Notes on Aberrant Mitochondrial DNA Synthesis in Macrophages and Atherosclerosis

Aberrant Mitochondrial DNA Synthesis in Macrophages Exacerbates Inflammation and Atherosclerosis

This study investigates the role of mitochondrial DNA (mtDNA) synthesis in macrophages in the context of inflammation and atherosclerosis. The key findings highlight that VCAM-1 induces mtDNA synthesis in macrophages, leading to STING-mediated inflammation and accelerated atherogenesis.

Introduction

• Cellular metabolism governs inflammation, which contributes to atherosclerosis.

• The role of macrophage mtDNA synthesis in atherosclerosis is not fully understood.

• This study demonstrates that mtDNA synthesis in atherosclerotic plaque macrophages is triggered by VCAM-1 under inflammatory conditions in both humans and mice.

• VCAM-1 activates C/EBPα, which upregulates CMPK2 and PGC-1α, triggering mtDNA synthesis and STING-mediated inflammation.

• Atherosclerosis and inflammation are less severe in Apoe−/− mice lacking Vcam1 in macrophages.

• Downregulation of macrophage-specific VCAM-1 decreases LYZ1 and FCOR expression, which are involved in STING signaling.

• VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA.

• The study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis, proposing a self-acerbating pathway involving increased mtDNA synthesis.

• Cellular metabolism regulates cell functions like energy production, growth, and proliferation.

• Non-canonical roles of metabolism include cell phenotypic switch, differentiation, senescence, neurogenesis, efferocytosis, memory formation, cell migration, and tissue repair.

• Pathways like fatty acid synthesis, fatty acid oxidation, and mitochondrial respiration control immune cell functions (CD8+ T, TH17, regulatory T cells).

• Cellular metabolism also shapes macrophage phenotype and function.

• ATP citrate lyase and fatty acid synthase are crucial for macrophage-mediated inflammation.

• Butyrate equips macrophages with enhanced anti-microbial properties.

• Uptake of triglycerides by macrophages via CD36 leads to alternative activation.

• Mitochondria regulate macrophage functions.

• The succinate receptor exerts anti-inflammatory reprogramming of macrophages in 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 contributions in shaping inflammation under disease conditions have not been explored.

Mitochondrial Homeostasis and Biogenesis

• Mitochondrial homeostasis is maintained by mitochondrial biogenesis and mitophagy.

• Mitochondrial biogenesis increases mitochondrial numbers to fulfill energy needs.

• The first step of mitochondrial biogenesis is mtDNA transcription.

• Genes orchestrating mtDNA synthesis: 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 NRF1 and NRF2, regulating electron transfer chain subunits.

• TFAM expression increases, which is essential for mitochondrial genome maintenance.

• CMPK2 is a rate-limiting enzyme for mtDNA synthesis, critical for cytidine triphosphate production.

• POLG gene (catalytic subunit) and POLG2 gene (auxiliary dimeric subunit) are crucial in mtDNA replication.

• mtDNA-encoded genes are translated into proteins with the help of mitochondrial initiation factors 2 and 3 (mtIF2, mtIF3), elongation factors Tu, Ts, and G1 (mtEFTu, mtEFTs, mtEFG1), translational release factor 1-like (mtRF1L), and recycling factors 1 and 2 (mtRRF1, mtRRF2).

• Aberrant mtDNA synthesis occurs in aging and diseases like neurodegeneration and cancer.

• mtDNA replication is an initial effect of the neurodegenerative process in neurons.

• Modulating mtDNA synthesis is a potential therapeutic strategy in neurodegenerative diseases (Alzheimer’s, Parkinson’s).

• PGC-1α levels increase in cancer invasion and metastasis. 2-methoxyestradiol reduces mtDNA synthesis in osteosarcoma cells.

• This study investigates myeloid VCAM-1-mediated regulation of mitochondrial metabolism and its role in atherogenesis.

Study Objectives and Key Findings

• The study aims to understand the role of aberrant mtDNA synthesis in macrophage-mediated inflammation and atherosclerosis.

• Increased VCAM-1 expression in human and mouse atherosclerotic plaque macrophages is demonstrated.

• 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.

• Selective silencing of mtDNA synthesis genes (Cmpk2, Pgc1a, and Polg) in macrophages in vivo causes a significant diminution in plaque burden and an increase in 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 to decrease the expression of FCOR and LYZ1 and augment inflammation.

• The findings demonstrate 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, this study establishes the pro-inflammatory effect of mtDNA in atherosclerotic macrophages and downstream signaling through the cGAS – STING pathway.

