Herman I M
Program in Cell, Molecular, and Developmental Biology, Tufts University Health Science Schools, Boston, Massachusetts 02111.
J Cardiovasc Pharmacol. 1993;22 Suppl 4:S25-36. doi: 10.1097/00005344-199322004-00005.
Vascular endothelial cell (EC) wound healing was characterized on an EC-synthesized extracellular matrix (ECM) previously treated with enzymes and antibodies specific for ECM components. Using a computer-assisted video-microscope recording system capable of automatic EC recognition, we learned whether components of the EC-synthesized matrix influenced post-injury migration and wound healing in vitro. Localization of actin and its encoded mRNA using isoform-specific antibodies and labeled cDNA probes allowed for a direct correlation of living-cell behavior with cytoskeletal form and distribution. Results of these studies indicate that the computer-assisted EC tracking system allows for an automatic and reproducible analysis of EC behavior following injury in vitro. EC migrate fastest immediately following injury and then achieve a new, slower migration rate that is maintained until EC from one edge of 200- to 300-microns-wide wound zone contact EC from the other wound face. Treatment of EC-synthesized matrices with antibodies against fibronectin and laminin has no effect on EC migration following injury (-0.25 microns/min) or on cytoskeletal array. Similarly, digestion of these matrices with heparinase and hyaluronidase has no effect on wound healing rates. Slowly spreading EC cytoplasm, which borders the intact and antibody-treated EC matrices, is rich in actin but lacks myosin II. Two different preparations of collagenase (bacterial and mammalian) each potentiate EC wound healing in vitro. Bacterial collagenase treatment of the EC-synthesized matrices potentiates EC migration fivefold (1 micron/min) while treatment of EC-matrices with mammalian cell collagenase stimulates EC migration following injury some twofold (0.4 micron/min) over control values. Whereas EC on control matrices migrate in unison as a tissue-like sheet, EC on the collagenase-treated EC matrices migrate as individuals. Concomitant with the increased rates of migration following injury on the collagenase-treated EC-matrices is a two- to fourfold increase in the steady-state levels of beta-actin mRNA. This increase in actin mRNA abundance is observable by its preferential localization (seen by in situ hybridization) in the lamellae bordering the wound edge in association with beta-actin, which is exclusively localized there. Because beta-actin and its encoded mRNA are positioned together in association with the plasma membrane in regions of moving cytoplasm, it seems likely that beta-actin filament assembly is required for motility following endothelial injury.
血管内皮细胞（EC）伤口愈合情况是在经酶和针对细胞外基质（ECM）成分的抗体预处理的EC合成细胞外基质上进行表征的。使用能够自动识别EC的计算机辅助视频显微镜记录系统，我们了解了EC合成基质的成分是否会影响体外损伤后的迁移和伤口愈合。使用同工型特异性抗体和标记的cDNA探针定位肌动蛋白及其编码的mRNA，可将活细胞行为与细胞骨架形态和分布直接关联起来。这些研究结果表明，计算机辅助的EC追踪系统能够对体外损伤后EC的行为进行自动且可重复的分析。EC在损伤后立即迁移速度最快，然后达到一个新的、较慢的迁移速率，并保持该速率，直到来自200至300微米宽伤口区域一侧边缘的EC与另一侧伤口面边缘的EC接触。用抗纤连蛋白和层粘连蛋白的抗体处理EC合成基质，对损伤后EC的迁移（-0.25微米/分钟）或细胞骨架排列没有影响。同样地，用肝素酶和透明质酸酶消化这些基质对伤口愈合速率也没有影响。与完整的和经抗体处理的EC基质相邻的缓慢伸展的EC细胞质富含肌动蛋白，但缺乏肌球蛋白II。两种不同制剂的胶原酶（细菌来源和哺乳动物来源）均能增强体外EC伤口愈合。用细菌胶原酶处理EC合成基质可使EC迁移速度提高五倍（1微米/分钟），而用哺乳动物细胞胶原酶处理EC基质可使损伤后EC的迁移速度比对照值提高约两倍（0.4微米/分钟）。对照基质上的EC作为类似组织的薄片一致迁移，而胶原酶处理的EC基质上的EC则单独迁移。与胶原酶处理的EC基质上损伤后迁移速率增加相伴的是β-肌动蛋白mRNA稳态水平增加两到四倍。通过原位杂交观察到，这种肌动蛋白mRNA丰度的增加表现为其在与伤口边缘相邻的片状伪足中优先定位，且与仅在该区域定位的β-肌动蛋白相关联。由于β-肌动蛋白及其编码的mRNA在移动细胞质区域与质膜一起定位，因此内皮损伤后的运动似乎需要β-肌动蛋白丝的组装。
