cAMP Phosphodiesterase Inhibitors Increases Nitric Oxide Production by Modulating Dimethylarginine DimethylaminohydrolasesClinical Perspective
Background—Pulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss of precapillary pulmonary vessel cross-sectional area. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) play important roles in endothelial dysfunction. We investigated whether combined phosphodiesterase (PDE) 3 and 4 inhibition ameliorates endothelial function by regulating the ADMA-DDAH axis.
Methods and Results—We investigated the effects of the PDE3/4 inhibitor tolafentrine in vitro on endothelial cell survival, proliferation, and apoptosis. Effects of tolafentrine on the endothelial nitric oxide synthase/nitric oxide pathway, DDAH expression, DDAH promoter activity, and cytokine release from endothelial cells and their subsequent influence on DDAH expression were investigated. In monocrotaline-induced pulmonary arterial hypertension in rats, the effects of inhaled tolafentrine on DDAH expression and activity were investigated. Real-time-polymerase chain reaction, immunocytochemistry, and PDE activity assays suggested high expression of PDE3 and PDE4 isoforms in endothelial cells. Treatment of endothelial cells with PDE3/4 inhibitor significantly decreased ADMA-induced apoptosis via a cAMP/PKA-dependent pathway by induction of DDAH2. Chronic nebulization of PDE3/4 inhibitor significantly attenuated monocrotaline-induced hemodynamic, gas exchange abnormalities, vascular remodeling, and right heart hypertrophy. Interestingly, PDE3/4 inhibitor treatment reduced ADMA and elevated nitric oxide/cGMP levels. Mechanistically, this could be attributed to direct modulatory effects of cAMP on the promoter region of DDAH2, which was consequently found to be increased in expression and activity. Furthermore, PDE3/4 inhibitor suppressed apoptosis in endothelial cells and increased vascularization in the lung.
Conclusion—Combined inhibition of PDE3 and 4 regresses development of pulmonary hypertension and promotes endothelial regeneration by modulating the ADMA-DDAH axis.
- dimethylarginine dimethylaminohydrolase (DDAH)
- endothelial regeneration
- hypertension, pulmonary
- nitric oxide synthase
- phosphodiesterase (PDE)
Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary vascular remodeling leading to right ventricular (RV) failure and death.1 The pathogenesis of PAH is complex and arises from a combination of pulmonary vasoconstriction, vascular wall remodeling, and thrombosis, resulting in severe loss of vessel cross-sectional area.2 Intimal changes in the vessel walls include endothelial injury, endothelial cell (EC) proliferation, invasion of the intima by (myo)fibroblast-like cells, enhanced matrix deposition, and in some cases obstruction of the vascular lumen by unique plexiform lesions.3 Despite the uncertainty of exact mechanisms causing pulmonary arteriolar obstructions, a growing body of evidence implicates EC apoptosis as the initiator of microvascular degeneration or apoptosis-resistant ECs.4 Thus, regeneration of the pulmonary vascular bed could be a novel therapeutic approach for reversal of pulmonary vascular disease in PAH patients.
Editorial see p 1156
Clinical Perspective on p 1204
Nitric oxide (NO) synthesized by endothelial NO synthase (eNOS) is a potent vasodilator and plays an important role in regulating pulmonary vascular tone. In addition, NO is a potent stimulator of EC proliferation and migration, and eNOS is a critical mediator of angiogenesis.5 Reduced bioavailability of NO has long been suspected to play a pathogenic role in PAH.6 Although several mechanisms have been postulated, impairment of NOS activity by endogenous NOS inhibitors such as asymmetrical dimethylarginine (ADMA) in endothelial dysfunction–associated diseases has gained substantial interest in recent years.7 Recently, we reported that ADMA levels are increased in plasma from PAH patients and in monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats, a preclinical model that mimics severe human PAH.8 This is linked to downregulation of the ADMA-metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) 2. Furthermore, several lines of evidence have revealed that the ADMA-DDAH pathway plays a crucial role in EC apoptosis, migration, and angiogenesis by regulating NO signaling.9,10 Recent studies have demonstrated that DDAH can activate cAMP-dependent signaling via a protein-protein interaction with protein kinase A (PKA). Hasegawa et al11 showed that PKA plays a stimulatory role in DDAH2-induced vascular endothelial growth factor (VEGF) transcription via an enhanced protein-protein interaction between DDAH2 and PKA. In addition, Tokuo et al12 reported interaction among PKA, DDAH1, and neurofibromin, suggesting a cross-talk between DDAH and cAMP signaling cascades. However, the effect of cAMP signaling on the DDAH signaling axis has not been explored so far.
