Tumor necrosis factor-α and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells
Tumor necrosis factor-α and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells
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DOI：
10.1046/j.1523-1755.1999.00473.x
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发表时间：
1999-06-01
影响因子：
19.6
通讯作者：
Pfeilschifter, J
中科院分区：
文献类型：
--
作者：
Messner, UK;Briner, VA;Pfeilschifter, J
背景肾小球内皮细胞是一种特殊的微血管细胞类型，参与肾小球超滤的调节。在革兰氏阴性脓毒症、肾小球肾炎和急性肾衰竭期间，细菌脂多糖（LPS）和肿瘤坏死因子-α（TNF-α）可引起严重的细胞损伤。我们的目的是研究和比较TNF-α和LPS对牛肾小球内皮细胞凋亡诱导的直接影响。用TNF-α或LPS刺激原代牛肾小球内皮细胞，通过DNA片段分析、形态学研究、细胞色素c流出和线粒体通透性转换、巴克、Bad、Bar、Bcl-2、Bcl-x（L）蛋白表达和caspase-3样蛋白酶活性的测量来研究细胞凋亡。TNF-α，以及LPS，引起凋亡细胞死亡的时间和浓度依赖性。沿着DNA梯状条带的形成，我们检测到50 kbp高分子量DNA片段的形成、核浓缩和线粒体通透性的转变。关于所有参数，LPS信号传导被证明比TNF-α更快。从机制上讲，TNF-α诱导的细胞死亡之前，线粒体细胞色素c流出到胞质溶胶中，随后，促凋亡蛋白巴克显著增加，抗凋亡Bcl-x（L）蛋白含量减少。用LPS观察到相当但更显著的效果。后来，caspase-3样蛋白酶活性在10小时后首次检测到，并在TNF-α和LPS刺激的细胞中持续增加至24小时。相应地，我们检测到一个扩展的裂解的核酶聚（ADP-核糖）聚合酶。半胱天冬酶抑制剂Z-Asp-CH 2-DCB和Z-VAD-fcB以类似的方式阻断TNF-α和LPS诱导的细胞凋亡。只有Z-Asp-CH 2-DCB能完全阻断凋亡细胞的死亡。细菌LPS和TNF-α均能有效诱导肾小球内皮细胞凋亡。因此，直接内毒素诱导的细胞凋亡可能与急性肾衰竭的进展有关，急性肾衰竭是革兰氏阴性脓毒症的常见并发症。
Background. The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration. During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) may cause severe cell damage. Our aim was to study and compare the direct effects of TNF-alpha and LPS on the induction of apoptosis in bovine glomerular endothelial cells.Methods. Primary bovine glomerular endothelial cells were stimulated with TNF-alpha or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bar, Bcl-2, Bcl-x(L) protein expression, and caspase-3-like protease activity.Results. TNF-alpha, as well as LPS, elicited apoptotic cell death both time and concentration dependently. Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition. Concerning all parameters, LPS signaling proved to be more rapid than TNF-alpha. Mechanistically, TNF-alpha-induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-x(L) protein content. Comparable but more pronounced effects were seen with LPS. Later, caspase-3-like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-alpha- and LPS-stimulated cells. Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase. Caspase inhibitors Z-Asp-CH2-DCB and Z-VAD-fmk blocked both TNF-alpha- and LPS-induced apoptosis in a comparable manner. Only Z-Asp-CH2-DCB was able to block apoptotic cell death completely.Conclusion. Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis.