Pulmonary Arterial Hypertension

Scientific Updates – April 2005

Complied by

United Therapeutics Corporation

Braz J Med Biol Res. 2005 Feb;38(2):185-95. Epub 2005 Feb 15.

One-year follow-up of the effects of sildenafil on pulmonary arterial hypertension and veno-occlusive disease.

Barreto AC, Franchi SM, Castro CR, Lopes AA.

Departamento de Cardiologia Pediatrica e Cardiopatias Congenitas, Instituto do Coracao, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brasil.

We hypothesized that chronic oral administration of the phosphodiesterase-5 inhibitor sildenafil could improve the exercise capacity and pulmonary hemodynamics in patients with pulmonary arterial hypertension (PAH) on the basis of previous short-term studies. We tested this hypothesis in 14 subjects with PAH, including seven patients with the idiopathic form and seven patients with atrial septal defects, but no other congenital heart abnormalities. Patients were subjected to a 6-min walk test and dyspnea was graded according to the Borg scale. Pulmonary flow and pressures were measured by Doppler echocardiography. Patients were given sildenafil, 75 mg orally three times a day, and followed up for 1 year. Sildenafil therapy resulted in the following changes: increase in the 6-min walk distance from a median value of 387 m (range 0 to 484 m) to 462 m (range 408 to 588 m; P < 0.01), improvement of the Borg dyspnea score from 4.0 (median value) to 3.0 (P < 0.01), and increased pulmonary flow (velocity-time integral) from a median value of 0.12 (range 0.08 to 0.25) to 0.23 (range 0.11 to 0.40; P < 0.01) with no changes in pulmonary pressures. In one patient with pulmonary veno-occlusive disease diagnosed by a lung biopsy, sildenafil had a better effect on the pulmonary wedge pressure than inhaled nitric oxide (15 and 29 mmHg, respectively, acute test). He walked 112 m at baseline and 408 m at one year. One patient died at 11 months of treatment. No other relevant events occurred. Thus, chronic administration of sildenafil improves the physical capacity of PAH patients and may be beneficial in selected cases of veno-occlusive disease.

J Heart Lung Transplant. 2005 Apr;24(4):498-500.

Novel use of sildenafil in the treatment of portopulmonary hypertension.

Chua R, Keogh A, Miyashita M.

St. Vincent's Hospital, Sydney, New South Wales, Australia.

Portopulmonary hypertension is a poorly understood and uncommon complication of advanced chronic liver disease. Current therapy is based largely on treatment options proven in idiopathic pulmonary hypertension. The severity of the portopulmonary hypertension should best be attenuated medically before attempting combined liver and lung transplantation to avoid increased peri-operative mortality. This case report describes the successful use of sildenafil to decrease the pulmonary vascular resistance in a patient with hepatitis-C cirrhosis who was preparing for liver transplantation.

Circ J. 2005 Apr;69(4):461-5.

Oral sildenafil improves primary pulmonary hypertension refractory to epoprostenol.

Kataoka M, Satoh T, Manabe T, Anzai T, Yoshikawa T, Mitamura H, Ogawa S.

Cardiopulmonary Division, Department of Medicine, Keio University School of Medicine.

