Introduction:
Accumulation of misfolded proteins in endothelial cells induced by cellular stress is a key factor to the development and progression of endothelial dysfunction and atherosclerosis. In addition to the well-known unfolded protein response of the endoplasmic reticulum, the mitochondrial unfolded protein response (UPRMT) represents a mitochondrial quality-control mechanism that maintains proteostasis under conditions of mitochondrial stress. Despite the established role of mitochondrial dysfunction, for example by pro-inflammatory mitochondrial DNA-release, in vascular disease, the contribution of UPRMT remains poorly understood.
The aim of our study was to characterize the role of UPRMT and mitochondrial DNA in endothelial cells.
Methods:
Human coronary artery endothelial cells (HCAECs) were exposed for 24 h to mitochondrial proteostasis stressors (CDDO, Gamitrinib-TPP) or the endoplasmic reticulum stressor tunicamycin. Cell viability was measured by alamarBlueTM assay. mRNA expression of mitochondrial quality-control proteins (LONP1, HSP60, HSP10) and inflammatory markers (NF-κB, ICAM-1, VCAM-1) was quantified by quantitative real-time PCR (qPCR). Mitochondrial membrane potential was determined using the TMRE assay. Cytosolic cell fractions were isolated using a digitonin-based buffer. Mitochondrial DNA was quantified by qPCR targeting MT-ND1. Mitochondrial depletion was induced using 2´,3´-Dideoxycytidine. Knockdown of ATF4, ATF5 and HSF1 was performed by siRNA-transfection.
Results:
Exposure of human coronary artery endothelial cells (HCAECs) to mitochondrial proteostasis stressors (CDDO, Gamitrinib-TPP (GTTP)) as well as the endoplasmic reticulum stressor tunicamycin significantly reduced cell viability, while expression of mitochondrial quality-control genes (LONP1, HSP60, HSP10) and inflammatory markers (NF-κB, ICAM-1, VCAM-1) were differentially regulated depending on the applied stimulus.
Importantly, the mitochondrial stressor GTTP induced a significant reduction in mitochondrial membrane potential which was associated with increased cytosolic mitochondrial DNA (mtDNA) levels, indicating mtDNA release. To evaluate the putative transcription factor, siRNA-mediated knockdown of ATF4, ATF5 and HSF1 was performed. However, there was no significant change in the expression of the investigated mitochondrial and inflammatory target genes.
Finally, using HCAECs with depleted mtDNA, we could show a trend to an attenuated inflammatory response upon stimulation with a mitochondrial stressor.
Conclusion:
Mitochondrial proteostasis stress in human coronary artery endothelial cells (HCAECs) can induce activation of the mitochondrial unfolded protein response (UPRMT) and an inflammatory response with cytosolic release of mitochondrial DNA (mtDNA). Depletion of mtDNA was associated with a trend toward reduced inflammatory response. To further elucidate the role of UPRMT activation and mtDNA in vascular inflammation future studies are required.
Funding:
Otto-Hess-Promotionsstipendium der Deutschen Gesellschaft für Kardiologie - Herz- und Kreislaufforschung e.V.
