Published online before print April 16, 2007, doi: 10.1161/CIRCULATIONAHA.106.676809
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2007;115:2331-2339.)
© 2007 American Heart Association, Inc.
Vascular Medicine |
Phosphodiesterase 1 Upregulation in Pulmonary Arterial Hypertension
Target for Reverse-Remodeling Therapy
From the University of Giessen Lung Centre (R.T.S., S.S.P., R.D., X.T., N.W., H.A.G., C.K., R.V., J.Z., A.S., W.S., F.G.), Giessen, Germany; Department of Pathology (G.K.) and Department of Anatomy and Cell Biology (R.P., W.K.), Justus Liebig University Giessen, Giessen, Germany; Department of Biochemistry 2 Inflammation (T.D., C.S.), Altana Pharma, Konstanz, Germany; and Department of Cardiothoracic Surgery (W.K.), University of Vienna, Vienna, Austria.
Correspondence to Ralph Schermuly, University of Giessen Lung Centre, Justus-Liebig-Universität Giessen, Klinikstrasse 36, 35392 Giessen, Germany. E-mail ralph.schermuly{at}uglc.de
Received November 16, 2006; accepted February 20, 2007.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Pulmonary arterial hypertension (PAH) is a life-threatening disease, characterized by vascular smooth muscle cell hyperproliferation. The calcium/calmodulin-dependent phosphodiesterase 1 (PDE1) may play a major role in vascular smooth muscle cell proliferation.
Methods and Results— We investigated the expression of PDE1 in explanted lungs from idiopathic PAH patients and animal models of PAH and undertook therapeutic intervention studies in the animal models. Strong upregulation of PDE1C in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was noted on the mRNA level by laser-assisted vessel microdissection and on the protein level by immunohistochemistry. In chronically hypoxic mouse lungs and lungs from monocrotaline-injected rats, PDE1A upregulation was detected in the structurally remodeled arterial muscular layer. Long-term infusion of the PDE1 inhibitor 8-methoxymethyl 3-isobutyl-1-methylxanthine in hypoxic mice and monocrotaline-injected rats with fully established pulmonary hypertension reversed the pulmonary artery pressure elevation, structural remodeling of the lung vasculature (nonmuscularized versus partially muscularized versus fully muscularized small pulmonary arteries), and right heart hypertrophy.
Conclusions— Strong upregulation of the PDE1 family in pulmonary artery smooth muscle cells is noted in human idiopathic PAH lungs and lungs from animal models of PAH. Inhibition of PDE1 reverses structural lung vascular remodeling and right heart hypertrophy in 2 animal models. The PDE1 family may thus offer a new target for therapeutic intervention in pulmonary hypertension.
Key Words: cardiovascular diseases • hypertension, pulmonary • muscle, smooth • phosphodiesterases • pharmacology
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Pulmonary arterial hypertension (PAH) is a severe disease with still largely unresolved pathogenesis. It is characterized by increased pulmonary vascular resistance and thus right ventricular (RV) afterload, which in the further course of the disease leads to RV failure and death. Both vasoconstriction and structural remodeling of the pulmonary vessels contribute to the progressive course of PAH, irrespective of different underlying causes.1,2 New treatment concepts in pulmonary hypertension include local and systemic administration of prostacyclin and its analogues, inhalation of nitric oxide (NO), and endothelin receptor antagonists.3,4 Recently, phosphodiesterase (PDE) 5 inhibitors have been demonstrated to be potent, selective pulmonary vasodilators.5–9
Clinical Perspective p 2339
PDEs hydrolyze the cyclic nucleotide second messengers cAMP and cGMP, which are known to play an important role in regulating vascular tone and smooth muscle cell (SMC) proliferation.10 Members of the PDE1 gene family are activated by calcium/calmodulin and are therefore termed "calcium/calmodulin–dependent PDEs." Three different PDE1 isoforms, namely, PDE1A, PDE1B, and PDE1C, have been reported thus far.11,12 Each of the calcium/calmodulin PDEs hydrolyzes both cAMP and cGMP but with different efficacy. PDE1A and PDE1B have higher affinity for cGMP than cAMP, whereas PDE1C hydrolyzes cAMP and cGMP with similar efficiency. Five different PDE1C splice variants, PDE1C1 to PDE1C5, have been reported in human and mouse species.13,14 Previous studies reported a high expression of PDE1C in proliferating human arterial SMCs,15 thereby linking PDE activity not only to regulation of vascular tone but also to control of proliferation. Furthermore, the inhibition of PDE1C in SMCs isolated from normal aorta or from atherosclerotic lesions with antisense oligonucleotides or the PDE1 inhibitor 8-methoxymethyl-isobutyl-1-methylxanthine (8MM-IBMX) resulted in suppression of SMC proliferation.16 Recently, it has been demonstrated that the addition of vinpocetine, another PDE1 inhibitor, enhanced pulmonary vasodilation and cGMP release induced by NO breathing without causing systemic vasodilation.17
The present study is the first to address a putative role of the PDE1 family in chronic PAH. We investigated the expression and function of PDE1 in 2 established models of experimental pulmonary hypertension and studied the expression of the PDE1 family in human lung explant material.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Patient Characteristics and Measurements
Human lung tissue was obtained from 5 donors and 5 patients with idiopathic PAH (IPAH) undergoing lung transplantation. Patient characteristics are given in the online Data Supplement. Lung tissue was snap-frozen directly after explantation for mRNA and protein extraction. The study protocol for tissue donation was approved by the ethics committee ("Ethik Kommission am Fachbereich Humanmedizin der Justus Liebig Universität Giessen") of the University Hospital Giessen (Giessen, Germany) in accordance with national law and with "Good Clinical Practice/International Conference on Harmonisation" guidelines. Written informed consent was obtained from each individual patient or the patient’s next of kin.
