Endothelial Cells and Culture Methods
Endothelial cells line the blood and lymphatic vessels of the body, providing important regulation of vessel integrity, blood pressure, wound healing, and formation of new vessels. In particular, many endothelial cell functions are mediated by the ability of endothelial cells to migrate, either to the site of new blood vessels or to repair a vessel wound. Interaction of cell surface molecules on endothelial cells with factors in the blood mediate the signals that regulate migration. The endothelium also acts as a barrier between contents in the blood and the internal environment by allowing the selective transport of cells and molecules between these two compartments.
Endothelial cells are regularly studied in vitro, and as such, multiple culture techniques have been developed to model endothelial cell function in culture. For example, under particular culture conditions, human umbilical vein endothelial cells (HUVECs) can form tube-like structure that resemble vessels in vitro to study angiogenesis. Additionally, the migratory activity of HUVECs (and other cell types) can be assessed using a scratch assay, in which a wound (a physical scratch, usually with a pipette tip) is introduced to a confluent layer of cells and migration into that wound is quantified. One study, described below, uses the scratch assay to evaluate the effects of cigarette and electronic cigarette smoke on HUVEC migration.
Recent Studies Using Lifeline® HUVECs
Cigarette smoking is a significant risk factor for the development of cardiovascular disease. In particular, smoking can directly affect endothelial cell function by inhibiting cell migration, which is critical for vascular repair. In recent years, electronic cigarettes have become an alternative to traditional cigarettes, and although long-term studies are still lacking, they are thought to decrease the adverse risks of toxic chemicals and carcinogens found in traditional cigarettes. Using a scratch assay, Taylor and colleagues assessed Lifeline® HUVEC migration in response to electronic cigarette smoke aqueous extract (AqE), compared to AqE from an established reference cigarette (3R4F).
As expected, the group found that 3R4F AqE inhibited HUVEC migration in a concentration-dependent manner. Next, the researchers tested the effects of AqE from two electronic cigarette devices. Interestingly, these AqEs did not inhibit HUVEC migration, even up to an AqE concentration of 100%. Therefore, although much research still remains, this study demonstrates that unlike traditional cigarette AqE, electronic cigarette AqE does not inhibit HUVEC migration in response to wounding in vitro.
Antioxidant and anti-inflammatory compounds are of great interest for use in many disease contexts. Oleanic acid is a naturally occurring compound whose synthetic derivative, CDDO-Im, was investigated by Bynum et al. for its effects on gene regulation. The group treated Lifeline® HUVECs with CDDO-Im and performed microarray analysis over the course of 24 hours (at 0.5, 1, 3, 6, and 24 hours). They found that early response genes were upregulated by CDDO-Im at 0.5 and 1 hour, which are responsible for growth, proliferation, and differentiation.
During the intermediate timepoints (3 and 6 hours), the researchers found that genes involved in cytoprotection were upregulated; this included genes associated with the NRF2 pathway, which is implicated in the protective response induced by CDDO-Im. Additionally, genes upregulated by 24 hours were considered to be a readout of the transcriptional activity of early genes. To obtain a more global evaluation of gene expression changes, the authors performed network analysis, and found that DUSP1 (a phosphatase regulated by MEK1) was a critical early node at 0.5 hours, while HMOX1 (an important cytoprotective gene) was a critical node at 3 hours of CDDO-Im treatment.
Finally, the researchers utilized Expression2Kinases analysis, which helped them generate a predicted list of transcription factors and kinases important for modulating the cellular response to CDDO-Im. Using this analysis, they identified MAP2K1, or MEK1, as a potential regulator of gene expression. Together, this study describes the effects of CDDO-Im at the transcriptional level, revealing a multi-faceted cellular response and candidate regulators of this response.
Lifeline® endothelial cells are optimized for growth in VascuLife® endothelial medium, and are derived from multiple sources, including:
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