There are multiple sources of reactive oxygen species (ROS) in the cell. As a major site of ROS production, mitochondria have drawn considerable interest because it was recently discovered that mitochondrial ROS (mtROS) directly stimulate the production of proinflammatory cytokines and pathological conditions as diverse as malignancies, autoimmune diseases, and cardiovascular diseases all share common phenotype of increased mtROS production above basal levels. Several excellent reviews on this topic have been published, but ever-changing new discoveries mandated a more up-to-date and comprehensive review on this topic. Therefore, we update recent understanding of how mitochondria generate and regulate the production of mtROS and the function of mtROS both in physiological and pathological conditions. In addition, we describe newly developed methods to probe or scavenge mtROS and compare these methods in detail. Thorough understanding of this topic and the application of mtROS-targeting drugs in the research is significant towards development of better therapies to combat inflammatory diseases and inflammatory malignancies.

Objective Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis. Approach and Results Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0 and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout (ApoE−/−) mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0 and 18:1 induced mtROS in primary human aortic ECs (HAECs), independently of the activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry-mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of AP-1 and inducing intercellular adhesion molecule 1 (ICAM-1) gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in ApoE−/− mice using intravital microscopy and flow cytometry methods. Conclusions ATP synthesis-uncoupled, but proton leak-coupled mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases.

It has been proposed that a mass transfer phenomenon called concentration polarization of low-density lipoproteins (LDLs) may occur in the arterial system and is likely involved in the localization of atherogenesis. To test the hypothesis that concentration polarization of LDL may be suppressed by the helical flow pattern in the human aorta, hence sparing the ascending aorta from atherosclerosis, the effects of aortic torsion, branching, curvature, and taper on blood flow and LDL transport in the lumen were simulated numerically under steady-state flow conditions using four aorta models constructed based on in vivo MRI slices. The results showed that it was the aortic torsion that induced the helical flow in the aortic arch, stabilizing the flow of blood in the aorta, and compensated the adverse effects of the aortic curvature on blood flow and LDL transport. The helical flow reduced the luminal surface LDL concentration in the aortic arch and probably played a role in suppressing severe polarization of LDL at the entrances of the three branches on the arch, hence, protecting them from atherogenesis. The taper of the aorta was another important feature of the aorta that further stabilized the flow of blood and delayed the attenuation of the helical flow, making it move beyond the arch and into the beginning part of the descending aorta. The results therefore may account for why the ascending aorta and the arch are relatively free of atherosclerosis.

Background— Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. Methods and Results— We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine β-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency ( Tg-hCBS apoE −/− Cbs −/− mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-α and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE −/− Cbs −/− mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE −/− Cbs −/− mice and another severe HHcy mouse model ( Tg-S466L Cbs −/− ) with a disease-relevant mutation ( Tg-S466L ) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6C hi and Ly-6C mid monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs −/− and Tg-hCBS apoE −/− Cbs −/− mice. These changes were exacerbated in Tg-S466L Cbs −/− mice with aging. Addition of l -homocysteine (100 to 500 μmol/L), but not l -cysteine, maintained the Ly-6C hi subset and induced the Ly-6C mid subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C mid subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. Conclusion— HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase–mediated oxidant stress.

IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation.

Background This study examined the causative role of hyperhomocysteinemia (HHcy) in atherogenesis and its effect on inflammatory monocyte (MC) differentiation. Methods and Results We generated a novel HHcy and hyperlipidemia mouse model, in which cystathionine β-synthase (CBS) and low-density lipoprotein receptor (LDLr) genes were deficient (Ldlr−/− Cbs−/+). Severe HHcy (plasma homocysteine (Hcy)=275 µM) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 µM from 244 µM by vitamin supplementation, which elevated plasma folate levels. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic mice after sub-lethal irradiation of the recipient. HHcy accelerated atherosclerosis and promoted Ly6Chigh inflammatory MC differentiation of both BM- and tissue-origins in the aortas and peripheral tissues. It also elevated plasma levels of TNF-α, IL-6 and MCP-1; increased vessel wall MC accumulation; and macrophage maturation. Hcy-lowering therapy reversed HHcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory MC (Ly6Chigh+middle) accumulation. Plasma Hcy levels were positively correlated with plasma levels of pro-inflammatory cytokines. In primary mouse splenocytes, L-Hcy promoted rIFNγ-induced inflammatory MC differentiation, as well as increased TNF-α, IL-6, and superoxide anion production in inflammatory MC subsets. Antioxidants and folic acid reversed L-Hcy-induced inflammatory MC differentiation and oxidative stress in inflammatory MC subsets. Conclusion HHcy causes vessel wall inflammatory MC differentiation and macrophage maturation of both BM- and tissue-origins leading to atherosclerosis via an oxidative stress related mechanism.