Cell Culture and PDE Activity
A description of cell culture techniques and measurements of PDE isoenzyme activities are provided in the online Data Supplement.
In Vivo Experiments
All experiments were performed according to institutional guidelines that complied with national and international regulations. Mice were exposed to chronic hypoxia (10% O2) in a ventilated chamber, as described previously.18 Rats were injected with 60 mg/kg monocrotaline (MCT) subcutaneously.18–20 A detailed description of the animal models is given in the online Data Supplement.
Hemodynamic Measurements
Animals were anesthetized with ketamine (6 mg/100 g IP) and xylazine (1 mg/100 g IP). The trachea was cannulated, and the lungs were ventilated with room air. Systemic arterial pressure was determined by catheterization of the carotid artery. For measurement of RV systolic pressure, a catheter was inserted into the RV via the right vena jugularis, as described previously.18
Isolated Mouse Lung Experiments
The isolated perfused lung model has been described in detail previously.21 (See the online Data Supplement.)
Pharmacological Treatments
To investigate the effects of the PDE1 inhibitor 8MM-IBMX on acute hypoxic vasoconstriction, 4 groups of mice (6 in each group) were studied in isolated lung experiments. Two groups were normoxic animals in which the effect of increasing doses of 8MM-IBMX or placebo on acute hypoxic pulmonary vasoconstriction was investigated. In these experiments, repetitive hypoxic challenges were performed, and 8MM-IBMX or placebo was applied in the normoxic periods. The other 2 groups consisted of chronically hypoxic mice (21 days at 10% O2) in which identical experiments with 8MM-IBMX or placebos were performed.
The long-termAQ effects of PDE1 inhibition were assessed in mice exposed to hypoxia for 35 days and rats injected with MCT for 35 days. After 21 days of either hypoxia or MCT injection, animals were randomized to receive either 8MM-IBMX or placebo via continuous infusion by implantation of osmotic minipumps. As described previously, animals were anesthetized with ketamine/xylazine, and a catheter was inserted into the jugular vein.19,22 The animals received either 20 µg of 8MM-IBMX per kilogram per minute or placebo for 14 days.
Assessment of Right Heart Hypertrophy and Vascular Remodeling
The RV was dissected from the left ventricle and septum (LV+S), and these dissected samples were weighed to obtain the RV to LV+S ratio [RV/(LV+S)].18–20 A detailed description of the methods is given in the online Data Supplement.
Laser-Assisted Microdissection
Microdissection was performed as described in detail previously.23–25 In brief, cryosections (10 µm) from lung tissue were mounted on glass slides. After hemalum staining for 30 seconds, the sections were subsequently immersed in 70% and 96% ethanol and stored in 100% ethanol until use. No more than 10 sections were prepared at once to reduce the storage time. Intrapulmonary arteries at a size between 50 and 200 µm were selected and microdissected under optical control with the Laser Microbeam System (P.A.L.M., Bernried, Germany; Data Supplement Figure I).
RNA Isolation, cDNA Synthesis, and Relative mRNA Quantification by Real-Time Polymerase Chain Reaction
RNA from laser-microdissected material, lung homogenate, and freshly isolated SMCs was isolated by RNeasy Micro and RNeasy Mini kits, respectively (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For cDNA synthesis, reagents and incubation steps were applied as described previously.24 (See the online Data Supplement.)
Statistical Analyses
Data are mean±SEM. For comparison of the pharmacological effects of 8MM-IBMX, 1-way ANOVA with the Student-Newman-Keuls post hoc test was performed. For comparison of 2 groups, a Student t test was performed. Statistical significance was assumed when P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
PDE1A, PDE1C, and PDE5A Expression in Patients With IPAH
The expression of PDE1A, PDE1C, and PDE5A was investigated by real-time reverse-transcription polymerase chain reaction in lung homogenate, microdissected pulmonary arteries (30 to 100 µm in diameter), and isolated SMCs (Figure 1A). A strong upregulation of PDE1C and PDE5A was evident in microdissected pulmonary arteries and SMCs of patients with IPAH. Furthermore, a stronger immunoreactivity of PDE1C and PDE5A in lung specimens from IPAH patients, along with
-smooth muscle actin colocalization, demonstrated that the site-specific changes of both PDE1C and PDE5A occur especially in the medial layer of pulmonary arteries (Figure 1B; supplemental Figure II). In contrast, only weak expression of PDE1C was detected in pulmonary vessels of healthy donor lung tissue. In addition, PDE1A expression was constitutively present in both donor and IPAH tissue, again located in the medial layer of pulmonary arteries. Immunoreactivity against PDE1A, PDE1C, and PDE5A was also noted for bronchial SMCs in small airways.
