Role of endothelial cells for nitric oxide production in periapical lesions

本研究旨在阐明内皮细胞在根尖周病变中产生一氧化氮的作用。分析根尖周渗出物和根尖周肉芽肿，并检测诱导型一氧化氮合酶（iNOS）和血管内皮（VE-）钙粘蛋白。从根尖周渗出液中分离的主要细胞是多形核白细胞（PMN），使用苏木精-伊红染色进行形态学测定。通过免疫细胞化学和RT-PCR分析证实了iNOS的蛋白表达和基因表达。双色免疫组化显示iNOS表达细胞为巨噬细胞、淋巴细胞、成纤维细胞、中性粒细胞和内皮细胞，而VE-钙粘蛋白表达细胞仅为内皮细胞。因此，内皮细胞共表达iNOS和VE-钙粘蛋白。真实的时间PCR检测根尖肉芽肿组织中iNOS和VE-cadherin mRNA的表达。iNOSmRNA的表达高于VE-cadherin的表达。在我们的体外研究中，人脐静脉内皮细胞（HUVEC）用白细胞介素-1和大肠杆菌衍生的LPS刺激2- 24小时。真实的时间PCR结果显示，诱导型一氧化氮合酶（iNOS）和VE-钙粘素（VE-cadherin）mRNA在刺激后的HUVEC中表达。VE-cadherin mRNA的表达水平高于iNOS。我们还使用HUVEC在体外研究中检查了iNOS抑制剂。诱导型一氧化氮合酶抑制剂可降低诱导型一氧化氮合酶mRNA的表达水平，从而证明了其药理学应用的可能性。这些数据与提示根尖周肉芽肿内皮细胞VE-钙粘蛋白表达与一氧化氮相关的假设一致。

The purpose of this study was to elucidate the role of endothelial cells for nitric oxide production in periapical lesions. Periapical exudates and periapical granulomas were analyzed, and inducible nitric oxide synthase (iNOS) and vascular endothelial (VE-) cadherin were examined in this study. Predominant cells isolated from periapical exudates were polymorphonuclear leukocytes (PMNs), as determined morphologically using hematoxylin-eosin stains. Protein expression and gene expression of iNOS was confirmed by immunocytochemical and RT-PCR analyses, respectively. Two-color immunohistochemistry using cryostat sections of periapical granulomas demonstrated that iNOS-expressing cells were macrophages, lymphocytes, fibroblasts, PMNs and endothelial cells whereas VE-cadherin-expressing cells were only endothelial cells. Thus, endothelial cells co-expressed iNOS and VE-cadherin. Real time PCR demonstrated the gene expression of iNOS and VE-cadherin mRNA in periapical granulomas. The expression levels of iNOS mRNA were higher than those of VE-cadherin. In our in-vitro study, human umbilical vein endothelial cells (HUVEC) were stimulated with interleukin-1 and Escherichia coli derived LPS for 2-24hr. Real time PCR demonstrated that iNOS and VE-cadherin mRNA was expressed in stimulated HUVEC. In addition, the expression levels of VE-cadherin mRNA were higher than that of iNOS. We also examined iNOS inhibitors in in-vitro study using HUVEC. The iNOS inhibitors decreased the expression levels of iNOS mRNA, therefore the possibility of pharmacological application was demonstrated. The data are consistent with a hypothesis suggesting that VE-cadherin expression of endothelial cells in periapical granulomas could be controlled in the association with nitric oxide.