Godder K, Vlodavsky I, Eldor A, Weksler B B, Haimovitz-Freidman A, Fuks Z
Department of Radiation Oncology, Memorial-Sloan Kettering Cancer Center, New York, New York 10021.
J Cell Physiol. 1991 Aug;148(2):274-80. doi: 10.1002/jcp.1041480213.
This study was undertaken to identify a heparan sulfate (HS) degradation endoglycosidase (heparanase) in cultured endothelial cells (EC) and to characterize the requirements for its release and subsequent degradation of HS side chains in the subendothelial extracellular matrix (ECM). Intact EC, EC lysates, or EC conditioned media from different sources were incubated with metabolically Na2(35)SO4-labeled ECM produced by bovine EC. The released sulfated products were analyzed by gel filtration on Sepharose 6B. Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) lysates expressed heparanase activity as indicated by release of most of the radioactivity from ECM as HS fragments that are one-fifth to one-sixth the size of the intact HS side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin. Rabbit coronary microvascular EC and bovine brain capillary EC lysates showed less heparanase activity (30-35%), whereas bovine aortic and corneal EC showed no activity. Intact HUVEC, plated directly on the labeled ECM, expressed low enzyme activity that was not changed when cells were exposed to various agents. Exposure of HUVEC to interleukin-1, phorbol myristate acetate, tumor necrosis factor, endotoxin, thrombin, calcium ionophore A23187, fibroblast growth factor, or radiation did not induce release of the enzyme to the medium or degradation of HS in the ECM, as long as the cells remained viable. EC differ from various normal and malignant cells that degrade HS by virtue of their inability to release the enzyme. We suggest that heparanase release during vessel wall injury may regulate the growth of EC and smooth muscle by release of HS degradation products in processes such as wound healing, neovascularization, and atherosclerosis.
本研究旨在鉴定培养的内皮细胞（EC）中的硫酸乙酰肝素（HS）降解内切糖苷酶（乙酰肝素酶），并确定其释放以及随后对内皮细胞外基质（ECM）中HS侧链进行降解的条件。将来自不同来源的完整EC、EC裂解物或EC条件培养基与经牛EC代谢产生的Na2(35)SO4标记的ECM一起孵育。通过在琼脂糖6B上进行凝胶过滤分析释放的硫酸化产物。人脐静脉内皮细胞（HUVEC）和人隐静脉内皮细胞（HSVEC）裂解物表现出乙酰肝素酶活性，这表现为ECM中大部分放射性以HS片段形式释放，这些片段的大小是完整HS侧链的五分之一到六分之一。这些片段对亚硝酸脱氨敏感，并且在肝素存在的情况下不会产生。兔冠状动脉微血管EC和牛脑毛细血管EC裂解物表现出较低的乙酰肝素酶活性（30 - 35%），而牛主动脉和角膜EC则无活性。直接接种在标记的ECM上的完整HUVEC表现出低酶活性，当细胞暴露于各种试剂时该活性没有变化。只要细胞保持存活，将HUVEC暴露于白细胞介素-1、佛波酯肉豆蔻酸酯乙酸酯、肿瘤坏死因子、内毒素、凝血酶、钙离子载体A23187、成纤维细胞生长因子或辐射，均不会诱导该酶释放到培养基中或ECM中HS的降解。EC与各种通过降解HS的正常和恶性细胞不同，因为它们无法释放该酶。我们认为，在血管壁损伤期间乙酰肝素酶的释放可能通过在伤口愈合、新生血管形成和动脉粥样硬化等过程中释放HS降解产物来调节EC和平滑肌的生长。