Background Epoprostenol (prostaglandin I(2)) has become recognized as a therapeutic breakthrough that can improve hemodynamics and survival in patients with primary pulmonary hypertension (PPH). However, a significant number of patients have PPH that is refractory to epoprostenol, and lung transplantation has been the only remaining treatment option. Methods and Results The study subjects included 20 consecutive patients with PPH (mean pulmonary arterial pressure: 65+/-15 mmHg) who had received epoprostenol for more than 12 months. The patients were divided into 2 groups; responders and non-responders. In the non-responders, New York Heart Association (NYHA) functional class did not improve and mean right atrial pressure (mRA) increased to 8 mmHg or more, and additional sildenafil, a phosphodiesterase-5 inhibitor, was started. Six patients were included in the non-responders, whose mRA was 9+/-5 mmHg before and significantly increased to 13+/-3 mmHg after epoprostenol administration (p<0.05). One patient died and the other 5 patients received oral sildenafil. The mRA of 12+/-4 mmHg (value before sildenafil) improved to 8+/-5 mmHg after sildenafil administration. Three patients were classified in the NYHA functional class 4 and improved to class 3, and 2 patients were in class 3 and remained in the same class after the addition of sildenafil. Conclusions In patients with severe PPH refractory to epoprostenol treatment, additional oral sildenafil can improve pulmonary hemodynamics and symptoms. The combination therapy of epoprostenol and sildenafil is a new medical treatment to attempt before progressing to lung transplantation for patients with PPH refractory to epoprostenol. (Circ J 2005; 69: 461 - 465).

High Alt Med Biol. 2005 Spring;6(1):43-9.

Effects of sildenafil on the human response to acute hypoxia and exercise.

Ricart A, Maristany J, Fort N, Leal C, Pages T, Viscor G.

Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain., Institut d'Estudis de Medicina de Muntanya, Barcelona, Spain.

Ricart, Antoni, Jaume Maristany, Nuria Fort, Conxita Leal, Teresa Pages, and Gines Viscor. Effects of sildenafil on the human response to acute hypoxia and exercise. High Alt. Med. Biol. 6:43-49, 2005.-We examined the effects of the 5-phosphodiesterase (5-PDE) inhibitor sildenafil on pulmonary arterial pressure and some oxygen transport and cardiopulmonary parameters in humans during exposure to hypobaric hypoxia at rest and after exercise. In a double-blind study, 100 mg sildenafil or placebo was administered orally to 14 healthy volunteers 45 min before exposure to 5,000 m of simulated altitude. Arterial oxygen saturation (Sa(O(2) )), heart rate (HR), tidal volume (VT), respiratory rate (RR), left ventricular ejection fraction (EF), and pulmonary arterial pressure (PAP) were measured first at rest in normoxia, at rest and immediately after exercise during hypoxia, and after exercise in normoxia. The increase in systolic PAP produced by hypoxia was significantly decreased by sildenafil at rest from 40.9 +/- 2.6 to 34.9 +/- 3.0 mmHg (-14.8%; p = 0.0046); after exercise, from 49.0 +/- 3.9 to 42.9 +/- 2.6 mmHg (-12.6%; p = 0.003). No significant changes were found in normoxia either at rest or after exercise. Measurements of the effect of sildenafil on exercise capacity during hypoxia did not provide conclusive data: a slight increase in Sa(O(2) ) was observed with exercise during hypoxia, and sildenafil did not cause significant changes in ventilatory parameters under any condition. Sildenafil diminishes the pulmonary hypertension induced by acute exposure to hypobaric hypoxia at rest and after exercise. Further studies are needed to determine the benefit from this treatment and to further understand the effects of sildenafil on exercise capacity at altitude.

Br J Anaesth. 2005 Mar 11; [Epub ahead of print]

Treatment of pulmonary hypertension in the general adult intensive care unit: a role for oral sildenafil?

Ng J, Finney SJ, Shulman R, Bellingan GJ, Singer M, Glynne PA.

Department of Critical Care, University College London Hospitals, Mortimer Street, London W1T 3AA, UK.

Use of inhaled nitric oxide for treatment of pulmonary hypertension in adult critical illness is limited by its mode of delivery and high costs, prompting evaluation of alternative therapies. We report the use of oral sildenafil in a patient with severe secondary pulmonary hypertension and right ventricular dysfunction. Following reduction in mean pulmonary artery pressure and pulmonary vascular resistance with inhaled nitric oxide, crossover to sildenafil therapy maintained control of pulmonary hypertension, facilitating discontinuation of respiratory and cardiovascular organ support. The relative pulmonary vascular specificity of oral sildenafil, and its low cost, makes it an attractive therapeutic alternative to inhaled nitric oxide, and warrants further study.

