Intralobar Pulmonary Arteries Research Articles

Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension

RationalePulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. MethodsTo test this hypothesis, we exposed global (Gpr75−/−) and endothelial cell (EC) GPR75 knockout (EC-Gpr75−/−) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. ResultsChronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75−/− or EC-Gpr75−/− mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75−/−, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. ConclusionThese results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

Comparison of signalling mechanisms underlying UTP-evoked vasoconstriction of rat pulmonary and tail arteries

The endogenous nucleotide, UTP, acts at smooth muscle P2Y receptors to constrict rat pulmonary and tail arteries, but the underlying signalling pathways are poorly understood. The aim was to characterise the contribution of Ca2+ release and influx, rho kinase and protein kinase C to these contractions. Isometric tension was recorded from endothelium-denuded rat intralobar pulmonary and tail artery rings mounted on a wire myograph. Contractions were evoked by UTP and peak amplitude measured. Thapsigargin (1 µM), but not ryanodine (10 µM), significantly depressed contractions in both by 30–40% (P < 0.05). Nifedipine (1 µM) significantly reduced contractions in tail artery by ~60% (P < 0.01). Y27632 (10 µM), a rho kinase inhibitor and GF109203X (10 µM), a protein kinase C inhibitor, each significantly reduced pulmonary vasoconstriction by ~20%, and tail artery contractions by ~80% and ~40%, respectively (P < 0.01). In pulmonary artery, Y27632, GF109203X and thapsigargin, acted in an additive manner, but nifedipine less so. Adding all four together abolished the UTP response. In tail artery, Y27632 plus thapsigargin or GF109203X or nifedipine abolished contractions. Thapsigargin, GF109203X and nifedipine, coapplied pair-wise, acted additively and applying all three together abolished UTP-evoked contractions. So, Ca2+ release from the sarcoplasmic reticulum and influx through Cav1.2 channels, but not ryanodine receptors, play significant roles in UTP-evoked vasoconstriction of rat pulmonary and tail arteries. Rho kinase and protein kinase C are also involved, but more so in tail artery. Thus UTP activates multiple signalling mechanisms that lead to vasoconstriction, but their relative importance differs in pulmonary compared with systemic arteries.

Connexin 43 Plays a Role in Pulmonary Vascular Reactivity in Mice.

Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using Cx43 heterozygous (Cx43+/−) mice and 37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male Cx43+/− mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female Cx43+/−mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female Cx43+/− mice or by pre-incubation of IPAs with 37,43Gap27. Nω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]−3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries.Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively.SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC.In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis.

Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis. We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis, and tested their functional and biomarker relevance. Anti-AT1R and -ETAR Abs were detected by ELISA in different cohorts of patients and tested in vitro and in an animal model for their pathophysiological effects. The Abs were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease-associated PAH (n = 110) compared with other forms of PAH/pulmonary hypertension (n = 106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and SSc-PAH-related mortality in a prospective analysis. Both Abs increased endothelial cytosolic Ca(2+) concentrations in isolated perfused rat lungs, which could be blocked by respective specific receptor antagonists. Ab-mediated stimulation of intralobar pulmonary rat artery ring segments increased vasoconstrictive responses to Ang II and ET-1, and implicated cross-talk between both pathways demonstrated by reciprocal blockade with respective antagonists. Transfer of SSc-IgG containing both autoantibodies into healthy C57BL/6J mice led to more abundant vascular and airway α-smooth muscle actin expression and inflammatory pulmonary vasculopathy. Anti-AT1R and -ETAR Abs are more frequent in SSc-PAH/connective tissue disease-PAH compared with other forms of pulmonary hypertension, and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy.

Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse

The calgranulin-like protein MTS1/S100A4 and the receptor for advanced glycation end-products (RAGE) have recently been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodelling in experimental pulmonary arterial hypertension (PH). Here, the effects of RAGE antagonism upon 2 weeks of hypobaric hypoxia (10% O2)-induced PH in mice were assessed. Treatment with sRAGE was protective against hypobaric hypoxia-induced increases in right ventricular pressure but distal pulmonary vascular remodelling was unaffected. Intralobar pulmonary arteries from hypobaric hypoxic mice treated with sRAGE showed protection against a hypoxia-induced reduction in compliance. However, a combination of sRAGE and hypoxia also dramatically increased the force of contractions to KCl and 5-HT observed in these vessels. The acute addition of sRAGE to the organ bath produced a small, sustained contraction in intralobar pulmonary vessels and produced a synergistic enhancement of the maximal force of contraction in subsequent concentration–response curves to 5-HT. sRAGE had no effect on 5-HT-induced proliferation of Chinese hamster lung fibroblasts (CCL39), used since they have a similar pharmacological profile to mouse pulmonary fibroblasts but, surprisingly, produced a marked increase in hypoxia-induced proliferation. These data implicate RAGE as a modulator of both vasoreactivity and of proliferative processes in the response of the pulmonary circulation to chronic-hypoxia.

Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease.

Protein kinases modulate store-operated channels in pulmonary artery smooth muscle cells

BackgroundThis study investigates whether protein kinase G (PKG), protein kinase A (PKA) and protein kinase C (PKC) are involved in the regulatory mechanisms of store-operated channel (SOC) in pulmonary arteries.MethodsPulmonary artery smooth muscle cells (PASMCs) were enzymatically dissociated from rat intralobar pulmonary arteries. Whole cell, cell-attached and inside-out patch-clamp electrophysiology were used to monitor SOCs in isolated PASMCs.ResultsInitially the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA, 10 μM) initiated a whole cell current that was reduced by the SOC blocker SKF-96365 (10 μM). Subsequent work using both cell-attached and whole cell configurations revealed that the PKG and PKA inhibitors, KT5823 (3 μM) and H-89 (10 μM), also stimulated SOC activity; this augmentation was attenuated by the SOC blockers SKF-96365 (10 μM) and Ni2+ (0.1 mM). Finally using the inside-out configuration, the PKC activator phorbol 12-myristate 13-acetate (PMA, 10 μM) was confirmed to modestly stimulate SOC activity although this augmentation appeared to be more substantial following the application of 10 μM inositol 1,4,5-triphosphate (Ins(1,4,5)P3).ConclusionsSOC activity in PASMCs was stimulated by the inhibition of PKG and PKA and the activation of PKC. Our findings suggest that the SOC could be a substrate of these protein kinases, which therefore would regulate the intracellular concentration of calcium and pulmonary arteriopathy via SOC.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats

BackgroundThe roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. MethodsPulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. ResultsHypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAPhypox) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAPhypox was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAPhypox by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAPhypox. Consistently, the TXA2 analogue U46619 increased ΔPAPhypox whereas prostacyclin abolished ΔPAPhypox. However, leukotriene D4 had no direct effect on ΔPAPhypox. In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. ConclusionsThe above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Lysophosphatidic acid (LPA) enhances barrier function in intralobar rat pulmonary artery endothelial cells

Lysophosphatidic acid (LPA) is a bioactive serum lipid whose levels are increased in atherosclerotic plaques. In endothelial cells (ECs), LPA promotes migration, adhesion, wound healing, and modulates barrier function, in part, via 6 receptors (LPA1–6). While its sphingolipid homologue sphingosine‐1‐phosphate, enhances PAEC barrier function, the LPA effects on PAECs are unknown. Here we hypothesized that LPA also enhances PAEC barrier function. To test this, we determined the expression of LPA1–6, GPR87 and P2Y10; and examined the LPA effects on PAEC transendothelial resistance (TER), cytosolic calcium ([Ca2+]i ), and [cAMP] in ECs isolated from 3rd/4th generation intralobar rat PAs. Intralobar PAECs express non‐Edg LPA4/p2y9/GPR23 and LPA6/p2y5. Low [LPA] (0.1uM) increased TER by 4.9±0.6 % (t=30m), while 10uM LPA initially decreased TER by 10.4±1.0% (t=10m) followed by a sustained rise of 14.4 ± 2.1 % (t=90m). LPA increased [Ca2+]i dose‐dependently, and [cAMP] in the presence of forskolin that was potentiated by pertussis toxin (PTX). Both entry and release mechanisms contribute to the LPA‐induced Ca transients since absence of extracellular Ca reduced the signal by 70±10% (p&lt;0.05). LPA‐induced Ca entry was PTX‐sensitive, while release was PTX‐insensitive. Based on these data, we conclude that circulating [LPA] enhance PAEC barrier function and modulates [Ca2+]i and [cAMP] via LPA4 activation.