Results

Macrophage VCAM-1 Expression and Correlation with Mitochondrial Volume and DNA Oxidative Stress

• VCAM-1, mainly expressed by endothelial cells, facilitates leukocyte extravasation.

• VCAM-1 is expressed by dendritic cells and macrophages, but its functions are understudied.

• Macrophages in human early (>20% blockade) and advanced (>70% blockade) carotid atherosclerotic plaques highly expressed VCAM-1 compared to healthy arteries.

• Analyses of published datasets confirmed increased VCAM-1 expression by atherosclerotic plaque macrophages.

• Single-cell analysis showed 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 were found in atherosclerotic macrophages in advanced plaques, indicated by heightened presence of 8-hydroxy-2’ -deoxyguanosine (8-OHdG).

• Atherosclerotic macrophages in early and advanced plaques had high expression of TOM20, a mitochondrial marker.

• VCAM-1 expression in atherosclerotic macrophages was positively correlated with TOM20 and 8-OHdG staining.

• Necrotic core area was also positively correlated with macrophage VCAM-1, 8-OHdG, and TOM20.

• Parallel increase in VCAM-1, TOM20, and 8-OHdG in mouse atherosclerotic macrophages in Apoe−/− mice fed an atherogenic diet (2 and 5 months).

• The atherogenic diet itself increased TOM20 and VCAM-1 levels, but not 8-OHdG, in aortic macrophages of C57BL/6 mice.

Characterization of VCAM-1+ Plaque Macrophages

• Bulk RNA sequencing on sorted aortic VCAM-1+ and VCAM-1− macrophages was performed.

• Genes involved in macrophage activation, differentiation, and stimulation of antigen-presenting cells were enriched in VCAM-1+ atherosclerotic macrophages.

• Upstream regulators (IL10RA, MAFB, MAPKAPK2) crucial in atherogenesis were upregulated in VCAM-1+ macrophages.

• mtROS levels were significantly greater in VCAM-1+ macrophages compared to VCAM-1− macrophages.

Macrophage VCAM-1 Expression, Inflammation, and Atherosclerosis

• Mice lacking Vcam1 in macrophages on the Apoe−/− background (Apoe−/− LyzMCre/+ Vcam1fl/fl) were generated and fed an atherogenic diet.

• Macrophages and monocytes in wildtype mice had detectable VCAM-1 expression, while only macrophages of Apoe−/− LyzMCre/+ Vcam1fl/fl mice exhibited reduction in VCAM-1 levels.

• Apoe−/− LyzMCre/+ Vcam1fl/fl mice had less atherosclerotic burden (decreased plaque and necrotic core areas) compared to Apoe−/− LyzM+/+ Vcam1fl/fl control mice.

• Serum cholesterol levels were unchanged, but Apoe−/− LyzMCre/+ Vcam1fl/fl mice had suppressed systemic inflammation: lower serum IL-5, IL-17, GM-CSF, and TNF-α with a high-fat diet and suppressed serum G-CSF on a regular diet.

• Mice with myeloid Vcam1 deficiency and fed with a regular diet exhibited higher levels of eotaxin and RANTES.

• Bone marrow-derived macrophages (BMDM) cultured from LyzMCre/+ Vcam1fl/fl mice secreted diminished amounts of pro-inflammatory cytokines and chemokines (IL-1α, IL-1β, IL-12, IL-17A, TNF-α, and MCP-1) upon exposure to oxidized LDL.

• Th2 cytokine levels (IL-4, IL-5) also decreased in the culture of Vcam1-deficient macrophages which also correlated with decreased eosinophil frequencies.

• Il6 and Ifnb mRNA expression was significantly lower in Vcam1−/− BMDM.

• Significant reduction in the expression of DNA damage and mitochondrial markers in Vcam-1−/− plaque macrophages.

• VCAM-1, 8-OHdG, and TOM20 correlations are not due to increased macrophage accumulation.

• The absence of Vcam1 did not affect the numbers of leukocyte subsets in the aorta, spleen, bone marrow, and blood.

• Oxidized LDL increased the expression of retention factors (Ccr7, Ntr1, Cd146, Sema3) in Vcam1+/+ macrophages; these were unaltered in Vcam1−/− macrophages.

• Vcam1 deficiency did not significantly change macrophage migration towards an Mcp-1 gradient in vitro.

Spatial Single-Cell Transcriptomics

• Spatial single-cell transcriptomics was performed in the aortic roots of Ldlr−/− LysM+/+ Vcam1fl/fl and Ldlr−/− LysMcre/+ Vcam1fl/fl mice to characterize macrophage subsets and transcriptional changes.