Int J Cardiol. 2005 Mar 10;99(1):91-5.

Related Articles, Links

Efficacy and optimal dose of sildenafil in primary pulmonary hypertension.

Chockalingam A, Gnanavelu G, Venkatesan S, Elangovan S, Jagannathan V, Subramaniam T, Alagesan R, Dorairajan S.

Department of Cardiology, Madras Medical College and Research Institute, Chennai 600 003, India. drcanands@yahoo.co.in

PURPOSE: We aimed to assess the effects of sildenafil and evaluate optimal dosing in primary pulmonary hypertension (PPH). Sildenafil selectively inhibits phosphodiesterase 5 (PDE5), which is abundant in pulmonary and penile tissue. This results in increasing nitric oxide (NO) at tissue level leading to pulmonary vasodilatation. SUBJECTS AND METHODS: Our study was a prospective study of sildenafil in 15 consecutive patients with severe symptomatic PPH of NYHA class III-IV. All patients were stabilized for a minimum period of 5 days with antifailure medications. Sildenafil was started at 50 mg twice daily for 4 weeks and increased to 100 mg bid for 4 more weeks in a step-up protocol. Primary end-points were change in Borg dyspnea index, NYHA class and 6-min walk distance, estimated at baseline 1, 2, 4 and 8 weeks. RESULTS: NYHA class (baseline 3.8 +/- 0.4 vs. 4 weeks 2.4 +/- 0.5, p = 0.002), Borg dyspnea index (8.1 +/- 1.7 vs. 4.4 +/- 1.9, p = 0.0007), 6-min walk distance (234 +/- 44 vs. 377 +/- 128 m, p = 0.001) and Pulmonary artery pressure (125 +/- 15 vs. 113 +/- 18 mm Hg p = 0.05) are significantly improved with sildenafil 50 mg bid at 4 weeks. Increasing the dose to 100 mg bid did not produce further benefit. Echocardiography parameters of right heart dimensions and functions did not change markedly in the study period. CONCLUSION: Sildenafil is well tolerated with no adverse effects in severe pulmonary hypertension. It reduces symptoms, improves effort tolerance and controls refractory heart failure significantly by 2 weeks in 70% of patients at 50 mg twice daily. Three patients (20%) failed to respond with sildenafil.

Am J Respir Crit Care Med. 2005 Mar 4; [Epub ahead of print]

Sildenafil versus Endothelin Receptor Antagonist for Pulmonary Hypertension (SERAPH) Study.

Wilkins MR, Paul GA, Strange JW, Tunariu N, Gin-Sing W, Banya W, Westwood MA, Stefanidis A, Ng LL, Pennell DJ, Mohiaddin RH, Nihoyannopoulos P, Gibbs JS.

Experimental Medicine and Toxicology, Imperial College London, Hammersmith Hospital, London, United Kingdom.

Rationale: Phosphodiesterase type 5 (PDE5) inhibition has been proposed for the treatment for pulmonary arterial hypertension (PAH). Objective: This study compared adding sildenafil, a PDE5 inhibitor, to conventional treatment with the current practice of adding bosentan, an endothelin receptor antagonist. Methods: Twenty-six patients with PAH, idiopathic or associated with connective tissue disease, WHO functional class III, were randomised, double-blind, to receive sildenafil (50mg twice daily for 4 weeks, then 50mg three times daily) or bosentan (62.5mg twice daily for 4 weeks, then 125mg twice daily) over 16 weeks. Measurements: Changes in right ventricle (RV) mass (using cardiovascular magnetic resonance), 6 minute walk distance, cardiac function, brain natriuretic peptide (BNP) and Borg dyspnoea index. Main results: When analysed by intention-to-treat, there were no significant differences between the two treatment groups. One patient on sildenafil died suddenly. Patients on sildenafil who completed the protocol showed significant changes from baseline - reductions in RV mass (-8.8g [95%CI -16, -2] n=13, p=0.015) and plasma BNP levels (-19.4fmol.ml(-1) [95%CI -5, -33] p=0.014) and improvements in 6 minute walk distance (114m [95%CI 67, 160] p=0.0002), cardiac index (0.3 [95%CI 0.1, 0.4] p=0.008) and systolic left ventricular eccentricity index (-0.2 [95%CI -0.02, -0.37) p=0.031). Bosentan improved 6 minute walk distance (59m [95%CI 29, 89] n=12, p=0.001) and cardiac index (0.3 [95%CI 0.1, 0.4] p=0.008). Conclusions: Sildenafil added to conventional treatment reduces RV mass and improves cardiac function and exercise capacity in patients with PAH, WHO functional class III. Safety monitoring is important until more experience is obtained.

