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Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension

Soni Pullamsetti^*, Ladislau Kiss^*, Hossein Ardeschir Ghofrani^*, Robert Voswinckel^*, Peter Haredza^*, Walter Klepetko, Clemens Aigner, Ludger Fink, Jai Prakash Muyal, Norbert Weissmann^*, Friedrich Grimminger^*, Werner Seeger^* and Ralph Theo Schermuly^*,1

^* Department of Internal Medicine, Justus-Liebig-University Giessen, Giessen, Germany;
Department of Cardiothoracic Surgery, University of Vienna, Vienna, Austria; and
Department of Pathology, Justus-Liebig-University Giessen, Giessen, Germany

¹ Correspondence: Medizinische Klinik, Klinikstrasse 36, Giessen 35392, Germany. E-mail: ralph.schermuly{at}innere.med.uni-giessen.de

SPECIFIC AIMS

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and life-limiting disorder which is associated with impaired bioactivity and/or synthesis of endogenous nitric oxide (NO). The mechanisms resulting in this impairment are multifactorial. Recently, the impact of endogenous NO-synthase inhibitors such as dimethylarginines (ADMA and SDMA) has come into the focus of attention. However, the precise mechanisms responsible for the biosynthesis and metabolism of these mediators are not yet completely understood. The aim of our study was to investigate the role of protein arginine methylation and/or alterations of the metabolizing enzyme DDAH in the course of chronic pulmonary hypertension. The studies were performed mainly on human lung tissue from patients suffering from IPAH as well as on tissue from pulmonary hypertensive rats.

PRINCIPAL FINDINGS

1. Elevated plasma concentrations of ADMA and SDMA in patients with IPAH and chronic PHT rat model
Free asymmetric and symmetric dimethylarginine (ADMA and SDMA) levels were assessed by HPLC in the serum of patients with IPAH and in that of monocrotaline (MCT) -induced pulmonary hypertensive rats. These results were compared with the levels measured in matched healthy controls. Compared with healthy subjects, free dimethylarginine concentrations were markedly increased (ADMA, 2.2-fold, P<0.01 and SDMA, 2.7-fold, P<0.05) in patients with IPAH. The human results were corroborated by the findings in pulmonary hypertensive rats in which plasma levels were elevated similarly (ADMA, 4.3-fold, P<0.001 and SDMA, 6.7-fold, P<0.001).

2. Increased biosynthesis of ADMA and SDMA in lungs from IPAH and chronic PHT rats
Since ADMA and SDMA are produced from the proteins that are methylated on arginine residues, relative changes in protein arginine methylation were determined using immunohistochemistry (IHC) and dot blot analyses. IHC staining of donor lungs showed localization of ADMA and SDMA proteins in all compartments of the lung, with a negligible difference in their overall distribution. In contrast, in lung tissue from patients with IPAH, ADMA, and SDMA, immunoreactivity was preferentially increased in the pulmonary vascular endothelium in areas of intimal fibrotic and plexiform lesions (Fig. 1 A). Differences were quantified using dot blot analysis. The relative abundance of ADMA and SDMA protein was increased 2.4- and 2.7-fold, respectively, in IPAH compared with donor lungs (Fig. 1B, C ; P<0.01). Likewise, in IPAH, lungs from pulmonary hypertensive rats showed an increased immunoreactivity and relative abundance (2.2- and 2.7-fold, respectively; P<0.001) of ADMA and SDMA protein compared with controls.

View larger version (63K): [in this window] [in a new window]   Figure 1. Increased biosynthesis of ADMA and SDMA in lungs from patients with IPAH. A) ADMA and SDMA immunoreactivity in lung sections from healthy donors and patients with IPAH. Lung section from a healthy donor with ADMA and SDMA immunoreactivity in pulmonary arterial endothelial cells and in alveolar epithelial cells. Lung sections from patients with IPAH: ADMA and SDMA immunoreactivity in the endothelial layer of pulmonary arteries. , endothelium; , epithelium. Scale bar: 100µm; PA: pulmonary artery. B) Expression of dimethylated arginine proteins in lung homogenates from healthy donors (n=7) and patients with IPAH (n=7). Dot blot analysis was performed with anti-ADMA and anti-SDMA antibodies. C) Quantification of the symmetric and asymmetric dimethylated proteins in each group. Values are expressed as percentage of expression found in donor tissue. **P<0.01 vs. donor (n=7 each).

3. Change in expression of DDAH isoforms in IPAH lungs
A family of enzymes that metabolizes ADMA levels is the dimethylarginine-dimethyl-amino-hydrolases (DDAH) which hydrolyze ADMA to L-citrulline and dimethylamine. To date, two isoforms of DDAH with a distinct tissue distribution (DDAH1 and DDAH2) have been found in mammals. To investigate alterations of the metabolism of ADMA levels in IPAH patients and in pulmonary hypertensive rats, localization and expression levels of DDAH isoforms were determined by IHC, real-time PCR, and Western blot analysis.

IHC staining of healthy donor lungs showed strong preferential localization of DDAH isoforms in the endothelium of all generations of arteries, with virtually no expression of DDAH1 and DDAH2 in either vascular smooth muscle or adventitial cells. When compared with healthy donor lungs, a marked reduction or even absence of DDAH2 immunoreactivity was observed in the endothelium of sections from patients with IPAH. In contrast, no significant difference was observed in DDAH1 immunostaining intensity between IPAH and healthy donor-lungs (Fig. 2 A).

