Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment.

Endothelin receptor antagonists are used to treat idiopathic pulmonary arterial hypertension (IPAH), but human pulmonary arterial endothelin receptor expression is not well defined. We hypothesised that disease and treatment would modify normal receptor distribution in pulmonary resistance arteries of children. Using immunohistochemistry and semiquantitative analysis, we investigated endothelin receptor subtypes A and B (ET(A) and ET(B), respectively), and endothelial nitric oxide synthase (eNOS) expression in peripheral pulmonary arteries of tissue from untreated children with IPAH (n=7), following extended combined bosentan and epoprostenol therapy (n=5) and from normal subjects (n=5). Clinical, haemodynamic and pathological abnormalities were severe and advanced in all IPAH cases. ET(A) was detected in pulmonary arterial endothelial cells of all normal and diseased tissue and cultured cells. Endothelial ET(A), ET(B) and eNOS expression was reduced in patent, plexiform and dilatation lesions of untreated cases, but in treated cases, ET(A) and ET(B) were normal and eNOS increased. In smooth muscle, ET(A) expression was reduced in treated cases but ET(B) expression increased in all arteries of both treated and untreated cases. In summary, ET(A) is expressed on human pulmonary arterial endothelium. In IPAH, combination treatment with bosentan and epoprostenol had a more marked influence on endothelin receptor expression of endothelial than smooth muscle cells.

The science of endothelin-1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension: current understanding and future studies.

Pathological vascular remodelling is a key contributor to the symptomatology of pulmonary arterial hypertension (PAH), and reversing this process may offer the best hope for improving this debilitating condition. The vascular remodelling process is believed to be due to endothelial cell dysfunction and to involve altered production of endothelial cell-derived vasoactive mediators. The observation that circulating plasma levels of the vasoactive peptide endothelin (ET)-1 are raised in patients with PAH, and that ET-1 production is increased in the pulmonary tissue of affected individuals, makes it a particularly interesting target for a therapeutic intervention in PAH. Clinical trials with ET receptor antagonists (ETRAs) show that they provide symptomatic benefit in patients with PAH, thereby proving the clinical relevance of the ET system as a therapeutic target. In this paper, we review the role of ET-1 together with the available data on the roles of the specific ET receptors and ETRAs in PAH. In particular, we discuss the possible role of ET receptor selectivity in the vascular remodelling process in PAH and whether selective ET(A) or nonselective ET(A)/ET(B) blockade offers the greatest potential to improve symptoms and alter the clinical course of the disease.

Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells.

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3-4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1-2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38(MAPK), but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and 6-keto-PGF(1alpha) release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 micro M), or the COX-2 antagonist, NS398 (10 micro M), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.

Differential adrenomedullin release and endothelin receptor expression in distinct subpopulations of human airway smooth-muscle cells.

Although adrenomedullin (ADM) is implicated in the control of airway tone, regulation of ADM release from airway smooth-muscle cells (ASMCs) has not been explored. Preliminary experiments have indicated that human ASMC populations were heterogeneous in their rate of ADM release and expression of endothelin (ET)(A) and ET(B) receptors. We isolated these phenotypically distinct ASMCs from explants derived from the same airway segment. ASMCs possessing exclusively ET(A) receptors appeared smaller and proliferated faster than ET(A)/ET(B) isolates. Macroautoradiographic analysis confirmed the presence of both receptors in human bronchi. ADM release and messenger RNA expression was greater in ET(A)/ET(B) isolates compared with ET(A) isolates. No measurable ET release was detected from ASMCs. Exogenous ET-1 (1 to 100 nM) more potently stimulated the release of ADM from ET(A)/ET(B) compared with ET(A) isolates. In addition, ET-3 (1 to 100 nM) stimulated ADM release only from ET(A)/ET(B) isolates, implicating the ET(B) receptor in this response. Exogenous ET-1 potentiated platelet- derived growth factor-stimulated [3H]thymidine uptake in ET(A)/ ET(B) but not ET(A) isolates. ET-3 did not affect [3H]thymidine uptake in either cell type. Possession of ET(A)/ET(B) receptors is associated with higher rates of ADM release and slower proliferation, but a capacity for ET-1 stimulated DNA synthesis via ET(A) receptors. These results support a paracrine role for the regulation of ADM release predominantly via the ET(B) receptor in human ASMCs.

Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations.