J Heart Lung Transplant. 2005 Apr;24(4):501-3.

Reversal of pulmonary hypertension and subsequent repair of atrial septal defect after treatment with continuous intravenous epoprostenol.

Frost AE, Quinones MA, Zoghbi WA, Noon GP.

Baylor College of Medicine, The Methodist Hospital, Houston, Texas, USA.

We report the first case in the world literature of a patient with an atrial septal defect, severe pulmonary hypertension, and equalization of pulmonary and systemic pressures, who underwent successful closure of an ASD following prolonged therapy with the intravenous vasodilator epoprostenol. Judicious use of continuous prostacyclin in apparently inoperable patients with congenital heart disease may be associated with significant reversal of pulmonary hypertension, and conversion to an operable state.

Pediatr Res. 2005 Mar 17; [Epub ahead of print]

Endothelin-A Receptor Blockade Prevents and Partially Reverses Neonatal Hypoxic Pulmonary Vascular Remodeling.

Ambalavanan N, Bulger A, Murphy-Ullrich J, Oparil S, Chen YF.

Department of Pediatrics, Department of Pathology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, 35233.

Hypoxia-induced pulmonary vascular remodeling (HPVR) may lead to persistent pulmonary hypertension of the newborn or cor pulmonale. Endothelin-1 (ET-1), via endothelin-A (ETA) receptor activation, mediates hypoxic pulmonary vasoconstriction. Our objectives were to develop a newborn mouse model of HPVR and to test the hypothesis that ETA blockade would prevent and reverse HPVR in this model. C57BL/6 mice (n = 64) were exposed to 12% oxygen (HYP group) or room air (RA group) from birth to 2 wk of age. The mice were injected intraperitoneally daily with either BQ-610 (ETA blocker) or vehicle (cottonseed oil) from birth (prevention study) or from 6 d of age (reversal study). HPVR was assessed histologically by pulmonary vascular morphometry by an examiner masked to study group, and by measurement of the right ventricle to left ventricle (RV/LV) thickness ratio. Hypoxia increased medial wall thickness (%WT) in pulmonary arteries <100 microm in diameter and RV/LV thickness ratio. BQ-610 prevented the hypoxia-induced increase in %WT and RV/LV thickness ratio when given from birth, and later therapy partially reversed the hypoxia-induced increase in %WT but not RV/LV thickness ratio. These data show that in the newborn mouse model, chronic hypoxia leads to HPVR that can be completely prevented and partially reversed by ETA blockade. These results indicate that ET-1, acting via ETA receptors, is a mechanism of pathophysiologic significance underlying neonatal HPVR. Development of this newborn mouse model of HPVR facilitates investigation of mechanisms underlying this important and severe disease entity in human infants.

Am J Health Syst Pharm. 2005 Mar 15;62(6):606-9.

Nesiritide for secondary pulmonary hypertension in patients with end-stage heart failure.

O'dell KM, Kalus JS, Kucukarslan S, Czerska B.

Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA.

