Glial‐cell‐mediated re‐induction of the blood‐brain barrier phenotype in brain capillary endothelial cells: A differential gel electrophoresis study

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood‐brain barrier (BBB) models. Hence, the re‐induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re‐induction remain largely unknown. Here, we used a DIGE‐based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re‐induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re‐induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by‐product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re‐induced BBB functions than in cells with limited BBB functions.     

检测瓜氨酸和非对称二甲基精氨酸的化学发光方法

非对称二甲基精氨酸（ADMA）是内源性一氧化氮合酶抑制剂,被公认是一种与心血管疾病、糖尿病、性功能障碍和肾功能衰竭等多种疾病密切相关的危险因子. 本文通过化学发光法检测瓜氨酸或ADMA经鸟氨酸氨基甲酰转移酶（ArcB）和氨基甲酰磷酸激酶（ArcC）偶联生成的ATP,并对该方法检测的灵敏度和动态范围进行了初步评价.1）从绿脓杆菌中克隆了鸟氨酸氨基甲酰转移酶和氨基甲酰磷酸激酶,转化大肠杆菌实现高效可溶性表达,用镍柱亲和纯化得到融合蛋白,TLC薄层层析法定性和测氨法定量验证了融合蛋白活性.2）用化学发光法检测了瓜氨酸或ADMA经相应酶偶联反应后的产物ATP,并且对实验进行了优化,结果表明两者都能在偶联酶作用下催化释放ATP,相应的底物浓度检测下限为01 μmol/L,检测结果接近正常生理血清中ADMA的浓度,且远低于正常生理血清中瓜氨酸浓度. 用正常尿液样品检测结果表明,该方法可行,为下一步血清和血浆样品的检测奠定了基础.     

ADMA-- 一个新的心血管疾病危险因子

非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)为内源性一氧化氮合酶(endogenous Nitric-Oxide Synthase,eNOS)竞争性抑制剂,可抑制NOS的活性,减少一氧化氮(nitric oxide,NO)的合成,致使内皮功能障碍。近年来研究认为ADMA是冠心病的独立预测因子,与心功能不全及心血管疾病死亡率明显相关。本文将针对ADMA与心血管疾病的相关性进行综述。     

Methionine metabolism and multiple sclerosis

Context: Methylation reactions are particularly important in the brain and their inhibition can lead to a number of serious pathologies. Multiple sclerosis is one of the most common neurological disorders caused by interaction of genetic and environmental factors, but little is known about its cause or factors that contribute to the disorder. Although multiple sclerosis is primarily regarded as demyelinating disorder, there are no many articles focusing on methionine determination.

Objective: The aim of this work was to investigate whether serum methionine and its related compounds like homocysteine, cysteine, glutathione and asymmetric dimethylarginine were changed in multiple sclerosis patients.

Materials and methods: Sulphur-containing compounds were determined by using high-performance liquid chromatography with electrochemical detection in a single run for providing more complex view on methionine metabolism and asymmetric dimetylarginine was measured by a commercial enzyme-linked immunosorbent assay kit.

Results: Methionine and glutathione were decreased, but homocysteine, asymmetric dimethylarginine and cysteine were unchanged in patients with multiple sclerosis compared with controls.

Conclusions: Methionine and glutathione seem to be potential biomarkers for prognosis of the disease.     

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels,VEGF expression,and cell phenotype

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.     

Nitric Oxide Synthesis Metabolites—As Potential Markers in Chronic Kidney Disease in Children

Nitric oxide (NO) is an important signaling molecule for many physiological and pathological processes. Diseases associated with abnormal NO synthesis include cardiovascular diseases, insulin-dependent diabetes, or chronic kidney disease (CKD). The aim of the paper was to evaluate NO synthesis metabolites, i.e., asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), dimethylamine (DMA), arginine, citrulline in plasma of patients with different severity of CKD and to seek possible links between these parameters and the development of this disease. Forty-eight CKD children and thirty-three age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III-stage III, Group IV-stage IV, and Group RRT children on dialysis). To determine the concentrations of the above-mentioned metabolites in plasma liquid chromatography-mass spectrometry was used. There were significant differences observed in levels of ADMA, SDMA, DMA, and citrulline between control vis CKD groups (p values ranging from <0.001 to 0.029). Plasma arginine concentration was also higher in CKD patients compared to the control group but statistically insignificant. ADMA levels in CKD children were statistically significantly higher in relation to particular stages of CKD (RRT vis II stage of CKD: p = 0.01; RRT vis III-IV stages of CKD: p < 0.046). Citrulline levels in CKD children were statistically significantly higher in RRT group vis control (p < 0.001). Children with CKD develop disturbances in most metabolites of NO synthesis. Dialysis children treated show the greatest disturbances of plasma ADMA and citrulline levels. ADMA seems to be a good indicator of the gradual progression of the CKD, which is proved by the negative correlation with eGFR.     

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.     

Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.