Fillinger M F, Sampson L N, Cronenwett J L, Powell R J, Wagner R J
Section of Vascular Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.
J Surg Res. 1997 Feb 1;67(2):169-78. doi: 10.1006/jsre.1996.4978.
Smooth muscle cell (SMC) growth characteristics are affected by endothelial cells (ECs) in vivo and in vitro. In this study, we compare a bilayer EC/SMC coculture model that allows cell contact with a model of SMCs growing in media continuously conditioned by ECs, but without physical contact. Bovine aortic SMCs were plated on one side of a 13-microns-thick, semipermeable membrane. Three models were compared: (1) SMCs cultured alone (with no cells on the opposite side of the membrane, O/SMC); (2) SMCs cultured with ECs on the opposite side of the membrane in a bilayer coculture system that allows physical contact between ECs and SMCs (EC/SMC); and (3) SMCs cultured in media continuously conditioned by adjacent ECs, without contact (conditioned media, CM). After cultures were established, SMCs were harvested at 7 and 14 days after plating (n = 5 cultures/day/group). SMC DNA and protein content and [3H]thymidine incorporation were measured in each group. On Days 7 and 14 after plating, ECs in both the EC/SMC and CM models stimulated SMC proliferation > 50% compared to O/SMC controls (P < 0.05). SMC density was similar for the EC/ SMC and CM models at Day 7, but SMC density was higher in the EC/SMC group at Day 14 in culture (P < 0.05). At Day 7, protein synthesis was similar in the three groups, but by Day 14, SMCs in the EC/SMC group had produced significantly less cellular protein/ DNA than SMCs in the CM group (P < 0.05), which in turn had less protein/DNA than the control (O/SMC) group (P < 0.05). SMCs in the EC/SMC and CM groups retained a thin, spindle shape with filamentous projections, compared to the hypertrophic appearance of SMCs in the absence of ECs. Electron microscopy revealed projections from SMCs which traversed the pores in the coculture membrane and made intimate contact with ECs. The degree of EC/SMC contact increased from 7 to 14 days (P < 0.05). Compared to SMCs alone, ECs in bilayer coculture or conditioned media altered SMCs growth characteristics similarly after 7 days in culture. By 14 days, however, the bilayer coculture had a significantly greater effect on SMC density and protein synthesis. The bilayer model is unique in terms of luminal/abluminal orientation of the cells, the proximity of the cell layers, and the presence of physical cell contact. Since the bilayer model amplifies the effect of ECs on SMCs, it may be more useful than conditioned media to study EC-SMC interactions.
体内和体外实验中，内皮细胞（ECs）均会影响平滑肌细胞（SMCs）的生长特性。本研究比较了双层EC/SMC共培养模型（该模型允许细胞接触）与SMC在由ECs持续调节的培养基中生长但无物理接触的模型。将牛主动脉平滑肌细胞接种在13微米厚的半透膜一侧。比较了三种模型：（1）单独培养的SMC（膜另一侧无细胞，O/SMC）；（2）在双层共培养系统中，膜另一侧与ECs共同培养的SMC（EC/SMC），该系统允许ECs与SMC进行物理接触；（3）在由相邻ECs持续调节的培养基中培养的SMC，无接触（条件培养基，CM）。培养建立后，接种后7天和14天收获SMC（每组每天n = 5个培养物）。测量每组中SMC的DNA、蛋白质含量以及[3H]胸苷掺入量。接种后第7天和第14天，与O/SMC对照组相比，EC/SMC和CM模型中的ECs均刺激SMC增殖超过50%（P < 0.05）。第7天时，EC/SMC和CM模型中的SMC密度相似，但培养至第14天时，EC/SMC组中的SMC密度更高（P < 0.05）。第7天时，三组中的蛋白质合成相似，但到第14天时，EC/SMC组中的SMC产生的细胞蛋白/DNA明显少于CM组中的SMC（P < 0.05），而CM组又少于对照组（O/SMC）（P < 0.05）。与无ECs时SMC的肥大外观相比，EC/SMC和CM组中的SMC保持细长的纺锤形，并带有丝状突起。电子显微镜显示SMC的突起穿过共培养膜上的孔并与ECs紧密接触。EC/SMC接触程度从7天到14天增加（P < 0.05）。与单独的SMC相比，双层共培养或条件培养基中的ECs在培养7天后对SMC生长特性的改变相似。然而，到第14天时，双层共培养对SMC密度和蛋白质合成的影响明显更大。双层模型在细胞的腔面/非腔面取向、细胞层的接近程度以及物理细胞接触的存在方面具有独特性。由于双层模型放大了ECs对SMCs的影响，因此在研究EC-SMC相互作用方面可能比条件培养基更有用。
