Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands.
Atherosclerosis. 2012 Mar;221(1):48-54. doi: 10.1016/j.atherosclerosis.2011.11.032. Epub 2011 Nov 28.
We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.
Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).
SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.
我们之前已经证明，同型半胱氨酸（Hcy）可诱导人内皮细胞中的磷脂酰丝氨酸（PS）暴露、凋亡和坏死。由于 S-腺苷同型半胱氨酸（SAH）被认为是 Hcy 诱导心血管疾病发病机制的主要致病因素，因此我们在此评估内皮细胞中 Hcy 的细胞毒性作用是否也依赖于 SAH。
将人脐静脉内皮细胞（HUVEC）暴露于以下条件：（1）未经处理的对照（导致细胞内 SAH 为 2.8 nM，细胞外 l-Hcy 为 3.1 μM）；并孵育于（2）50 μM 腺苷-2,3-二醛（ADA；导致细胞内 SAH 为 17.7 nM，细胞外 l-Hcy 为 3.1 μM），（3）2.5 mM Hcy（导致细胞内 SAH 为 20.9 nM，细胞外 l-Hcy 为 1.8 mM）和（4）1、10 和 100 μM SAH。然后，我们确定了处理对内质网 PS 阳性率、半胱天冬酶-3 活性、细胞色素 c 释放（亚）细胞表达的 NOX2、NOX4、p47(phox)和硝基酪氨酸以及 H2O2 的影响。Hcy 和 ADA 均可显著增加 PS 暴露（n=5）、半胱天冬酶-3 活性（n=6）和细胞色素 c 释放（n=3）。单独孵育细胞外 SAH 不会影响细胞活力。Hcy 和 ADA 还诱导了类似的核 NOX2 和（核周）NOX4 的增加，与（核周）p47(phox)表达和局部活性氧物种（ROS）（n=3）同时发生。 flavoenzyme 抑制剂二苯基碘（DPI）抑制 NOX 介导的 ROS 可显著降低细胞凋亡诱导（n=3）和 ROS 产生（n=3）。
SAH 可诱导内皮细胞中的 PS 暴露和凋亡，而与 Hcy 无关。因此，我们的研究表明，在所使用的细胞模型中，Hcy 介导的内皮功能障碍主要归因于 SAH 的积累。