|
PDE1C Activity Is the Major Activity in Human Pulmonary SMCs, and Inhibition of PDE1 Inhibits DNA Synthesis
Human pulmonary artery SMCs obtained from PromoCell (Heidelberg, Germany) were processed to analyze the cAMP/cGMP hydrolysis activity of different PDE family members. As shown in Figure 2A, a hydrolytic activity of PDEs 3, 4, and 5 was detected, whereas no obvious PDE2 activity was observed. Incubation of the cellular lysate with calcium/calmodulin induced cAMP and cGMP hydrolysis strongly and to an equal degree, which must be attributed to PDE1C expression. In addition, the PDE1 inhibitor PI79 dose dependently inhibited the DNA synthesis of human pulmonary artery SMCs (Figure 2B).
|
Expression of PDE1A, PDE1C, and PDE5A in Animal Model of Pulmonary Hypertension
The expression of PDE1A, PDE1C, and PDE5A was investigated in lung homogenate, microdissected pulmonary arteries, and freshly isolated SMCs from MCT-injected rats (Figure 3A) and chronically hypoxic mice (21 days, 10% O2; supplemental Figure IIIA) by quantitative reverse-transcription polymerase chain reaction. No significant upregulation of any of the 3 genes was detected in lung homogenate, whereas PDE1A was significantly upregulated in pulmonary arterial SMCs of MCT-treated rats and chronically hypoxic mice. These expression changes were specific for the pulmonary circulation, because no expression changes were found in aortic tissue from MCT rats (supplemental Figure IV). Immunohistological staining of PDE1A, PDE1C, PDE5A, and -smooth muscle actin in serial sections is given in Figure 3B (MCT rats) and supplemental Figure IIIB (chronically hypoxic mice). Strong immunoreactivity of PDE1A and PDE5A, which colocalizes with smooth muscle actin, confirmed medial expression of these enzymes in experimental pulmonary hypertension. Similar data were obtained from chronically hypoxic mice.
|
8MM-IBMX Improves Hemodynamics, Right Heart Hypertrophy, and Vascular Remodeling in MCT-Treated Rats
The injection of MCT resulted in severe pulmonary hypertension within 21 days, which was sustained until day 35. RV systolic pressure was increased significantly compared with the saline-challenged group (Figure 4A). The PDE1 inhibitor 8MM-IBMX was infused continuously from day 21 to 35 and reversed chronic pulmonary hypertension. Mean systemic arterial pressure (Figure 4B), total systemic resistance (Figure 4D), and left ventricular pressures (not shown) did not change in any of the treatment groups. Compared with control animals, total pulmonary resistance (0.98±0.05 mm Hg · mL–1 · min–1 per 100 g of body weight) was increased in the MCT group at day 21 (2.14±0.26 mm Hg · mL–1 · min–1 per 100 g of body weight; P<0.05) and 35 (3.23±0.35 mm Hg · mL–1 · min–1 per 100 g of body weight; P<0.05). In the 8MM-IBMX–treated animals, total pulmonary resistance decreased significantly compared with sham treatment (1.32±0.33 mm Hg · mL–1 · min–1 per 100 g of body weight; P<0.05; Figure 4C). In addition to the hemodynamic changes, structural changes occurred that were characterized by right heart hypertrophy, measured as RV/(LV+S) (Figure 5A), and muscularization of normally nonmuscularized small pulmonary arteries (Figure 5B). The muscularization of pulmonary arteries at a size of 20 to 70 µm is depicted in an exemplary fashion in Figure 5C through 5E. The continuous infusion of 8MM-IBMX (20 µg · kg–1 · min–1) via implanted osmotic pumps from day 21 to day 35 reversed right heart hypertrophy (P<0.05 versus MCT at day 21 and day 35) and increased the portion of nonmuscularized pulmonary arteries (P<0.05 versus MCT at day 21 and day 35).
|
P<0.05 vs MCT at day 21; 
P<0.05 vs MCT at day 35.
|
8MM-IBMX Reverses Acute Hypoxia–Induced Vasoconstriction in Isolated Mouse Lungs
The PDE1 inhibitor 8MM-IBMX dose dependently reversed acute pulmonary vasoconstriction in isolated lungs from mice that were kept under normoxic conditions (Figure 6, closed circles). Notably, when investigating lungs isolated from mice that were kept for 21 days under hypoxic conditions (10% O2), we noted a significantly enhanced sensitivity to 8MM-IBMX inhibition, as is obvious from the leftward shift of the dose-response curve in these lungs (open circles). This leftward shift signaled higher PDE1 activity in the chronically hypoxic lungs.
