Regulation of bone morphogenetic protein signalling in human pulmonary vascular development.

The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH.

Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension.

Clinical pulmonary hypertension is characterized by a sustained elevation in pulmonary arterial pressure. Pulmonary vascular remodeling involves structural changes in the normal architecture of the walls of pulmonary arteries. The process of vascular remodeling can occur as a primary response to injury, or stimulus such as hypoxia, within the resistance vessels of the lung. Alternatively, the changes seen in more proximal vessels may arise secondary to a sustained increase in intravascular pressure. To withstand the chronic increase in intraluminal pressure, the vessel wall becomes thickened and stronger. This "armouring" of the vessel wall with extra-smooth muscle and extracellular matrix leads to a decrease in lumen diameter and reduced capacity for vasodilatation. This maladaptive response results in increased pulmonary vascular resistance and consequently, sustained pulmonary hypertension. The process of pulmonary vascular remodeling involves all layers of the vessel wall and is complicated by the finding that cellular heterogeneity exists within the traditional compartments of the vascular wall: intima, media, and adventitia. In addition, the developmental stage of the organism greatly modifies the response of the pulmonary circulation to injury. This review focuses on the latest advances in our knowledge of these processes as they relate to specific forms of pulmonary hypertension and particularly in the light of recent genetic studies that have identified specific pathways involved in the pathogenesis of severe pulmonary hypertension.

Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension.

Pulmonary vascular remodelling is an important pathological feature of pulmonary hypertension, leading to increased pulmonary vascular resistance and reduced compliance. It involves thickening of all three layers of the blood vessel wall (due to hypertrophy and/or hyperplasia of the predominant cell type within each layer), as well as extracellular matrix deposition. Neomuscularisation of non-muscular arteries and formation of plexiform and neointimal lesions also occur. Stimuli responsible for remodelling involve transmural pressure, stretch, shear stress, hypoxia, various mediators [angiotensin II, endothelin (ET)-1, 5-hydroxytryptamine, growth factors, and inflammatory cytokines], increased serine elastase activity, and tenascin-C. In addition, there are reductions in the endothelium-derived antimitogenic substances, nitric oxide, and prostacyclin. Intracellular signalling mechanisms involved in pulmonary vascular remodelling include elevations in intracellular Ca2+ and activation of the phosphatidylinositol pathway, protein kinase C, and mitogen-activated protein kinase. In animal models of pulmonary hypertension, various drugs have been shown to attenuate pulmonary vascular remodelling. These include angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, ET receptor antagonists, ET-converting enzyme inhibitors, nitric oxide, phosphodiesterase 5 inhibitors, prostacyclin, Ca2+ -channel antagonists, heparin, and serine elastase inhibitors. Inhibition of remodelling is generally accompanied by reductions in pulmonary artery pressure. The efficacy of some of the drugs varies, depending on the animal model of the disease. In view of the complexity of the remodelling process and the diverse aetiology of pulmonary hypertension in humans, it is to be anticipated that successful anti-remodelling therapy in the clinic will require a range of different drug options.

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion.

1. Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO*; NO gas solution) and nitroxyl ion (NO(-); from Angeli's salt). 2. The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 microM), concentration-dependently inhibited responses to all agents. 10 microM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3. The NO* scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 microM) and hydroxocobalamin (100 microM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4. The NO(-) inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO* and NO(-). Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Comparison of pulmonary vascular function and structure in early and established hypoxic pulmonary hypertension in rats.

In pulmonary hypertension, changes in pulmonary vascular structure and function contribute to the elevation in pulmonary artery pressure. The time-courses for changes in function, unlike structure, are not well characterised. Medial hypertrophy and neomuscularisation and reactivity to vasoactive agents were examined in parallel in main and intralobar pulmonary arteries and salt-perfused lungs from rats exposed to hypoxia (10% O2) for 1 and 4 weeks (early and established pulmonary hypertension, respectively). After 1 week of hypoxia, in isolated main and intralobar arteries, contractions to 5-hydroxytryptamine and U46619 (thromboxane-mimetic) were increased whereas contractions to angiotensins I and II and relaxations to acetylcholine were reduced. These alterations varied quantitatively between main and intralobar arteries and, in many instances, regressed between 1 and 4 weeks. The alterations in reactivity did not necessarily link chronologically with alterations in structure. In perfused lungs, constrictor responses to acute alveolar hypoxia were unchanged after 1 week but were increased after 4 weeks, in conjunction with the neomuscularisation of distal alveolar arteries. The data suggest that in hypoxic pulmonary hypertension, the contribution of altered pulmonary vascular reactivity to the increase in pulmonary artery pressure may be particularly important in the early stages of the disease.