Phosphodiesterase (PDE) isoenzymes, predominantly PDE3 and 4, are essential coregulators of cAMP catabolism in many organs, including the lung, and are upregulated in experimental PAH models.13 In addition, cAMP-elevating agents have been shown to enhance various EC functions, including angiogenesis.14 On the other hand, direct activation of PKA by cAMP was shown to inhibit EC survival and angiogenesis.15 However, there is scant information on the molecular mechanisms and signaling pathways by which cAMP-elevating agents or a combined PDE3/4 inhibitor regulates endothelial degeneration. Furthermore, the effect of cAMP-elevating agents or a combined PDE3/4 inhibitor on endothelial degeneration and ADMA-DDAH signaling pathway has not been delineated.
We postulated that cAMP production induced by inhibition of PDE3 and PDE4 leads to microvascular regeneration by regulating ADMA and DDAH. In particular, we studied the influence of combined PDE3/4 inhibitor tolafentrine on (1) EC survival and proliferation; (2) serum starvation– and ADMA-mediated apoptosis in vitro followed by deciphering the signaling events involved; (3) the eNOS/NO pathway, DDAH expression, and promoter activity; (4) cytokine release from ECs and their subsequent influence on DDAH expression; (5) hemodynamics and structural remodeling associated with MCT-induced PH; and (6) the regulation of ADMA, DDAH, apoptosis, proliferation, and vascularization in vivo.
For details of the experimental setup, see the online-only Data Supplement.
EC Isolation and Culture
Primary human umbilical vein ECs (HUVECs) were isolated from umbilical veins. Human pulmonary arterial ECs and ECV304 cells were obtained from Lonza (Basel, Switzerland) and ATCC (Manassas, VA).
Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA was reverse transcribed with ImProm-II Reverse Transcription System, followed by real-time polymerase chain reaction analysis of PDE3, PDE4, and DDAH isoforms using the primers described in Table I in the online-only Data Supplement.
Assessment of Cell Viability, Apoptosis, and Proliferation of ECs
Influence of PDE3/4 inhibitor on EC viability, apoptosis, and proliferation was assessed with MTT, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL), and BrdU incorporation assays.
PDE Activity, DDAH Activity, and Intracellular NO/NOS Activity Measurements
PDE activity was determined via radioimmunoassay, DDAH activity by colorimetric assay, and NO/NOS activity by the DAF-2/DA method.
DDAH Promoter Activity Assay
ECV304 cells were transfected with the DDAH2 promoter/reporter gene construct in the absence/presence of PDE3/4 inhibitor, 8-Br-cAMP, and KT5720 and measured for luciferase activity.
Measurement of ADMA, l-Arginine, Nitrite and Nitrate, and cGMP
Plasma ADMA and l-arginine were measured with high-performance liquid chromatography/fluorescence detection, nitrate and nitrate (NOx) with colorimetry, and cGMP with radioimmunoassay.
MCT Treatment, Nebulization of Tolafentrine, and Hemodynamic Measurements
Two weeks after a single MCT injection, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole-body aerosol exposure system. After 4 weeks, hemodynamics, cardiac output, and RV hypertrophy were assessed.