|
8MM-IBMX Improves Hemodynamics, Right Heart Hypertrophy, and Vascular Remodeling in Chronically Hypoxic Mice
Mice kept at hypoxia developed severe pulmonary hypertension within 21 days, which was sustained until day 35. Consequently, RV systolic pressure was increased significantly compared with normoxic animals (Figure 7). The continuous infusion of 8MM-IBMX (20 µg · kg–1 · min–1) via implanted osmotic pumps from day 21 to day 35 reversed chronic pulmonary hypertension to near normal values (24.0±2.9 mm Hg, P<0.05 versus hypoxia at 21 and 35 days). In hypoxic animals, a significant RV hypertrophy developed as a consequence of increased pulmonary arterial pressures. RV/LV+S increased from 0.24±0.05 (controls) to 0.38±0.04 (21 days of hypoxia) and 0.42±0.06 (35 days of hypoxia), respectively (both P<0.05 versus controls). 8MM-IBMX caused a reduction of this ratio to 0.33±0.02 (P<0.05 versus hypoxia for 35 days). We then quantitatively assessed the degree of muscularization of pulmonary arteries with a diameter between 20 and 70 µm. In controls, the majority of vessels of this diameter are nonmuscularized (54%), with lower percentages of partially muscularized (37%) and fully muscularized (9%) vessels (Figure 8). In hypoxic animals, both at day 21 and at day 35, a significant decrease in nonmuscularized pulmonary arteries occurred, with a concomitant increase in fully muscularized pulmonary arteries. Treatment with 8MM-IBMX resulted in a significant reduction of fully muscularized arteries compared with both hypoxia groups (21 days [ie, before start of 8MM-IBMX treatment] and 35 days), and increased the percentage of nonmuscularized pulmonary arteries.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Abnormal media proliferation and de novo muscularization are characteristic features of PAH. In the present study, strong vascular upregulation of PDE1C was demonstrated by laser-assisted microdissection followed by quantitative reverse-transcription polymerase chain reaction in lung explants and isolated pulmonary SMCs from patients with IPAH. Immunostaining consistently demonstrated strong expression of PDE1C in the pulmonary arterial SMCs of the diseased but not the control lungs. In the present study, we also demonstrate that PDE1A is constitutively expressed in the vessel wall. Recently, Wharton et al26 demonstrated a significant upregulation of PDE5A in lungs from patients with pulmonary hypertension, and the present study confirms the strong pulmonary vascular upregulation of PDE5A in IPAH patients.
Chronic hypoxia and MCT injection are well-recognized stimuli for pulmonary vasoconstriction and structural remodeling of the precapillary lung resistance vessels. Hypertrophy and proliferation of vascular SMCs, with complete muscularization of physiologically partially muscularized vessels and de novo muscularization of physiologically nonmuscularized vessel, represent key features of the structural remodeling process. When PDE1A and PDE1C expression was assessed in such remodeled pulmonary vasculature, striking differences to the control rats and mouse vasculature became apparent. The PDE1A isoenzyme was strongly expressed in the pulmonary arteries of lungs from MCT-injected rats and chronically hypoxic mice, with predominant localization in pulmonary arterial SMCs. This was clearly demonstrated by immunostaining in serial sections that showed colocalization with the SMC marker -smooth muscle actin. Moreover, a functional role of this upregulated PDE1 in the control of vasoregulation was demonstrated in isolated lungs from chronically hypoxic mice: The 8MM-IBMX dose-effect curve was significantly shifted leftward, which indicates enhanced sensitivity to this inhibitor and thus a major contribution of PDE1 to the control of lung vascular tone in the chronically hypoxic lungs. A recent study from Evgenov et al17 demonstrated that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute thromboxane-induced pulmonary hypertension, which demonstrates the contribution of PDE1 to pulmonary vascular tone. On the selectivity of 8MM-IBMX for PDE1 versus other PDEs, previously published data from our own group27 and from others16 suggest a 10- to 30-fold more selective inhibition of PDE1 versus PDE3 and PDE4, respectively. Thus, at the currently chosen dose, no relevant inhibition of other PDE isoforms is to be expected to contribute to the findings presented here.
