Schnaper H W, Grant D S, Stetler-Stevenson W G, Fridman R, D'Orazi G, Murphy A N, Bird R E, Hoythya M, Fuerst T R, French D L
Laboratory of Developmental Biology, National Institute of Dental Research, Bethesda, Maryland 20892.
J Cell Physiol. 1993 Aug;156(2):235-46. doi: 10.1002/jcp.1041560204.
It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.
有人提出蛋白酶在内皮细胞行为中起重要作用。我们在一个内皮细胞分化的体外模型中研究了明胶酶/IV型胶原酶系统的作用。人脐静脉内皮细胞在基底膜制剂Matrigel上培养时会迅速排列并形成管状网络。培养上清液的酶谱显示有72-kD和92-kD的明胶酶活性；细胞产生大部分的72-kD明胶酶，而92-kD活性完全来自Matrigel。添加抗IV型明胶酶/胶原酶的抗体可减少管状网络的面积。金属蛋白酶组织抑制剂TIMP-1和TIMP-2添加到培养物中时同样会减少管状结构的形成。相反，外源性重组72-kD明胶酶可增加管状形成活性。抗明胶酶抗体和TIMP的作用不是相加的。当在培养开始时添加抗体或TIMP时，抑制作用最大，这表明蛋白酶活性在形态发生的早期步骤中很重要。然而，细胞在Matrigel上培养并不会增加72-kD明胶酶mRNA的早期表达。在测定过程中，该酶的信使RNA表达实际上会下降，而TIMP的mRNA转录会增加，这进一步支持了胶原酶促进管状形成早期事件的概念。这些数据表明明胶酶/IV型胶原酶活性在Matrigel上的内皮细胞形态发生中很重要，并提示胶原酶在体内新毛细血管形成中起作用。