PURPOSE: The impact of adding nesiritide to standard therapy and positive inotropic agents in patients with end-stage heart failure and secondary pulmonary hypertension (PH) was studied. METHODS: Patients included in this retrospective study were 18 years of age or older, admitted to the hospital with PH secondary to end-stage heart failure (New York Heart Association functional class IV), had received a pulmonary artery catheter, had been treated with nesiritide because of inadequate hemodynamic response to previous therapy (pulmonary capillary wedge pressure [PCWP], >18 mm Hg), and had shown minimal symptomatic benefit from standard heart-failure therapy, continuous infusions of loop diuretics, and positive inotropic agents (milrinone or dobutamine or both). The primary endpoint was change in PCWP. Secondary endpoints included change in mean pulmonary artery pressure (MPAP), change in cardiac index (CI), change in mean arterial pressure (MAP), change in serum creatinine (SCr) concentration, and occurrence of symptomatic hypotension. RESULTS: The study included 33 patients. Mean PCWP was reduced by 31.1% with the addition of nesiritide to previous therapy (p < 0.0001). Significant improvements in other hemodynamic variables, including MPAP (15.6% reduction) and CI (13.0% increase), were also observed. MAP was reduced significantly (by 15.2%), but SCr concentration did not change. There were five episodes of symptomatic hypotension. All patients exhibited relief of dyspnea symptoms. CONCLUSION: The addition of nesiritide to standard therapy and positive inotropic agents improved hemodynamic measures and clinical symptoms in patients with end-stage heart failure and secondary pulmonary hypertension.

Pediatr Pulmonol. 2005 Mar 23; [Epub ahead of print]

Pathophysiologic mechanisms of persistent pulmonary hypertension of the newborn.

Dakshinamurti S.

Departments of Pediatrics and Physiology, University of Manitoba, Manitoba Institute of Child Health, Winnipeg, Canada.

Persistent pulmonary hypertension of the newborn (PPHN), among the most rapidly progressive and potentially fatal of vasculopathies, is a disorder of vascular transition from fetal to neonatal circulation, manifesting as hypoxemic respiratory failure. PPHN represents a common pathway of vascular injury activated by numerous perinatal stresses: hypoxia, hypoglycemia, cold stress, sepsis, and direct lung injury. As with other multifactorial diseases, a single inciting event may be augmented by multiple concurrent/subsequent phenomena that result in differing courses of disease progression. I review the various mechanisms of vascular injury involved in neonatal pulmonary hypertension: endothelial dysfunction, inflammation, hypoxia, and mechanical strain, in the context of downstream effects on pulmonary vascular endothelial-myocyte interactions and myocyte phenotypic plasticity. (c) 2005 Wiley-Liss, Inc.

Genet Med. 2005 Mar;7(3):169-74.

Gross BMPR2 gene rearrangements constitute a new cause for primary pulmonary hypertension.

Cogan JD, Vnencak-Jones CL, Phillips JA 3rd, Lane KB, Wheeler LA, Robbins IM, Garrison G, Hedges LK, Loyd JE.

From the 1Department of Pediatrics, 2Department of Pathology, and 3Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.

PURPOSE:: Approximately 50% of patients with familial primary pulmonary hypertension (FPPH) have been reported to have mutations within the bone morphogenic protein receptor type 2 (BMPR2) gene. The vast majority of these mutations were identified by PCR amplification and sequencing of individual exons. The aim of our study was to determine if additional BMPR2 mutations not found by exon sequencing alone could account for a significant portion of these negative cases. METHODS:: We examined DNA samples from 12 families, previously found to be negative for BMPR2 mutations, to identify any large BMPR2 gene rearrangements. RESULTS:: Southern blot analysis found large gene rearrangements in four (33%) unrelated kindreds. Further analysis by reverse transcriptase PCR (RT-PCR) of BMPR2 transcripts from two of these kindreds found one to be heterozygous for a exon 10 duplication and the second to be heterozygous for a deletion of exons 4 to 5. Nonhomologous recombination is believed to be the cause of these large insertions/deletions. CONCLUSION:: Our results demonstrate the inherent problems associated with exon-by-exon sequencing and the importance of other screening methods such as Southern blot and RT-PCR in the identification of BMPR2 mutations.