In analogy with these findings in the experimental models, very low levels of PDE1C expression were noted in the "quiescent" vascular SMC of the healthy human donor lungs, whereas this isoenzyme was strongly expressed at the mRNA and protein level in the IPAH lungs, confirmed to be localized predominantly in the media of the small pulmonary arteries by immunohistochemistry. In addition to contributing to the regulation of vascular tone in the IPAH lungs, this localization suggests a role of PDE1C in the structural remodeling process underlying the development of severe pulmonary hypertension. Both cAMP and cGMP, targeted by this PDE isoenzyme, have been found to counteract several pathways involved in SMC proliferation.28,29 For example, cAMP was shown to inhibit cyclin-dependent kinase 4, extracellular signal-regulated kinase activation,30 and upregulation of the cyclin-dependent kinase 2 inhibitor p27kip1.31 In line with these observations, prostacyclin, a potent stimulus of vascular cAMP generation, can inhibit proliferation of aortic SMCs by blocking cell cycle progression from the G1 to the S phase, largely via occupancy of the cAMP response elements in the promoter region of the cyclin A gene, along with an increase in the level of cAMP response element-binding protein transcription factor.32,33
Because PDE1C, in contrast to PDE1A and PDE1B, is strongly upregulated during SMC proliferation,10 it may act as a major cAMP-hydrolyzing PDE in SMCs under these conditions. This does not cast doubt on the role of PDE3 and PDE4, which are also expressed in the medial layer of the pulmonary artery and which also hydrolyze cAMP,34 but the specific link of PDE1C to the proliferative state of the SMC renders this PDE a particularly interesting candidate for therapeutic intervention aimed at antiremodeling or reversal of remodeling. The PDE activity profile of human pulmonary SMCs demonstrated significant PDE1 activity, which supports the role of PDE 1 in cyclic nucleotide hydrolysis in vascular SMCs. The PDE5 inhibitor sildenafil does possess some inhibitory potency toward PDE1C, although less than PDE5, but some of the strong beneficial effects of sildenafil in PAH patients35–38 might also be linked to some extent to its effect on PDE1C. Sildenafil is a PDE 1/5/6 inhibitor, with IC50 values of 280, 3.5, and 37 nmol/L for the different PDE subtypes.39,40 The IC50 of sildenafil against PDE1 is 
280 nmol/L, and these plasma levels are clearly reached after sildenafil application in men. Paul et al41 reported plasma levels in the range of 750 ng/mL after oral intake of a single dose of 100 mg of sildenafil in patients with PAH. In addition, plasma levels of 380 ng/mL of the active metabolite desmethylsildenafil were reached. These plasma levels translate into a concentration of 1.2 µmol/L, which clearly indicates the PDE1 inhibitory capacity of sildenafil. In line with these results, another study reported a plasma level of 1070 ng/mL after a single application of 100 mg of sildenafil,42 which corresponds to 1.3 µmol/L. In both studies, plasma levels above the IC50 value of sildenafil for PDE1 were maintained over a range of 4 hours. Taken together, we hereby highlight the evidence that in common clinical doses, sildenafil, in addition to its PDE5 inhibitory effects, concomitantly achieves plasma levels capable of inhibiting PDE1. Along with the first proof of PDE1 upregulation in the pulmonary circulation provided in the present report, a strong rationale to explain potential antiproliferative effects via PDE1 inhibition is provided. Moreover, the present findings may add to the understanding of why certain hemodynamic differences between clinically available PDE5 inhibitors were found7 and explain the strong synergism between sildenafil and cAMP-increasing compounds, eg, inhaled iloprost, which has been demonstrated experimentally27 and clinically.43–45 Using another PDE1 inhibitor, PI79, we demonstrated the inhibition of DNA synthesis of pulmonary SMCs in a dose-dependent manner. Similar results were obtained by inhibition of PDE1C in aortic SMCs with antisense oligonucleotides or a PDE1 inhibitor.16 More recently it has been shown by Murray et al46 in pulmonary arterial SMCs from pulmonary hypertensive patients that PDE1C is upregulated on mRNA, protein, and activity levels. Intervention studies on the in vitro level nicely corroborate the present findings in the currently used in vivo disease models. To address the putative antiproliferative potential of PDE1 interference in the pulmonary vasculature in a direct fashion, continuous infusion of the PDE1 inhibitor 8-MM-IBMX via osmotic minipumps, implanted in rats injected with the plant alkaloid MCT and mice undergoing chronic hypoxia, was undertaken. The therapeutic intervention started after pulmonary hypertension, structural remodeling of the lung vasculature, and right heart hypertrophy had fully developed. Most impressively, animals treated intravenously with 8MM-IBMX, but not vehicle-treated controls, showed not only a stoppage of further progression but significant regression of all changes within the next 2 weeks. In particular, the pattern of small pulmonary artery muscularization (nonmuscularized compared with partially muscularized and fully muscularized vessels) nearly approached the profile of control lungs. Thus, reversal of pulmonary artery remodeling was achieved in the 14 day-treatment period, accompanied by regression of right heart hypertrophy.
In conclusion, strong upregulation of PDE1C in hyperproliferative pulmonary artery SMCs was noted in clinical PAH, both on the mRNA and protein level, and was corroborated on the functional level in the animal model. Long-term infusion of a PDE1 inhibitor reversed pulmonary hypertension, lung vascular remodeling, and right heart hypertrophy in MCT-injected rats and chronically hypoxic mice, in which PDE1A appears to be the responsible PDE1 variant. We suggest that upregulation of PDE1C plays an important role in the structural remodeling process underlying severe pulmonary hypertension, thus offering a target for therapeutic intervention aimed at reversing lung vascular remodeling and subsequent right heart hypertrophy.
|
Acknowledgments |
|---|
We acknowledge the excellent technical support offered by Ewa Bieneck and Anke Voigt for this study. Part of Dr Samidurai’s thesis work was included in this manuscript.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 547 (SFB 547), projects C6 and B7, and by the European Commission under the Sixth Framework Programme (contract No. LSHM-CT-2005-018725, PULMOTENSION).
Disclosures
Dr Dunkern and Dr Schudt are employed by Altana Pharma AG. The remaining authors report no conflicts.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, Christman BW, Weir EK, Eickelberg O, Voelkel NF, Rabinovitch M. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol. 2004; 43: 13S–24S.
