Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension

Abstract

Rationale: Prostacyclin analogs, used to treat idiopathic pulmonary arterial hypertension (IPAH), are assumed to work through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, although the potential to signal through peroxisome proliferator-activated receptor-γ (PPARγ) exists.

Objectives: IP receptor and PPARγ expression may be depressed in IPAH. We wished to determine if pathways remain functional and if analogs continue to inhibit smooth muscle proliferation.

Methods: We used Western blotting to determine IP receptor expression in peripheral pulmonary arterial smooth muscle cells (PASMCs) from normal and IPAH lungs and immunohistochemistry to evaluate IP receptor and PPARγ expression in distal arteries.

Measurements and main results: Cell proliferation and cAMP assays assessed analog responses in human and mouse PASMCs and HEK-293 cells. Proliferative rates of IPAH cells were greater than normal human PASMCs. IP receptor protein levels were lower in PASMCs from patients with IPAH, but treprostinil reduced replication and treprostinil-induced cAMP elevation appeared normal. Responses to prostacyclin analogs were largely dependent on the IP receptor and cAMP in normal PASMCs, although in IP(-/-) receptor cells analogs inhibited growth in a cAMP-independent, PPARγ-dependent manner. In IPAH cells, antiproliferative responses to analogs were insensitive to IP receptor or adenylyl cyclase antagonists but were potentiated by a PPARγ agonist and inhibited (∼ 60%) by the PPARγ antagonist GW9662. This coincided with increased PPARγ expression in the medial layer of acinar arteries.

Conclusions: The antiproliferative effects of prostacyclin analogs are preserved in IPAH despite IP receptor down-regulation and abnormal coupling. PPARγ may represent a previously unrecognized pathway by which these agents inhibit smooth muscle proliferation.

Figure 1.

Cell proliferation rates in pulmonary arterial smooth muscle cells (A and B) derived from normal and idiopathic pulmonary arterial hypertension (IPAH) patients and (C) HEK-293 cells expressing the IP receptor (HEK-293-IP) or the pcDNA3.1Zeo vector alone. Starved cells were grown in media containing 10% FBS. Results are presented as cell number (A) or expressed as mean fold increase in proliferation (B and C). HEK-293-IP cells were also treated with the IP receptor antagonist (1 μM; RO1183452), present throughout the 5-day period (C). Data are expressed as mean ± SEM, with growth assays repeated two to four times for each isolate. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to adult IPAH or HEK-293-IP.

Figure 2.

(A) Subconfluent pulmonary arterial smooth muscle cells (PASMCs) from different isolates stained with the IP receptor antibody using FITC-conjugated Alexa fluor 488 (green stain) as the secondary. TO-PRO-3 (red) was used to stain nuclei, and all images were taken under identical conditions. (B) Western blotting showing IP receptor expression in crude homogenates from PASMCs and HEK-293 cells expressing the IP receptor (HEK-IP) or empty vector (HEK-Con). For PASMC and HEK-293 lysates, 10 and 20 μg of protein, respectively, was loaded to match β-actin levels. (C) Box-whisker-plot of IP receptor expression in PASMCs shown relative to β-actin and normalized with respect to control. Bands were measured by densitometry using NIH Image software. Samples for each isolate (12 in total) were run on three or four separate occasions with blots always containing normal and disease samples. The passage number used was P4-P7 for controls, P3-P6 for adult idiopathic pulmonary arterial hypertension, and P3-P4 for child idiopathic pulmonary arterial hypertension. ***P < 0.001.

Figure 3.

(A) Immunohistochemical staining for the IP receptor (a, b, c) and peroxisome proliferator-activated receptor-gamma (PPARγ) (d, e, f) in serial sections of pulmonary arteries from a normal and from an untreated or treated child with idiopathic pulmonary arterial hypertension. Asterisk depicts regions of intimal proliferation and the scale bar represents 50 μm for all panels. Average IP receptor (B) and PPARγ (C) expression in medial preacinar and intraacinar arteries and in intimal proliferative cells. Staining was scored blinded in one to three sections per sample where 0, 1, 2, 3 are equivalent to no, weak, intermediate, or strong staining. *P < 0.05 compared with normal and ^#P < 0.05 compared with untreated intraacinar.

