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Rationale: MicroRNAs are small non-coding RNAs involved in the regulation of gene expression and have recently been implicated in the development of pulmonary arterial hypertension (PAH). Previous work established that miR-451 is up-regulated in rodent models of PAH.\ud Objectives: The role of miR-451 in the pulmonary circulation is unknown. We therefore sought to assess the involvement of miR-451 in the development of pulmonary arterial hypertension.\ud Methods: Silencing of miR-451 was performed in vivo using miR-451 knockout mice and an antimiR targeting mature miR-451 in rats. Coupled with exposure to hypoxia, indices of pulmonary arterial hypertension were assessed. The effect of modulating miR-451 on human pulmonary artery smooth muscle cell proliferation and migration was analysed.\ud Measurements and Main Results: We observed a reduction in systolic right ventricular pressure in hypoxic rats pre-treated with antimiR-451 compared to hypoxia alone (47.7 ± 1.36mmHg and 56.0 ± 2.03mmHg respectively, p\ud Conclusions: Transient inhibition of miR-451 attenuated the development of pulmonary arterial hypertension in hypoxia exposed rats. Genetic deletion of miR-451 had no beneficial effect on indices of pulmonary arterial hypertension, potentially due to pathway redundancy compensating for the loss of miR-451.

1. 5-Hydroxytryptamine (5-HT)(1)-receptor-induced contraction is enhanced, or uncovered, by elevated vascular tone in many arteries including pulmonary arteries. In hypoxia-induced pulmonary hypertension, the endogenous tone of pulmonary arteries is elevated and this may contribute to increased 5-HT(1)-receptor-induced contraction. Here we investigate the influence of vascular tone induced by endothelin-1 (ET-1), neuropeptide Y (NPY), KCl, 4-aminopyridine (inactivator of K(v) channels, 4-AP) or the calcium ionophore A23187 on contractile responses to the 5-HT(1)-receptor agonist 5-carboxamidotryptamine (5-CT) in small muscular pulmonary arteries from control rats and rats exposed to chronic hypoxia. The influence of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) was also studied. These conditions were chosen to mimic those that influence pulmonary vascular tone in hypoxia-induced pulmonary hypertension. 2. In control rat small pulmonary arteries, only high concentrations of 5-CT (1 microM) induced vasoconstriction. Tone induced by NPY, 4-AP and A23187 had no effect on responses to 5-CT whilst responses to 5-CT were increased by ET-1- and KCl-induced tone. In the presence of L-NAME these responses to 5-CT were enhanced further. 3. Responses to 5-CT were enhanced 3 - 4 fold in small pulmonary arteries from hypoxia-exposed, pulmonary hypertensive rats and neither L-NAME nor increasing tone with NPY, 4-AP, A23187, ET-1 or KCl had any further effect on responses to 5-CT. 4. The results suggest that inhibition of nitric oxide synthase combined with KCl- or ET-1-induced vascular tone potentiates responses to 5-HT(1)-receptor-induced contraction in pulmonary arteries in a synergistic fashion and this mimics the effects of chronic hypoxic exposure.

1. 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). 2. 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. 3. Ketanserin (5-HT2A-selective antagonist, 0.1 microM) inhibited 5-HT-induced contractions only at non-physiological/pathological concentrations of 5-HT (0.1 microM) whilst GR55562 (5-HT1B/1D-selective antagonist, 1 microM) 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 microM) inhibited sumatriptan-induced contractions (estimated pKB=8.4+/-0.1) whilst these were unaffected by the 5-HT1D-selective antagonist BRL15572 (0.5 microM) suggesting that the 5-HT1B receptor mediates vasoconstriction in this vessel. 4. 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. 5. 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.

1. Vasoconstrictor responses to 5-HT, 5-carboxamidotryptamine (5-CT, 5-HT1 receptor agonist), alpha-methyl-5-HT (5-HT2 receptor agonist) and sumatriptan (5-HT1D/1B receptor agonist) were studied in fetal, 0-24 h, 4 day, 7 day and adult rabbit pulmonary resistance arteries (PRAs), alone and in the presence of the NO synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME). The effect of the selective 5-HT receptor antagonists ketanserin (5-HT2A receptor) and GR55562 (5-HT1B/1D receptor) on vasoconstrictor responses to 5-HT were studied in the presence of L-NAME. Vasodilator responses to 5-CT were also studied in pre-contracted PRAs. 3. 5-HT and alpha-methyl-5-HT were equipotent in causing contraction in the PRAs at each age (e.g. pEC50s for 5-HT and alpha-methyl-5-HT were 6.74+/-0.13 and 6.63+/-0.22 respectively in adult vessels). In the perinatal PRAs, sumatriptan and 5-CT produced negligible contractions, but in adult PRAs, 5-CT and sumatriptan were potent agonists with pEC50s of 6.05+/-0.3 and 5.70+/-0.20 respectively. 4. L-NAME markedly increased the maximum response to 5-HT in the 0-24 h, 4 day and 7 day vessels and increased 5-HT potency in the 4-, 7-day-old and adult rabbit vessels. 5. In perinatal vessels, responses to 5-HT, with L-NAME present, were antagonized by ketanserin (30 nM and 0.1 microM) but not GR55562 (1 microM). A small ketanserin-resistant, GR55562-sensitive component was observed at 0-24 h. In adult vessels, both ketanserin and GR55562 inhibited 5-HT-induced responses. 7. Vasodilator responses to 5-CT were observed in pre-contracted PRAs from 4- and 7-day-old rabbits but not in the fetus, 0-24 h old or adult rabbit vessels. At 4 days the vasodilator response was inhibited both by L-NAME and GR55562. At 7 days the response was only partly blocked by L-NAME and resistant to GR55562. The L-NAME resistant component was antagonized by the 5-HT7 receptor antagonist spiperone (1 microM). 8. The results suggest that 5-HT2A-receptors mediate vasoconstriction in perinatal vessels whilst the 5-HT1D or 5-HT1B receptor contributes in adult rabbit vessels. The 5-HT1D or 5-HT1B receptor mediates NO-dependent vasodilation in vessels from rabbits at 4 days of age whilst 5-HT7 receptors mediate NO-independent vasodilation by 7 days.