How cellular metabolism facilitates tissue-resident macrophage maintenance remains elusive. Here we show that visceral adipose tissue (VAT)-resident macrophages, unlike monocyte-derived macrophages, are enriched with mitochondrial-specific antioxidant enzymes restraining inflammation and promoting VAT homeostasis and insulin sensitivity. Additionally, VAT resident macrophages express high levels of plasminogen activator inhibitor type 2, encoded by SerpinB2, which is involved in the blood coagulation cascade. SerpinB2 promotes adipose resident macrophage survival by regulating mitochondrial oxidative phosphorylation and preventing the release of pro-apoptotic cytochrome c from the mitochondria into the cytoplasm via antioxidant glutathione production. Chronic inflammation, such as obesity, diminishes SerpinB2 expression in VAT macrophages in patients and mice, leading to the decline of this macrophage subset. Mechanistically, interferon-γ elevation in diabetes induces Ikaros, a transcriptional suppressor, which binds to the SerpinB2 promoter and decreases SerpinB2 expression. Congruently, selective depletion of the IFN-γ receptor in myeloid cells or supplementation of macrophage-specific SerpinB2 deficient mice with N-acetylcysteine, a glutathione precursor, restores VAT resident macrophage survival, decreases adipocyte size, and improves glucose tolerance and insulin sensitivity. Our data thus reveal an unexpected function of SerpinB2 in the regulation of mitochondrial function and survival of tissue-resident macrophages.

Our post-GWAS functional analysis revealed that cathepsin L (CTSL) is an upstream regulator of CUX1, and it induces p16INK4a-dependent and atherosclerosis-associated senescence by indirectly activating CUX1 transcription in a process that requires its proteolytic activity. This suggests an unidentified transcription regulator between CTSL and CUX1, and CTSL-mediated cleavage of this regulator could transcribe CUX1, inducing senescence. Here, in search of this transcriptional regulator, we discovered that Notch1 is a substrate of CTSL, and CTSL can proteolytically activate Notch1 in a ligand-independent fashion, liberating NICD. NICD, after complexing with RBPJ in the nuclei, induces CUX1/p16INK4a-dependent senescence. Consistently, an upregulation of both CTSL and NICD, along with elevated cellular senescence in the plaques isolated from patients with atherosclerosis, was observed. In addition, we showed that endothelial deletion of CUX1 in the atherosclerosis-prone ApoE-/- mice blocks high-fat diet-induced senescence throughout the entire plaques, and these ApoE-/- mice exhibit similar phenotypes as the atherosclerosis-prone models with CTSL and Notch1/RBPJ inactivation including attenuated atherosclerotic lesion, intact and well-organized elastin fibers, and reduced macrophage content of plaque. This further supports our findings that both CTSL and Notch1/RBPJ are upstream regulators of CUX1, regulating senescence. Thus, while our studies identify a non-canonical Notch1 pathway that can be activated by CTSL in a ligand-independent fashion to induce senescence, our findings also reveal a role of senescence in the development of atherosclerosis. This provides new insight into developing drugs aimed to target cellular senescence for atherosclerosis.

Abstract Mitochondrial DNA (mtDNA) released into the cytosol activates innate immune signaling and promotes inflammation, yet its role in macrophages following sterile tissue injury remains poorly understood. Here, we show that cardiac macrophages from both patients and mice with myocardial infarction (MI) exhibit increased mitochondrial biogenesis, mitochondrial content, membrane potential, and expression of mitochondrial nucleases that facilitate mtDNA release. Consistently, macrophage-specific silencing of genes regulating mitochondrial biogenesis or mtDNA processing attenuated adverse cardiac remodeling after MI. Unexpectedly, despite the role of mtDNA in activating the cGAS–STING pathway, myeloid deletion or macrophage-specific silencing of Sting or cGas exacerbated ventricular dilation, fibrosis, and contractile dysfunction following MI. Single-cell transcriptomic and cell communication analyses identified amyloid precursor protein (APP) as a key downstream effector of STING in cardiac macrophages. Macrophage-specific in vivo App silencing rescued the detrimental effects of myeloid Sting deficiency, establishing APP as a critical mediator of adverse remodeling. Mechanistically, STING interacted with the transcriptional repressor MZF1, promoted its nuclear localization, facilitated its binding to the App promoter, and suppressed App transcription to restrain adverse cardiac remodeling. Together, our findings uncover an unexpected cardioprotective function of myeloid STING and identify the STING–MZF1–APP axis as a previously unrecognized mechanism governing cardiac repair after myocardial infarction.