J Appl Physiol. 2005 Mar 31; [Epub ahead of print]

Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.

Fike CD, Zhang Y, Kaplowitz MR.

The pulmonary vasoconstrictor, thromboxane, may contribute to the development of pulmonary hypertension. Our objective was to determine if a combined thromboxane synthase inhibitor/receptor antagonist, terbogrel, prevents pulmonary hypertension and the development of aberrant pulmonary arterial responses in newborn piglets exposed to 3 days hypoxia. Piglets were maintained in room air (control) or 11% O2 (hypoxic) for 3 days. Some hypoxic piglets received terbogrel, 10 mg/kg po bid. Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. A cannulated artery technique was used to measure responses to acetylcholine. Pulmonary vascular resistance for terbogrel-treated hypoxic piglets was almost one-half the value of untreated hypoxic piglets but remained greater than values for control piglets. Dilation to acetylcholine in preconstricted pulmonary arteries was greater for terbogrel-treated hypoxic than for untreated hypoxic piglets, but was less for pulmonary arteries from both groups of hypoxic piglets than for control piglets. Terbogrel may ameliorate pulmonary artery dysfunction and attenuate the development of chronic hypoxia-induced pulmonary hypertension in newborns.

Crit Care. 2005 Apr;9(2):R132-8. Epub 2005 Feb 11.

Related Articles, Links

Pulmonary capillary pressure in pulmonary hypertension.

Souza R, Amato MB, Demarzo SE, Deheinzelin D, Barbas CS, Schettino GP, Carvalho CR.

Pulmonary Division, Respiratory ICU - Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil. rgrsz@uol.com.br

INTRODUCTION: Pulmonary capillary pressure (PCP), together with the time constants of the various vascular compartments, define the dynamics of the pulmonary vascular system. Our objective in the present study was to estimate PCPs and time constants of the vascular system in patients with idiopathic pulmonary arterial hypertension (IPAH), and compare them with these measures in patients with acute respiratory distress syndrome (ARDS). METHODS: We conducted the study in two groups of patients with pulmonary hypertension: 12 patients with IPAH and 11 with ARDS. Four methods were used to estimate the PCP based on monoexponential and biexponential fitting of pulmonary artery pressure decay curves. RESULTS: PCPs in the IPAH group were considerably greater than those in the ARDS group. The PCPs measured using the four methods also differed significantly, suggesting that each method measures the pressure at a different site in the pulmonary circulation. The time constant for the slow component of the biexponential fit in the IPAH group was significantly longer than that in the ARDS group. CONCLUSION: The PCP in IPAH patients is greater than normal but methodological limitations related to the occlusion technique may limit interpretation of these data in isolation. Different disease processes may result in different times for arterial emptying, with resulting implications for the methods available for estimating PCP.

Thromb Haemost. 2005 Mar;93(3):512-6.

Medical conditions increasing the risk of chronic thromboembolic pulmonary hypertension.

Bonderman D, Jakowitsch J, Adlbrecht C, Schemper M, Kyrle PA, Schonauer V, Exner M, Klepetko W, Kneussl MP, Maurer G, Lang I.

Medical University of Vienna, Department of Cardiology, Vienna, Austria.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by organized thromboemboli that obstruct the pulmonary vascular bed. Although CTEPH is a serious complication of acute symptomatic pulmonary embolism in 4% of cases, signs, symptoms and classical risk factors for venous thromboembolism are lacking. The aim of the present study was to identify medical conditions conferring an increased risk of CTEPH. We performed a case-control-study comparing 109 consecutive CTEPH patients to 187 patients with acute pulmonary embolism that was confirmed by a high probability lung scan. Splenectomy (odds ratio=13, 95% CI 2.7-127), ventriculo-atrial (VA-) shunt for the treatment of hydrocephalus (odds ratio=13, 95% CI 2.5-129) and chronic inflammatory disorders, such as osteomyelitis and inflammatory bowel disease (IBD, odds ratio=67, 95% CI 7.9-8832) were associated with an increased risk of CTEPH.

