Midline-1 regulates effector T cell motility in experimental autoimmune encephalomyelitis via mTOR/microtubule pathway.

Background: Effector T cell activation, migration, and proinflammatory cytokine production are crucial steps in autoimmune disorders such as multiple sclerosis (MS). While several therapeutic approaches targeting T cell activation and proinflammatory cytokines have been developed for the treatment of autoimmune diseases, there are no therapeutic agents targeting the migration of effector T cells, largely due to our limited understanding of regulatory mechanisms of T cell migration in autoimmune disease. Here we reported that midline-1 (Mid1) is a key regulator of effector T cell migration in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS. Methods: Mid1-/- mice were generated by Crispr-Cas9 technology. T cell-specific Mid1 knockout chimeric mice were generated by adoptive transfer of Mid1-/- T cells into lymphocyte deficient Rag2-/- mice. Mice were either immunized with MOG35-55 (active EAE) or received adoptive transfer of pathogenic T cells (passive EAE) to induce EAE. In vitro Transwell® assay or in vivo footpad injection were used to assess the migration of T cells. Results: Mid1 was significantly increased in the spinal cord of wild-type (Wt) EAE mice and disruption of Mid1 in T cells markedly suppressed the development of both active and passive EAE. Transcriptomic and flow cytometric analyses revealed a marked reduction in effector T cell number in the central nervous system of Mid1-/- mice after EAE induction. Conversely, an increase in the number of T cells was observed in the draining lymph nodes of Mid1-/- mice. Mice that were adoptively transferred with pathogenic Mid1-/- T cells also exhibited milder symptoms of EAE, along with a lower T cell count in the spinal cord. Additionally, disruption of Mid1 significantly inhibited T-cell migration both in vivo and in vitro. RNA sequencing suggests a suppression in multiple inflammatory pathways in Mid1-/- mice, including mTOR signaling that plays a critical role in cell migration. Subsequent experiments confirmed the interaction between Mid1 and mTOR. Suppression of mTOR with rapamycin or microtubule spindle formation with colcemid blunted the regulatory effect of Mid1 on T cell migration. In addition, mTOR agonists MHY1485 and 3BDO restored the migratory deficit caused by Mid1 depletion. Conclusion: Our data suggests that Mid1 regulates effector T cell migration to the central nervous system via mTOR/microtubule pathway in EAE, and thus may serve as a potential therapeutic target for the treatment of MS.

Exposure to Air Pollution Disrupts Circadian Rhythm through Alterations in Chromatin Dynamics.

Particulate matter ≤2.5µm (PM2.5) air pollution is a leading environmental risk factor contributing disproportionately to the global burden of non-communicable disease. We compared impact of chronic exposure to PM2.5 alone, or with light at night exposure (LL) on metabolism. PM2.5 induced peripheral insulin resistance, circadian rhythm (CR) dysfunction, and metabolic and brown adipose tissue (BAT) dysfunction, akin to LL (with no additive interaction between PM2.5 and LL). Transcriptomic analysis of liver and BAT revealed widespread but unique alterations in CR genes, with evidence for differentially accessible promoters and enhancers of CR genes in response to PM2.5 by ATAC-seq. The histone deacetylases 2, 3, and 4 were downregulated with PM2.5 exposure, with increased promoter occupancy by the histone acetyltransferase p300 as evidenced by ChIP-seq. These findings suggest a previously unrecognized role of PM2.5 in promoting CR disruption and metabolic dysfunction through epigenetic regulation of circadian targets.

Differential contribution of bone marrow-derived infiltrating monocytes and resident macrophages to persistent lung inflammation in chronic air pollution exposure.

Chronic exposure to particulate matter < 2.5µ (PM2.5) has been linked to cardiopulmonary disease. Tissue-resident (TR) alveolar macrophages (AΦ) are long-lived, self-renew and critical to the health impact of inhalational insults. There is an inadequate understanding of the impact of PM2.5 exposure on the nature/time course of transcriptional responses, self-renewal of AΦ, and the contribution from bone marrow (BM) to this population. Accordingly, we exposed chimeric (CD45.2/CD45.1) mice to concentrated PM2.5 or filtered air (FA) to evaluate the impact on these end-points. PM2.5 exposure for 4-weeks induced an influx of BM-derived monocytes into the lungs with no contribution to the overall TR-AΦ pool. Chronic (32-weeks) PM2.5 exposure on the other hand while associated with increased recruitment of BM-derived monocytes and their incorporation into the AΦ population, resulted in enhanced apoptosis and decreased proliferation of TR-AΦ. RNA-seq analysis of isolated TR-AΦ and BM-AΦ from 4- and 32-weeks exposed mice revealed a unique time-dependent pattern of differentially expressed genes. PM2.5 exposure resulted in altered histological changes in the lungs, a reduced alveolar fraction which corresponded to protracted lung inflammation. Our findings suggest a time-dependent entrainment of BM-derived monocytes into the AΦ population of PM2.5 exposed mice, that together with enhanced apoptosis of TR-AΦ and reorganization of transcriptional responses, could collectively contribute to the perpetuation of chronic inflammation.

Metabolic effects of air pollution exposure and reversibility.