Abstract Background Myocardial ischemia drives adverse cardiac remodeling, metabolic inflexibility, and progression to heart failure. Mitochondrial dysfunction and impaired substrate utilization contribute to cardiomyocyte death and fibrosis, particularly with aging. Humanin (HNG), a mitochondria-derived peptide, has been shown to reduce acute ischemic injury, but its role in chronic ischemia and cardiac remodeling remains unknown. Methods We investigated the effects of HNG treatment in young and aged murine models of myocardial ischemia without reperfusion. Cardiac function and structure were assessed by echocardiography and molecular markers of remodeling. Myocardial metabolism was interrogated using targeted metabolomics, gene expression, substrate uptake assays, and metabolic flux analyses. Mechanistic studies examined glucose transporter trafficking and protein–protein interactions. Results HNG treatment improved cardiac function and significantly attenuated adverse remodeling in both young and old mice. HNG treatment induced marked metabolic reprogramming characterized by reduced myocardial fatty acid content, downregulation of fatty acid uptake and oxidation pathways, and decreased oxidative stress. Importantly, these changes were accompanied by enhanced glucose oxidation, increased tricarboxylic acid cycle flux, improved coupling of glycolysis to mitochondrial oxidation, and increased ATP production. Time-course studies demonstrated that increased glucose oxidation preceded reductions in fatty acid oxidation, indicating a primary role for glucose metabolism in HNG-mediated cardioprotection. Mechanistically, we identified vesicle-associated membrane protein 7 (VAMP7) as a novel binding partner of HNG, and that this interaction is required for GLUT4 translocation to the plasma membrane and HNG-induced ATP generation. Conclusions HNG protects the ischemic heart by promoting metabolic reprogramming that shifts substrate utilization from fatty acids to glucose and limiting maladaptive remodeling. These findings identify HNG as a novel regulator of cardiac metabolism and a potential therapeutic strategy for ischemic heart failure. Graphical abstract: What are the clinical implications? Heart failure (HF) is a major global health concern, affecting over 6.7 million adults in the United States alone, with projections to exceed 11 million by 2050. Myocardial infarction (MI) is a leading cause of HF. Despite substantial advances in acute MI care, survivors remain at high risk for adverse cardiac remodeling and chronic HF, especially in the elderly. We report here that treatment with a potent analog of Humanin (HN), an endogenous mitochondria-associated peptide, decreases infarct size, decreases fibrosis and improves cardiac function following cardiac ischemia induced by permanent ligation of coronary artery in both young and aged mice. These effects are associated with changes in cardiac metabolism, oxidative stress, and remodeling. HN and analogs have been shown to be beneficial in many age-related diseases. The endogenous origin of Humanin, its favorable safety profile in preclinical studies and its pleiotropic effects support targeting HNG as a promising therapeutic strategy for ischemic heart disease and post–myocardial infarction heart failure in humans.