Pulmonary Hypertension/Vascular Remodeling

March 2005

Citation: Lin MJ, Leung GPH, Zhang WM, Yang XR, Yip KP, Tse CM, Sham JSK. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca²+ channels in pulmonary arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension. Circ Res. 2004;95(5):496-505.

Link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15256480

Background: The article in focus examines a novel mechanism by which chronic hypoxia (CH) elevates basal pulmonary arterial smooth muscle cell (PASMC) intracellular free Ca²+ concentration ([Ca²+]i) and arterial tone in rats. Specifically, the authors provide evidence suggesting a role for enhanced Ca²+ entry through store-operated cation channels (SOCs) in these responses to CH, and suggest that the resultant increase in pulmonary arterial tone contributes to the vasoconstrictor component of CH-induced pulmonary hypertension.

Chronic hypoxia has traditionally been considered to mediate pulmonary hypertension through at least three primary responses, namely hypoxic pulmonary vasoconstriction, pulmonary arterial remodeling, and polycythemia. However, findings that basal tone is elevated in endothelium-denuded pulmonary arterial rings isolated from CH rats studied under normoxic conditions (1) suggests the vasoconstrictor response to CH is multifaceted. Consistent with this increase in arterial tone, resting [Ca²+]i is greater in cultured PASMCs derived from CH rats compared to controls (1). Some investigators have suggested that these responses to CH result from enhanced Ca²+ influx through L-type voltage-gated Ca²+ channels secondary to downregulation of delayed rectifier K+ channels, decreased outward K+ current, and resultant membrane depolarization. However, at odds with this hypothesis is evidence that the selective L-channel inhibitor, nifedipine, does not alter resting PASMC [Ca²+]i or basal tone in pulmonary arterial rings from CH rats (1).

Based on these previous findings, the authors of the present study sought to examine the potential contribution of both SOCs and receptor-operated cation channels (ROCs) to elevated PASMC [Ca²+]i following CH. The best candidates for SOCs and ROCs are members of the canonical transient receptor potential (TRPC) family of cation channels. The rationale for this study is further supported by evidence of store-operated Ca²+ entry (SOCE) in rat PASMCs (2), and by the identification of several TRPC isoforms (2) that have been implicated in mediating both SOCE and receptor-operated Ca²+ entry (ROCE) (3).

Hypothesis: The authors address the hypothesis that elevated basal PASMC [Ca²+]i and arterial tone following CH is mediated by increased Ca²+ influx through SOCs or ROCs.

Methods:

1. To determine effects of CH on TRPC channel expression in pulmonary arterial smooth muscle, the expression profile of TRPC isoforms was assessed in transiently cultured (16-24 hr) PASMCs and in endothelium-denuded intrapulmonary arteries (300-800 m diameter) isolated from both control and CH rats (3-4 wk @ 10% O₂) using RT-PCR, Western blotting, and immunohistochemical techniques.
2. To examine Influences of CH on SOCE, cultured PASMCs cells from each group of rats were treated with the sarcoplasmic reticulum Ca²+-ATPase inhibitor, thapsigargin, to deplete intracellular Ca²+ stores and thereby activate SOCs, and the [Ca²+]i response to restoration of extracellular Ca²+ measured using the Ca²+-sensitive fluorophore, fluo-3 AM. A second approach was to measure the rate of Mn²+-quenching of fura-2 fluorescence as an index of Mn²+ influx following activation of SOCs in PASMCs from control and CH rats using protocols similar to those described above.
3. Effects of CH on ROCE were assessed in similar experiments using the ROC agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol (DAG).
4. To demonstrate a causal relationship between altered expression of PASMC TRPC isoforms and changes in SOCE and ROCE following CH, siRNA techniques were employed to selectively knockdown TRPC1 and TRPC6 expression, and functional consequences of decreased TRPC expression on both SOCE and ROCE determined as before.
5. Finally, to assess whether enhanced Ca²+ entry through SOCs or ROCs contributes to elevated basal [Ca²+]i and increased pulmonary arterial tone following CH, the authors examined effects of ROC/SOC channel inhibitors (La³+, SKF-96365) on both resting PASMC [Ca²+]i and basal tone in pulmonary arterial rings from each group of rats.

