Dept. of Pharmacology & Toxicology, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 638, Little Rock, AR 72205, USA.
Am J Physiol Heart Circ Physiol. 2011 Jun;300(6):H1997-2004. doi: 10.1152/ajpheart.01311.2010. Epub 2011 Apr 1.
End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.
终末期肾病是慢性肾脏病的终末期，与心血管疾病的高发率有关。心血管疾病通常是由尿素衍生的异氰酸酯修饰 LDL（cLDL）引起的内皮损伤引起的。我们之前的数据表明，cLDL 诱导有丝分裂原激活蛋白激酶依赖性有丝分裂 DNA 片段化和细胞死亡。然而，该途径的机制尚不清楚。本研究表明，cLDL 诱导的内皮有丝分裂细胞死亡不依赖于半胱天冬酶-3。内切核酸酶 G（EndoG）的表达，即涉及半胱天冬酶非依赖性 DNA 片段化的核酸酶，在细胞暴露于 cLDL 时显著增加。通过 RNAi 抑制 EndoG 可保护 cLDL 诱导的 DNA 片段化，而 EndoG 的过表达可诱导内皮细胞中更多的 DNA 片段化。从野生型（WT）和 EndoG 敲除（KO）小鼠分离的原代内皮细胞的离体实验表明，与 WT 细胞相比，EndoG KO 细胞对 cLDL 毒性有部分保护作用。为了确定 cLDL 在体内的毒性，我们将 cLDL 或天然 LDL（nLDL）静脉内给予 WT 和 EndoG KO 小鼠，然后使用流式细胞术测量血液中的漂浮内皮细胞。结果表明，与 nLDL 注射相比，WT 小鼠注射 cLDL 后漂浮内皮细胞的数量增加，但 EndoG KO 小鼠中则没有。最后，MEK-ERK1/2 和 JNK-c-jun 途径的抑制剂减少了 cLDL 诱导的 EndoG 过表达和 DNA 片段化。总之，我们的数据表明，cLDL 诱导的内皮毒性不依赖于半胱天冬酶，而是由依赖于 EndoG 的 DNA 片段化引起的。
