Wagener F A, da Silva J L, Farley T, de Witte T, Kappas A, Abraham N G
Department of Pharmacology, New York Medical College, Valhalla, New York, USA.
J Pharmacol Exp Ther. 1999 Oct;291(1):416-23.
Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme-induced ICAM-1 expression. Endothelial cells pretreated with a glutathione precursor, N-acetylcysteine, or glutathione ester, showed a decrease in heme-induced ICAM-1 expression of 37 and 44%, respectively, suggesting that the mechanism of ICAM-1 induction by heme may be partly dependent on the levels of antioxidant. It is possible that amelioration of the heme-induced oxidative stress and expression of ICAM-I is due, in part, to the induction of HO-1 activity. Regulation of HO activity in this manner may have clinical applications.
血红素加氧酶（HO）通过将血红素分解为胆色素，下调细胞中的血红素蛋白、血红蛋白和血红素；后者会产生包括自由基在内的促氧化产物。已描述了两种HO同工酶，它们是不同基因的产物；HO-1是可诱导的同工型，而HO-2被认为是组成性表达的。我们研究了几种金属化合物（氯化钴、中卟啉锡和血红素）对HO活性的诱导作用。此外，我们研究了血红素在内皮细胞中产生的黏附分子表达实验模型中HO-1的表达，以及HO-1表达与诱导的黏附分子之间的关系。流式细胞术分析表明，血红素以浓度（10 - 100 microM）和时间（1 - 24小时）依赖性方式诱导人脐静脉内皮细胞中细胞间黏附分子1（ICAM-1）的表达。用HO活性抑制剂中卟啉锡预处理导致血红素诱导的ICAM-1表达增加2倍。相反，氯化钴诱导的HO使血红素诱导的ICAM-1表达降低33%。为了研究HO-1和HO-2对内皮细胞HO活性的贡献，测试了每种同工型的特异性反义寡核苷酸（ODN）对暴露于血红素的细胞中抑制HO活性的特异性。暴露于血红素的内皮细胞HO活性增加，而HO-1反义ODN可阻止（70%）这种增加。HO-2反义ODN抑制血红素诱导的HO活性达21%。添加HO-1反义ODN可阻止血红素降解并导致微粒体血红素升高。蛋白质印迹分析表明，HO-1反义ODN选择性抑制HO-1蛋白而不抑制HO-2蛋白。用HO-1反义寡核苷酸孵育内皮细胞可增强血红素依赖性的ICAM-1增加。相反，向内皮细胞中添加HO-2反义寡核苷酸不会增加黏附分子。研究了重要抗氧化剂谷胱甘肽对血红素诱导的ICAM-1表达的作用。用谷胱甘肽前体N-乙酰半胱氨酸或谷胱甘肽酯预处理的内皮细胞显示，血红素诱导的ICAM-1表达分别降低37%和44%，这表明血红素诱导ICAM-1的机制可能部分取决于抗氧化剂水平。血红素诱导的氧化应激和ICAM-1表达的改善可能部分归因于HO-1活性的诱导。以这种方式调节HO活性可能具有临床应用价值。