Results: The major findings of this study are as follows:

1. TRPC1 contributes to SOCE, whereas TRPC6 appears to be a ROC in transiently cultured rat PASMCs.
2. CH upregulates TRPC1 and TRPC6 expression and thereby enhances SOCE and ROCE in PASMCs.
3. SOCs appear to contribute to elevated basal PASMC [Ca²+]i following CH.
4. Increased resting tone in pulmonary arteries from CH rats may be a function of greater Ca²+ influx through SOCs.

Discussion: The authors speculate that CH-induced increases in SOCE and basal [Ca²+]i in PASMCs may represent a novel mechanism of vasoconstriction, that along with hypoxic vasoconstriction, contributes to the development of pulmonary hypertension. It is further possible that greater Ca²+ entry through both SOCs and ROCs may function to enhance pulmonary vasoconstrictor reactivity to receptor-mediated agonists following CH as demonstrated by other investigators (4). However, a recent study by Nagaoka and colleagues (5) suggests that an additional mechanism of vasoconstriction involving enhanced Rho kinase activity may represent a major component of the pulmonary hypertensive response to CH in rats. These authors found that iv administration of the Rho kinase inhibitor, Y-27632, dose-dependently reduced both mean pulmonary arterial pressure and total pulmonary resistance in conscious, CH rats that had been acutely returned to normoxia. Interestingly, the highest dose of Y-27632 normalized total pulmonary resistance between control and CH rats, suggesting that vasoconstrictor mechanisms involving Rho kinase are of greater importance in mediating CH-induced pulmonary hypertension in rats than fixed components of hypertension, i.e. arterial remodeling and polycythemia. These findings are further consistent with recent observations by our laboratory that CH augments RhoA/Rho kinase-induced pulmonary vascular smooth muscle Ca²+-sensitization, but not PKC-dependent Ca²+-sensitization, a response associated with enhanced pulmonary arterial RhoA and Rho kinase activity, and increased Rho kinase expression (6). These previous findings, together with those of the article in focus, suggest that CH-induced pulmonary vasoconstriction may be multifactorial, with hypoxic vasoconstriction, increased basal tone resulting from greater SOCE, and enhanced RhoA/Rho kinase-dependent PASMC Ca²+-sensitization functioning independently to mediate pulmonary hypertension.

Potential changes in PASMC phenotype and intracellular Ca²+-handling resulting from cell isolation and culture represent a limitation of this study, which could be addressed in similar studies using acutely isolated pulmonary arteries. It further remains to be determined whether Ca²+ influx pathways in PASMCs from the relatively large diameter arteries used in the present study are reflective of those from more distal arteries that likely provide a greater contribution to pulmonary vascular resistance in the setting of CH.

References:

1. Shimoda et al. L-type Ca2+ channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes. Am J Physiol. 279:L884-L894, 2000.
2. Wang et al. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol. 286:L848-L858, 2004.
3. Hofmann et al. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397:259-263, 1999.
4. McMurtry et al. Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. Am J Physiol. 235:H104-H109, 1978.
5. Nagaoka et al. Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol. 287:L665-L672, 2004.
6. Jernigan et al. Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling. Am J Physiol. 287:L1220-L1220, 2004.