Importance of extracellular matrix and growth state for the EA.hy926 endothelial cell response to polyunsaturated...

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.