Recently, we have identified rs1333046 as one of the candidate functional single nucleotide polymorphisms (fSNPs) on the atherosclerosis-associated CDKN2A/B locus. However, how rs1333046 influences the pathogenesis of and susceptibility to atherosclerosis is unknown. In this work, we demonstrate that rs1333046 is part of a cis-regulatory element (cis-RE) that regulates p16INK4a and p16INK4a-dependent cellular senescence in human endothelial cells (ECs). This is achieved by recruiting poly(rC)-binding protein 2 (PCBP2), a member of the poly-cytosine binding protein family. We also reveal that PCBP2 is an upstream regulator of CD40, which regulates the expression of senescence-associated secretory phenotype (SASP) genes through NF-κB signaling. Moreover, consistent with PCBP2 being an iron chaperone, we discover that iron can induce cellular senescence by regulating both p16INK4a and CD40-mediated SASP gene expression through PCBP2. Notably, iron dynamically regulates p16INK4a expression by altering the binding of PCBP2 to rs1333046. In addition, reducing intracellular labile iron by overexpressing both iron storage protein ferritin light chain (FTL) and iron exporter ferroportin 1 (FPN1) in ECs suppresses cellular senescence, and overexpression of PCBP2 in both FTL- and FPN1-overexpressing cells restores cellular senescence. Thus, our studies suggest that iron could be a potential environmental factor regulating atherosclerosis-associated cellular senescence, and this is achieved by modulating PCBP2-dependent p16INK4a and CD40 expression. This study shows the mechanism by which iron affects the pathology of atherosclerosis.

Metabolic pathways, such as fatty acid oxidation and oxidative phosphorylation, can modulate inflammatory cells. However, little is known about the effects of the fatty acid synthesis pathway in macrophages on inflammation and cardiac remodeling after myocardial infarction (MI). Using spatial metabolomics, here we show that cardiac macrophages residing in the infarct synthesize de novo fatty acids and increase the production of fatty acid enzymes including ACLY and FASN. Mice deficient in myeloid Acly and Fasn have improved cardiac function after MI and reduced fibrosis. Combining Cleavage Under Targets and Release Using Nuclease (CUT&RUN), RNA sequencing analysis of Acly -/- macrophages, and macrophage-specific in vivo gene silencing, we demonstrate that ACLY acetylates the promoter region of the upstream regulator Krt17 , which drives the production of pro-fibrotic cytokines, including IL-33. Single-cell RNA sequencing of cardiac fibroblasts shows that the expansion of a population of fibroblasts (Fibroblast 5) expressing high levels of extracellular matrix genes after MI is confined in the absence of macrophage Acly . Finally, the analysis of spatial multi-omics data of human hearts with MI uncovers myofibroblasts with the Fibroblast 5 gene signature. These myofibroblasts are located near cardiac macrophages expressing high levels of ACLY. In summary, we show that macrophage ACLY and FASN are deleterious in MI pathogenesis.

BACKGROUND:Emergency myelopoiesis by bone marrow hematopoietic stem and progenitor cells (HSPCs) exacerbates disease pathology in various chronic diseases, including myocardial infarction (MI) and atherosclerosis. However, the mechanisms triggering myelopoiesis in the bone marrow after a distant organ injury, such as MI, remain unknown. METHODS:We ligated the left descending coronary artery to induce MI in mice. Platelet-derived extracellular vesicles (pEVs) were detected and enumerated in mice and patients with MI using NanoSight, ImageStream, and flow cytometry. microRNA in pEVs was quantified using a microRNA array. We used parabiosis, flow cytometry, adoptive transfer experiments, and transgenic mice to assess the effects of pEVs and microRNA on HSPC lineage commitment and inflammatory cell generation. In addition, we carried out RNA sequencing, luciferase assay, lentivirus-mediated in vivo gene overexpression, and echocardiography to evaluate the merit of lactoferrin/lactotransferrin in post-MI pathogenesis. RESULTS:In this study, we demonstrate that patients and mice with MI and mice with hindlimb ischemia exhibit an increased number of circulating pEVs, which, in turn, augment HSPC number and proliferation in the bone marrow, leading to inflammatory myeloid cell generation and disease progression. S100A8/9 (S100 calcium-binding protein A8/A9), an alarmin complex produced by cardiomyocytes after MI, induced pEV secretion. In vivo and in vitro inhibition of platelet activation and exocytosis, and HSPC endocytosis, markedly lessened the production of pEV, HSPC proliferation, and myeloid cell generation in emergency hematopoiesis. A microRNA array revealed that pEVs isolated after MI had elevated cargo levels of miR-499 and miR-184, which were enriched in reticulated platelets after MI. miR-499 and miR-184 overexpression in mouse and human HSPCs resulted in enhanced hematopoiesis and myelopoiesis. miR-499-deficient pEVs were inefficient in stimulating emergency myelopoiesis and inducing cardiac remodeling after MI. RNA sequencing analysis, luciferase assay, and lentivirus-mediated in vivo gene overexpression demonstrated that miR-499 bound to the 3' region of lactoferrin/lactotransferrin in HSPC to downregulate this gene, promoting myelopoiesis and unleashing inflammation. CONCLUSIONS:Our study suggests that pEVs generated after MI induce HSPC proliferation and inflammatory cell generation. These discoveries uncover several therapeutic targets to reduce cardiac inflammation and remodeling after MI.

