Notes on Aberrant Mitochondrial DNA Synthesis in Macrophages and Atherosclerosis

Introduction

• Cellular metabolism significantly influences inflammation, which in turn contributes to the progression of atherosclerosis.
• The understanding of how mitochondrial DNA (mtDNA) synthesis affects macrophage function and atherosclerosis pathology is still incomplete.
• This study reveals that mtDNA synthesis in macrophages within atherosclerotic plaques is triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice.
• Mechanistically, VCAM-1 activates C/EBPα, which then binds to the promoters of key mitochondrial biogenesis genes, CMPK2 and PGC-1α.
• Increased expression of CMPK2 and PGC-1α leads to mtDNA synthesis, activating STING-mediated inflammation.
• The study demonstrates that atherosclerosis and inflammation are less severe in Apoe−/− mice lacking Vcam1 in macrophages.
• Downregulation of macrophage-specific VCAM-1 in vivo results in decreased expression of LYZ1 and FCOR, which are involved in STING signaling.
• VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative DNA damage.
• The study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis, proposing a self-exacerbating pathway involving increased mtDNA synthesis.

Cellular Metabolism and Macrophage Function

• Cellular metabolism regulates energy production, growth, and proliferation and also plays non-canonical roles in cell phenotypic switch, differentiation, cellular senescence, neurogenesis, efferocytosis, memory formation, cell migration, and tissue repair.
• Pathways such as fatty acid synthesis and oxidation, and mitochondrial respiration control immune cell functions, including CD8+ T cells, TH17 cells, and regulatory T cells.
• ATP citrate lyase and fatty acid synthase, crucial enzymes in de novo fatty acid production, are important for macrophage-mediated inflammation by promoting histone acetylation and atherosclerosis propagation.
• Macrophages uptake triglycerides via the scavenger receptor CD36, leading to alternative activation.
• Mitochondria regulate macrophage functions, with succinate (a TCA cycle metabolite) exerting anti-inflammatory reprogramming in a mouse model of obesity.
• Inhibition of mitochondrial fission decreases efferocytosis and exacerbates atherosclerosis.
• Macrophage-specific mitochondrial complex deficiency results in inflammatory macrophages with poor efferocytosis capacity, leading to defective cardiac healing after myocardial infarction.
• Mitochondrial function correlates with the severity of heart failure with preserved ejection fraction.

mtDNA Synthesis and Inflammation

• The contributions of mtDNA synthesis in shaping inflammation under disease conditions have not been fully explored.
• Mitochondrial homeostasis is maintained by mitochondrial biogenesis and mitophagy (removal of damaged mitochondria).
• Mitochondrial biogenesis is a multi-step process, starting with mtDNA transcription.
• Genes orchestrating mtDNA synthesis include peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC1A), Cytidine/Uridine monophosphate kinase 2 (CMPK2), and DNA polymerase γ (POLG).
• PGC1A induces transcription of nuclear-encoded mitochondrial genes and interacts with nuclear respiratory factors NRF1 and NRF2, regulating the expression of electron transfer chain subunits.
• TFAM, a mtDNA binding protein, is essential for mitochondrial genome maintenance.
• CMPK2 is a rate-limiting enzyme for mtDNA synthesis, critical for cytidine triphosphate production.
• The catalytic subunit encoded by the POLG gene and the auxiliary dimeric subunit encoded by the POLG2 gene are crucial in mtDNA replication.
• Aberrant mtDNA synthesis occurs in aging and diseases such as neurodegeneration and cancer.
• Modulating mtDNA synthesis is a potential therapeutic strategy in neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases.
• PGC-1α levels are increased in cancer invasion and metastasis, and 2-methoxyestradiol has been shown to reduce mtDNA synthesis in osteosarcoma cells.

