Involvement of asymmetric dimethylarginine and Rho kinase in the vascular remodeling in monocrotaline-induced pulmonary hypertension

Abstract

Recent studies have shown that the plasma level of asymmetric dimethylarginine (ADMA) was increased accompanied by the decreased dimethylarginine dimethylaminohydrolase (DDAH) activity in pulmonary hypertension (PH) and ADMA was able to regulate pulmonary endothelial cells mobility through increasing the activity of Rho kinase (ROCK). This work was conducted to explore the role of ADMA/DDAH pathway in vascular remodeling in PH and the underlying mechanisms. The rat model of PH was established by a single injection of monocrotaline (60 mg/kg, s.c.). The pulmonary arterial pressure, the remodeling of pulmonary artery, the hypertrophy of right ventricle, the plasma levels of ADMA and NO, the expression of DDAH2, ROCK1 or ROCK2 and the ROCK activity were determined. In vitro studies, the pulmonary artery smooth muscle cells (PASMCs) were isolated and cultured. The effect of ADMA on PASMCs proliferation and ROCK activation was investigated. The results showed that the injection of monocrotaline successfully induced PH characterized by the increased pulmonary arterial pressure, vascular remodeling and right ventricle hypertrophy. The plasma level of ADMA was elevated concomitantly with the increased ROCK activity and ROCK1 expression as well as the decreased DDAH2 expression in pulmonary arteries. In the cultured PASMCs, ADMA promoted cellular proliferation accompanied by the increased ROCK1 expression and ROCK activity, which was attenuated by the ROCK inhibitor or by the intracellular antioxidant. These results suggest that ADMA could promote the proliferation of PASMCs through activating ROCK pathway, which may account for, at least partially, the vascular remodeling in monocrotaline-induced PH.

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.