Introduction:

Endoplasmic reticulum stress resulting in accumulation of misfolded proteins and activation of the unfolded protein response (UPR^ER) is a major contributor to endothelial dysfunction and atherosclerosis.

The mitochondrial unfolded protein response (UPR^Mito) is also activated in endothelial dysfunction, but thus far an only poorly understood signalling pathway.

Aim of our study is to characterize activation, regulation and possible cytoprotective or cytotoxic effects of the UPR^Mito in endothelial cell dysfunction.

Methods:

Human coronary artery endothelial cells (HCAEC) were treated for 6 and 24 hours with stressors of ER protein homeostasis (Tunicamycin, Thapsigargin) or mitochondrial protein homeostatis (CDDO, MitoBloCK-6, OligomycinA), and atherosclerotic stimuli (TNF-alpha, IL-1b, oxLDL) in different concentrations. Viability was measured by alamarblue^® assay. mRNA expression of the UPR-regulators ATF4, CHOP and ATF5 as well as ER/Mito chaperones (Mito: HSP10, HSP 60, LONP1; ER: GRP78) were quantified by quantitative PCR. Apoptosis was measured by using a Caspase-Glo® 3/7 Assay. Mitochondrial reactive oxygen species (ROS) were analyzed by fluorescence microscopy (MitoSOX™). DCFDA was used to detect general ROS.

Results:

We found profound differences in cell viability and gene expression depending on time and concentration of the stimuli. UPR^ER and UPR^Mito stressors consistently increase the transcription of ATF4, CHOP and LONP1. The other genes show a heterogeneous transcription response to the stimuli, even within the respective groups of stimuli. TNF-alpha increases transcription of ATF5. IL-1b and oxLDL increase CHOP mRNA concentration. When analyzing cell viability, we found that UPR^ER-activators uniformly impair cell viability while UPR^Mito-activators mostly increase viability.

Interestingly, UPR^Mito stressors attenuate general ROS formation. In addition to this effect, MitoBloCK-6 increases mitochondrial ROS. We found no effects of UPR^ER-activators on mitochondrial or general ROS formation.

SiRNA-mediated gene silencing of the UPR^Mito regulators ATF4 and ATF5 promotes mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPR^Mito in human coronary artery endothelial cells.

Conclusion:

ATF5 is important for the regulation of the UPR^Mito in HCAEC and is important in reducing ROS formation during cellular stress. UPR^Mito and UPR^ER lead to a partly different mRNA expression pattern. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPR^Mito in endothelial dysfunction and atherosclerosis.