Involvement of asymmetric dimethylarginine and Rho kinase in the vascular remodeling in monocrotaline-induced pulmonary hypertension
Graphical abstract
Introduction
Pulmonary hypertension (PH) is a serious condition characterized by the elevated pulmonary arterial pressures. Although the pathogenesis of PH has not been fully understood, it is well accepted that vascular remodeling leading to the stegnosis and stiffness of pulmonary artery accounts for the elevated pressure, and the proliferation and migration of vascular smooth muscle cells (VSMCs) play a major role in vascular remodeling (Dahal et al., 2010, Lykouras et al., 2008).
Rho kinase (ROCK), a downstream kinase of Rho GTPases family, plays an important role in cell motility, angiogenesis, proliferation and migration (Chapados et al., 2006). The baseline activity of ROCK is important for fetal lung development and vascular adaptation to the changes in oxygen levels (McMurtry et al., 2003). However, over activation of ROCK in the lung may lead to vascular remodeling and contribute to the development of PH (Guilluy et al., 2009, Oka et al., 2008, Tawara et al., 2007). It has been shown that activation of ROCK pathway is involved in endothelin-1 (ET-1) or angiotensin II (Ang II)-induced VSMCs proliferation because the effect of ET-1 or Ang II was attenuated in the presence of the selective inhibitor of ROCK, fasudil or Y27632 (Kanda et al., 2005). Recently, a clinical study revealed that the expression of ROCK (ROCK1 and ROCK2)and its activity in isolated lung tissues were significantly increased in PH patients compared with the controls (Do e et al., 2009, Guilluy et al., 2009). These results support the important role of ROCK in vascular remodeling and PH development.
Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, can competitively inhibit the activity of NOS and decrease the synthesis of NO (Pope et al., 2009). There is growing evidence that endogenous ADMA is emerging as a novel risk factor contributing to vascular dysfunction and the elevation of ADMA level is associated with the development of many cardiovascular diseases (Boger, 2003, Boger et al., 2009, Duckelmann et al., 2007). There are reports that the plasma levels of ADMA were elevated in patients with chronic thromboembolic pulmonary hypertension or with systemic sclerosis-related pulmonary hypertension (Dimitroulas et al., 2008, Skoro-Sajer et al., 2007), indicating that ADMA is closely related to the development of PH. In vivo, ADMA is in part cleared by renal excretion whereas most of ADMA clearance is dependent on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to l-citrulline and dimethylamine (Teerlink, 2005). Accumulating evidence suggests that decrease of DDAH activity is a key factor contributing to the elevation of ADMA level under many pathophysiological conditions (Sasaki et al., 2007, Wilcken et al., 2007, Yuan et al., 2007). Recent studies have shown that the plasma level of ADMA was significantly increased accompanied by the decreased DDAH activity in PH (Arrigoni et al., 2003, Pullamsetti et al., 2005), and ADMA was able to regulate pulmonary endothelial cells mobility through increasing ROCK activity (Wojciak-Stothard et al., 2007). We therefore hypothesize that the increased ADMA in PH may have an important role in regulating the proliferation and migration of VSMCs through activation of ROCK pathway.
In this study, by using the model of monocrotaline-induced PH, we first evaluate the correlations among PH, ADMA, DDAH and ROCK. Then by using the cultured primary pulmonary artery smooth muscle cells (PASMCs), we examined the effect of ADMA on the proliferation of VSMCs and the possible role of ROCK in mediating the effect of ADMA. Since ADMA has been reported to increase the intracelluar reactive oxygen species (ROS) production in cultured endothelial cells (Bode-Boger et al., 2005, Yuan et al., 2007), therefore, the effect of ADMA on ROS production in PASMCs was also determined.
Section snippets
Animals
Male Sprague–Dawley rats weighting 180–220 g were obtained from Laboratory Animal Center, Xiang-Ya School of Medicine, Central South University, China. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1996) and with the approval of the Central South University Veterinary Medicine Animal Care and Use Committee.
Animal experiments
The animals were either received a single injection
Pulmonary hypertension was induced by monocrotaline
As shown in Fig. 1, the pulmonary artery pressure in monocrotaline group was significantly higher than that in control group (41.5 ± 9.6 Vs 18.9 ± 1.8, n = 8, P < 0.01, Fig. 1A). The ratio of right ventricle (RV) weight to that of left ventricle (LV) + interventricular septum (IVS), a key marker of pulmonary hypertension, was dramatically increased in the monocrotaline group (19 ± 3.82 Vs 10 ± 1.94 %, n = 8, P < 0.01, Fig. 1B). HE staining showed that there was proliferation of PASMCs in the vascular media of
Discussion
In the present study, we explored the correlations among PH, ADMA, DDAH and ROCK in vivo or in vitro. The major findings of this work include 1) in the animal model of monocrotaline-induced PH, the plasma level of ADMA was elevated concomitantly with the increase in ROCK activity and ROCK1 expression, and the decrease in DDAH2 expression in pulmonary arteries; 2) ADMA, in a dose-dependent manner, promoted the proliferation of the primary cultured PASMCs, which was attenuated by ROCK inhibitor;
Acknowledgements
This work was supported by grants from the National Basic Research Program of China (973 Program, No. 2007CB512007), the National Natural Science Foundation of China (No. 30971194; No. 30873061) and the Hunan Innovation Project for Postgraduate (No. CX2009B059).
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