Patton W F, Yoon M U, Alexander J S, Chung-Welch N, Hechtman H B, Shepro D
Biological Science Center, Boston University, Massachusetts 02215.
J Cell Physiol. 1990 Apr;143(1):140-9. doi: 10.1002/jcp.1041430119.
Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.
通过二维凝胶电泳对牛主动脉、肺动脉和肺微血管内皮细胞以及血管平滑肌细胞和视网膜周细胞的多肽进行了评估。所有这些细胞类型中的主要细胞骨架蛋白是肌动蛋白、波形蛋白、原肌球蛋白和微管蛋白。培养的肺微血管内皮细胞还表达了12种独特的多肽，包括一种41kd的酸性I型和两种52kd的碱性II型简单上皮细胞角蛋白的同工型。简单上皮细胞角蛋白在肺微血管内皮细胞中的表达在视网膜衍生生长因子存在或不存在的情况下，以及无论细胞是在明胶、纤连蛋白、I型胶原、IV型胶原、层粘连蛋白、基底膜蛋白或塑料上培养时，均可维持。细胞角蛋白的表达在培养中至少通过50次群体倍增得以维持。发现细胞角蛋白的表达受细胞密度调节。以2.5×10(5)个细胞/cm2接种（汇合接种）的肺微血管内皮细胞表达的细胞角蛋白比以1.25×10(4)个细胞/cm2接种（稀疏接种）的细胞多3.5倍。波形蛋白的表达不受细胞密度的影响。通过间接免疫荧光显微镜确定，细胞角蛋白在亚汇合细胞密度时分布于细胞质中，但细胞汇合后细胞角蛋白19有时定位于细胞-细胞接触区域。波形蛋白无论细胞密度如何均呈细胞质分布。这些结果表明，与来自大血管的内皮细胞相比，肺微血管内皮细胞具有独特的细胞骨架，这可能赋予它们功能上独特的特性。结合传统的内皮细胞标志物，简单上皮细胞角蛋白的存在可能是鉴定肺微血管内皮细胞的重要生化标准。