Assessment of Vascular Remodeling, Microvessel Density, Apoptosis, and Proliferation In Vivo
Paraffin-embedded lung sections double-stained with α-smooth muscle actin and von Willebrand factor antibodies for assessing the degree of muscularization of peripheral pulmonary arteries. In addition, lung sections were stained for Elastin–Nuclear Fast Red to assess medial wall thickness. Vessels were detected by staining for von Willebrand factor, proliferation by staining for proliferating cell nuclear antigen, and apoptosis by staining with an in situ cell death detection kit.
Data are presented as mean±SEM. Unless otherwise stated, statistical comparisons of samples were performed by ANOVA followed by the Dunnett posthoc test. Residuals were checked for normal distribution by normal quantile-quantile plots and Shapiro-Wilk tests; homogeneity of variances was checked with the Bartlett test. Dose dependency was checked by the Spearman correlation. Statistical comparisons for real-time polymerase chain reaction experiments are based on ΔCT values. Samples were compared by use of Student t tests with pooled variances. P values were corrected for multiple testing with the Holm procedure.
PDE Isoform Expression in ECs
Using real-time polymerase chain reaction, we found that the PDE isoforms 3A, 3B, 4C, and 4D but not 4A and 4B are expressed in HUVECs (Figure 1A). These results were confirmed by immunostainings (Figure 1B and 1C). Furthermore, we found that PDE3 and PDE4 were the major contributors to total cAMP-PDE activity in HUVECs (Figure I in the online-only Data Supplement). Similar expression profiles of PDE3 and PDE4 isoforms were also observed in human pulmonary arterial ECs and in HUVECs (Figure II in the online-only Data Supplement).
Effect of a PDE3/4 Inhibitor on EC Survival and Proliferation
To determine whether PDE3/4 inhibitor has a general effect on cell survival and proliferation, we stimulated quiescent HUVECs with 0.1% FCS, 10% FCS, or VEGF in the absence/presence of PDE3/4 inhibitor. We first examined the effects of PDE3/4 inhibitor on EC survival. Both 10% FCS and VEGF increased the viability and proliferation of HUVECs compared with serum-starved cells. However, viability of 0.1% FCS– or 10% FCS–stimulated HUVECs was not significantly affected by various concentrations of PDE3/4 inhibitor (0.1, 0.5, or 1 μmol/L; Figure 2A and 2C). In contrast, PDE3/4 inhibitor significantly increased the viability of VEGF-stimulated HUVECs (Figure 2B). Notably, PDE3/4 inhibitor significantly increased DNA synthesis of both 10% FCS– and VEGF-stimulated HUVECs in a dose-dependent manner (P<0.0001; Figure 2D and 2E).
Effect of PDE3/4 Inhibitor on EC Apoptosis
To address apoptosis, cell death was induced in quiescent HUVECs by exposure to 0.1% FCS medium or ADMA (NOS inhibitor), and the effect of PDE3/4 inhibitor (0.1, 0.5, or 1 μmol/L) was investigated. Exposure of HUVECs to 0.1% FCS or ADMA (10 or 20 μmol/L) for 24 hours dose-dependently increased the number of TUNEL-positive cells (P=0.004; Figure 3A through 3C). Incubation with various concentrations of PDE3/4 inhibitor did not influence apoptosis induced by 0.1% FCS (Figure 3A). However, PDE3/4 inhibitor treatment dose-dependently inhibited apoptosis induced by ADMA in ECs (P<0.0001; Figure 3B and 3C).
cAMP Analogs and cAMP-Elevating Agents Mimic the Effect of PDE3/4 Inhibition on ADMA-Induced Apoptosis
We hypothesized that PDE3/4 inhibitors would be able to inhibit ADMA-induced EC apoptosis by increasing intracellular cAMP level. For this purpose, we examined the effects of forskolin, a potent and unique activator of adenylyl cyclase that elevates the intracellular cAMP levels, and 8-Br-cAMP, a cell-permeable and nonhydrolyzable cAMP analog, on ADMA-induced EC apoptosis. Similar to PDE3/4 inhibitor, both forskolin (10 or 50 μmol/L) and 8-Br-cAMP (0.01, 0.1, or 1 mmol/L) decreased ADMA-induced apoptosis in a concentration-dependent manner (P<0.0001; Figure 3D).
