Phosphodiesterase type 4 expression and anti-proliferative effects in human pulmonary artery smooth muscle cells

Abstract

Background: Pulmonary arterial hypertension is a proliferative vascular disease, characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Prostacyclin (PGI2) analogues have anti-proliferative effects on distal human pulmonary artery smooth muscle cells (PASMCs), which are dependent on intracellular cAMP stimulation. We therefore sought to investigate the involvement of the main cAMP-specific enzymes, phosphodiesterase type 4 (PDE4), responsible for cAMP hydrolysis.

Methods: Distal human PASMCs were derived from pulmonary arteries by explant culture (n = 14, passage 3-12). Responses to platelet-derived growth factor-BB (5-10 ng/ml), serum, PGI2 analogues (cicaprost, iloprost) and PDE4 inhibitors (roflumilast, rolipram, cilomilast) were determined by measuring cAMP phosphodiesterase activity, intracellular cAMP levels, DNA synthesis, apoptosis (as measured by DNA fragmentation and nuclear condensation) and matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) production.

Results: Expression of all four PDE4A-D genes was detected in PASMC isolates. PDE4 contributed to the main proportion (35.9 +/- 2.3%, n = 5) of cAMP-specific hydrolytic activity demonstrated in PASMCs, compared to PDE3 (21.5 +/- 2.5%), PDE2 (15.8 +/- 3.4%) or PDE1 activity (14.5 +/- 4.2%). Intracellular cAMP levels were increased by PGI2 analogues and further elevated in cells co-treated with roflumilast, rolipram and cilomilast. DNA synthesis was attenuated by 1 microM roflumilast (49 +/- 6% inhibition), rolipram (37 +/- 6%) and cilomilast (30 +/- 4%) and, in the presence of 5 nM cicaprost, these compounds exhibited EC50 values of 4.4 (2.6-6.1) nM (Mean and 95% confidence interval), 59 (36-83) nM and 97 (66-130) nM respectively. Roflumilast attenuated cell proliferation and gelatinase (MMP-2 and MMP-9) production and promoted the anti-proliferative effects of PGI2 analogues. The cAMP activators iloprost and forskolin also induced apoptosis, whereas roflumilast had no significant effect.

Conclusion: PDE4 enzymes are expressed in distal human PASMCs and the effects of cAMP-stimulating agents on DNA synthesis, proliferation and MMP production is dependent, at least in part, on PDE4 activity. PDE4 inhibition may provide greater control of cAMP-mediated anti-proliferative effects in human PASMCs and therefore could prove useful as an additional therapy for pulmonary arterial hypertension.

Figure 1

Phosphodiesterase type 4 (PDE4) expression in human pulmonary artery smooth muscle cells. RT-PCR demonstration of PDE4A (546 bp), PDE4B (506 bp), PDE4C (410 bp), PDE4D (479 bp) expression in a PASMC isolate, which is representative of 4 separate cell lines. Controls included expression of β-actin and absence of reverse transcriptase (- RT) or RNA (- RNA).

Figure 2

Characterisation of cAMP phosphodiesterase (PDE) activity in human PASMCs. Total cAMP hydrolytic activity and contribution of PDE enzyme families to cAMP hydrolysis in the cytosol (A) and membrane fractions (B) of human PASMCs (n = 5 isolates). Activity inhibited by 10^-3} M EGTA (PDE1), 10^-5 M EHNA (PDE2), 10^-5 M cilostamide (PDE3) and 10^-6 M roflumilast (PDE4).

Figure 3

Effect of roflumilast on intracellular cAMP levels. Increase in cAMP levels following PDE4 inhibition with 10^-6 M roflumilast (A) and dual treatment with iloprost (B). Data (mean ± SEM) from 6 PASMC isolates (A) and four replicates, which is representative of three experiments with distinct isolates (B).

Figure 4

Anti-mitogenic effects of PDE4 inhibition in PASMCs. Effects of PDE4 (cilomilast, rolipram, roflumilast) and PDE3 (cilostamide) inhibitors on PDGF-BB (5 ng/ml) stimulated DNA synthesis (A). Concentration-dependent effect of roflumilast, combined with a sub-maximal concentration of cicaprost, on [methyl-³H]-thymidine incorporation (B). Effect of roflumilast (10^-6 M, open squares) on serum-stimulated (5% FBS) cell growth (closed squares) (C) and combined inhibitory effect of iloprost (10^-7 M) after 10 days serum-stimulated growth (D). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control cells stimulated with either PDGF-BB (A), cicaprost (B) or serum (C-D). Data represent mean ± SEM from four-six distinct isolates (A-B, D) and four replicates (C).

Figure 5

Pro-apoptotic effects of cAMP elevating agents. Concentration-dependent effect of iloprost on apoptosis, as demonstrated by the proportion of Hoechst-stained cells showing characteristic condensed nuclear fluorescence (A) and measurement of DNA fragmentation in human PASMCs (B). Effects of PDE4 inhibition (10^-6 M roflumilast) and adenylyl cyclase activation (10^-5 M forskolin) on DNA fragmentation (C). Data represent mean ± SEM of four replicates in three distinct isolates. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus untreated control cells in serum free (SF) medium.

Figure 6

Gelatin zymography of matrix-metalloproteinase (MMP) in conditioned medium from human PASMCs. Representative zymograms showing the effects of phorbol 12-myristate 13-acetate (PMA, 10^-7 M) and 10 ng/ml TNF-α, IL-1β or TGF-β1 on inducible MMP-9 activity after 48 h (A) and the concentration-dependent inhibitory effect of cicaprost (B). MMP-9 (proMMP-9, 92 kDa); MMP-2 (proMMP-2, 72 kDa; active isoforms, 66 kDa and 62 kDa); MMP-2+, APMA-activated MMP-2; Dex, dexamethasone; Mif, mifepristone; 4α-PMA, inactive phorbol ester.

Figure 7

Inhibitory effect of cAMP elevating agents on MMP-9 activity. ELISA data of total MMP-9 activity in conditioned medium after 48 h, showing stimulation following treatment of PASMCs with PMA (10^-7 M) and TNF-α (10 ng/ml) and its inhibition by cicaprost and forskolin (A-B). Inhibitory effect of roflumilast, both alone (C) and in combination with a sub-maximal concentration of cicaprost (D). Data represent mean ± SEM of four replicates (A-B) and three-four distinct PASMC isolates (C-D). * P < 0.05, ** P < 0.01 and *** P < 0.001 versus medium from control cells in serum free (SF) medium (A) or PMA and TNF-α treatment (B-D).

Figure 8

Inhibitory effect of cAMP elevating agents on MMP-2 activity. ELISA data showing the inhibitory effect of roflumilast (10^-6 M) and iloprost (10^-7 M) on PMA (10^-7 M) and TNF-α (10 ng/ml) stimulated MMP-2 activity in PASMC conditioned medium after 48 h. * P < 0.05, versus medium from control cells in serum free (SF) medium