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe−/− mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis. Macrophages and their metabolism are known to contribute to inflammation in the atherosclerotic plaques, but the underpinning molecular level regulatory processes are lesser known. Here authors show that under inflammatory conditions, macrophages express VCAM-1 within the atherosclerotic plaques, which leads to increased mitochondrial DNA synthesis via activation of the transcription factor C/EBPα, which in turn triggers inflammation by STING signalling.

The development of coronary artery disease (CAD) depends heavily on platelet activation, and inflammation plays a major role in all stages of atherosclerosis. Platelet-specific soluble triggering receptor expressed on myeloid cells like transcript 1 (sTLT-1) facilitate clot formation and have been linked to chronic inflammation. In this study, we explored the role of platelet-derived sTLT-1 in platelet-mediated inflammation in CAD patients. Plasma levels of sTLT-1 were measured using enzyme-linked immunosorbent assay in CAD patients (n = 163) and healthy controls (n = 99). Correlation analysis was performed to determine the circulatory sTLT-1 levels with platelet activation markers, immune cells, and inflammatory cytokines/chemokines. Increased plasma sTLT-1 levels were observed in CAD patients compared with those in healthy controls (p < 0.0001). A positive correlation was observed between sTLT-1 and platelet activation markers (P-selectin, PAC-1), CD14++ CD16- cells (classical monocytes), Natural killer T (NKT) cells, and platelet-immune cell aggregates with monocytes, neutrophils, dendritic cells, CD11c+ cells, and NKT cells. In contrast, a significant negative correlation was observed with CD8 cells. Furthermore, a significant positive correlation was observed between sTLT-1 and inflammatory markers (TNF-α, IL-1β, IL-2, IL-6, IL-12p70, IL-18, CXCL-12, and CCL-11). Logistic regression analysis identified sTLT-1 and triglycerides as predictors of CAD. Receiver operating characteristic curve (ROC) analysis showed that sTLT-1 had a higher sensitivity and specificity for predicting CAD. Our findings suggest that platelet activation induces the release of sTLT-1 into the circulation in CAD patients, which aggregates with immune cells and enhances inflammatory responses.

BACKGROUND:Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown.METHODS:We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 mu g/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction.RESULTS:Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NF kappa B (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NF kappa B, or CCR5.CONCLUSIONS:Our data demonstrate that CCL5/CCR5, through activation of NF kappa B and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.