Testosterone acts as an efficacious vasodilator in isolated human pulmonary arteries and veins: Evidence for a biphasic effect at physiological and supra-physiological concentrations

Testosterone is recognized to elicit vasodilatation in numerous vascular beds, however to date no study has investigated whether testosterone has this effect in the human pulmonary vasculature. To determine whether isolated human pulmonary arteries and veins dilate in response to testosterone and whether the response differs in relation to gender, endothelial function or location with the pulmonary vasculature. Intralobar pulmonary arteries [no.=44, diameter =581 (349) microm] and veins [no.=27, diameter =573 (302) microm] were dissected from lobectomy samples obtained from male and female patients [no.=40, age =69 (8) yr]. Vessels were mounted in an automated wire myograph, bathed in physiological saline at 37 C and pH 7.4, and loaded to their in vivo pressure. Vessels were preconstricted with noradrenaline (10 microM) and exposed to acetylcholine (1 microM) to assess endothelial function, washed and then preconstricted with potassium chloride (1-100 mM) followed by either cumulative concentrations of testosterone (1 nM-100 microM) or ethanol vehicle (<0.1%). Significant marked vasodilatation was seen in all vessels, irrespective of size, gender and endothelial function at micromolar concentrations. Testosterone triggered significant vasodilatation at concentrations > or = 10 nM in pulmonary arteries obtained from males, a response which was not observed in vessels from females. The maximal response at 100 microM was also significantly greater in male pulmonary arteries. Significant vasodilatation was only observed at physiological (nM) concentrations in pulmonary resistance arteries and pulmonary arteries with good endothelial function. Testosterone acts as an efficacious vasodilator in the human pulmonary vasculature, with dilatation observed at physiological concentrations in the male arterial resistance bed, dependent on the presence of an intact endothelium.

Giant aneurysmal dilation of an intralobar pulmonary sequestration artery

Bronchopulmonar intralobar sequestrations receive their arterial blood supply through anomalous arteries from the systemic circulation. Usually the lumen of the aberrant artery can be oversized, but it is unusual to find a true arterial aneurysm. Here, we report a case of a 40-year-old woman with this unusual evolution. Because of the potential risk of rupture, she was treated with a lobectomy associated with a resection of the aneurysm.

Endobronchial Ultrasound-Facilitated Video-Assisted Lobectomy With Wedge Bronchoplasty for Typical Carcinoid Tumor of the Right Middle Lobe

A 19-year-old man presented with pneumonia, cough, and occasional dyspnea. Chest CT scan and bronchoscopy with biopsy revealed a typical carcinoid tumor obstructing the orifice of the right middle lobe, leading to lobar collapse. Preoperative surgical planning included radial endobronchial ultrasound, which confirmed that the tumor was not invasive into the bronchus intermedius. With that information, a video-assisted right middle lobectomy was performed with a wedge bronchoplasty in order to preserve the right lower lobe. The operation was performed completely thoracoscopically with three 1.2-cm ports and one 3.5-cm utility incision. With the intralobar pulmonary artery retracted, the bronchus was divided with a scalpel in wedge fashion to obtain a margin on the endobronchial tumor, and the defect was closed with absorbable suture. The patient recovered without complication and was doing well at 8-month follow-up, without evidence of recurrent disease.

Impairment of NO-Dependent Relaxation in Intralobar Pulmonary Arteries: Comparison of Urban Particulate Matter and Manufactured Nanoparticles

Background and ObjectivesBecause pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO–cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries.MethodsWe used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release.ResultsEndothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-α, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2.ConclusionIn contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress–independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM–induced cardiovascular dysfunction.