PDE3/4 Inhibitor Attenuates ADMA-Induced Apoptosis in a PKA-Dependent Manner
To demonstrate cAMP-mediated downstream signaling,15 we examined the effect of the PKA inhibitor KT5720 on ADMA-induced apoptosis. Interestingly, cotreatment of KT5720 (5 μmol/L) with PDE3/4 inhibitor did not alter ADMA-induced EC apoptosis (Figure 3E).
Since peroxisome proliferator–activated receptor-γ was shown to antagonize ADMA levels by inducing DDAH expression16 and cAMP-elevating agents were shown to be involved in peroxisome proliferator–activated receptor activation,17 we treated ADMA-stimulated HUVECs with GW9662, a potent antagonist of PPARγ (10 or 20 μmol/L). Treatment with GW9662 did not alter ADMA-induced EC apoptosis. In contrast, cotreatment with PDE3/4 inhibitor reduced ADMA-induced EC apoptosis (Figure 3F).
Effect of PDE3/4 Inhibitor on eNOS/NO Pathway
To examine whether the attenuation of effects of ADMA by PDE3/4 inhibitor is via regulation of eNOS/NO pathway, we evaluated intracellular NO production/NOS activity, eNOS expression, and eNOS phosphorylation. Direct measurement of intracellular NO levels/NOS activity by the DAF-2/DA method showed elevated NO production/NOS activity by treatment of HUVECs with PDE3/4 inhibitor (Figure 4A and 4B). However, Western blot analysis revealed that PDE3/4 inhibitor did not influence either eNOS expression or eNOS phosphorylation (Figure 4C and 4D).
Effect of PDE3/4 Inhibitor on DDAH Expression and DDAH Promoter Activity
To investigate whether the attenuation of the effects of ADMA by PDE3/4 inhibitor is via regulation of DDAH, we evaluated changes in the expression of DDAH isoforms. As shown in Figure 4E and 4F, PDE3/4 inhibitor caused a 3-fold induction of DDAH2 but not DDAH1 mRNA compared with nontreated cells. Western blotting likewise disclosed increased expression of DDAH2 protein in HUVECs exposed to PDE3/4 inhibitor (Figure 4G and 4H).
Based on the presence of 6 putative cAMP response element binding (CREB) response element sequences in the DDAH2 promoter (Figure III in the online-only Data Supplement), we examined whether PDE3/4 inhibitor regulates DDAH2 promoter activity by transient transfection of DDAH2 promoter/reporter plasmid containing human DDAH2 promoter sequences in ECV304 cells. PDE3/4 inhibitor treatment significantly increased luciferase activity (≈60.7±8.7%) compared with the controls, which was significantly inhibited by cotreatment with the PKA inhibitor KT5720. Similarly, treatment with 8-Br-cAMP significantly increased DDAH2 promoter activity (Figure 4I).
Effect of Cytokines on DDAH Expression and the Effect of PDE3/4 Inhibitor on Cytokine Release From ECs
To determine which growth factors/cytokines induce DDAH2 expression, HUVECs were stimulated with different concentrations of platelet-derived growth factor, transforming growth factor-β1, tumor necrosis factor-α, interferon-γ, interleukin (IL)-1β, IL-4, IL-6, and IL-8. We found that tumor necrosis factor-α and interferon-γ stimulation significantly reduced DDAH2 expression, whereas IL-1β and IL-4 induced DDAH2 expression (Figure 5A through 5D). On the other hand, PDE3/4 inhibitor caused a decrease in tumor necrosis factor-α–stimulated IL-1β and IL-8 secretion from HUVECs (Figure IVA through IVD in the online-only Data Supplement).
