoradiographic film. The autoradiography, densitometry and normalization processes were performed as described in the methods chapter.

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