[Abstract/Free Full Text] - Simonneau G, Galie N, Rubin LJ, Langleben D, Seeger W, Domenighetti G, Gibbs S, Lebrec D, Speich R, Beghetti M, Rich S, Fishman A. Clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2004; 43: 5S–12S.
[Abstract/Free Full Text] - Olschewski H, Rose F, Schermuly R, Ghofrani HA, Enke B, Olschewski A, Seeger W. Prostacyclin and its analogues in the treatment of pulmonary hypertension. Pharmacol Ther. 2004; 102: 139–153.[CrossRef][Medline]
[Order article via Infotrieve]
- Ghofrani HA, Friese G, Discher T, Olschewski H, Schermuly RT, Weissmann N, Seeger W, Grimminger F, Lohmeyer J. Inhaled iloprost is a potent acute pulmonary vasodilator in HIV-related severe pulmonary hypertension. Eur Respir J. 2004; 23: 321–326.
[Abstract/Free Full Text] - Ghofrani HA, Wiedemann R, Rose F, Schermuly RT, Olschewski H, Weissmann N, Gunther A, Walmrath D, Seeger W, Grimminger F. Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial. Lancet. 2002; 360: 895–900.[CrossRef][Medline]
[Order article via Infotrieve]
- Ghofrani HA, Rose F, Schermuly RT, Olschewski H, Wiedemann R, Kreckel A, Weissmann N, Ghofrani S, Enke B, Seeger W, Grimminger F. Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol. 2003; 42: 158–164.
[Abstract/Free Full Text] - Ghofrani HA, Voswinckel R, Reichenberger F, Olschewski H, Haredza P, Karadas B, Schermuly RT, Weissmann N, Seeger W, Grimminger F. Differences in hemodynamic and oxygenation responses to three different phosphodiesterase-5 inhibitors in patients with pulmonary arterial hypertension: a randomized prospective study. J Am Coll Cardiol. 2004; 44: 1488–1496.
[Abstract/Free Full Text] - Ghofrani HA, Reichenberger F, Kohstall MG, Mrosek EH, Seeger T, Olschewski H, Seeger W, Grimminger F. Sildenafil increased exercise capacity during hypoxia at low altitudes and at Mount Everest base camp: a randomized, double-blind, placebo-controlled crossover trial. Ann Intern Med. 2004; 141: 169–177.
[Abstract/Free Full Text] - Galie N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D, Fleming T, Parpia T, Burgess G, Branzi A, Grimminger F, Kurzyna M, Simonneau G. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med. 2005; 353: 2148–2157.
[Abstract/Free Full Text] - Koyama H, Bornfeldt KE, Fukumoto S, Nishizawa Y. Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation. J Cell Physiol. 2001; 186: 1–10.[CrossRef][Medline]
[Order article via Infotrieve]
- Loughney K, Martins TJ, Harris EA, Sadhu K, Hicks JB, Sonnenburg WK, Beavo JA, Ferguson K. Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases. J Biol Chem. 1996; 271: 796–806.
[Abstract/Free Full Text] - Fidock M, Miller M, Lanfear J. Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants. Cell Signal. 2002; 14: 53–60.[CrossRef][Medline]
[Order article via Infotrieve]
- Yan C, Zhao AZ, Bentley JK, Beavo JA. The calmodulin-dependent phosphodiesterase gene PDE1C encodes several functionally different splice variants in a tissue-specific manner. J Biol Chem. 1996; 271: 25699–25706.
[Abstract/Free Full Text] - Yan C, Zhao AZ, Bentley JK, Loughney K, Ferguson K, Beavo JA. Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons. Proc Natl Acad Sci U S A. 1995; 92: 9677–9681.
[Abstract/Free Full Text] - Rybalkin SD, Bornfeldt KE, Sonnenburg WK, Rybalkina IG, Kwak KS, Hanson K, Krebs EG, Beavo JA. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest. 1997; 100: 2611–2621.[Medline]
[Order article via Infotrieve]
- Rybalkin SD, Rybalkina I, Beavo JA, Bornfeldt KE. Cyclic nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cell proliferation. Circ Res. 2002; 90: 151–157.
[Abstract/Free Full Text] - Evgenov OV, Busch CJ, Evgenov NV, Liu R, Petersen B, Falkowski GE, Petho B, Vas A, Bloch KD, Zapol WM, Ichinose F. Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol. 2006; 290: L723–L729.
[Abstract/Free Full Text] - Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest. 2005; 115: 2811–2821.[CrossRef][Medline]
[Order article via Infotrieve]
- Dumitrascu R, Weissmann N, Ghofrani HA, Dony E, Beuerlein K, Schmidt H, Stasch JP, Gnoth MJ, Seeger W, Grimminger F, Schermuly RT. Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling. Circulation. 2006; 113: 286–295.
[Abstract/Free Full Text] - Schermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W, Grimminger F. Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res. 2004; 94: 1101–1108.
