Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.     

Plasminogen activator inhibitor type 1 inhibits smooth muscle cell proliferation in pulmonary arterial hypertension

RATIONALE: Pulmonary arterial smooth muscle cells (PASMCs) in the medial layer of the vessel wall are responsible for vessel homeostasis, but also for pathologic vascular remodelling in diseases, such as idiopathic pulmonary arterial hypertension (IPAH). Vascular remodelling in IPAH results in vessel stiffness, occlusion, and increased vascular resistance, but its underlying mechanisms remain to be fully elucidated. In this study, we investigated the expression and function of plasminogen activator inhibitor (PAI)-1, an inhibitor of the plasminogen activator system and target gene of the transforming growth factor (TGF)-beta1 signalling cascade, in PASMC in IPAH. METHODS AND RESULTS: RNA and protein analysis from lung tissues of donors and patients with IPAH (n=7 each) revealed a significant downregulation of PAI-1 in IPAH lungs. Immunohistochemical analysis localised PAI-1 to the bronchial and alveolar epithelium, as well as to vascular and airway smooth muscle cells. PAI-1 was also downregulated in primary PASMC derived from IPAH lungs as compared with donor-derived PASMC. In order to elucidate PAI-1 function, primary PASMC were stimulated with active recombinant (r)PAI-1, or transfected with PAI-1-specific siRNA. Stimulation with rPAI-1 led to decreased PASMC proliferation and adhesion to vitronectin, and increased PASMC migration. In contrast, PAI-1 knock-down with siRNA increased PASMC proliferation and decreased PASMC migration. CONCLUSIONS: PAI-1 is significantly downregulated in PASMC in IPAH, on the mRNA and protein level. PAI-1 negatively regulates PASMC proliferation, while it increases PASMC migration. Thus, its loss in IPAH may therefore contribute to pathologic vascular remodelling in IPAH.     

LRP1 promotes synthetic phenotype of pulmonary artery smooth muscle cells in pulmonary hypertension

Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways.Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a β1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures.In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.     

Phosphodiesterase 10A upregulation contributes to pulmonary vascular remodeling

Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%-50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH.     

cAMP phosphodiesterase inhibitors potentiate effects of prostacyclin analogs in hypoxic pulmonary vascular remodeling

We investigated the effects of prostacyclin analogs and isoform-selective phosphodiesterase (PDE) inhibitors, alone and in combination, on pulmonary vascular remodeling in vitro and in vivo. Vascular smooth muscle cells (VSMC) isolated from pulmonary (proximal and distal) and systemic circulations demonstrated subtle variations in expression of PDE isoform mRNA. However, using biochemical assays, we found PDE3 and PDE4 isoforms to be responsible for the majority of cAMP hydrolysis in all VSMC. In growth assays, the prostacyclin analogs cicaprost and iloprost inhibited mitogen-induced proliferation of VSMC in a cAMP-dependent manner. In addition, isoform-selective antagonists of PDEs 1, 3, or 4 inhibited VSMC proliferation, an effect that synergized with the effect of prostacyclin analogs. The inhibitory effects were greater in cells isolated from pulmonary circulation. In an in situ perfused rat lung preparation, administration of prostacyclin analogs or the PDE inhibitors vinpocetine (PDE1), cilostamide (PDE3), or rolipram (PDE4), but not EHNA (PDE2), attenuated acute hypoxic vasoconstriction (HPV). Combinations of agents led to a greater reduction in HPV. Furthermore, during exposure to hypoxia for 13 days, Wistar rats were treated with iloprost, rolipram, cilostamide, or combinations of these agents. Compared with normoxic controls, hypoxic animals developed pulmonary hypertension and distal pulmonary artery muscularization. These parameters were attenuated by iloprost+cilostamide, iloprost+rolipram, and cilostamide+rolipram but were not significantly affected by single agents. Together, these findings provide a greater understanding of the role of cAMP PDEs in VSMC proliferation and provide rationale for combined use of prostacylcin analogs plus PDE3/4 inhibitors in treatment of pulmonary vascular remodeling.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

Upregulation of Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation     

High‐mobility group box‐1 induces vascular remodelling processes via c‐Jun activation

Extracellular high‐mobility group box‐1 (HMGB1) acts as a signalling molecule during inflammation, cell differentiation and angiogenesis. Increased abundance of HMGB1 is associated with several pathological disorders such as cancer, asthma and chronic obstructive pulmonary disease (COPD). In this study, we investigated the relevance of HMGB1 in the pathological remodelling present in patients with idiopathic pulmonary arterial hypertension (IPAH) and pulmonary hypertension (PH) associated with COPD. Remodelled vessels present in COPD with PH and IPAH lung samples were often surrounded by HMGB1‐positive cells. Increased HMGB1 serum levels were detected in both patient populations compared to control samples. The effects of physiological HMGB1 concentrations were then examined on cellular responses in vitro. HMGB1 enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC) and primary human arterial endothelial cells (PAEC). HMGB1 stimulated p38, extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) phosphorylation. Furthermore, activation of the downstream AP‐1 complex proteins c‐Fos and c‐Jun was observed. Silencing of c‐Jun ablated the HMGB1‐induced proliferation in PASMC. Thus, an inflammatory component such as HMGB1 can contribute to PASMC and PAEC proliferation and therefore potentially to vascular remodelling and PH pathogenesis.     