Several preclinical studies have focused on the beneficial effects of garlic on cardiovascular diseases, but the results were inconsistent. We performed a systematic review and meta-analysis on the effect of garlic powder tablets and aged garlic extract (AGE) in CAD patients, mainly focusing on blood pressure, coronary artery calcification, lipid profile, and inflammatory markers. We searched PubMed, Cochrane CENTRAL, and Google Scholar to identify randomized controlled trials which examined garlic's effect on CAD patients. The standardized mean difference with 95% CI was calculated using fixed-effect or random-effect models. Garlic has shown statistically significant changes of HDL (SMD = 0.18; 95% CI = -0.00 to 0.37; p = .05); LDL (SMD = -0.27; 95% CI = -0.46 to -0.08; p = .004), apolipoprotein-A (SMD = 0.68; 95% CI = 0.24 1.13; p = .002), C-RP (SMD = -0.59; 95% CI = -0.92 to -0.25; p = .0007), IL-6 (SMD = -1.08; 95% CI = -2.17 to 0.01; p = .05), homocysteine (SMD = -0.66; 95% CI = -1.04 to -0.28; p = .0007) and CAC score (SMD = -1.61; 95% CI = -2.66 to -0.57; p = .003). In the case of subgroup analysis, the overall effect was significantly effective in reducing TC, LDL levels and improving HDL levels in CV risk patients. Our study findings provide consistent evidence that intake of garlic reduces CVD risk factors. However, garlic could be considered a safe natural medicine to debilitate inflammation in CAD patients.

Insulin resistance is a compromised response to insulin in target tissues such as liver. Emerging evidence shows that vascular endothelial cells (ECs) are critical in mediating glucose metabolism. However, how liver ECs can regulate inflammation in the setting of insulin resistance is still unknown. Using genome-wide transcriptome analysis of ECs isolated from diabetic mice, we found enrichment of the genes involved in epidermal growth factor receptor (Egfr) signaling. In line with this, hepatic sinusoidal ECs in diabetic mice had elevated levels of Egfr expression. Interestingly, we found an increased number of hepatic myeloid cells, especially macrophages, and systemic glucose intolerance in Cdh5Cre/+Egfrfl/fl mice lacking Egfr in ECs compared with littermate control mice with type II diabetes. Egfr deficiency upregulated the expression of MCP-1 in hepatic sinusoidal ECs. This resulted in augmented monocyte recruitment and macrophage differentiation in Cdh5Cre/+Egfrfl/fl mice compared with littermate control mice as determined by a mouse model of parabiosis. Finally, MCP-1 neutralization and hepatic macrophage depletion in Cdh5Cre/+Egfrfl/fl mice resulted in a reduced number of hepatic macrophages and ameliorated glucose intolerance compared with the control groups. Collectively, these results demonstrate a protective endothelial Egfr signaling in reducing monocyte-mediated hepatic inflammation and glucose intolerance in type II diabetic mice.

Background: Peripheral ischemia caused by peripheral artery disease is associated with systemic inflammation, which may aggravate underlying comorbidities such as atherosclerosis and heart failure. However, the mechanisms of increased inflammation and inflammatory cell production in patients with peripheral artery disease remain poorly understood. Methods: We used peripheral blood collected from patients with peripheral artery disease and performed hind limb ischemia (HI) in Apoe −/− mice fed a Western diet and C57BL/6J mice with a standard laboratory diet. Bulk and single-cell RNA sequencing analysis, whole-mount microscopy, and flow cytometry were performed to analyze hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and relocation. Results: We observed augmented numbers of leukocytes in the blood of patients with peripheral artery disease and Apoe −/− mice with HI. RNA sequencing and whole-mount imaging of the bone marrow revealed HSPC migration into the vascular niche from the osteoblastic niche and their exaggerated proliferation and differentiation. Single-cell RNA sequencing demonstrated alterations in the genes responsible for inflammation, myeloid cell mobilization, and HSPC differentiation after HI. Heightened inflammation in Apoe −/− mice after HI aggravated atherosclerosis. Surprisingly, bone marrow HSPCs expressed higher amounts of the receptors for IL (interleukin)-1 and IL-3 after HI. Concomitantly, the promoters of Il1r1 and Il3rb had augmented H3K4me3 and H3K27ac marks after HI. Genetic and pharmacological inhibition of these receptors resulted in suppressed HSPC proliferation, reduced leukocyte production, and ameliorated atherosclerosis. Conclusions: Our findings demonstrate increased inflammation, HSPC abundance in the vascular niches of the bone marrow, and elevated IL-3Rb and IL-1R1 (IL-1 receptor 1) expression in HSPC following HI. Furthermore, the IL-3Rb and IL-1R1 signaling plays a pivotal role in HSPC proliferation, leukocyte abundance, and atherosclerosis aggravation after HI.

