Margaret R. MacLean

Cellular and molecular basis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is caused by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance. The process of pulmonary vascular remodeling is accompanied by endothelial dysfunction, activation of fibroblasts and smooth muscle cells, crosstalk between cells within the vascular wall, and recruitment of circulating progenitor cells. Recent findings have reestablished the role of chronic vasoconstriction in the remodeling process. Although the pathology of PAH in the lung is well known, this article is concerned with the cellular and molecular processes involved. In particular, we focus on the role of the Rho family guanosine triphosphatases in endothelial function and vasoconstriction. The crosstalk between endothelium and vascular smooth muscle is explored in the context of mutations in the bone morphogenetic protein type II receptor, alterations in angiopoietin-1/TIE2 signaling, and the serotonin pathway. We also review the role of voltage-gated K(+) channels and transient receptor potential channels in the regulation of cytosolic [Ca(2+)] and [K(+)], vasoconstriction, proliferation, and cell survival. We highlight the importance of the extracellular matrix as an active regulator of cell behavior and phenotype and evaluate the contribution of the glycoprotein tenascin-c as a key mediator of smooth muscle cell growth and survival. Finally, we discuss the origins of a cell type critical to the process of pulmonary vascular remodeling, the myofibroblast, and review the evidence supporting a contribution for the involvement of endothelial-mesenchymal transition and recruitment of circulating mesenchymal progenitor cells.

Dynamic Changes in Lung MicroRNA Profiles During the Development of Pulmonary Hypertension due to Chronic Hypoxia and Monocrotaline

Objective—MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding “seed” sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown. Methods and Results—We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH. Conclusion—Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention.

Serotonin Increases Susceptibility to Pulmonary Hypertension in BMPR2-Deficient Mice

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II (BMPR-II) receptor underlie the majority (>70%) of cases of familial pulmonary arterial hypertension (FPAH), and dysfunction of BMP signaling has been implicated in other forms of PAH. The reduced disease gene penetrance in FPAH indicates that other genetic and/or environmental factors may also be required for the clinical manifestation of disease. Of these, the serotonin pathway has been implicated as a major factor in PAH pathogenesis. We investigated the pulmonary circulation of mice deficient in BMPR-II (BMPR2+/− mice) and show that pulmonary hemodynamics and vascular morphometry of BMPR2+/− mice were similar to wild-type littermate controls under normoxic or chronic hypoxic (2- to 3-week) conditions. However, chronic infusion of serotonin caused increased pulmonary artery systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling in BMPR2+/− mice compared with wild-type littermates, an effect that was exaggerated under hypoxic conditions. In addition, pulmonary, but not systemic, resistance arteries from BMPR2+/− mice exhibited increased contractile responses to serotonin mediated by both 5-HT2 and 5-HT1 receptors. Furthermore, pulmonary artery smooth muscle cells from BMPR2+/− mice exhibited a heightened DNA synthesis and activation of extracellular signal-regulated kinase 1/2 in response to serotonin compared with wild-type cells. In vitro and in vivo experiments suggested that serotonin inhibits BMP signaling via Smad proteins and the expression of BMP responsive genes. These findings provide the first evidence for an interaction between BMPR-II-mediated signaling and the serotonin pathway, perturbation of which may be critical to the pathogenesis of PAH.

Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat.