Effect of PDE3/4 Inhibitor on Hemodynamics and Gas Exchange
To assess therapeutic potential of PDE3/4 inhibitor in MCT-induced PH (MCT-PH), MCT-injected rats were treated with aerosolized PDE3/4 inhibitor or saline for 2 weeks. MCT-PH rats treated with saline have increased RV systolic pressure (66.5±3.2 versus 25.9±4.0 mm Hg) and RV hypertrophy (0.53±0.04 versus 0.29±0.02) compared with control rats (Figure 6A). In contrast, MCT-PH rats treated with saline have reduced cardiac index (data not shown) and body weight but have no significant changes in systemic arterial pressure compared with control rats (Figure 6B and 6C). Aerosolized PDE3/4 inhibitor treatment compared with saline treatment for 2 weeks significantly lowered RV systolic pressure to 43.4±2.1 mm Hg but with no significant effects on systemic arterial pressure, cardiac index, or body weight (Figure 6A through 6C). Furthermore, the partial arterial oxygenation and central venous oxygen saturation that were decreased in MCT-PH rats treated with saline became normalized (≈70%) in PDE3/4 inhibitor–treated rats (Figure 6D and 6E). Importantly, inhaled PDE3/4 inhibitor reduced established RV hypertrophy to 0.37±0.03 (Figure 6F).
Effect of PDE3/4 Inhibitor on Pulmonary Vascular Remodeling
To assess PDE3/4 inhibitor effects on pulmonary vascular remodeling, we quantitatively assessed the degree of muscularization of pulmonary resistance arteries. In MCT-PH rats treated with saline, a dramatic increase in muscularization and medial wall thickness of pulmonary arteries occurred compared with control rats (Figure 6G and 6H). Aerosolized PDE3/4 inhibitor treatment compared with saline treatment for 2 weeks significantly lowered fully muscularized pulmonary resistance arteries (24.1±4.6% versus 49.6±7.5%) and medial wall thickness (25.0±0.4% versus 31.4±0.5%; Figure 6G and 6H).
Effects of Aerosolized PDE3/4 Inhibitor on Total cAMP-Specific PDE Activity in Different Tissues
To study the widespread/tissue-specific effects of aerosol containing PDE3/4 inhibitor in different tissues, total cAMP-specific PDE activity was measured in the lung, kidney, and liver. Interestingly, aerosolized PDE3/4 inhibitor treatment significantly reduced total cAMP-specific PDE activity only in lung tissues compared with kidney and liver tissues of MCT-PH rats (Figure VA through VC in the online-only Data Supplement).
Effect of PDE3/4 Inhibitor on Methylarginine Production
Two weeks of treatment of MCT-PH rats with PDE3/4 inhibitor caused a substantial and significant decrease in the plasma ADMA levels. Plasma ADMA but not l-arginine levels were decreased in PDE3/4 inhibitor–treated MCT-PH rats compared with saline-treated MCT-PH rats from 1.65±0.15 to 0.31±0.04 (Figure 7A and 7B).
Effect of PDE3/4 Inhibitor on DDAH Expression and Activity
Two weeks of treatment of MCT-PH rats with PDE3/4 inhibitor increased DDAH2 mRNA levels compared with saline-treated MCT-PH rats (Figure 7C). Western blot analysis confirmed that DDAH2 expression also increased with PDE3/4 inhibitor treatment in MCT-PH rat lungs (Figure 7D and 7E). Furthermore, DDAH activity decreased by 4-fold in saline-treated MCT-PH rat lungs compared with control lungs and in organs other than lung, ie, in primary organs responsible for ADMA metabolism (kidney and liver; Figure 7F and Figure VIA and VIB in the online-only Data Supplement). Importantly, 2 weeks of PDE3/4 inhibitor administration in MCT-PH rat lungs restored DDAH expression and activity to a nearly normal level.