VCAM-1 and Atherosclerosis

• Myeloid vascular cell adhesion molecule-1 (VCAM-1) augments the production of CMPK2 and PGC1A, increasing mtDNA synthesis in atherosclerosis.
• VCAM-1 in endothelial cells facilitates monocyte migration into the intima and is triggered by pro-inflammatory cytokines, reactive oxygen species (ROS), oxidized low-density lipoprotein (ox-LDL), and shear stress.
• VCAM-1 binds to very late antigen-4 (VLA-4), an integrin, which is required for firm adhesion of monocytes to the luminal surface of the endothelium.
• Macrophages, particularly foam cells in atherosclerotic plaques of humans and mice, express VCAM-1 at high levels.
• The study aims to understand the role of aberrant mtDNA synthesis in macrophage-mediated inflammation and atherosclerosis, examine myeloid VCAM-1-mediated regulation of mitochondrial metabolism, and discern the role of myeloid VCAM-1 in atherogenesis.
• Mice lacking Vcam1 in macrophages exhibit reduced atherosclerotic plaque and necrotic core areas.
• Vcam1 silencing in macrophages diminishes oxidized LDL-mediated inflammation, oxidative phosphorylation, and mtDNA synthesis in vitro.
• Silencing of mtDNA synthesis genes Cmpk2, Pgc1a, and Polg in macrophages in vivo diminishes plaque burden and increases plaque stability in Ldlr−/− mice on an atherogenic diet.
• VCAM-1-mediated oxidized and fragmented DNA signals through the stimulator of interferon genes (STING) pathway, decreasing the expression of FCOR and LYZ1 and augmenting inflammation.
• The study demonstrates the role of macrophage VCAM-1 in encouraging mtDNA synthesis to increase inflammation and aggravate atherosclerosis.
• Increased mitochondrial DNA damage has been documented in CVD, and this study establishes the pro-inflammatory effect of mtDNA in atherosclerotic macrophages and downstream signaling through the cGAS – STING pathway.

Macrophage VCAM-1 Expression and Mitochondrial Stress

• VCAM-1 is an adhesion molecule, mainly expressed by endothelial cells, facilitating the extravasation of leukocytes into the intima.
• Macrophages in human early (>20% blockade) and advanced (>70% blockade) carotid atherosclerotic plaques highly express VCAM-1 compared to healthy artery parts.
• Analyses of published datasets confirm increased VCAM-1 expression by atherosclerotic plaque macrophages.
• Single-cell analysis shows that mouse and human macrophage subsets express high levels of Itga4 and Itgb1, encoding the VCAM-1 ligand VLA-4.
• Elevated levels of oxidative stress in atherosclerotic macrophages in advanced plaques are indicated by the heightened presence of 8-hydroxy-2’ -deoxyguanosine (8-OHdG), a form of oxidant-induced oxidative lesions in nuclear and mtDNA.
• Atherosclerotic macrophages in early and advanced plaques have high expression of TOM20, a mitochondrial marker.
• VCAM-1 expression in atherosclerotic macrophages is positively correlated with TOM20 and 8-OHdG staining.
• Necrotic core area is also positively correlated with macrophage VCAM-1, 8-OHdG, and TOM20.
• VCAM-1, TOM20, and 8-OHdG are increased in mouse atherosclerotic macrophages in Apoe−/− mice fed an atherogenic diet for 2 and 5 months.

Macrophage VCAM-1 and Its Impact

• The atherogenic diet itself increases the levels of TOM20 and VCAM-1 but not 8-OHdG in aortic macrophages of C57BL/6 mice.
• Bulk RNA sequencing on sorted aortic VCAM-1+ and VCAM-1− macrophages reveals enrichment of genes involved in macrophage activation, differentiation, and stimulation of antigen-presenting cells in VCAM-1+ atherosclerotic macrophages.
• Upstream regulators, such as IL10RA, MAFB, and MAPKAPK2, crucial in atherogenesis, are upregulated in VCAM-1+ macrophages.
• mtROS levels are significantly greater in VCAM-1+ macrophages compared to VCAM-1− macrophages.

VCAM-1, Inflammation, and Atherosclerosis

• Mice lacking Vcam1 in macrophages on the Apoe−/− background (Apoe−/− LyzMCre/+ Vcam1fl/fl) and fed an atherogenic diet have less atherosclerotic burden, as shown by decreased plaque and necrotic core areas.
• Serum cholesterol levels remain unchanged in these mice.
• Apoe−/− LyzMCre/+ Vcam1fl/fl mice have suppressed systemic inflammation, shown by lower serum levels of IL-5, IL-17, GM-CSF, and TNF-α when fed a high-fat diet.
• Mice with myeloid Vcam1 deficiency exhibit higher levels of eotaxin and RANTES.
• Vcam1-deficient macrophages secrete reduced amounts of pro-inflammatory cytokines and chemokines such as IL-1α, IL-1β, IL-12, IL-17A, TNF-α, and MCP-1 upon exposure to oxidized LDL.
• Levels of Th2 cytokines, such as IL-4 and IL-5, also decrease in the culture of Vcam1-deficient macrophages.
• Eosinophil frequencies and numbers decrease, and the amount of plaque malondialdehyde, an oxidation-specific epitope, is lower in myeloid Vcam1-deficient mice.
• Th2 frequencies and numbers increase in myeloid Vcam1-deficient mice.
• $Il6$ and $Ifnb$ mRNA expression is significantly lower in Vcam1−/− BMDM.
• VCAM-1 correlations: Significant reduction in expression of DNA damage and mitochondrial markers in Vcam-1−/− plaque macrophages.
• Absence of Vcam1 does not affect the numbers of leukocyte subsets in the aorta, spleen, bone marrow, and blood.

