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Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice
Published on: October 19, 2013
Phosphodiesterase type 4 expression and anti-proliferative effects in human pulmonary artery smooth muscle cells
Ellena J Growcott1, Karen G Spink, Xiaohui Ren
1Section on Experimental Medicine and Toxicology, Imperial College London, Hammersmith Campus, London W12 0NN, UK. ellena.growcott@imperial.ac.uk
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.
Insights
Phosphodiesterase type 4 (PDE4) enzymes are present in pulmonary artery smooth muscle cells and influence cell proliferation. Inhibiting PDE4 enhances the anti-proliferative effects of prostacyclin analogues, suggesting a potential therapy for pulmonary arterial hypertension.
Area of Science:
- Vascular Biology
- Pharmacology
- Cellular Signaling
Background:
- Pulmonary arterial hypertension (PAH) is a vascular disease involving abnormal smooth muscle cell proliferation and apoptosis.
- Prostacyclin (PGI2) analogues reduce proliferation in human pulmonary artery smooth muscle cells (PASMCs) via cyclic adenosine monophosphate (cAMP) signaling.
- The role of phosphodiesterase type 4 (PDE4), a key enzyme in cAMP hydrolysis, in PAH was investigated.
Purpose of the Study:
- To determine the presence and activity of PDE4 enzymes in human PASMCs.
- To investigate the effect of PDE4 inhibition on PASMC proliferation, apoptosis, and matrix metalloproteinase (MMP) production.
- To assess the potential of PDE4 inhibitors as an adjunct therapy for PAH.
Main Methods:
- Human PASMCs were cultured from pulmonary arteries.
- Cells were treated with growth factors, PGI2 analogues, and PDE4 inhibitors (roflumilast, rolipram, cilomilast).
- Measurements included PDE activity, intracellular cAMP levels, DNA synthesis, apoptosis, and MMP-2/MMP-9 production.
Main Results:
- All four PDE4 subtypes (A-D) were expressed in PASMCs, with PDE4 accounting for the majority of cAMP hydrolytic activity.
- PDE4 inhibitors increased intracellular cAMP levels and attenuated DNA synthesis and proliferation, particularly when combined with PGI2 analogues.
- Roflumilast inhibited cell proliferation and MMP production, while PDE4 inhibitors did not induce apoptosis.
Conclusions:
- PDE4 enzymes are functionally expressed in human PASMCs and contribute to regulating cAMP-mediated effects on proliferation and MMP production.
- PDE4 inhibition potentiates the anti-proliferative actions of cAMP-stimulating agents in PASMCs.
- Targeting PDE4 may offer a novel therapeutic strategy for managing pulmonary arterial hypertension by enhancing anti-proliferative responses.