Rationale Chronic kidney disease (CKD) patients develop hyperhomocysteinemia (HHcy) and have a higher cardiovascular mortality than those without HHcy by 10-fold. Objective We investigated monocyte (MC) differentiation in human CKD and cardiovascular disease (CVD). Methods and Results We identified CD40 as a CKD-related MC activation gene using CKD-MC-mRNA array analysis and classified CD40 MC (CD40+CD14+) as a stronger inflammatory subset than the intermediate MC (CD14++CD16+) subset. We recruited 27 CVD/CKD patients and 14 healthy subjects and found that CD40/CD40 classical/CD40 intermediate MC (CD40+CD14+/CD40+CD14++CD16−/CD40+CD14++CD16+), plasma homocysteine (Hcy), S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) levels were higher in CVD and further elevated in CVD+CKD. CD40 and CD40 intermediate subsets were positively correlated with plasma/cellular Hcy levels and SAH and SAM but negatively correlated with estimated glomerular filtration rate (eGFR). HHcy was established as a likely mediator for CKD-induced CD40 intermediate MC, and reduced SAH/SAM was established for CKD-induced CD40/CD40 intermediate MC. Soluble CD40 ligand (sCD40L), TNFα/IL-6/IFNγ levels were elevated in CVD/CKD. CKD serum/Hcy/CD40L/TNFα/IL-6/IFNγ-induced CD40/CD40 intermediate MC in PBMC. Hcy and CKD serum-induced CD40 MC were prevented by neutralizing antibodies against CD40L/TNFα/IL-6. DNA hypomethylation was found on NFκB consensus element in CD40 promoter in WBC from CKD patients with lower SAM/SAH ratios. Finally, Hcy inhibited DNA methyltransferase-1 activity and promoted CD40 intermediate MC differentiation which was reversed by folic acid in PBMC. Conclusion CD40 MC is a novel inflammatory MC subset that appears to be a biomarker for CKD severity. HHcy mediates CD40 MC differentiation via sCD40L induction and CD40 DNA hypomethylation in CKD.

Innate immune cells express danger-associated molecular pattern (DAMP) receptors, T-cell costimulation/coinhibition receptors, and major histocompatibility complex II (MHC-II). We have recently proposed that endothelial cells can serve as innate immune cells, but the molecular mechanisms involved still await discovery. Here, we investigated whether human aortic endothelial cells (HAECs) could be transdifferentiated into innate immune cells by exposing them to hyperlipidemia-up-regulated DAMP molecules, lysophospholipids. Performing RNA-seq analysis of lysophospholipid-treated HAECs, we found that lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) regulate largely distinct gene programs as revealed by principal component analysis. Metabolically, LPC up-regulated genes that are involved in cholesterol biosynthesis, presumably through sterol regulatory element-binding protein 2 (SREBP2). By contrast, LPI up-regulated gene transcripts critical for the metabolism of glucose, lipids, and amino acids. Of note, we found that LPC and LPI both induce adhesion molecules, cytokines, and chemokines, which are all classic markers of endothelial cell activation, in HAECs. Moreover, LPC and LPI shared the ability to transdifferentiate HAECs into innate immune cells, including induction of potent DAMP receptors, such as CD36 molecule, T-cell costimulation/coinhibition receptors, and MHC-II proteins. The induction of these innate-immunity signatures by lysophospholipids correlated with their ability to induce up-regulation of cytosolic calcium and mitochondrial reactive oxygen species. In conclusion, lysophospholipids such as LPC and LPI induce innate immune cell transdifferentiation in HAECs. The concept of prolonged endothelial activation, discovered here, is relevant for designing new strategies for managing cardiovascular diseases.