The vasodilator peptide adrenomedullin is elevated in patients with pulmonary hypertension and has been implicated in the inhibition of vascular remodeling. We questioned whether adrenomedullin is released by human pulmonary artery smooth muscle cells (PASMCs) and inhibits PASMC growth and release of endothelin, a known smooth muscle cell mitogen. The majority of PASMCs isolated from proximal pulmonary arteries and all PASMCs from distal pulmonary arteries released adrenomedullin, although at differing rates (mean, 177 +/- 28 and 62 +/- 11 fmol/10(5) cells/24 h, respectively). These cells were designated ADM+. However, some proximal PASMC isolates did not release adrenomedullin, designated ADM-. Northern blot analysis confirmed adrenomedullin expression in proximal ADM+ but not ADM- isolates. ADM- and distal ADM+ PASMCs proliferated faster in serum than did proximal ADM+ cells. Adrenomedullin potently and dose-dependently (mean EC(50) = 2.2 +/- 0.5 nM) increased intracellular cyclic adenosine monophosphate (cAMP) in ADM- isolates via specific adrenomedullin receptors. In contrast, both adrenomedullin and calcitonin gene-related peptide modestly elevated cAMP in 50% of ADM+ isolates. Adrenomedullin dose-dependently inhibited platelet-derived growth factor-stimulated [3H]thymidine incorporation and endothelin release in ADM- cells but did not affect [3H]thymidine uptake in ADM+ isolates. We conclude that distinct subpopulations of human PASMCs release and respond to adrenomedullin. The heterogeneity of adrenomedullin release and the inhibition of PASMC DNA synthesis and endothelin release suggest that adrenomedullin may function as a paracrine mediator in the inhibition of pulmonary vascular remodeling.

Prostacyclin analogues differentially inhibit growth of distal and proximal human pulmonary artery smooth muscle cells.

BACKGROUND: Prostacyclin has proved to be a beneficial treatment for patients with severe pulmonary hypertension. We postulated that the response may reflect, at least in part, inhibition of pulmonary artery smooth muscle cell (PASMC) growth. METHODS AND RESULTS: Human PASMCs were derived from distal (<1-mm external diameter, n=8) and proximal (>8-mm external diameter, n=12) pulmonary arteries obtained at transplant surgery and pneumonectomy. The effects of the stable prostacyclin analogues on [methyl-(3)H]thymidine incorporation and cell proliferation were investigated by using immunohistochemically characterized cells. Distal cells proliferated faster than did proximal PASMCs and displayed a distinct sensitivity to cicaprost and iloprost. Both analogues inhibited thymidine uptake over 24 hours (20% to 60%, P<0.001; n=8) and abolished stimulation of DNA synthesis by platelet-derived growth factor-BB (10 ng/mL) in distal but not proximal cells. The inhibitory effect of cicaprost was mimicked by isoproterenol (10(-5) mol/L), forskolin (10(-5) mol/L), and dibutyryl cAMP (5x10(-4) mol/L) and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (5x10(-5) mol/L). Cicaprost (10(-10) to 10(-6) mol/L) inhibited the proliferation of PASMCs, which had been stimulated with either platelet-derived growth factor-BB or serum, and increased cAMP production. These effects were potentiated by 3-isobutyl-1-methylxanthine and attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10(-5) to 10(-4) mol/L). CONCLUSIONS: ++Cicaprost and iloprost inhibit DNA synthesis and proliferation to a greater extent in distal compared with proximal human PASMCs, acting at least in part via a cAMP-dependent mechanism. The results are consistent with the hypothesis that prostacyclin analogues inhibit vascular remodeling in pulmonary hypertension and demonstrate heterogeneity among human PASMCs.

Expression and function of angiotensin converting enzyme, chymase, and angiotensin II in the human radial artery and internal thoracic artery.

BACKGROUND: The potential role of the local renin-angiotensin system to differentially affect radial artery and internal thoracic artery graft performance has not been examined. METHODS: Contractile responses to angiotensin I and II in the radial artery and the internal thoracic artery were examined in vitro. The expression function, and localization of angiotensin receptors, angiotensin converting enzyme, and chymase were studied in radial artery and internal thoracic artery segments. RESULTS: Angiotensin I and II contractions were significantly greater (p < 0.05) in the radial artery compared to the internal thoracic artery. In both arteries, angiotensin II responses were mediated via the AT1 receptor. Messenger RNA transcripts for angiotensin-converting enzyme and chymase were detected in both arteries. Angiotensin-converting enzyme was localized to luminal and vaso vasorum endothelial cells and smooth muscle cells in both vessels, while chymase was colocalized with mast cells in adventitial and medial layers. An angiotensin converting enzyme or a chymase inhibitor singularly had no effect on angiotensin I contractions, however, when combined, a marked inhibition of the angiotensin I response was observed in both vessels. CONCLUSIONS: Our results illustrate the complexities which exist within the local renin angiotensin system and suggest that clinical trials which may modulate the system are warranted.