RESULTS

3.4.1 TRAIL expression is upregulated on PA-SMCs from patients with PAH.

Prior work from our laboratory has observed TRAIL immunostaining in concentric and plexiform lesions from patients with idiopathic PAH (Lawrie, Waterman et al. 2008). Given that a major role for PASMCs in the vascular remodeling of PAH is well established and TRAIL expression has been demonstrated on systemic VSMCs we first sought to confirm if TRAIL was also expressed on PASMCs from patients with PAH.

Using Taqman PCR, there was an approx. three-fold upregulation in TRAIL gene expression in PASMCs from patients with PAH was observed compared to controls (Fig. 3A). The PASMCs had previously been isolated from explanted lungs of patients, with advanced idiopathic PAH who had undergone lung transplantation and grown ex-vivo. Patients undergoing lung surgery for either emphysema (COPD) or operable lung cancer served as disease controls. Clinical details of the patients from whom PASMCs had been isolated are shown in Table 3.1.

Table 3.1 Clinical details for human cells used in experiments outlined in Fig. 3

Sample Diagnosis Gender Age (yr) mPAP (mm Hg) CO (l/min)

1 IPAH Female 38 56 2.8

2 IPAH Female 23 70 2.0

3 hPAH (R491M BMPR2) Female 45 50 2.4

4 Emphysema Female 58 n/a n/a

5 Centrilobular Emphysema Male 62 n/a n/a 6 Squamous cell carcinoma Female 60 n/a n/a

IPAH – Idiopathic pulmonary arterial hypertension, hPAH – hereditary pulmonary arterial hypertension, mPAP – mean pulmonary artery pressure, CO – cardiac output, n/a – not available.

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Figure 3. TRAIL induces proliferation and migration of PA-SMCs. (A) TaqMan expression of TRAIL

in explanted PASMC from patients with IPAH normalised using ΔΔCT with 18S rRNA as the endogenous control gene. Human PASMCs were serum starved for 48 hours before stimulation with recombinant TRAIL (1-100 ng/ml) or 10 ng/ml PDGF-BB. (B) Proliferation was assessed by cell counting at 72 hours;

(C) migration was measured at 6 hours using a Boyden Chamber assay and normalized relative to

unstimulated cells (0.2% FCS). (D) Time course of p42/44 / ERK1/2 phosphorylation in PASMC using 30 ng/ml of TRAIL. Treatment of PASMC with the ERK1/2 inhibitor PD98059 inhibited TRAIL (30 ng/ml) induced proliferation (E) and migration (F). TaqMan expression of TRAIL receptors in explanted PASMC from patients with IPAH normalised using ΔΔCT with 18S rRNA as the endogenous control gene (G). Human PASMCs stimulated with recombinant TRAIL (30 ng/ml) or 20 ng/ml PDGF-BB with blocking antibodies for TRAIL-R1, TRAIL-R3 or an IgG control. Proliferation was assessed by cell counting at 72 hours (H) Bars represent mean +/- SEM, all experiments were performed in triplicate. For data using

patient material (A & G) n=3. All remaining figures n=5. *=p<0.05, **=p<0.01, ***=p<0.001 compared to 0.2% FCS, control or 0h samples. These experiments were done by staff listed on p82.

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3.4.2 Receptors for TRAIL are upregulated on PA-SMCs from patients with PAH

All four membrane TRAIL receptors are expressed and upregulated on both human and rat aortic VSMCs (Secchiero, Zerbinati et al. 2004; Kavurma, Schoppet et al. 2008; Song, Choi et al. 2011). Using TaqMan PCR, PASMCs from both disease controls and IPAH expressed all five TRAIL receptors, although only the surface receptors TRAIL-R1 (two-fold) and in particular TRAIL R3 (six-fold) were both significantly upregulated (Fig. 3G). The soluble decoy receptor Osteoprotegerin (OPG:TRAIL-R5, was also upregulated three fold (data not shown).

3.4.3 TRAIL stimulates proliferation & migration of human PA-SMCs in-vitro.

As TRAIL induces the proliferation of aortic vascular smooth cells, we next determined if a similar response would occur with pulmonary arterial SMCs. Growth starved hPASMCS were stimulated with recombinant human TRAIL (rhTRAIL) for 72 hours and cell counts were determined using the CoulterTM cell count method. Recombinant human TRAIL (rhTRAIL) induced the proliferation of normal human PASMCs in cell culture with a significant response observed with a concentration of 30ng/ml (performed in duplicate with n=5 experiments) (Fig.

3B).

TRAIL naturally forms a homo-trimer and experimentally, the effects (apoptotic) of rTRAIL have been be shown to be enhanced by oligimerisation with a cross linking antibody. Thus In separate experiments we crosslinked TRAIL (using an anti-histidine antibody) and stimulated hPASMCs in-vitro for 72 hours (Performed in duplicate, n=6 individual experiments). However no significant difference in human PASMC was observed when compared to non linked rhTRAIL (data not shown).

To determine if TRAIL displayed chemotactic properties towards PASMCs, a Boyden chamber migration assay was performed. rhTRAIL induced the migration of human PASMCs in a dose dependant manner, when compared to control (0.2% FCS treated) cells (performed in duplicate with n=5 experiments) (Fig. 3C).

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3.4.4 TRAIL receptors and PA-SMC proliferation in-vitro

There are 4 cell surface receptors for TRAIL and previous work has identified that the proliferative effects of TRAIL on aortic VSMCs were mediated via receptors TRAIL-R1 and R3 (Kavurma, Schoppet et al. 2008). Our data demonstrate a similar upregulation of TRAIL R1 and R3 on PA-SMCs in PAH (Fig. 3G). In cultured human PASMCs, antibody blockade of TRAIL-R3, but not R1 or R4 significantly, although modestly attenuated the proliferative response to TRAIL (Fig. 3H). We were unable to evaluate the contribution of TRAIL R2 to cell proliferation as no commercially available blocking antibody to TRAIL R2 was available at the time of this work.

3.4.5 TRAIL induced proliferation of PA-SMCs is mediated through ERK signaling.

MAPK (Mitogen Activate Protein Kinase) signaling via ERK (Extracellular signal Regulated Kinase, ERK1/2) is an established downstream signaling pathway in PASMCs. Furthermore the pro-survival effects of TRAIL on VSMCs have previously been demonstrated to activate ERK signaling (Secchiero, Gonelli et al. 2003; Secchiero, Zerbinati et al. 2004). A dose of 30ng/ml of rhTRAIL induced a rapid and significant upregulation of ERK1/2 signaling as evidence by an increase in the ratio of phosporylated P44/42 / total 44/42 levels in hPASMC lysates (Fig. 3D). Moreover the pro-proliferative and pro-migratory effects of rhTRAIL were inhibited by the MAPKK inhibitor- PD98059 when compared to vehicle (DMSO) treated cells (Figs. 3E and 3F). PD98059 inhibits MEK1/2 which sits immediately upstream of ERK1/2 and activates it through phosphorylation (Cargnello and Roux 2011).

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