Atherosclerotic cardiovascular disease is a leading cause of adult mortality worldwide. Macrophages play a key role in atherogenesis by sustaining the local inflammatory response, facilitating accumulation of immune cells in atheromas that contribute to plaque development. Mitochondrial metabolism plays a key role in governing inflammatory gene expression in macrophages. Yet, the role of mitochondrial biogenesis and mitochondrial DNA synthesis in regulating macrophage inflammation is not known. Vascular cell adhesion protein 1 (VCAM-1) expressed by endothelial cells mediates monocyte adhesion and extravasation in developing atherosclerotic plaques. We observed increased VCAM-1 expression in human and murine plaque macrophages. However, the function of this cell adhesion molecule expressed by macrophages is not known. Here, we evaluated if and how myeloid-VCAM-1 drive mitochondrial biogenesis, inflammation, and atherosclerosis pathology. Increased VCAM-1 expression in plaque macrophages correlated with oxidative DNA damage and mitochondrial volume. To understand the role of myeloid-Vcam1 in atherogenesis, we generated Apoe -/- LyzM cre/+ VCAM-1 fl/fl mice. Consistently, Apoe -/- mice lacking Vcam1 in macrophages exhibited reduced atherosclerosis severity and inflammation. Vcam1-deficient macrophages exhibited decreased inflammation, oxidative phosphorylation, mitochondrial biogenesis, and mitochondrial DNA synthesis genes, including Cmpk2. Cmpk2 deletion in macrophages after oxidized LDL treatment reduced inflammatory mediators that aggravate atherosclerosis. RNA sequencing analysis of Vcam-1 -deficient plaque macrophages and analysis of macrophages lacking Cmpk2 identified Fcor and Lyz1 as the target genes of Vcam1 and Cmpk2 . Interestingly, atherosclerotic plaque macrophages deficient of Sting , which mediates inflammatory signaling in response to oxidized mitochondrial DNA, had increased levels of Fcor and Lyz1 . Our data suggest that VCAM-1 in macrophages signals via CMPK2 and POLG to promote mitochondrial biogenesis, oxidation, and fragmentation, mediating atherogenesis.

Background Recently, our group identified increased platelet-mediated inflammation in type 2 diabetes (T2DM) patients, and it is a well-established risk factor for diabetes complications, particularly for the development of cardiovascular diseases (CVD). Furthermore, vitamin D is reported to play an important role in the modulation of platelet hyperactivity and immune function, although the effect of vitamin D on platelet-mediated inflammation is not well studied. Hence, we aimed to investigate the effect of vitamin D supplementation on platelet-mediated inflammation in T2DM patients. Methods After screening a total of 201 subjects, our randomized, double-blind, placebo-controlled trial included 59 vitamin-D-deficient T2DM subjects, and the participants were randomly assigned to placebo (n = 29) or vitamin D3 (n = 30) for 6 months. Serum vitamin D metabolite levels, immunome profiling, platelet activation, and platelet–immune cell aggregate formation were measured at baseline and at the end of the study. Similarly, the serum levels of inflammatory cytokines/chemokines were assessed by a multiplex assay. Results Six months of vitamin D supplementation increases the serum vitamin D3 and total 25(OH)D levels from the baseline (p < 0.05). Vitamin D supplementation does not improve glycemic control, and no significant difference was observed in immune cells. However, platelet activation and platelet immune cell aggregates were altered after the vitamin D intervention (p < 0.05). Moreover, vitamin D reduces the serum levels of IL-18, TNF-α, IFN-γ, CXCL-10, CXCL-12, CCL-2, CCL-5, CCL-11, and PF-4 levels compared to the baseline levels (p < 0.05). Our ex vivo experiment confirms that a sufficient circulating level of vitamin D reduces platelet activation and platelet intracellular reactive oxygen species. Conclusion Our study results provide evidence that vitamin D supportive therapy may help to reduce or prevent the disease progression and cardiovascular risk in T2DM patients by suppressing oxidative stress and platelet-mediated inflammation. Clinical Trial Registration Clinical Trial Registry of India: CTRI/2019/01/016921.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