• A murine single-cell RNA-seq reference dataset from aortic tissue served as the basis for cell type deconvolution to identify macrophage niches.

• Aortic macrophages in Ldlr−/− LysM+/+ Vcam1fl/fl mice expressed higher levels of mt-Nd5 and Tspo, which are critical in mitochondrial metabolism, compared to those in Ldlr−/− LysMCre/+ Vcam1fl/fl mice.

• Dimensionality reduction analyses identified four clusters of macrophages in the aortic roots (Clusters 0, 2, 5, and 12).

• Pathways such as the immune system, neutrophil degranulation, adaptive immune system, and antigen processing and cross-presentation were suppressed in aortic macrophages in the absence of Vcam1.

• The transcriptional and phenotypic alterations seen in the absence of Vcam1 transcend macrophage subsets.

Macrophage VCAM-1 and Mitochondrial Metabolism

• Atherosclerosis is linked to mitochondrial dysfunction and increased mitochondrial ROS generation.

• VCAM-1 expression is correlated with TOM20 levels, suggesting it modulates mitochondrial metabolism.

• LyzM+/+ Vcam1fl/fl macrophages treated with oxidized LDL had higher oxygen consumption rate (OCR), demonstrating higher basal and maximal respiration and spare capacity, unlike Vcam1-deficient (LyzMcre/+ Vcam1fl/fl) macrophages.

• LyzM+/+ Vcam1fl/fl macrophages treated with oxidized LDL had higher mitochondrial volume and membrane potential.

• Vcam1-deficient macrophages exhibited diminished maximal and glucose-dependent maximal glycolysis after oxidized LDL treatment.

• Vcam1−/− macrophages had reduced amount of lactate.

• Mitochondrial complexes III and IV activities were diminished in macrophages lacking Vcam1.

• Expression of complexes II and III was reduced in these macrophages.

• Metabolomics experiment revealed that oxidized LDL exposure decreased 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 the wildtype macrophages.

• 13C glucose labeling experiment showed elevated 13C-labeled TCA metabolites in LyzM+/+ Vcam1fl/fl macrophages, suggesting accelerated usage of metabolites in the TCA cycle.

• Macrophage VCAM-1 increases mitochondrial metabolism.

VCAM-1, mtDNA Synthesis, and Inflammation

• Macrophages in atheromas of patients and mice have elevated VCAM-1 and TOM20.

• VCAM-1 deficiency limits oxidized LDL-mediated expansion of mitochondrial volume, suggesting VCAM-1 amplifies mtDNA synthesis.

• Oxidized LDL treatment triggered mtDNA synthesis and increased cytoplasmic mtDNA in Vcam1+/+ BMDM, while Vcam1 deficiency abrogated this response.

• VCAM-1 was dispensable for mitophagy.

• mtDNA synthesis relies on genes including Cmpk2, Pgc1a, Polg, Diacylglycerol kinase (Dgk), and Adenylate kinase 2 (Ak2).

• Expression of these genes was heightened in Vcam1+/+ BMDM in response to oxidized LDL, but the absence of Vcam1 dampened the expression of the genes.

• Vcam1 deficiency decreased mtDNA synthesis and inflammation.

• Both mitochondrial and nuclear DNA transfection augmented the expression of genes encoding pro-inflammatory cytokines in Vcam1−/− BMDM, while mtDNA depletion curtailed oxLDL-mediated inflammation.

• Silencing Polg and Cmpk2 in Vcam1+/+ BMDM exposed to native or oxidized LDL abrogated the inflammatory response to oxLDL.

• CMPK2 overexpression increased IL1B, IL6, and TNFA expression in THP-1 macrophages treated with either siControl or siVCAM1.

• Cmpk2 silencing decreased D-loop/Tert ratio in BMDM treated with oxidized LDL.

• Vcam1−/− BMDM had significantly suppressed levels of mitochondrial ROS, and inhibition of mitochondrial ROS in Vcam1+/+ BMDM led to suppressed inflammatory gene expression.

• OxLDL treatment significantly increased STING expression in macrophages, and the ratio of pSTING/STING was significantly suppressed in absence of Vcam1.

• Both Cmpk2 and Pgc1a but not Polg silencing decreased p-Sting/Sting ratios.

• Expression of pro-inflammatory cytokines in response to oxidized LDL was depressed in BMDM after Sting knock down.