Pulmonary vascular remodelling in hypoxic rats: effects of amlodipine, alone and with perindopril.

This study investigated whether pulmonary vascular remodelling in hypoxic pulmonary hypertensive rats (10% oxygen; 4 weeks) could be prevented by treatment, during hypoxia, with amlodipine (10 mg/kg/day, p.o.), either alone or in combination with the angiotensin converting enzyme inhibitor, perindopril (30 mg/kg/day, p.o.). Medial thickening of pulmonary arteries (30-500 microm o.d.) was attenuated by amlodipine whereas it was totally prevented by the combination treatment (amlodipine plus perindopril); neomuscularisation of small alveolar arteries (assessed from critical closing pressure in isolated perfused lungs) was not affected. Pulmonary vascular resistance (isolated perfused lungs) was reduced by both treatment regimes but only combination treatment reduced right ventricular hypertrophy. Thus, amlodipine has anti-remodelling properties in pulmonary hypertensive rats. The finding that combining amlodipine with another anti-remodelling drug produced effects on vascular structure that were additive raises the question of whether combination therapy with two different anti-remodelling drugs may be of value in the treatment of patients with hypoxic (and possibly other forms of) pulmonary hypertension.

Specific uptake of 5-hydroxytryptamine is reduced in lungs from hypoxic pulmonary hypertensive rats.

In this study, the aim was to determine whether 5-hydroxytryptamine (5-HT) removal by the pulmonary endothelium is reduced in 1-week hypoxic, pulmonary hypertensive rats by directly measuring [3H]5-HT uptake in isolated lungs. In lungs from hypoxic rats, specific 5-HT uptake was reduced. This was due to a 50% decrease in the maximal initial rate of uptake rather than a decrease in affinity of 5-HT for its transporter. It is possible that reduced removal of 5-HT may contribute to the elevation in plasma levels of this vasoactive amine in pulmonary hypertension.

Perindopril, an angiotensin converting enzyme inhibitor, in pulmonary hypertensive rats: comparative effects on pulmonary vascular structure and function.

1. Hypoxic pulmonary hypertension in rats (10% O2, 4 weeks) is characterized by changes in pulmonary vascular structure and function. The effects of the angiotensin converting enzyme inhibitor perindopril (oral gavage, once daily for the 4 weeks of hypoxia) on these changes were examined. 2. Perindopril (30 mg kg-1 d-1) caused an 18% reduction in pulmonary artery pressure in hypoxic rats. 3. Structural changes (remodelling) in hypoxic rats included increases in (i) critical closing pressure in isolated perfused lungs (remodelling of arteries <50 microm o.d.) and (ii) medial wall thickness of intralobar pulmonary arteries, assessed histologically (vessels 30 - 100 and 101 - 500 microm o.d.). Perindopril 10 and 30 mg kg-1 d-1 attenuated remodelling in vessels < or = 100 microm (lungs and histology), 30 mg kg-1 d-1 was effective in vessels 101 - 500 microm but neither dose prevented hypertrophy of main pulmonary artery. 3 mg kg-1 d-1 was without effect. 4. Perindopril (30 mg kg-1 d-1) prevented the exaggerated hypoxic pulmonary vasoconstrictor response seen in perfused lungs from hypoxic rats but did not prevent any of the functional changes (i.e. the increased contractions to 5-HT, U46619 (thromboxane-mimetic) and K+ and diminished contractions to angiotensins I and II) seen in isolated intralobar or main pulmonary arteries. Acetylcholine responses were unaltered in hypoxic rats. 5. We conclude that, in hypoxic rats, altered pulmonary vascular function is largely independent of remodelling. Hence any drug that affects only remodelling is unlikely to restore pulmonary vascular function to normal and, like perindopril, may have only a modest effect on pulmonary artery pressure.

