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


CONCLUSIONS
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.
Growcott
Ellena J
EJ
Section on Experimental Medicine and Toxicology, Imperial College London, Hammersmith Campus, London W12 0NN, UK. ellena.growcott@imperial.ac.uk
Spink
Karen G
KG
Ren
Xiaohui
X
Afzal
Saliha
S
Banner
Kathy H
KH
Wharton
John
J
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000631:Aminopyridines / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003521:Cyclopropanes / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D015870:Gene Expression; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D045726:Metalloproteases / Q000096:biosynthesis; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology*
D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; D010726:Phosphodiesterase Inhibitors; C424423:Roflumilast; D004247:DNA; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human; D045726:Metalloproteases; D016285:Iloprost