• STING overexpression in THP-1 macrophages magnified oxidized LDL-induced inflammatory cytokine expression. Macrophage VCAM-1 augments mtDNA synthesis in the presence of an inflammatory stimulus and escalates inflammation via the STING pathway.

mtDNA Synthesis Genes in Macrophages and Atherosclerosis

• Selective silencing of Polg, Ppargc1a (Pgc1a), and Cmpk2 using a macrophage-specific in vivo siRNA delivery approach in Ldlr-/- mice fed an atherogenic diet led to significant decreases in plaque size and necrotic core area relative to the control group, while the fibrous cap thickness increased.

• Significant elevation was observed in splenic B cell numbers after Polg and Pgc1a silencing, and bone marrow T cells after Cmpk2, Polg, and Pgc1a knockdown compared to the control siRNA group.

• Macrophages in atherosclerotic plaques of mice treated with siRNA against the mtDNA synthesis genes significantly reduced the expression of TOM20 and 8-OHdG in atherosclerotic plaque macrophages. iNos expression was significantly decreased in atherosclerotic plaque macrophages after the silencing of the mitochondrial biogenesis genes.

• Cmpk2 silencing in macrophages in mice with atherosclerosis resulted in the most 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 Downstream Genes and Inflammation

• Female Ldlr−/− mice were lethally irradiated, transplanted with either LyzM+/+ Vcam1fl/fl or LyzMcre/+ Vcam1fl/fl bone marrow cells, and fed with a high fat diet for four months. RNA sequencing was performed with aortic macrophages isolated from these mice.

• 25 genes were differentially expressed in plaque macrophages lacking Vcam1, referred to as VCAM-1 downstream genes (Fcor, Nid1, Wisp2, Zfp300, Zfp940, Cxcr6, Nid1, Apoc4, and Itgb7).

• Silencing these genes in BMDM significantly increased overall inflammation.

• The VCAM-1 downstream genes are controlled by the mtDNA synthesis genes, with Cmpk2 and Polg silencing upregulating them in BMDM in the presence of oxidized LDL.

• VCAM1 silencing increased the expression of the VCAM-1 downstream genes compared to the control, and CMPK2 and STING overexpression rescinded the upregulation of the VCAM-1 downstream genes after VCAM1 silencing.

• The VCAM-1 downstream genes are regulated by CMPK2 and STING, and mtDNA transfection in BMDM lessened the expression of the VCAM-1 downstream genes.

• VCAM-1 dampens the expression of downstream genes via mtDNA synthesis and the STING pathway, and the VCAM-1 downstream genes curb inflammation.

• CEBP/α is a transcription factor regulated by VCAM-1, and its expression was downregulated in BMDM lacking Vcam1.

• Rac1 expression was significantly reduced in response to oxLDL in Vcam1-deficient macrophages, and Cebpα levels were significantly reduced after Rac1 silencing in BMDM.

• CEBP/α has a binding motif on the promoter regions of Cmpk2, Pgc1a, and Polg, binding to Cmpk2 and Pgc1a was reduced in the absence of Vcam1.

• Cebpα silencing significantly dampened the expression of Cmpk2 and Pgc1a but not Polg in BMDM. Macrophage VCAM-1 increases the expression of Cebpα, which binds to the promoter regions of Cmpk2 and Pgc1a, elevating their expression.

VCAM-1, FCOR, LYZ1, and Atherosclerosis

• Atherogenesis due to macrophage VCAM-1 is mediated by low Fcor and Lyz1 expression.

• The expression of Fcor, Lyz1, and Wisp2 was detectable in atherosclerotic plaque macrophages, their expression was downregulated in aortic macrophages present in atherosclerotic lesions.

• Generation of bone marrow chimeric Ldlr−/− mice lacking Vcam1 in macrophages, with Fcor, Lyz1, and Wisp2 silenced in macrophages using siRNA incorporated in DOTAP nanoparticles, showed that Fcor and Lyz1 silencing resulted in larger atherosclerotic plaques without changing fibrous cap thickness compared to control siRNA-treated mice, with siFcor significantly increasing necrotic core areas.

• Wisp2 knockdown in macrophages thickened fibrous caps without changing plaque and necrotic core areas. Silencing of these genes did not significantly alter the numbers and frequencies of leukocytes in the aorta, blood, bone marrow, and spleen, and HDL, LDL+vLDL, and total cholesterol levels. This is supported by the observation that myeloid STING deficiency rose Fcor and Lyz1, but not Wisp2, expression in atherosclerotic plaque macrophages. Analysis of aortic macrophages obtained from siFcor and siLyz1-treated mice showed upregulation of Akt2, Chkb, and Fkbp5, and downregulation of Mmp14, Rrp9 and Arg1 in both groups compared to siControl. The protective roles of Fcor and Lyz1 but not Wisp2 against atherosclerosis.