There is a large body of evidence that cellular metabolism governs inflammation. However, whether mitochondrial biogenesis alters macrophage function and disease pathogenesis is not understood. We observed augmented mitochondrial DNA synthesis and biogenesis in macrophages of human and mouse atherosclerotic plaques. Unexpectedly, mitochondrial biogenesis in macrophages was triggered by vascular cell adhesion molecule 1 (VCAM-1) expressed by this myeloid cell subset in inflammatory conditions. RNA sequencing, bone marrow chimera, and gene silencing and overexpression experiments revealed that VCAM-1 in macrophages signals via CMPK2 and POLG to promote mitochondrial DNA synthesis, oxidation, and fragmentation. In vivo macrophage-specific gene downregulation experiments demonstrated that oxidized mitochondrial DNA released in the cytoplasm unleashes inflammation by controlling two downstream genes- LYZ1 and FCOR, which depend on the cGAS-STING signaling. Finally, VCAM-1 expression in human carotid plaque macrophages was correlated with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Consistently, Apoe-/- mice lacking Vcam1 in macrophages exhibited reduced atherosclerosis severity and inflammation.R01HL143967, R01HL142629, R01AG069399, and R01DK129339 to P.D.; the AHA Transformational Project Award (19TPA34910142), AHA Innovative Project Award (19IPLOI34760566) and ALA Innovation Project Award (IA-629694) to P.D.; the VMI Postdoctoral Training Program in Translational Research and Entrepreneurship in Pulmonary and Vascular Biology T32 funded by the National, Heart, Lung and Blood Institute (NHLBI) and United Therapeutics Jenesis award to J.F; and NHLBI F32HL146000 to N.N. cDNA generation, library preparation, and sequencing were performed by the University of Pittsburgh Health Science Sequencing Core at the UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA. We thank the NIH-supported microscopy resources at the Center for Biologic Imaging (NIH grant 1S10OD019973-01). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All animal experiments were performed according to the NIH guidelines, and the protocols of the animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Background Type 2 diabetes mellitus (T2DM) is a well-established risk factor for the development of atherosclerotic coronary artery disease. Platelet hyperactivity and inflammation are associated with the development of coronary artery disease (CAD) in T2DM patients. We investigated the status of immune cells, platelet activation, and platelet-immune cell interactions in T2DM_CAD patients. Methodology The study population consisted of four groups of subjects, healthy control (CT, n = 20), T2DM (n = 44), CAD (n = 20) and T2DM_CAD (n = 38). Platelet activation, immunome profiling and platelet-immune cell interactions were analysed by flow cytometry. The circulatory levels of inflammatory cytokines/chemokines were assessed using multiplex assay. Results Increased platelet activation and increased platelet-immune cell aggregate formation were observed in T2DM and T2DM_CAD groups compared to the control and CAD groups (p < 0.05). Our immunome profile analysis revealed, altered monocyte subpopulations and dendritic cell populations in T2DM, CAD and T2DM_CAD groups compared to the control group (p < 0.05). Furthermore, significantly increased IL-1β, IL-2, IL-4, IL-6, IL-8, IL12p70, IL-13 IL-18, CCL2, and decreased CXCL1, CCL5 levels were observed in T2DM_CAD group compared to the control group. Our ex-vivo study increased platelet-monocyte aggregate formation was observed upon D-glucose exposure in a time and concentration dependent manner. Conclusion Our data suggests that T2DM, CAD and T2DM_CAD are associated with altered immune cell populations. Furthermore, it has been confirmed that hyperglycemia induces platelet activation and forms platelet-immune cell aggregation which may lead to the release of inflammatory cytokines and chemokines and contribute to the complexity of CAD and type 2 diabetes.