Mechanisms

• Oxidized LDL increases the expression of retention factors, such as $Ccr7$, $Ntr1$, $Cd146$, and $Sema3$ in Vcam1+/+ macrophages.
• Spatial single-cell transcriptomics in aortic roots reveals higher levels of mt-Nd5 and Tspo, critical in mitochondrial metabolism, in aortic macrophages of Ldlr−/− LysM+/+ Vcam1fl/fl mice compared to Ldlr−/− LysMCre/+ Vcam1fl/fl mice.
• Dimensionality reduction reveals four macrophage clusters in aortic roots (Clusters 0, 2, 5, and 12), with canonical marker genes having similar expression levels across clusters but corresponding to different spatial microniches.
• Pathways such as the immune system, neutrophil degranulation, adaptive immune system, and antigen processing and cross-presentation are suppressed in aortic macrophages in the absence of Vcam1.
• Atherosclerosis is linked with mitochondrial dysfunction and increased mitochondrial ROS generation, with macrophage metabolism playing a substantial role in atherosclerosis.
• VCAM-1 expression is correlated with TOM20 levels.
• LyzM+/+ Vcam1fl/fl macrophages treated with oxidized LDL have higher oxygen consumption rate (OCR) compared to native LDL-treated group, while Vcam1-deficient (LyzMcre/+ Vcam1fl/fl) macrophages do not exhibit a similar increase in OCR.
• LyzM+/+ Vcam1fl/fl macrophages treated with oxidized LDL have higher mitochondrial volume and membrane potential, while LyzMcre/+ Vcam1fl/fl macrophages do not exhibit increases in these parameters upon oxidized LDL exposure.
• Vcam1-deficient macrophages treated with oxLDL have diminished maximal and glucose-dependent (2-DG-sensitive) maximal glycolysis and reduced lactate amounts.
• Mitochondrial complexes III and IV activities are diminished in macrophages lacking Vcam1, and the expression of complexes II and III is reduced in these macrophages.
• Oxidized LDL exposure decreases the amount of most TCA metabolites in the culture of LyzM+/+ Vcam1fl/fl macrophages compared to LyzMcre/+ Vcam1fl/fl macrophages, indicating accelerated usage of the TCA cycle metabolites in wildtype macrophages.

VCAM-1 and mtDNA Synthesis

• Macrophages in atheromas of patients and mice have elevated VCAM-1 and TOM20 mean fluorescence intensity (MFI).
• VCAM-1 deficiency limits oxidized LDL-mediated expansion of mitochondrial volume.
• oxidized LDL treatment triggers mtDNA synthesis and increases cytoplasmic mtDNA in Vcam1+/+ BMDM, while Vcam1 deficiency abrogates this response.
• VCAM-1 is dispensable for mitophagy.
• Aberrant mtDNA synthesis: In response to oxidized LDL, genes including $Cmpk2$, $Pgc1a$, $Polg$, Diacylglycerol kinase (Dgk), and Adenylate kinase 2 (Ak2)$are heightened in Vcam1+/+ BMDM, while the absence of Vcam1 dampens the expression of these genes.</li> <li>Vcam1 deficiency decreases mtDNA synthesis and inflammation.</li> <li>Mitochondrial and nuclear DNA transfection augment the expression of the genes encoding pro-inflammatory cytokines in Vcam1−/− BMDM.</li> <li>mtDNA depletion in BMDM significantly curtails oxLDL-mediated inflammation.</li> <li>Silencing of$Polg$and$Cmpk2$expression in Vcam1+/+ BMDM abrogates the inflammatory response to oxLDL.</li> <li>CMPK2 overexpression increases$IL1B$,$IL6$, and$TNFA$expression in THP-1 macrophages treated with either siControl or siVCAM1.</li> </ul> <h3 id="mitochondrialroleininflammation">Mitochondrial Role in Inflammation</h3> <ul> <li>Dysfunctional mitochondria: Vcam1−/− BMDM have significantly suppressed levels of mitochondrial ROS, and inhibition of mitochondrial ROS in Vcam1+/+ BMDM leads to suppressed inflammatory gene expression.</li> <li>Damaged mtDNA binds to cGAS, activating the production of cGAMP, which activates the adaptor STING.</li> <li>oxLDL treatment significantly increases expression of STING in macrophages.</li> <li>The ratio of pSTING/STING is significantly suppressed in absence of Vcam1.</li> <li>$Cmpk2$and$Pgc1a$, but not$Polg$silencing, decreases p-Sting/Sting ratios.</li> <li>Expression of pro-inflammatory cytokines in response to oxidized LDL is depressed in BMDM after Sting knockdown, while STING overexpression in THP-1 macrophages magnifies oxidized LDL-induced inflammatory cytokine expression.</li> </ul> <h3 id="mtdnasynthesisandatherosclerosis">mtDNA Synthesis and Atherosclerosis</h3> <ul> <li>mtDNA synthesis: Selective silencing of Polg, Ppargc1a (Pgc1a), and Cmpk2 in macrophages of Ldlr-/- mice leads to significant decreases in plaque size and necrotic core area relative to the control group while fibrous cap thickness increases.</li> <li>Minor effects to immune cell population detected in the spleen and bone marrow after silencing.</li> <li>Macrophages in atherosclerotic plaques of mice treated with siRNA against the mtDNA synthesis genes significantly reduce expression of TOM20 and 8-OHdG.</li> <li>iNos expression is significantly decreased in atherosclerotic plaque macrophages after the silencing of mitochondrial biogenesis genes.</li> <li>Cmpk2 silencing resulted in the highest numbers of differentially expressed genes, such as$Gdf15$,$Ndufa5$,$Ndufv2$,$Ndufb3$,$Atp6v1e1$,$Map3k6$,$Lrp8$,$Ifnlr1$,$Acly$,$Gpr82$,$Ccl6,8,9$, and$Ncoa4$$, which are critical in inflammation, atherosclerosis, and cellular metabolism.