Effect of PDE3/4 Inhibitor on cGMP and NO Production
Parallel to alteration in DDAH activity, PDE3/4 inhibitor also increased NO synthesis. Four weeks after MCT injection, rats showed a remarkable decrease in NOx levels. Compared with saline treatment, treatment of MCT-PH rats with PDE3/4 inhibitor significantly elevated plasma NOx levels and plasma cGMP levels (Figure 8A and 8B).
Effects of PDE3/4 Inhibitor on Endothelial Regeneration
Quantitative analysis of von Willebrand factor–positive blood vessels in lung sections indicated a lower vascular density in MCT-injected rat lungs compared with control rat lungs. Interestingly, the vascular density was increased in PDE3/4 inhibitor–treated MCT-PH rats compared with saline-treated MCT-PH rats (Figure 8C and 8D). The number of proliferating (proliferating cell nuclear antigen–positive) ECs increased significantly in the PDE3/4 inhibitor–treated group compared with the saline-treated group (Figure 8E). In contrast, the number of apoptotic ECs increased significantly 2 weeks after MCT injection. PDE3/4 inhibitor inhalation significantly decreased the number of apoptotic pulmonary ECs (Figure 8F).
This study has 4 salient findings. First, the combined PDE3/4 inhibitor tolafentrine increases DDAH2 promoter activity, upregulates DDAH2 expression, and inhibits ADMA-induced apoptosis in ECs via a cAMP-PKA–dependent pathway. Second, inhalation therapy of PDE3/4 inhibitor improved pulmonary hemodynamics and reversed structural changes in MCT-PH rats. Third, PDE3/4 inhibitor restores DDAH expression and activity in MCT-PH rats, reduces ADMA levels, and elevates NO levels. Fourth, treatment alters pulmonary EC apoptosis, proliferation, and vascularization, which contribute to the regeneration of pulmonary ECs (Figure 8G).
Impairment of pulmonary vascular and endothelial homeostasis plays a significant role in the pathobiology of PAH. Despite the uncertainty of the exact mechanisms causing the pulmonary arteriolar obstruction, EC apoptosis could initiate microvascular degeneration.4 Thus, regeneration of the pulmonary vascular bed could be a novel therapeutic approach for the treatment of PAH. In this scenario, although it is generally accepted that cAMP signaling is most likely involved in various EC events,14 there is currently a paucity of information regarding the role of cAMP elevation in regulating the function of these cells. We found here that the PDE3 and PDE4 isoforms are expressed in ECs. Inhibition of these isoforms by combined PDE3/4 inhibitor inhibited ADMA-induced apoptosis and augmented VEGF-stimulated proliferation.
ADMA, an analog of l-arginine present in blood of both humans and animals, can inhibit NOS18 and is the übermarker of endothelial dysfunction–associated diseases.8,19 In addition, endogenous ADMA and exogenous ADMA inhibit acetylcholine-induced vascular endothelium–dependent relaxation.20 ADMA also increases EC motility, oxidative stress, and upregulated genes encoding endothelial adhesion molecules that are redox sensitive.21 The present study shows that ADMA induces apoptosis in ECs, in agreement with a study from Jiang et al.9 Furthermore, treatment with the PDE3/4 inhibitor forskolin and 8-Br-cAMP reduced ADMA-induced apoptosis in ECs by accumulating cAMP. This event was inhibited by inhibition of PKA activity, indicating that PDE3/4 inhibitor inhibits ADMA-mediated endothelial apoptosis in a cAMP/PKA-dependent manner. The effective concentrations of ADMA used in in vitro studies are higher than the plasma level of ADMA achieved in patients and experimental models of PH.8,22 However, the intracellular ADMA level in endothelium has been reported to be much more concentrated, even 10-fold higher than the reported range for plasma values. The intracellular levels of ADMA in patients with PAH and its apoptotic effects on ECs in vivo remain to be determined.
