Protein arginine methylation represents a posttranslational modification undertaken by protein arginine methyltransferases (PRMT) that results in production of protein-incorporated omega-NG-monomethylarginine (L-NMMA), asymmetric omega-NG, NG-dimethylarginine (ADMA), or omega-NG, N G-dimethylarginine (SDMA). Free cellular L-NMMA, ADMA and SDMA can be generated via the proteolytic cleavage of intracellular proteins, thereby also affecting methylarginine content in the plasma. Free methylarginines can be cleared from the body by renal excretion. L-NMMA and ADMA, but not SDMA, can be degraded via enzymes called NG, NG dimmethylarginine dimethylaminohydrolase (DDAH).
ADMA is an endogenous inhibitor of nitric oxide synthases (NOS) and a marker of endothelial dysfunction. Increased plasma ADMA levels have been reported in patients with cardiovascular disorders including pulmonary arterial hypertension (PAH), a fatal disease characterized by elevated blood pressure in the pulmonary circulation, due to increased resistance of pulmonary arterioles. The major pathophysiologic hallmark of PAH is pulmonary arterial smooth muscle cell (PASMC) hypertrophy and proliferation, leading to the occlusion of pulmonary arterioles. The interplay between methylarginine synthesis and degradation in vivo, as well as specific alterations to intrapulmonary ADMA levels or distorted generation of ADMA in PAH, however, remains to be elucidated.
In the current study, we hypothesized that methylarginine production and degradation is tissue-specific and that the lung has a significant impact on serum/plasma ADMA levels, possibly leading to endothelial dysfunction observed in PAH. To this end, we sought to address the following specific aims: 1) to develop a novel, HPLC-based method to assess protein-incorporated and free cellular methylarginine content in biological samples, 2) to analyze the tissue-specific methylarginine metabolism in normal subjects, and 3) to analyze the methylarginine content in the lungs of patients with PAH compared with healthy donors.
First, to analyze tissue-specific methylarginine metabolism in the normal physiological state, we performed high performance liquid chromatography (HPLC)-driven assessment of protein-incorporated and free cellular methylarginine levels, together with Western blot analyses of PRMT and DDAH expression, in organs of the cardiovascular system. Our results revealed that pulmonary expression of type I PRMT was correlated with enhanced protein arginine methylation in the lung. Moreover, our studies also revealed that the kidney and the liver provide complementary routes for clearance and metabolic conversion of circulating ADMA.
To address the impact of intrapulmonary ADMA metabolism in pathogenic conditions, we next analyzed lung homogenates of PAH patients. HPLC analysis revealed significantly lower levels of protein-incorporated ADMA in the lungs of PAH patients (n=12), compared with controls (n=10, transplant donors). Western Blot analyses confirmed a significantly decreased content of asymmetrically dimethylated proteins in PAH lungs. The expression of PRMT, in particular PRMT1, was decreased in PAH. Immunohistochemical staining of IPAH and control lungs localized PRMT1 to pulmonary arterial vascular smooth muscle cells (PASMC). Moreover, PRMT1 knockdown in primary PASMC by siRNA technology significantly increased PASMC proliferation.
Our results demonstrate that, in the normal physiological state, methylarginine metabolism by the pulmonary system significantly contributes to circulating methylarginine levels. In pathogenic conditions, protein-incorporated ADMA concentrations do not reflect free cellular levels of ADMA in the lung. This may be explained by the alterations of DDAH activity in the lung, which, consequently, regulate ADMA content in the serum of IPAH patients. In addition, our studies demonstrated a novel regulatory role of PRMT1 in progression of PAH, by the alteration of PASMC proliferation, a major characteristic of PAH. This led to conclusions that protein arginine methylation plays a pivotal role in the pathogenesis of PAH.
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