[Abstract/Free Full Text] - Weissmann N, Akkayagil E, Quanz K, Schermuly RT, Ghofrani HA, Fink L, Hanze J, Rose F, Seeger W, Grimminger F. Basic features of hypoxic pulmonary vasoconstriction in mice. Respir Physiol Neurobiol. 2004; 139: 191–202.[CrossRef][Medline]
[Order article via Infotrieve]
- Schermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W, Grimminger F. Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res. 2004; 94: 1101–1108.
[Abstract/Free Full Text] - Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med. 1998; 4: 1329–1333.[CrossRef][Medline]
[Order article via Infotrieve]
- Fink L, Kohlhoff S, Stein MM, Hanze J, Weissmann N, Rose F, Akkayagil E, Manz D, Grimminger F, Seeger W, Bohle RM. cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue. Am J Pathol. 2002; 160: 81–90.
[Abstract/Free Full Text] - Kwapiszewska G, Wilhelm J, Wolff S, Laumanns I, Koenig IR, Ziegler A, Seeger W, Bohle RM, Weissmann N, Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension. Respir Res. 2005; 6: 109.[CrossRef][Medline]
[Order article via Infotrieve]
- Wharton J, Strange JW, Moller GM, Growcott EJ, Ren X, Franklyn AP, Phillips SC, Wilkins MR. Antiproliferative effects of phosphodiesterase type 5 inhibition in human pulmonary artery cells. Am J Respir Crit Care Med. 2005; 172: 105–113.
[Abstract/Free Full Text] - Schermuly RT, Inholte C, Ghofrani HA, Gall H, Weissmann N, Weidenbach A, Seeger W, Grimminger F. Lung vasodilatory response to inhaled iloprost in experimental pulmonary hypertension: amplification by different type phosphodiesterase inhibitors. Respir Res. 2005; 6: 76.[CrossRef][Medline]
[Order article via Infotrieve]
- Bornfeldt KE, Campbell JS, Koyama H, Argast GM, Leslie CC, Raines EW, Krebs EG, Ross R. The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells: dependence on the availability of downstream targets. J Clin Invest. 1997; 100: 875–885.[Medline]
[Order article via Infotrieve]
- Bornfeldt KE, Krebs EG. Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle. Cell Signal. 1999; 11: 465–477.[CrossRef][Medline]
[Order article via Infotrieve]
- Paternot S, Coulonval K, Dumont JE, Roger PP. Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells. J Biol Chem. 2003; 278: 26533–26540.
[Abstract/Free Full Text] - Coulonval K, Bockstaele L, Paternot S, Dumont JE, Roger PP. The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis. Exp Cell Res. 2003; 291: 135–149.[CrossRef][Medline]
[Order article via Infotrieve]
- Kothapalli D, Stewart SA, Smyth EM, Azonobi I, Pure E, Assoian RK. Prostacyclin receptor activation inhibits proliferation of aortic smooth muscle cells by regulating cAMP response element-binding protein- and pocket protein-dependent cyclin A gene expression. Mol Pharmacol. 2003; 64: 249–258.
[Abstract/Free Full Text] - Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, Reusch JE. cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration 9. J Biol Chem. 2001; 276: 46132–46141.
[Abstract/Free Full Text] - Pauvert O, Salvail D, Rousseau E, Lugnier C, Marthan R, Savineau JP. Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery. Biochem Pharmacol. 2002; 63: 1763–1772.[CrossRef][Medline]
[Order article via Infotrieve]
- Ghofrani HA, Wiedemann R, Rose F, Olschewski H, Schermuly RT, Weissmann N, Seeger W, Grimminger F. Combination therapy with oral sildenafil and inhaled iloprost for severe pulmonary hypertension. Ann Intern Med. 2002; 136: 515–522.
[Abstract/Free Full Text] - Ghofrani HA, Wiedemann R, Rose F, Schermuly RT, Olschewski H, Weissmann N, Gunther A, Walmrath D, Seeger W, Grimminger F. Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial. Lancet. 2002; 360: 895–900.[CrossRef][Medline]
[Order article via Infotrieve]
- Ghofrani HA, Rose F, Schermuly RT, Olschewski H, Wiedemann R, Kreckel A, Weissmann N, Ghofrani S, Enke B, Seeger W, Grimminger F. Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol. 2003; 42: 158–164.
[Abstract/Free Full Text] - Ghofrani HA, Schermuly RT, Rose F, Wiedemann R, Kohstall MG, Kreckel A, Olschewski H, Weissmann N, Enke B, Ghofrani S, Seeger W, Grimminger F. Sildenafil for long-term treatment of nonoperable chronic thromboembolic pulmonary hypertension. Am J Respir Crit Care Med. 2003; 167: 1139–1141.