A central finding of the present study is that PDE3/4 inhibitor increases DDAH2 expression, and in silico analysis demonstrates 6 putative CREB-binding elements in DDAH2 promoter. In addition, increased DDAH2 promoter/reporter activity in the presence of PDE3/4 inhibitor confirmed the function role of these CREB responsive elements. This supposition is strengthened by the additional finding that treatment with 8-Br-cAMP increased DDAH2 promoter/reporter activity and inhibited it by cotreatment with PKA inhibitor. In addition, because eNOS appears to be the central source of NO in endothelium, we evaluated the expression and activation of eNOS followed by direct measurement of intracellular NO.23 Interestingly, the PDE3/4 inhibitor increased NO production/NOS activity with no changes in the expression/phosphorylation of eNOS, indicating that PDE3/4 inhibitor increases NO production as a result of changed DDAH expression and activity.
Because it has been well demonstrated that PDE3 and/or PDE4 inhibitors block synthesis of proinflammatory cytokines,24 we assessed the influence of PDE3/4 inhibitor on the synthesis/release of inflammatory cytokines by ECs in vitro. We observed that tolafentrine caused a decrease in tumor necrosis factor-α–stimulated IL-8 secretion. Vice versa, proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ mediated DDAH2 downregulation. These observations in vitro may have important implications for the pathogenesis and treatment of PH. First, they show the involvement of ADMA and DDAH in MCT-induced endothelial dysfunction. From our results and previous reports,8,25 we postulate that MCT induces oxidative stress and inflammatory milieu by downregulating DDAH, which then induces ADMA production, thus causing endothelial apoptosis, a process that precedes progressive PH and vascular remodeling. Second, the fact that tolafentrine modulated DDAH, which is dysregulated in both experimental and human PAH,8,22 suggests a therapeutic potential of PDE3/4 inhibitors.
Interestingly, an increased serum- and VEGF-stimulated proliferation was observed in ECs after treatment with PDE3/4 inhibitor. This observation can be explained by augmented VEGF signaling in these settings either by adenylyl cyclase/PKA-dependent VEGF induction26 or by induction of DDAH2, which in turn can induce VEGF, resulting in increased proliferation and migration of ECs.11
To address the therapeutic potential of PDE3/4 inhibition, MCT-PH rats were treated with tolafentrine from day 14 to 28 by repetitive inhalations. We aimed to achieve a selective pulmonary vasodilation by using a 15-fold lower dose of tolafentrine compared with the intravenous dose used in our previous studies.27 In accordance, analysis of total cAMP-specific PDE activity in different tissues demonstrated significant PDE inhibition in lungs but not in kidney and liver after tolafentrine treatment, suggesting a less widespread inhibition of PDE activity after aerosolized tolafentrine treatment. Hemodynamics and RV hypertrophy were significantly improved, as were the structural changes in the lung vasculature evoked by MCT. These most impressive beneficial effects of PDE3/4 inhibitor on structural remodeling may be explained in part by cAMP-mediated inhibition of proliferation, cell cycle progression, and migration of pulmonary arterial smooth muscle cells.28
In line with these hemodynamic data, a strong impact of PDE3/4 inhibitor on plasma ADMA, an endogenous NOS inhibitor of MCT-challenged animals, was noted. PDE3/4 inhibitor treatment of MCT-PH rats for 2 weeks reduced ADMA levels, which was paralleled by increased DDAH expression and activity in lungs and, to a lesser extent, in kidneys that become dysregulated during development of MCT-induced PH.8 Because DDAH is key regulator of endogenous ADMA levels, increased DDAH expression and activity may accelerate the degradation of endogenous ADMA, enhancing the activity of eNOS and eventually augmenting the synthesis of NO. In line with this hypothesis, NOx levels returned to close to control values in PDE3/4 inhibitor–treated PH rats. To the best of our knowledge, this is the first study to demonstrate cAMP-PDE regulation of NO/cGMP signaling by reducing the endogenous NOS inhibitor ADMA. Interestingly, our results with tolafentrine identify cAMP-elevating agents in general and combined PDE3/4 inhibitor specifically as transcriptional modulators of DDAH2, in addition to the previously recognized all-transretinoic acid.29 In addition to transcriptional regulation, combined PDE3/4 inhibitor may directly influence DDAH activity by its potent antiinflammatory properties. Thus, these 2 pathways together enhance the bioavailability of NO, thereby reducing the impairment of endothelium-dependent relaxation/endothelial degeneration induced by MCT.
