Futagami Seiji, Hiratsuka Tetsuro, Shindo Tomotaka, Hamamoto Tatsuhiko, Horie Akane, Ueki Nobue, Kusunoki Masafumi, Gudis Katya, Miyake Kazumasa, Tsukui Taku, Sakamoto Choitsu
Division of Gastroenterology, Internal Medicine, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.
J Gastroenterol Hepatol. 2008 Dec;23 Suppl 2:S222-8. doi: 10.1111/j.1440-1746.2008.05442.x.
Recent studies have shown that CD40, a key player in angiogenesis and tubular formation, is an extracellular receptor of the heat shock protein 70 (HSP70)-peptide complex in endothelial cells. The aim of the present study was to determine the effect of extracellular HSP70 treatment on CD40L-suppressed apoptosis and CD40L-induced tubular formation in human umbilical vein endothelial cells (HUVEC).
The apoptotic index of CD40L-stimulated HUVEC with or without recombinant human HSP70 was evaluated using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay analysis. Binding of HSP70-peptide complex to CD40 on HUVEC was determined by double-labeling immunofluorescence methods. To evaluate the biological activity of CD40 engagement pretreated with rhHSP70 (0.5, 1 and 3 ng/mL), the extent of new capillary-like networking structure (tubular formation) formation in HUVEC was counted using an Olympus digital camera. Vascular invasion into MNK-28 cell clusters was assessed by counting the number of tubular structures extending from the HUVEC into growth factor-depleted Matrigel. Scores for CD34, HSP70 and CD40L expression levels in gastric cancer tissues were determined by immunostaining.
CD40L stimulation inhibited vincristine-induced apoptosis of HUVEC in a dose-dependent manner. Extracellular HSP70 treatment significantly blocked the inhibition of apoptosis by CD40L in HUVEC exposed to vincristine. HSP70-peptide complex bound to CD40 on HUVEC. Extracellular HSP70 treatment also significantly reduced CD40L-induced tubular formation in a dose-dependent manner. HSP70 treatment also suppressed invasive tubular formation into MKN-28 cells clusters by CD40L-activated HUVEC. There was a significant relationship between CD40L expression levels and microvessel density; however, the relationship between HSP70 expression level and microvessel density in gastric cancer tissues was not significant.
Extracellular HSP70 treatment blocks CD40L inhibition of apoptosis and CD40L induction of tubular formation in HUVEC.
近期研究表明，CD40作为血管生成和管状结构形成的关键因子，是内皮细胞中热休克蛋白70（HSP70）-肽复合物的细胞外受体。本研究旨在确定细胞外HSP70处理对人脐静脉内皮细胞（HUVEC）中CD40L抑制的细胞凋亡和CD40L诱导的管状结构形成的影响。
使用末端脱氧核苷酸转移酶生物素-dUTP缺口末端标记分析评估有无重组人HSP70时CD40L刺激的HUVEC的凋亡指数。采用双标记免疫荧光法测定HSP70-肽复合物与HUVEC上CD40的结合。为评估经rhHSP70（0.5、1和3 ng/mL）预处理的CD40参与的生物学活性，使用奥林巴斯数码相机对HUVEC中新的毛细血管样网络结构（管状结构形成）的形成程度进行计数。通过计算从HUVEC延伸到生长因子耗尽的基质胶中的管状结构数量来评估血管向MNK-28细胞簇的侵袭。通过免疫染色确定胃癌组织中CD34、HSP70和CD40L表达水平的评分。
CD40L刺激以剂量依赖方式抑制长春新碱诱导的HUVEC细胞凋亡。细胞外HSP70处理显著阻断了CD40L对暴露于长春新碱的HUVEC细胞凋亡的抑制作用。HSP70-肽复合物与HUVEC上的CD40结合。细胞外HSP70处理也以剂量依赖方式显著减少了CD40L诱导的管状结构形成。HSP70处理还抑制了CD40L激活的HUVEC向MKN-28细胞簇的侵袭性管状结构形成。CD40L表达水平与微血管密度之间存在显著关系；然而，胃癌组织中HSP70表达水平与微血管密度之间的关系不显著。
细胞外HSP70处理可阻断CD40L对HUVEC细胞凋亡的抑制作用以及CD40L对管状结构形成的诱导作用。