Phosphodiesterase III and V inhibitors on pulmonary artery from pulmonary hypertensive rats: differences between early and established pulmonary hypertension.

Milrinone and 6-bromo-8(methylamino)imidazo[1,2a]pyrazine-2-carbonitrile [SCA40; phosphodiesterase (PDE) III inhibitors], zaprinast (PDE V inhibitor), and 3-isobutyl-1-methyl xanthine (IBMX; nonselective PDE inhibitor) were examined on main pulmonary arteries from control rats and rats exposed to hypoxia (10% O2; 1 or 4 weeks) to induce pulmonary hypertension. Each drug fully relaxed preparations precontracted submaximally with phenylephrine. In the absence of endothelium or the presence of the nitric oxide synthase inhibitor, L-NAME, responses to zaprinast, but not the other drugs, were reduced but not abolished. The potencies [negative log median effective concentration (EC50)] of the drugs in 4-week hypoxic rats (established pulmonary hypertension; zaprinast, 5.60; milrinone, 5.64; SCA40, 6.41; IBMX, 5.38) were not different from corresponding control values (6.05; 5.88; 6.65; 5.64) but in early pulmonary hypertension (1-week hypoxic rats), all except IBMX had reduced potency. The potency of the adenylate cyclase activator, forskolin, was reduced in arteries from both groups of rats. In early, but not established, pulmonary hypertension, arteries had inherent tone, spontaneous contractions, and diminished endothelial function. In established, but not early, pulmonary hypertension, arteries had increased overall contractile ability. It is concluded that (a) PDE V inhibitors require cyclic guanosine monophosphate (cGMP) produced by endothelial nitric oxide for optimal effect, (b) the potencies of PDE III and V inhibitors are not compromised in established pulmonary hypertension, and (c) data on pulmonary vascular function obtained in 1-week hypoxic rats do not necessarily reflect data in rats exposed to hypoxia for longer periods.

Recognition and management of pulmonary hypertension.

Pulmonary hypertension (mean pulmonary arterial pressure > 20mm Hg at rest or > 30mm Hg during exercise) occurs (i) as primary pulmonary hypertension (no known underlying cause), (ii) as persistent pulmonary hypertension of the newborn or (iii) secondary to a variety of lung and cardiovascular diseases. In the last 10 to 15 years there have been significant advances in the medical management of this debilitating and life-threatening disorder. The main drugs in current use are anticoagulants (warfarin, heparin) and vasodilators, especially oral calcium antagonists, intravenous prostacyclin (prostaglandin I2; epoprostenol) and inhaled nitric oxide. Calcium antagonists, (e.g. nifedipine, diltiazem) are used chiefly in primary pulmonary hypertension. They are effective in patients who give a pulmonary vasodilator response to an acute challenge with a short acting vasodilator (e.g. prostacyclin, nitric oxide or adenosine), and are used in doses greater than are usual in the treatment of other cardiovascular disorders. Prostacyclin, given by continuous intravenous infusion, is effective in patients even if they do not respond to an acute vasodilator challenge. The long term benefit in these patients is thought to reflect the antiproliferative effects of the drug and/or its ability to inhibit platelet aggregation. It is used either as long term therapy or as a bridge to transplantation. Inhaled nitric oxide, which is used mainly in persistent pulmonary hypertension of the newborn, has the particular benefit of being pulmonary selective, due to its route of administration and rapid inactivation. Anticoagulants have a specific role in the treatment of pulmonary thromboembolic pulmonary hypertension and are also used routinely in patients with primary pulmonary hypertension. Nondrug treatments for pulmonary hypertension include (i) supplemental oxygen (> or = 15 h/day), which is the primary therapy in patients with pulmonary hypertension secondary to chronic obstructive pulmonary disease and (ii) heart-lung or lung transplantation, which nowadays is regarded as a last resort. Different types of pulmonary hypertension require different treatment strategies. Future advances in the treatment of pulmonary hypertension may come from the use of drug combinations, the development of new drugs, such as endothelin antagonists, nitric oxide donors and potassium channel openers, or the application of gene therapy.