VCAM-1 and Inflammation

• In plaque macrophages lacking Vcam1, 25 genes were differentially expressed.
• The expression of Fcor, Lyz1, Nid1, Wisp2, Zfp300, and Zfp940 (referred to as the VCAM-1 downstream genes) was heightened in Vcam1-deficient macrophages, suggesting their roles in suppressing inflammation.
• Silencing these genes in BMDM significantly increases overall inflammation.
• The VCAM-1 downstream genes are regulated by CMPK2 and STING.
• Cmpk2 and Polg silencing upregulates the VCAM-1 downstream genes in BMDM in presence of oxidized LDL.
  -Overexpression of CMPK2 and STING, rescinded the upregulation of the VCAM-1 downstream genes after VCAM1 silencing.
• mtDNA transfection in BMDM lessens the expression of the VCAM-1 downstream genes.
• VCAM-1 dampens expression of downstream genes via mtDNA synthesis & STING pathway; VCAM-1 downstream genes curb inflammation.
• CEBP/α, a transcription factor regulated by VCAM-1, is downregulated in BMDM lacking Vcam1.
• Reduced Rac1 expression in response to oxLDL in Vcam1-deificient macrophages. Reduction in Cebpα level after Rac1 silencing in BMDM.

Mechanism for VCAM-1 regulating CMPK2

• VCAM-1 increases expression of Cebpα, which binds to the promoter regions of Cmpk2 and Pgc1a, elevating their expression.
  -Binding to Cmpk2 and Pgc1a but not Polg was reduced in absence of Vcam1

VCAM-1 and Atherosclerosis

• VCMA-1 exacerbates atherosclerosis by suppressing Fcor and Lyz1
• Fcor, Lyz1, and Wisp2 expression was detectable in atherosclerotic plaque macrophages and their expression was downregulated in aortic macrophages present in atherosclerotic lesions.
• Silencing Fcor and Lyz1, resulted in larger atherosclerotic plaques without changing fibrous cap thickness compared to control siRNA treated mice, while only siFcor increased necrotic core areas in atheromas.
  -Wisp2 knockdown in macrophages thickened fibrous caps without changing plaque and necrotic core areas.
  -Myeloid STING deficiency rose Fcor and Lyz1, but not Wisp2, expression in atherosclerotic plaque macrophages

Expression

• Increased Akt2, Chkb, and Fkbp5 and downregulation of Mmp14, Rrp9, and Arg1 in siFcor and siLyz1-treated mice vs. siControl.
• Data indicate atherogenesis due to macrophage VCAM-1 is mediated by low Fcor and Lyz1 expression.

Atherosclerosis Causation

• Inflammatory macrophages are the most prevalent leukocytes within plaques via differentiation of circulating inflammatory monocytes and local proliferation of plaque resident macrophages.
• Developing plaque macrophages take up lipoproteins and differentiate into foam cells, with plaque microenvironment orchestrating metabolic rewiring of foam cells.
• Atherosclerotic plaque macrophages have VCAM-1-mediated high mtDNA synthesis, which amplifies inflammation and accelerates atherosclerosis progression.
• The adhesion molecule VCAM-1 increases the expression of C/EBPa that binds to the promoter region of Cmpk2, augmenting its expression upon oxidized and fragmented mtDNA, triggering the activation of cGAS-STING pathway-dependent inflammation which aggravates atherosclerosis.
• Targeting mtDNA synthesis in atherosclerosis is worth doing.