Figure 4.

Effect of treprostinil (TREP) on cAMP generation and proliferation in distal pulmonary arterial smooth muscle cells derived from (A) normal subjects and (B) patients with idiopathic pulmonary arterial hypertension and in (C) HEK-293-IP cells. For cAMP measurements, TREP (0.1 or 1 μM) was applied for 30 minutes with or without the IP receptor antagonist (RO1183452 or RO11; 1 μM) or 2′5′dideoxyadenosine (DDA; 100 μM). Inhibitors were given 1 hour before the addition of treprostinil. Results expressed as fold increase above basal in pmol of cAMP per mg of total protein. Growth-arrested cells were stimulated with 10% FBS ± treprostinil or cicaprost and counted at various time points. All data are expressed as mean ± SEM (n = 9–15). **P < 0.01; ***P < 0.001.

Figure 5.

Growth-arrested distal human pulmonary arterial smooth muscle cells (PASMCs) (A, C, and D) or HEK-293-IP cells (B) were incubated in media containing 10% FBS and left untreated (Con) or treated with the IP receptor antagonist (RO1183452), DMSO (0.1%), the IP receptor agonist (TREP, treprostinil or ILO, iloprost), or a combination. Data, expressed as % cell proliferation relative to growth response mediated by 10% FBS alone, are shown as mean ± SEM (n = 9). **P < 0.01 and ***P < 0.001 compared with control or as shown.

Figure 6.

Time-dependent effects of cicaprost (1 μM), iloprost (1 μM), and forskolin (10 μM) on intracellular cAMP concentration in (A) wild-type and (B) IP^−/− cultured distal pulmonary arterial smooth muscle cells (PASMCs) expressed as pmol of cAMP per 10⁶ cells. (C) ³H-thymidine incorporation in IP receptor wild-type (^+/+) mice. (D) ³H-thymidine incorporation in IP receptor deficient (^−/−) mice. All data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.0.01, and ***P < 0.001.

Figure 7.

Role of cAMP in mediating treprostinil (TREP) effects. Growth-arrested (A and B) distal pulmonary arterial smooth muscle cells (PASMCs) or (C) HEK-293-IP cells were stimulated with 10% FBS ± TREP (100 nM or 1 μM) in combination with 2′5′dideoxyadenosine (DDA; 100 μM). Cells were pretreated with 2′5′dideoxyadenosine 1 hour before stimulation with TREP. Data, expressed as % cell proliferation relative to the proliferative response mediated by 10% FBS alone, are shown as mean ± SEM (n = 9). ***P < 0.001.

Figure 8.

Effect of peroxisome proliferator-activated receptor-gamma (PPARγ) modulators on cell growth. (A) Concentration-response curve to the PPARγ agonist rosiglitazone (ROSI) in human pulmonary arterial smooth muscle cells (PASMCs) and HEK-293-IP cells. Growth-arrested cells were stimulated with 10% FBS with and without agonist. Cells were counted, and results are expressed as % cell proliferation relative to FBS alone. (B) Time-course of treprostinil effects on the luciferase activity. HEK-293-IP were transfected with GAL5TKpGL3 (reporter construct), the control renilla vector pMLuc2, and GAL4-hPPARγ-pcDNA3 (containing PPARγ fusion protein). Results are expressed as luciferase light units normalized to renilla activity. Effect of the PPARγ antagonist GW9662 (1 μM) on the antiproliferative effects of treprostinil (TREP; 100 nM) in (C) normal and in (D) IPAH PASMCs. (E) Concentration-response to TREP in the absence and presence of rosiglitazone (300 nM) in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with DMSO (0.05%) or ROSI alone. (F) Effect of cicaprost (1 μM) and GW9662 (0.1 μM) on ³H-thymidine incorporation in cultured distal PASMC from IP receptor–deficient mice. In C and F, cells were pretreated with GW9662 for 1 hour before addition of the agonist. Data are expressed as mean ± SEM (n = 9–15 for A, D, and E; n = 3–5 for C and F). *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to appropriate control or as indicated.