Characterization of the Human EA.hy926 Endothelial Cell Response to Dietary Polyunsaturated Fatty Acids as a Function of Growth Conditions
Authors: Du Youjia , Taylor Carla , Aukema Harold , Zahradka Peter 
Published: November 2023

Background

Endothelial cells line the luminal surface of blood vessels and play an important role in maintaining vascular homeostasis. Inappropriate elevated levels of non‐esterified fatty acids in the blood may negatively affect endothelial cell function. The development of endothelial dysfunction is believed to be the initial step leading to atherosclerosis.

Objective

This study compared the effects of different polyunsaturated fatty acids (PUFAs) on the viability and proliferation of subconfluent and confluent human EA.hy926 cells grown with and without Matrigel (a mixture of extracellular matrix proteins that provide a surface for cellular attachment).

Methods

EA.hy926 were seeded (9000 cells/cm2) on plastic dishes with and without Matrigel coating and cultured in Dulbecco's Modified Eagle Medium and 10% fetal bovine serum for up to 12 days without changing the media. Dietary PUFAs (linoleic acid, arachidonic acid – AA, α‐linolenic acid, eicosapentaenoic acid – EPA, docosahexaenoic acid – DHA) were prepared in phosphate‐buffered saline containing 5% fatty acid‐free bovine serum albumin and then added at final concentrations of 1 to 150 μM to subconfluent (4 d after seeding) and confluent cells (8 d after seeding) for 24 h. Viability was assessed using the Cell Counting Kit‐8 (CCK) while proliferation was assessed with a BrdU cell proliferation assay.

Results

Cells reached a maximum number 8 days after seeding on both plain and Matrigel coated dishes. However, BrdU incorporation peaked on day 7 when cells were grown on Matrigel and then started decreasing, whereas BrdU incorporation by cells grown on uncoated dishes continued for up to 10 days. On uncoated dishes, AA and DHA decreased the viability of subconfluent cells in a dose‐dependent manner, while EPA only had a slight effect. PUFA treatment had no effect on confluent cells. No decrease in viability was observed upon PUFA treatment of subconfluent and confluent cells when grown on Matrigel coated plates. On the other hand, the BrdU assay indicated that AA and DHA decrease the proliferation of both subconfluent and confluent cells in a dose‐dependent manner on Matrigel coated dishes.

Conclusions

EA.hy926 cells reach confluence 8 day after seeding on Matrigel coated and uncoated plates, but became quiescent only on Matrigel coated plates after becoming confluent suggesting extracellular matrix is important for normal function. AA and DHA showed an adverse effect on viability of endothelial cells, and their effects were greatest in subconfluent cells and in the absence of extracellular matrix. Survival of subconfluent cells exposed to high concentrations of PUFAs was greatest on Matrigel. The proliferative potential of both subconfluent and confluent cells was decreased by exposure to high concentration of AA and DHA on Matrigel.

Significance

Subconfluent and confluent endothelial cells have different responses to different concentration of different dietary PUFAs in relation to viability and proliferation. However, growth on Matrigel, which resembles the complex extracellular environment of blood vessels, promotes cell survival under high concentration of PUFAs, particularly AA and DHA, compared to the low survival rate of cells grown on uncoated dishes. This implies that Matrigel, by providing a more physiological relevant physical and chemical substrate, may also affect the response of endothelial cells to dietary PUFAs on other aspects, thus warranting further investigation.

Support or Funding Information

Funding from the Canadian Institute of Health Research (CIHR) and the University of Manitoba Graduate Enhancement of Tri‐Council Stipends (GETS) program.