Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, Oezatalay D, Uhal B D
The Cardiovascular Institute, Michael Reese Hospital and Medical Center, Chicago, Illinois, USA.
J Cell Physiol. 2000 Nov;185(2):253-9. doi: 10.1002/1097-4652(200011)185:2<253::AID-JCP10>3.0.CO;2-#.
Recent work from this laboratory demonstrated that apoptosis of pulmonary alveolar epithelial cells (AEC) in response to Fas requires angiotensin II (ANGII) generation de novo and binding to its receptor (Wang et al., 1999b, Am J Physiol Lung Cell Mol Physiol 277:L1245-L1250). These findings led us to hypothesize that a similar mechanism might be involved in the induction of AEC apoptosis by TNF-alpha. Apoptosis was detected by assessment of nuclear and chromatin morphology, increased activity of caspase 3, binding of annexin V, and by net cell loss inhibitable by the caspase inhibitor ZVAD-fmk. Purified human TNF-alpha induced dose-dependent apoptosis in primary type II pneumocytes isolated from rats or in the AEC-derived human lung carcinoma cell line A549. Apoptosis in response to TNF-alpha was inhibited in a dose-dependent manner by the nonselective ANGII receptor antagonist saralasin or by the nonthiol ACE inhibitor lisinopril; the inhibition of TNF-induced apoptosis was maximal at 50 microgram/ml saralasin (101% inhibition) and at 0.5 microgram/ml lisinopril (86% inhibition). In both cell culture models, purified TNF-alpha caused a significant increase in the mRNA for angiotensinogen (ANGEN), which was not expressed in unactivated cells. Transfection of primary cultures of rat AEC with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of TNF-stimulated apoptosis by 72% (P < 0.01). Exposure to TNF-alpha increased the concentration of ANGII in the serum-free extracellular medium by fivefold in A549 cell cultures and by 40-fold in primary AEC preparations; further, exposure to TNF-alpha for 40 h caused a net cell loss of 70%, which was completely abrogated by either the caspase inhibitor ZVAD-fmk, lisinopril, or saralasin. Apoptosis in response to TNF-alpha was also completely inhibited by neutralizing antibodies specific for ANGII (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by TNF-alpha requires a functional renin/angiotensin system (RAS) in the target cell. They also suggest that therapeutic control of AEC apoptosis in response to TNF-alpha is feasible through pharmacologic manipulation of the local RAS.
该实验室最近的研究表明，肺泡上皮细胞（AEC）对Fas作出反应时发生的凋亡需要从头生成血管紧张素II（ANGII）并与它的受体结合（Wang等人，1999b，《美国生理学杂志：肺细胞与分子生理学》277：L1245 - L1250）。这些发现使我们推测，类似的机制可能参与肿瘤坏死因子-α（TNF-α）诱导的AEC凋亡。通过评估细胞核和染色质形态、半胱天冬酶3活性增加、膜联蛋白V结合以及半胱天冬酶抑制剂ZVAD - fmk可抑制的净细胞丢失来检测凋亡。纯化的人TNF-α在从大鼠分离的原代II型肺细胞或AEC来源的人肺癌细胞系A549中诱导剂量依赖性凋亡。非选择性ANGII受体拮抗剂沙拉新或非巯基血管紧张素转换酶（ACE）抑制剂赖诺普利以剂量依赖性方式抑制对TNF-α的凋亡反应；在50微克/毫升沙拉新（抑制率101%）和0.5微克/毫升赖诺普利（抑制率86%）时，对TNF诱导凋亡的抑制作用最大。在两种细胞培养模型中，纯化的TNF-α均使血管紧张素原（ANGEN）的mRNA显著增加，而未活化细胞中不表达该mRNA。用针对ANGEN mRNA的反义寡核苷酸转染大鼠AEC原代培养物可使随后TNF刺激的凋亡诱导减少72%（P < 0.01）。在A549细胞培养物中，暴露于TNF-α使无血清细胞外培养基中的ANGII浓度增加5倍，在原代AEC制剂中增加40倍；此外，暴露于TNF-α 40小时导致净细胞丢失70%，半胱天冬酶抑制剂ZVAD - fmk、赖诺普利或沙拉新都可完全消除这种丢失。对ANGII特异的中和抗体也完全抑制对TNF-α的凋亡反应（P < 0.01），但同型匹配的非免疫球蛋白无显著作用。这些数据表明，TNF-α诱导AEC凋亡需要靶细胞中有功能性肾素/血管紧张素系统（RAS）。它们还提示，通过对局部RAS进行药理调控来治疗性控制TNF-α诱导的AEC凋亡是可行的。