Responses to human urotensin‐II (hU‐II) were investigated in human and rat pulmonary arteries. Rat pulmonary arteries: hU‐II was a potent vasoconstrictor of main pulmonary arteries (2–3 mm i.d.) (pEC50, 8.55±0.08, n=21) and was ∼4 fold more potent than endothelin‐1 [ET‐1] (P<0.01), although its Emax was considerably less (∼2.5 fold, P<0.001). The potency of hU‐II increased 2.5 fold with endothelium removal (P<0.05) and after raising vascular tone with ET‐1 (P<0.01). Emax was enhanced ∼1.5 fold in the presence of Nω‐nitro‐L‐arginine methylester (L‐NAME, 100 μM, P<0.01) and ∼2 fold in vessels from pulmonary hypertensive rats exposed to 2 weeks chronic hypoxia (P<0.05). hU‐II did not constrict smaller pulmonary arteries. Human pulmonary arteries (∼250 μm i.d.): in the presence of L‐NAME, 3 out of 10 vessels contracted to hU‐II and this contraction was highly variable. hU‐II is, therefore, a potent vasoconstrictor of rat main pulmonary arteries and this response is increased by endothelial factors, vascular tone and onset of pulmonary hypertension. Inhibition of nitric oxide synthase uncovers contractile responses to hU‐II in human pulmonary arteries.

A Role for miR-145 in Pulmonary Arterial Hypertension Evidence From Mouse Models and Patient Samples

Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective: We assessed the role of miR-145 in PAH. Methods and Results: We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR–mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2 -deficient mice. Conclusions: miR-145 is dysregulated in mouse models of PAH. Downregulation of miR-145 protects against the development of PAH. In patient samples of heritable PAH and idiopathic PAH, miR-145 is expressed in remodeled vessels and mutations in BMPR2 lead to upregulation of miR-145 in mice and PAH patients. Manipulation of miR-145 may represent a novel strategy in PAH treatment. # Novelty and Significance {#article-title-37}Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective: We assessed the role of miR-145 in PAH. Methods and Results: We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR–mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2-deficient mice. Conclusions: miR-145 is dysregulated in mouse models of PAH. Downregulation of miR-145 protects against the development of PAH. In patient samples of heritable PAH and idiopathic PAH, miR-145 is expressed in remodeled vessels and mutations in BMPR2 lead to upregulation of miR-145 in mice and PAH patients. Manipulation of miR-145 may represent a novel strategy in PAH treatment.

5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats.

1 We investigated 5‐hydroxytryptamine (5‐HT)‐receptor mediated vasoconstriction in the main, first branch and resistance pulmonary arteries removed from control and pulmonary hypertensive rats. Contractile responses to 5‐HT, 5‐carboxamidotryptamine (5‐CT, non‐selective 5‐HT1 agonist), and sumatriptan (5‐HT1D‐like receptor agonist) were studied. The effects of methiothepin (non‐selective 5‐HT1+2‐receptor antagonist) and ketanserin (5‐HT2A receptor antagonist) and GR55562 (a novel selective 5‐HT1D receptor antagonist) on 5‐HT‐mediated responses were also studied. Basal levels of adenosine 3′:5′‐cyclic monophosphate ([cyclic AMP]i) and guanosine 3′:5′‐cyclic monophosphate ([cyclic GMP]i) were determined and we assessed the degree of inherent tone in the vessels under study. 2 5‐HT was most potent in the resistance arteries. pEC50 values were 5.6 ± 0.1, 5.3 ± 0.1, 5.0 ± 0.2 in the resistance arteries, pulmonary branch and main pulmonary artery, respectively (n = at least 5 from 5 animals). The sensitivity to, and maximum response of, 5‐HT was increased in all the arteries removed from the chronic hypoxic (CH) rats. In CH rats the pEC50 values were 5.9 ± 0.2, 6.3 ± 0.2, 6.4 ± 0.2 and the increase in the maximum response was 35%, 51% and 41% in the resistance arteries, pulmonary branch and main pulmonary artery, respectively. Sumatriptan did not contract any vessel from the control rats whilst 5‐CT did contract the resistance arteries. In the CH rats, however, they both contracted the resistance arteries (responses to sumatriptan were small) (pEC50: 5‐CT; 5.4 ± 0.2) and the pulmonary artery branches (pEC50: sumatriptan, 5.4 ± 0.2; 5‐CT, 5.4 ± 0.2). 5‐CT also caused a contraction in the main pulmonary artery (pEC50: 6.0 ± 0.3). 3 Ketanserin (1 nM‐1 μm) caused a competitive antagonism of the 5‐HT response in all vessels tested. In control rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 8.3, 7.8 and 9.2, respectively. Methiothepin (1 nM‐1 μm) inhibited responses to 5‐HT in the first branch (estimated pKb value: 7.8) and main pulmonary artery. In CH rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 7.7, 8.3 and 9.6, respectively. Methiothepin also inhibited contractions to 5‐HT in the pulmonary artery branch and main pulmonary artery with estimated pKb values of 7 and 9.5, respectively. In control animals, GR55562 had no effect on responses to 5‐HT in any of the vessels tested. In the CH rats the estimated pKb values for GR55562 were 6.5, 7.8 and 7.0 in the pulmonary resistance arteries, first branch and main pulmonary artery, respectively. 4 Large pulmonary arteries from controls demonstrated inherent tone and this was increased three fold in the CH rats. The resistance arteries from controls demonstrated little inherent tone though this was enhanced in those from the CH rats. 5 [Cyclic AMP]i was 259 ± 23 pmol mg−1 protein in the pulmonary artery branches removed from control rats and decreased to 192 ± 11 pml mg−1 protein in the CH rats (P < 0.01, n = 8). [Cyclic GMP]i also decreased in the pulmonary artery branches (from 550 ± 15, control to 462 ± 31 pmol mg−1 protein in CH vessels, n = 8, P < 0.01) and in the main pulmonary arteries (from 566 ± 33, control to 370 ± 25 pmol mg−1 protein in CH vessels, n = 8, P < 0.001). No changes in either [cyclic AMP]i or [cyclic GMP]i were observed in the resistance arteries. 6 The results suggest that the increased vasoconstrictor response to 5‐HT in CH rat pulmonary arteries is due to an increase in 5‐HT2A‐receptor mediated contraction combined with an increase in r5‐HT1B‐like receptor‐mediated contraction. An increase in vascular tone and decreased levels of [cyclic GMP]i in the large pulmonary arteries may contribute to the observed increase in activity of r5‐HT1B‐like receptors.

5-Hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries : importance of the 5-HT1B receptor

The 5‐hydroxytryptamine (5‐HT) receptors mediating vasoconstriction in isolated human small muscular pulmonary arteries (SMPAs) were determined using techniques of wire myography and reverse transcription‐polymerase chain reaction (RT–PCR). The agonists 5‐HT, 5‐carboxamidotryptamine (5‐CT, unselective for 5‐HT1 receptors) and sumatriptan (selective for 5‐HT1B/D receptors) all caused contraction and were equipotent (pEC50s: 7.0±0.2, 7.1±0.3 and 6.7±0.1, respectively) suggesting the presence of a 5‐HT1 receptor. Ketanserin (5‐HT2A‐selective antagonist, 0.1 μM) inhibited 5‐HT‐induced contractions only at non‐physiological/pathological concentrations of 5‐HT (>0.1 μM) whilst GR55562 (5‐HT1B/1D‐selective antagonist, 1 μM) inhibited 5‐HT‐induced contractions at all concentrations of 5‐HT (estimated pKB=7.7±0.2). SB‐224289 (5‐HT1B‐selective antagonist, 0.2 μM) inhibited sumatriptan‐induced contractions (estimated pKB=8.4±0.1) whilst these were unaffected by the 5‐HT1D‐selective antagonist BRL15572 (0.5 μM) suggesting that the 5‐HT1B receptor mediates vasoconstriction in this vessel. RT–PCR confirmed the presence of substantial amounts of mRNA for the 5‐HT2A and 5‐HT1B receptor subtypes in these arteries whilst only trace amounts of 5‐HT1D receptor message were evident. These findings suggest that a heterogeneous population of 5‐HT2A and 5‐HT1B receptors co‐exist in human small muscular pulmonary arteries but that the 5‐HT1B receptor mediates 5‐HT‐induced vasoconstriction at physiological and pathophysiological concentrations of 5‐HT. These results have important implications for the treatment of pulmonary hypertension in which the 5‐HT1B receptor may provide a novel and potentially important therapeutic target.

Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease

Heightened expression of the S100 calcium–binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100&bgr;, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT–induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal–regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4–mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

Increased expression of the cGMP‐inhibited cAMP‐specific (PDE3) and cGMP binding cGMP‐specific (PDE5) phosphodiesterases in models of pulmonary hypertension

Chronic hypoxic treatment of rats (to induce pulmonary hypertension, PHT) for 14 days increased cGMP‐inhibited cAMP specific phosphodiesterase (PDE3) and cGMP binding cGMP specific phosphodiesterase (PDE5) activities in pulmonary arteries. The objective of this study was to establish the molecular basis for these changes in both animal and cell models of PHT. In this regard, RT–PCR and quantitative Western blotting analysis was applied to rat pulmonary artery homogenates and human pulmonary ‘artery’ smooth muscle cell (HPASMC) lysates. PDE3A/B gene transcript levels were increased in the main, first, intrapulmonary and resistance pulmonary arteries by chronic hypoxia. mRNA transcript and protein levels of PDE5A2 in the main and first branch pulmonary arteries were also increased by chronic hypoxia, with no effect on PDE5A1/A2 in the intra‐pulmonary and resistance vessels. The expression of PDE3A was increased in HPASMCs maintained under chronic hypoxic conditions for 14 days. This may be mediated via a protein kinase A‐dependent mechanism, as treatment of cells with Br‐cAMP (100 μM) mimicked chronic hypoxia in increasing PDE3A expression, while the PKA inhibitor, H8 peptide (50 μM) abolished the hypoxic‐dependent increase in PDE3A transcript. We also found that the treatment of HPASMCs with the inhibitor of κB degradation Tosyl‐Leucyl‐Chloro‐Ketone (TLCK, 50 μM) reduced PDE5 transcript levels, suggesting a role for this transcription factor in the regulation of PDE5 gene expression. Our results show that increased expression of PDE3 and PDE5 might explain some changes in vascular reactivity of pulmonary vessels from rats with PHT. We also report that NF‐κB might regulate basal PDE5 expression.

Endothelin ETA- and ETB-receptor-mediated vasoconstriction in rat pulmonary arteries and arterioles.

Summary: We investigated the endothelin (ET) receptors involved in the vasoconstrictor responses to ET-1 in rat pulmonary arteries and arterioles and the effect of endo-thelium removal, nitric oxide (NO) synthase inhibition, and hypoxia on ET-1-induced responses in the arteries. In isolated rat pulmonary artery rings (2–3 mm ID) prepared from the pulmonary artery branch before its entry into the lung, ET-1-induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were competitively antagonized by the ETA receptor antagonist FR 139317, and the ETB-receptor agonist sarafotoxin S6c (SXS6c) was a very weak vasoconstrictor in these vessels, inducing maximum contractions only 9% of those of ET-1. In contrast, in rat intrapulmonary resistance arteries (100–150 μm ID), SXS6c induced FR 139317-resistant contractions, and these vessels were more sensitive to SXS6c than to ET-1. SXS6c produced maximum contractions 92% those of ET-1, suggesting that ET-1-induced contractions were mediated by the ETB receptor in these resistance vessels. In the larger pulmonary arteries, the NO synthase inhibitor L-N ω ni-troarginine methyl ester (L-NAME) (100 μM) potentiated responses to ET-1, an effect that was reversed by FR 139317. Endothelium removal also potentiated response to ET-1, and L-NAME had no effect on ET-1 responses in endothelium-denuded vessels, suggesting that in these vessels the ETA receptor-mediated responses to ET-1 are normally suppressed by endothelium-derived NO. Hypoxia did not affect the sensitivity of the vessels to ET-1, but did increase the ability of FR 139317 to antagonise these responses. L-NAME did not affect responses to SXS6c in pulmonary resistance vessels.