Discussion

• Atherosclerosis is the dominant cause of cardiovascular disease, and inflammatory macrophages are the most prevalent leukocytes within plaques.

• This cell population rises by differentiation of circulating inflammatory monocytes and local proliferation of plaque-resident macrophages.

• Plaque microenvironment (cholesterol, hypoxia, apoptotic debris) orchestrates metabolic rewiring of foam cells.

• Atherosclerotic plaque macrophages have VCAM-1-mediated high mtDNA synthesis, which amplifies inflammation and accelerates atherosclerosis progression.

• VCAM-1 and its ligand VLA-4 facilitate leukocyte entry into the intima.

• Endothelial cells express VCAM-1 at high levels, and hematopoietic cells, including monocytes, tissue macrophages, and dendritic cells, express this adhesion molecule in inflammatory conditions. However, the functions of leukocyte VCAM-1 are understudied, VCAM-1-mediated mtDNA synthesis also contributes to inflammation, which subsequently amplifies atherosclerosis.

• Aortic samples from atherosclerotic patients had greater mtDNA oxidative damage than nonatherosclerotic aortic samples.

• mtDNA drives noncanonical inflammation via the cyclic cGAS-STING signaling pathway in various diseases, leading to the production of interferon-stimulated genes and inflammatory cytokinesExcessive mtDNA synthesis is a prerequisite step in this process, there are several genes, which orchestrate mtDNA synthesis, such as PGC1A, CMPK2, and POLG. Mutations in POLG have been identified in patients with mitochondrial diseases such as Alpers syndrome and ataxia-neuropathy syndromes, CMPK2 supports the generations of oxidized mtDNA, then triggers NLRP3 inflammasome activation, Metformin was successfully used to reduce mtDNA synthesis, Oxidized and fragmented mtDNA can be crucial into subsequent inflammatory step,

• Besides activates STING, breaks in mtDNA trigger to RIG-I-MAVS-dependent immune response, and enhance the immune surveillanceIn the present study, VCAM-1 also increase of C/EBPa that helps to binds the promoter region of Cmpk2, therefore enhance the expression, oxidized and fragmented mtDNA triggers promotes the activation of cGAS-STING pathway-dependent inflammation aggravates atherosclerosis in C/EBPa-dependent manner.

• Furthermore, the VCAM-1-amplified mitochondrial metabolism stimulates inflammation and atherogenesis, independent of mtDNA sensing by STING.

• The study shows that the adhesion molecule VCAM-1 increases the expression of C/EBPa that binds to the promoter region of Cmpk2, augmenting its expression, then oxidized and fragmented mtDNA triggers the activation of cGAS-STING pathway thus aggravates atherosclerosis.

• The atheroprotective effects of VCAM-1 deficiency were due to elevated expression of FCOR and LYZ1, resulting in dampened inflammation; Fcor regulates insulin sensitivity and energy metabolism by acting to fine-tune FOXO1 activity, and LYZ1 has antibacterial properties, that is why these genes are controlled by the genes regulating mtDNA synthesis, such as POLG and CMPK2. Furthermore, macrophages lacking these genes are less inflammatory

• Targeting the VCAM-1 expression by plaque macrophage is an essential contributor of inflammation and atherosclerosis development and proposes an urgent need in targeting mtDNA synthesis in atherosclerosis.

Methods

• This section provides details on the experimental procedures, including animal models, cell culture, molecular biology techniques, and data analysis methods used in the study.

• It covers aspects such as animal handling, tissue preparation, flow cytometry, histology, western blotting, qPCR, metabolomics, bioenergetic measurements, and statistical analysis.

Conclusions

• The study concludes that macrophage VCAM-1 plays a crucial role in promoting mtDNA synthesis, which leads to STING-mediated inflammation and, consequently, accelerated atherogenesis.

• By demonstrating that VCAM-1 induces mtDNA synthesis in macrophages under inflammatory conditions, the research underscores a self-acerbating pathway that intensifies atherosclerosis.

• The findings suggest that targeting mtDNA synthesis could serve as a promising therapeutic strategy for managing and treating atherosclerosis and other inflammatory diseases.

• This approach may help mitigate the inflammatory responses driven by excessive mtDNA synthesis, thus reducing the progression and severity of these conditions.