MCT and environmental stress induce pulmonary EC injury and decrease the number of pulmonary capillaries,30 contributing to the development of PH, suggesting that regeneration of lung microvasculature may be a novel and effective therapeutic strategy for restoring pulmonary hemodynamics in experimental and clinical PH. In fact, recent studies have demonstrated that transplantation of somatic cells or endothelium-like progenitor cells transduced with eNOS rescues MCT-induced PH,31 suggesting that NO-elevating agents such as PDE3/4 inhibitors may be involved in endothelial regeneration. In line with this reasoning, inhalation of PDE3/4 inhibitor enhanced expression of proliferating cell nuclear antigen, a marker for cell proliferation, in pulmonary ECs. Interestingly, PDE3/4 inhibitor also increased the number of pulmonary capillaries in MCT-injected rats, suggesting attenuation of MCT-induced PH by PDE3/4 inhibitor, possibly by protecting against EC apoptosis or by inducing microvascular angiogenesis.
Therapeutic inhalation of combined PDE3/4 inhibitor tolafentrine reduced MCT-induced PH, ie, improved hemodynamic values, and reduced vascular remodeling and endothelial degeneration by modulating ADMA-DDAH axis. Thus, regeneration of the pulmonary vascular bed could be a novel therapeutic approach; hence, PDE3/4 inhibitors may be useful for the treatment of pulmonary vascular disease.
Sources of Funding
This work was supported by Sonderforschungsbereich 547 Kardiopulmonales Gefäßsystem, Excellence Cluster Cardio Pulmonary System, European Union FP6 PULMOTENSION (LSHM-CT-2005-018725), and Else-Kroener-Fresenius Foundation.
We thank Andrea Mohr and Katharina Weidl for technical assistance.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.941484/DC1.
- Received August 8, 2008.
- Accepted December 27, 2010.
- © 2011 American Heart Association, Inc.
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Endothelial dysfunction is a key feature of chronic systemic and pulmonary vascular disorders. The nitric oxide pathway plays a central role in maintaining physiological organ function. Alterations of this pathway have been attributed to be centrally involved in the course of diseases like chronic heart failure, systemic and pulmonary arterial hypertension, and arteriosclerosis. The activity of nitric oxide synthases, particularly nitric oxide synthase-3, was found to be suppressed by its endogenous inhibitor asymmetrical dimethylated arginine (ADMA). In the present study, we show that dimethylarginine dimethylaminohydrolase (DDAH), the key regulator of ADMA levels, is downregulated in experimental pulmonary hypertension. In untreated animals, endothelial dysfunction, pulmonary vascular pruning, and right heart dysfunction were associated with reduced nitric oxide. We show a mechanistic link between cAMP-increasing agents and the restoration of endothelial function in progressive pulmonary hypertension. Administration of the phosphodiesterase 3/4 inhibitor tolafentrine led to an increase in the expression of DDAH2 in endothelial cells via a protein kinase A–dependent activation of the DDAH2 promoter. This resulted in decreased ADMA levels and subsequent increased nitric oxide production. In addition, this cAMP-elevating agent prevented vascular pruning and decreased right heart hypertrophy. Prostanoids are one mainstay of the treatment of pulmonary hypertension that operate mainly via elevation of cAMP and subsequent downstream signaling. However, their clinical utility is hampered in part by their immanent side effect profile and/or the route of administration (eg, inhaled, subcutaneous, intravenous). Thus, phosphodiesterase 3/4 inhibitors could represent an independent new class of drugs that warrant further investigation in pulmonary vascular disorders.