[Abstract/Free Full Text] - Ghofrani HA, Osterloh IH, Grimminger F. Sildenafil: from angina to erectile dysfunction to pulmonary hypertension and beyond. Nat Rev Drug Discov. 2006; 5: 689–702.[CrossRef][Medline]
[Order article via Infotrieve]
- Gresser U, Gleiter CH. Erectile dysfunction: comparison of efficacy and side effects of the PDE-5 inhibitors sildenafil, vardenafil and tadalafil: review of the literature. Eur J Med Res. 2002; 7: 435–446.[Medline]
[Order article via Infotrieve]
- Paul GA, Gibbs JS, Boobis AR, Abbas A, Wilkins MR. Bosentan decreases the plasma concentration of sildenafil when coprescribed in pulmonary hypertension. Br J Clin Pharmacol. 2005; 60: 107–112.[CrossRef][Medline]
[Order article via Infotrieve]
- Lee M, Min DI. Determination of sildenafil citrate in plasma by high-performance liquid chromatography and a case for the potential interaction of grapefruit juice with sildenafil citrate. Ther Drug Monit. 2001; 23: 21–26.[CrossRef][Medline]
[Order article via Infotrieve]
- Ghofrani HA, Wiedemann R, Rose F, Olschewski H, Schermuly RT, Weissmann N, Seeger W, Grimminger F. Combination therapy with oral sildenafil and inhaled iloprost for severe pulmonary hypertension. Ann Intern Med. 2002; 136: 515–522.
[Abstract/Free Full Text] - Ghofrani HA, Rose F, Schermuly RT, Olschewski H, Wiedemann R, Kreckel A, Weissmann N, Ghofrani S, Enke B, Seeger W, Grimminger F. Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol. 2003; 42: 158–164.
[Abstract/Free Full Text] - Wilkens H, Guth A, Konig J, Forestier N, Cremers B, Hennen B, Bohm M, Sybrecht GW. Effect of inhaled iloprost plus oral sildenafil in patients with primary pulmonary hypertension. Circulation. 2001; 104: 1218–1222.
[Abstract/Free Full Text] - Murray F, Patel HH, Suda RY, Zhang S, Thistlethwaite P, Yuan JX, Insel PA. Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1. Am J Physiol Lung Cell Mol Physiol. 2007; 292; L294–L303.
[Abstract/Free Full Text]
CLINICAL PERSPECTIVE
Pulmonary arterial hypertension (PAH), a life-threatening disease, is characterized by aberrant pulmonary vascular remodeling; at a cellular level, evidence exists of abnormal smooth muscle cell proliferation in the medial wall, neointima formation, and endothelial cell dysfunction. Cyclic nucleotide phosphodiesterases (PDEs) control the effects induced by several mediators such as nitric oxide and prostanoids via hydrolyzing their second messengers, cGMP and cAMP. PDE5 is known to be upregulated in the lung vasculature, and the PDE5 inhibitor sildenafil has been approved for the treatment of PAH. To date, little is known about the antiproliferative effects observed with sildenafil treatment. Acknowledging the considerable PDE1 inhibitory capacity of sildenafil, we hypothesized that PDE1 may play a role in pulmonary vascular smooth muscle cell proliferation. The expression of PDE1 in explanted lungs from patients with idiopathic PAH and animal models of pulmonary hypertension was investigated. Therapeutic application of a selective PDE1 inhibitor was performed in 2 animal models of PAH. In essence, strong upregulation of PDE1C in pulmonary arterial vessels from idiopathic PAH patients compared with healthy donor lungs was noted. Long-term PDE1 inhibitor treatment in the experimental models resulted in reduced pulmonary artery pressure, reversed remodeling of the lung vasculature, and reduction in right heart hypertrophy. Our results, therefore, could explain in part the antiremodeling effects of sildenafil by its fractional PDE1 inhibitory capacity. Furthermore, our results open speculation about the therapeutic efficacy of selective PDE1 inhibitors in pulmonary vascular disorders.
|
Footnotes |
|---|
This publication reflects only the authors’ views, and the European Community is in no way liable for any use that may be made of the information contained herein.
*The first 2 authors contributed equally to this article. 
The online-only Data Supplement, consisting of Methods, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.676809/DC1.
This article has been cited by other articles:
 |
|
 |
|
  Y.-J. Lai, S. S. Pullamsetti, E. Dony, N. Weissmann, G. Butrous, G.-A. Banat, H. A. Ghofrani, W. Seeger, F. Grimminger, and R. T. Schermuly Role of the Prostanoid EP4 Receptor in Iloprost-mediated Vasodilatation in Pulmonary Hypertension Am. J. Respir. Crit. Care Med., July 15, 2008; 178(2): 188 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Wilkins, J. Wharton, F. Grimminger, and H. A. Ghofrani Phosphodiesterase inhibitors for the treatment of pulmonary hypertension Eur. Respir. J., July 1, 2008; 32(1): 198 - 209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-C. Peyter, V. Muehlethaler, L. Liaudet, M. Marino, S. Di Bernardo, G. Diaceri, and J.-F. Tolsa Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice Am J Physiol Lung Cell Mol Physiol, July 1, 2008; 295(1): L201 - L213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Klein, R. T. Schermuly, P. Ellinghaus, H. Milting, B. Riedl, H. A. Ghofrani, F. Grimminger, and S. Schafer Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure Eur. Respir. Rev., June 1, 2008; 17(108): 72 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kirsch, B. Kemp-Harper, N. Weissmann, F. Grimminger, and H. H. H. W. Schmidt Sildenafil in hypoxic pulmonary hypertension potentiates a compensatory up-regulation of NO-cGMP signaling FASEB J, January 1, 2008; 22(1): 30 - 40. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||