Cyclooxygenase (COX)-1 and COX-2 Participate in 5,6-Epoxyeicosatrienoic Acid-Induced Contraction of Rabbit Intralobar Pulmonary Arteries

Epoxyeicosatrienoic acids (EETs) have been reported to contract intralobar pulmonary arteries (PA) of the rabbit in a cyclooxygenase (COX)-dependent manner. In the present study, we observed that COX-1 and COX-2 isoforms were expressed in freshly isolated PA of healthy rabbits. We examined the hypothesis that both COX isoforms participate in 5,6-EET-induced contraction of rabbit intralobar PA. Selective inhibition of COX-1 with 300 nM 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560) prevented 5,6-EET (1x10(-8)-1x10(-5) M)-induced contractions of isolated intralobar rabbit PA rings in a manner similar to that observed with the nonselective cyclooxygenase inhibitor indomethacin at 10 microM. Selective inhibition of COX-2 with either 100 nM 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DUP-697) or 3 microM N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) shifted the EC50 value of 5,6-EET-induced PA contraction to the right but with considerably lower efficacy than SC-560. In rabbit PA, 5,6-EET-induced contraction was primarily dependent on COX-1 activity. Differential metabolism of 5,6-EET by COX-1 and COX-2 does not explain the primary dependence of PA contraction on COX-1 activity because 5,6-EET was metabolized similarly by both COX isoforms. COX-1 and -2 were expressed primarily in PA endothelium where COX-1 expression was dense and uniform, whereas COX-2 expression was sparse and nonuniform. 5,6-EET-induced PA contraction was endothelium-dependent. These results suggest that 5,6-EET-induced contraction is primarily dependent on COX-1 activity.

Integrin Ligands Mobilize Ca2+ from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary Arterial Smooth Muscle Cells

Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.

Acute Normoxia Increases Fetal Pulmonary Artery Endothelial Cell Cytosolic Ca2+ Via Ca2+-Induced Ca2+ Release

To test the hypothesis that an acute increase in O(2) tension increases cytosolic calcium ([Ca(2+)](i)) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (<10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (Po(2), 25 Torr). Using the calcium-sensitive dye fura-2, we demonstrated that acute normoxia (Po(2) = 120 Torr) increased PAECs [Ca(2+)](i) by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca(2+)](i) Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)-sensitive dye, DAF - FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O(2) tension had no effect on either [Ca(2+)](i) or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca(2+)](i) and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores.

Selective Inhibitors of Cyclooxygenase 1 &amp; 2 Decrease 5,6‐EET‐Induced Contraction of Rabbit Intralobar Pulmonary Arteries

Previously, we reported that 5,6-epoxyeicosatrienoic acid (EET) increased tension in small intralobar rabbit pulmonary arteries (IPAs). The non-selective cyclooxygenase (COX) inhibitor indomethacin decreased EET-induced IPA contraction. COX-1 and COX-2 isoforms have been reported to be expressed constitutively in rabbit lung. We hypothesized that both COX isoforms are expressed in rabbit pulmonary vasculature and contribute to the 5,6-EET-induced IPA responses. IPA (1-3 mm od) rings were suspended from force displacement transducers in physiological salt solution gassed with 95% O2, 5% CO2. The presence of COX-1 and COX-2 in IPAs was verified by Western immunoblotting. COX-1 and COX-2 metabolized [14C]-5,6-EET at the rates of 0.8 ± 0.2 and 0.5 ± 0.3 μmole · protein −1 · min−1, respectively (n=3). Administration of 5,6-EET resulted in IPA contractions of 0.35 ± 0.21 g at 0.3 μM and 1.17 ± 0.23 g at 3 μM. In the presence of a selective COX-1 inhibitor, SC-560 (300 nM), these contractions were completely abolished (n=6, P<0.05). In contrast, in the presence of a selective COX-2 inhibitor, NS-398 (3 μM) contractions to 0.3 μM 5,6-EET were also abolished, but contractions to 3 μM 5,6-EET were only reduced from 1.13 ± 0.4 to 0.23 ± 0.05 g (n=7, P<0.05), suggesting a greater dependence of 5,6-EET-induced contraction on COX-1 than COX-2 activity. We conclude that IPAs constitutively express both COX isoforms, COX-1 and COX-2 metabolize 5,6-EET at similar rates, and 5,6-EET-induced contraction of rabbit IPA is dependent on the activities of both COX-1 and COX-2. Supported by NIH grants: HL-52675 and HL-64180.