Fenyves A M, Behrens J, Spanel-Borowski K
Anatomisches Institut, Universität Basel, Switzerland.
J Cell Sci. 1993 Nov;106 ( Pt 3):879-90. doi: 10.1242/jcs.106.3.879.
Endothelial cells are known to undergo transitions in cell shape during long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovine corpus luteum represent constitutively different cell strains cannot automatically be made. For this reason, particular morphological qualities from four of five reported MVEC types were studied. Confluent cultures of MVEC types 1, 3, 4 and 5 were either left untreated or exposed to recombinant bovine interferon-gamma (IFN-gamma; 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were permeabilized with Triton X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filaments, microtubules, E- and N-cadherins as molecules of cell adhesion plaques, and fibronectin filaments were localized by the application of specific antibodies in combination with epifluorescence microscopy. Cells from untreated single cultures uniformly and reproducibly showed an actin cytoskeleton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin filaments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominent circular band of thick microfilament bundles from which short filaments radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vimentin filaments occurred as a dense network constricted to the central area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent in MVEC type 5. Fibronectin assembled into two differently shaped layers at the basal cell side of each MVEC type. Under IFN-gamma treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cadherin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear contour. The upregulation of E-cadherin by IFN-gamma treatment could also be demonstrated by western blotting, which revealed a 135 kDa full-sized molecule and a 95 kDa tryptic fragment characteristic of cadherins. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.
已知内皮细胞在长期培养过程中会发生细胞形态转变。因此，不能想当然地认为最近从牛黄体中分离出的微血管内皮细胞（MVEC）的不同表型代表了组成型不同的细胞株。出于这个原因，我们研究了五种已报道的MVEC类型中四种的特定形态特征。将1型、3型、4型和5型MVEC的汇合培养物要么不进行处理，要么暴露于重组牛干扰素-γ（IFN-γ；200单位/0.5毫升培养基）中3天。在用鬼笔环肽-FITC检测丝状肌动蛋白之前，用多聚甲醛固定的单层细胞用Triton X-100进行通透处理。波形蛋白丝、细胞角蛋白丝、微管、作为细胞粘附斑分子的E-钙粘蛋白和N-钙粘蛋白以及纤连蛋白丝通过应用特异性抗体结合落射荧光显微镜进行定位。来自未处理的单一培养物的细胞一致且可重复地显示出区分特定MVEC类型的肌动蛋白细胞骨架。1型MVEC呈现出一条由细肌动蛋白丝组成的环形带。3型MVEC优先形成了一种星爆状的肌动蛋白模式。4型MVEC主要表现为多边形网络。5型MVEC显示出一条由粗微丝束组成的突出环形带，短丝从该环形带辐射而出。仅在1型MVEC中观察到细胞角蛋白丝。波形蛋白丝在1型MVEC中以密集网络的形式局限于中央区域，而在3型和4型MVEC中则分散分布。在5型MVEC中可以看到一条与微管走向相当的波浪状路径。纤连蛋白在每种MVEC类型的细胞基底侧组装成两层不同形状的层。在IFN-γ处理下，MVEC类型之间的细胞骨架多样性得以维持，但每种MVEC类型对其细胞骨架和纤连蛋白基质都表现出特定的调节。在1型MVEC中检测到抗E-钙粘蛋白标记的上调，显示出线性轮廓的荧光细胞边界。通过蛋白质印迹法也可以证明IFN-γ处理对E-钙粘蛋白的上调作用，该方法揭示了一种135 kDa的全长分子和一种95 kDa的钙粘蛋白特征性胰蛋白酶片段。5型MVEC的抗N-钙粘蛋白标记明显，产生荧光点状细胞边缘。我们的研究支持真正不同的MVEC类型的存在。