磷酸二酯酶的心血管功能调节作用

磷酸二酯酶(phosphodiesterase,PDE)是细胞内第二信使cAMP和cGMP降解的关键酶,作为药物研发的靶点受到广泛关注。近年研究发现,PDE在心肌细胞中能与β-肾上腺素受体及一些与兴奋收缩相关的蛋白形成复合物而使细胞内信号传递区室化分布,该现象可能为PDE抑制剂治疗慢性心力衰竭提供新的启示。血管平滑肌功能调节主要为血管张力和表型的调控,PDE5抑制剂舒张血管的作用已成功应用到勃起障碍的治疗。PDE4和PDE1C等在增殖的平滑肌细胞中表达量增高,单独抑制PDE的某一亚型将为治疗与平滑肌增殖有关的疾病(如肺动脉高压、血管成行术后再狭窄)提供新的途径。本文将重点阐述近年来PDE在心血管系统功能调节研究中的主要进展,以及PDE抑制剂在心血管系统疾病治疗中的应用。     

High levels of hyaluronan in idiopathic pulmonary arterial hypertension

Hyaluronan (HA), a large glycosaminoglycan found in the ECM, has major roles in lung and vascular biology and disease. However, its role in idiopathic pulmonary arterial hypertension (IPAH) is unknown. We hypothesized that HA metabolism is abnormal in IPAH. We measured the plasma levels of HA in IPAH and healthy individuals. We also evaluated HA synthesis and the expression of HA synthases and hyaluronidases in pulmonary artery smooth muscle cells (PASMCs) from explanted lungs. Plasma HA levels were markedly elevated in IPAH compared with controls [HA (ng/ml, mean +/- SD): IPAH 325 +/- 80, control 28 +/- 9; P = 0.02]. In vitro, unstimulated IPAH PASMCs produced high levels of HA compared with control cells [HA in supernatant (microg/ml, mean +/- SD): IPAH 12 +/- 2, controls 6 +/- 0.9; P = 0.04]. HA levels were also higher in IPAH PASMC lysates. The increased HA was biologically relevant as shown by tissue staining and increased HA-specific binding of mononuclear cells to IPAH compared with control PASMCs [number of bound cells x 10(4) (mean +/- SD): IPAH 9.5 +/- 3, control 3.0 +/- 1; P = 0.01]. This binding was abrogated by the addition of hyaluronidase. HA synthase-2 and hyaluronidase-2 were predominant in control and IPAH PASMCs. Interestingly, the expressions of HA synthase-2 and hyaluronidase-2 were approximately 2-fold lower in IPAH compared with controls [HA synthase-2 (relative expression mean +/- SE): IPAH 4.3 +/- 0.02, control 7.8 +/- 0.1; P = 0.0004; hyaluronidase-2 (relative expression mean +/- SE): IPAH 4.2 +/- 0.06, control 7.6 +/- 0.07; P = 0.008]. Thus patients with IPAH have higher circulating levels of HA, and PASMCs derived from IPAH lungs produce more HA compared with controls. This is associated with increased tissue levels and increased binding of inflammatory cells suggesting a role for HA in remodeling and inflammation in IPAH.     

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca²⁺ influx in response to endothelin‐1 (ET‐1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH‐derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho‐antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET‐1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET‐1 receptor antagonist therapy to PAH patients. J. Cell. Physiol. 228: 322–329, 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of cAMP degradation by phosphodiesterases in the accessory olfactory system

To characterize the potential role of cAMP in pheromone transduction, we have examined the occurrence of cyclic nucleotide phosphodiesterases (PDEs) in the mouse vomeronasal organ (VNO). We show that the cAMP-specific isoforms PDE4A and PDE4D are found preferentially in the apical and basal layers, respectively, of the VNO neuroepithelium and in the rostral (PDE4A) and caudal (PDE4D) portions of the accessory olfactory bulb glomerular layer. Assays for cAMP hydrolysis showed that PDE activity in VNO homogenates was about half that measured in the cerebral cortex and olfactory epithelium, and the proportion of total activity inhibited by rolipram, a PDE4-specific inhibitor, was approximately 40%. Activity in the VNO was enhanced 60% by Ca(2+) and calmodulin (CaM), implicating the presence of Ca(2+)/CaM-dependent PDE1. Zaprinast, which is known to inhibit PDE1C isoforms, completely suppressed Ca(2+)/CaM-stimulated activity and, together, zaprinast and rolipram inhibited cAMP hydrolysis by approximately 70%. Our results suggest that PDE1 and PDE4 isoforms are the primary source of cAMP degradation in the VNO.     

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.     

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E(2) (PGE(2)) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A(2)/COX-driven autocrine effect whereupon PGE(2) is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.     

Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α(1)- and β(1)-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H(2)O(2)). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively (P < 0.01 vs. controls). Hyperoxia or H(2)O(2) enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.