View larger version (55K): [in this window] [in a new window]   Figure 2. Change in expression of DDAH isoforms in lungs from patients with IPAH. A) DDAH1 and DDAH2 immunoreactivity in lung sections from healthy donors and patients with IPAH. Lung sections from a healthy donor with a moderate to strong DDAH1 and DDAH2 immunoreactivity in pulmonary arterial endothelial cells and alveolar epithelial cells. Lung sections from patients with IPAH: DDAH1 immunoreactivity appears even stronger, in endothelial and alveolar epithelial cells. No DDAH2 immunostaining detected in endothelial layer of pulmonary arteries of IPAH patients lung sections. , endothelium; , epithelium. Scale bar: 100µm; PA: pulmonary artery. B) Expression of DDAH1 and DDAH2 in lung homogenates from patients with IPAH (n=7) and healthy donors (n=7). Western blot analysis was performed with anti-DDAH1 and anti-DDAH2 antibodies. The specific antibodies recognize protein at a molecular mass of 34 and 42 kDa. C) Quantification of the DDAH1 and DDAH2 signal in each group. Values are expressed as percentage of expression found in donor tissue. ***P< 0.001 vs. donor.

Real time PCR analysis of DDAH isoforms revealed a significant reduction in DDAH2/GAPDH expression compared with DDAH1/GAPDH. Accordingly, Western blot analysis showed a significant down-regulation of DDAH2/GAPDH (77%, P<0.001) as opposed to DDAH1/GAPDH (17%, P<0.001) in IPAH lungs compared with healthy donor lungs (Fig. 2B, C ).

Results seen in human tissue were corroborated by the findings in tissue of pulmonary hypertensive rats. DDAH2 expression at mRNA and protein levels was reduced compared with DDAH1. A notable inhibition of DDAH function, measured by activity assay, was found in the lung tissue of chronically ill animals (79% vs. controls, P<0.001).

CONCLUSIONS AND SIGNIFICANCE

An increase in levels of ADMA, an endogenous inhibitor of nitric oxide synthases (Fig. 3 A), is known to contribute to the pathogenesis and progression of various cardiovascular diseases associated with endothelial dysfunction. Marked elevation in plasma ADMA levels was noted in IPAH patients and chronic PHT rats. Such enhanced ADMA levels may well result in substantial NOS inhibition, and it is in line with this reasoning that pulmonary endothelial dysfunction was previously noted in response to MCT treatment (Fig. 3B ). Plasma levels of SDMA were also found to be markedly increased in IPAH patients and chronic PHT rats. The role of SDMA in vascular dysfunction is not yet clear; however, since SDMA is an inhibitor of the human cationic amino acid transporter hCAT-2B, it may indirectly inhibit NO synthesis by interfering with arginine uptake. Thus, increases in SDMA levels can synergize with enhanced ADMA levels in causing endothelial NOS inhibition. As a consequence of this inhibitory pathway, prolonged pulmonary vasoconstriction and lung vascular remodeling in pulmonary hypertension can be provoked and maintained. As demonstrated by dot blot and immunohistochemistry analyses, asymmetric and symmetric dimethylated arginine proteins are highly expressed in lung tissues from patients suffering from idiopathic pulmonary arterial hypertension as well as from PHT rats. The sources of asymmetric and symmetric dimethylated arginines are protein arginine N-methyltransferases (PRMTs) which were previously shown to be up-regulated in response to oxidative stress in human endothelial cells. Moreover, the main endothelial ADMA metabolizing enzyme, DDAH2, was found to be drastically reduced in IPAH patients and PHT rat tissue, as shown on the mRNA, protein level, and by immunohistochemistry in this study. This loss of DDAH2 activity may result in local (intracellular) ADMA levels and may even surpass those in the blood compartment, thereby aggravating the suppression of endothelial NO formation. This notion is in line with previous experimental studies in hypoxic lungs, where reduced DDAH expression was also observed. However, the present study demonstrates for the first time that idiopathic pulmonary arterial hypertension is associated with reduced pulmonary expression of the ADMA-metabolizing enzyme DDAH2. The molecular mechanisms underlying DDAH suppression in pulmonary hypertension remain to be elucidated, but may include the impact of oxidative stress, viral infection, and the nuclear receptor PPAR on the transcriptional and translational control of DDAH.

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram describing a central role of ADMA, SDMA, and DDAH in the pathogenesis of pulmonary hypertension. A) Overview explaining basic mechanism of NO mediated vasodilation in physiological conditions. B) Overview showing that ADMA and SDMA levels elevation is due to increased dimethylation and decreased metabolism by DDAH, thus causing endothelial NOS impairment in pathophysiology of pulmonary hypertension.

The consistency of results seen in human disease and in an established experimental animal model of pulmonary hypertension strongly suggests that changes in dimethylarginine regulation may contribute considerably to the course of the disease. The current study might stimulate development of novel therapeutics which target the signaling pathway of endogenous NOS inhibitors and promote the functional capacity of DDAH.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3223fje; doi: 10.1096/fj.04-3223fje

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