Department of Physiology & Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Albrechtsen Research, Winnipeg, Manitoba, Canada.
PLoS One. 2018 May 15;13(5):e0197613. doi: 10.1371/journal.pone.0197613. eCollection 2018.
Consumption of different PUFAs (polyunsaturated fatty acids) can induce functional changes in blood vessels via endothelial cells, which interact with dietary factors in the circulation. The basement membrane that separates the endothelium from the smooth muscle cells of the medial layer can also influence the functional state of endothelial cells. However, the effect of basement membrane on the endothelial response to dietary PUFAs in relation to growth state (e.g. proliferation versus quiescence) has never been investigated. We therefore compared the viability (CCK kit) and proliferation (bromodeoxyuridine incorporation) of EA.hy926 endothelial cells grown on Matrigel or collagen versus non-coated plates. EA.hy926 viability and proliferation were also assessed after treatment with 0-150 μM of PUFAs [linoleic acid (LA), arachidonic acid (AA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)]. Our study showed that only cells grown on Matrigel-coated plates reached quiescence after becoming confluent with a decreased level of MCM2 and p-cyclin D1 (T286), increased levels of p27kip1 and a low level of apoptosis and senescence. AA, EPA and DHA decreased the viability and proliferation of subconfluent cells grown on plastic dishes in a dose-dependent manner, while the presence of Matrigel made the cells resistant to these adverse effects. Confluent cell viability was less sensitive to higher concentrations of AA, EPA and DHA than subconfluent cells, and a significant increase in caspase-3 cleavage was only observed in confluent cells treated with DHA. Higher concentrations of AA, EPA and DHA suppressed DNA synthesis by both subconfluent and confluent cells, while precursor C18 PUFAs (LA and ALA) had no negative effects on viability and proliferation. Our study is the first to show that extracellular matrix and growth state are important factors in the EA.hy926 cell response to PUFAs, and that the mechanisms by which individual PUFAs operate may be growth state-dependent.
不同的多不饱和脂肪酸（PUFAs）的消耗可以通过内皮细胞在血管中引起功能变化，这些变化与循环中的饮食因素相互作用。将内皮细胞与中膜层的平滑肌细胞分开的基底膜也可以影响内皮细胞的功能状态。然而，基底膜对内皮细胞对饮食 PUFAs 的反应的影响，与生长状态（例如增殖与静止）有关，这一点从未被研究过。因此，我们比较了在 Matrigel 或胶原蛋白上生长的 EA.hy926 内皮细胞与非涂层板上生长的 EA.hy926 内皮细胞的活力（CCK 试剂盒）和增殖（溴脱氧尿苷掺入）。还评估了用 0-150μM 的 PUFAs[亚油酸（LA）、花生四烯酸（AA）、α-亚麻酸（ALA）、二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）]处理后 EA.hy926 细胞的活力和增殖。我们的研究表明，只有在 Matrigel 涂层板上生长的细胞在达到汇合后才会进入静止状态，此时 MCM2 和 p-cyclin D1（T286）的水平降低，p27kip1 的水平升高，细胞凋亡和衰老的水平较低。AA、EPA 和 DHA 以剂量依赖的方式降低了在塑料盘上生长的亚汇合细胞的活力和增殖，而 Matrigel 的存在使细胞对这些不利影响具有抗性。与亚汇合细胞相比，汇合细胞对较高浓度的 AA、EPA 和 DHA 的活力不太敏感，只有在用 DHA 处理的汇合细胞中才观察到 caspase-3 切割的显著增加。较高浓度的 AA、EPA 和 DHA 抑制了亚汇合和汇合细胞的 DNA 合成，而前体 C18 PUFAs（LA 和 ALA）对活力和增殖没有负面影响。我们的研究首次表明，细胞外基质和生长状态是 EA.hy926 细胞对 PUFAs 反应的重要因素，而个体 PUFAs 作用的机制可能依赖于生长状态。
