Multi-Omics identify the mitochondrial unfolded protein response as a key regulator of endothelial function and pro-Inflammatory mtDNA release

Introduction:
Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the role of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction remains unclear. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of its role in endothelial cell function.

Methods:
Human coronary artery endothelial cells (HCAEC) were subjected to mitochondrial (MitoBlock-6, CDDO, GTTP, Oligomycin A) and ER (Tunicamycin) stressors. RNA sequencing and quantitative proteomic analysis were conducted to assess changes in the transcriptome and proteome. Functional assessments of cell viability, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane integrity were performed through resazurin-based assays, caspase 3/7 activity, MitoSox staining, and TMRE fluorescence. Pro-inflammatory mitochondrial DNA (mtDNA) release was quantified by qPCR after cell fractionation, and circulating cell-free mtDNA levels were measured in serum samples of 226 patients undergoing coronary angiography, including 133 with confirmed coronary artery disease (CAD).

Results:
The multi-omics analysis identified 12,745 unique transcripts and 7,891 unique proteins (Fig 1A-B). Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts. Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes showed that 21 proteins were commonly upregulated by mitochondrial stressors, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. 

Mitochondrial stressors induced dose-dependent changes in HCAEC viability, apoptosis, and ROS formation. Low doses and short incubation with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged exposure led to unresolvable mitochondrial stress (Fig 2A-C). siRNA-mediated silencing of the UPRMito regulators ATF4 and ATF5 enhanced mitochondrial ROS formation in stressed cells. Moreover, all tested UPRMito stressors impaired mitochondrial membrane integrity in a dose-dependent manner, resulting in cytosolic release of pro-inflammatory mtDNA (Fig 3A-B). Finally, CAD patients demonstrated significantly higher circulating cell-free mtDNA levels than non-CAD patients, suggesting a potential link between UPRMitoinduced mtDNA release and atherosclerosis (Fig 4A-B).

Conclusion: 
UPRMito and UPRER lead to a different stress response. Depending on the context, both responses can exert cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.