Air pollution involving particulate matter smaller than 2.5 µm in size (PM2.5) is the world's leading environmental risk factor contributing to mortality through cardiometabolic pathways. In this study, we modeled early life exposure using chow-fed C57BL/6J male mice that were exposed to real-world inhaled, concentrated PM2.5 (~10 times ambient levels/~60-120 µg/m3) or filtered air over a 14-week period. We investigated the effects of PM2.5 on phenotype, the transcriptome, and chromatin accessibility and compared these with the effects of a prototypical high-fat diet (HFD) as well as cessation of exposure on phenotype reversibility. Exposure to PM2.5 impaired glucose and insulin tolerance and reduced energy expenditure and 18FDG-PET uptake in brown adipose tissue. Multiple differentially expressed gene clusters in pathways involving metabolism and circadian rhythm were noted in insulin-responsive tissues. Although the magnitude of transcriptional change detected with PM2.5 exposure was lower than that observed with a HFD, the degree of alteration in chromatin accessibility after PM2.5 exposure was significant. The novel chromatin remodeler SMARCA5 (SWI/SNF complex) was regulated in response to PM2.5 exposure, the cessation of which was associated with a reversal of insulin resistance and restoration of chromatin accessibility and nucleosome positioning near transcription start sites, as well as a reversal of exposure-induced changes in the transcriptome, including SMARCA5. These changes indicate pliable epigenetic control mechanisms following cessation of exposure.

Novel Long Noncoding RNA, Macrophage Inflammation-Suppressing Transcript (MIST), Regulates Macrophage Activation During Obesity.

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.

CITED2 Restrains Proinflammatory Macrophage Activation and Response.

Macrophages are strategically distributed in mammalian tissues and play an essential role in priming the immune response. However, macrophages need to constantly strike a balance between activation and inhibition states to avoid a futile inflammatory reaction. Here, we identify the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) as a potent repressor of macrophage proinflammatory activation. Gain- and loss-of-function studies revealed that CITED2 is required for optimal peroxisome proliferator-activated receptor gamma (PPARγ) activation and attendant select anti-inflammatory gene expression in macrophages. More importantly, deficiency of CITED2 resulted in significant attenuation of rosiglitazone-induced PPARγ activity, PPARγ recruitment to target gene promoters, and anti-inflammatory target gene expression in macrophages. Interestingly, deficiency of Cited2 strikingly heightened proinflammatory gene expression through stabilization of hypoxia-inducible factor 1 alpha (HIF1α) protein in macrophages. Further, overexpression of Egln3 or inhibition of HIF1α in Cited2-deficient macrophages completely reversed elevated proinflammatory cytokine/chemokine gene expression. Importantly, mice bearing a myeloid cell-specific deletion of Cited2 were highly susceptible to endotoxin-induced sepsis symptomatology and mortality. Collectively, our observations identify CITED2 as a novel negative regulator of macrophage proinflammatory activation that protects the host from inflammatory insults.

Noncoding RNAs in Cardiovascular Disease: Pathological Relevance and Emerging Role as Biomarkers and Therapeutics.

Noncoding RNAs (ncRNA) include a diverse range of functional RNA species-microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) being most studied in pathophysiology. Cardiovascular morbidity is associated with differential expression of myriad miRNAs; miR-21, miR-155, miR-126, miR-146a/b, miR-143/145, miR-223, and miR-221 are the top 9 most reported miRNAs in hypertension and atherosclerotic disease. A single miRNA may have hundreds of messenger RNA targets, which makes a full appreciation of the physiologic ramifications of such broad-ranging effects a challenge. miR-21 is the most prominent ncRNA associated with hypertension and atherosclerotic disease due to its role as a "mechano-miR", responding to arterial shear stresses. "Immuno-miRs", such as miR-155 and miR-223, affect cardiovascular disease (CVD) via regulation of hematopoietic cell differentiation, chemotaxis, and activation in response to many pro-atherogenic stimuli. "Myo-miRs", such as miR-1 and miR-133, affect cardiac muscle plasticity and remodeling in response to mechanical overload. This in-depth review analyzes observational and experimental reports of ncRNAs in CVD, including future applications of ncRNA-based strategies in diagnosis, prediction (e.g., survival and response to small molecule therapy), and biologic therapy.

The Role of the Mineralocorticoid Receptor in Inflammation: Focus on Kidney and Vasculature.

BACKGROUND: The remarkable success of clinical trials in mineralocorticoid receptor (MR) inhibition in heart failure has driven research on the physiological and pathological role(s) of nonepithelial MR expression. MR is widely expressed in the cardiovascular system and is a major determinant of endothelial function, smooth muscle tone, vascular remodeling, fibrosis, and blood pressure. An important new dimension is the appreciation of the role MR plays in immune cells and target organ damage in the heart, kidney and vasculature, and in the development of insulin resistance. SUMMARY: The mechanism for MR activation in tissue injury continues to evolve with the evidence to date suggesting that activation of MR results in a complex repertoire of effects involving both macrophages and T cells. MR is an important transcriptional regulator of macrophage phenotype and function. Another important feature of MR activation is that it can occur even with normal or low aldosterone levels in pathological conditions. Tissue-specific conditional models of MR expression in myeloid cells, endothelial cells, smooth muscle cells and cardiomyocytes have been very informative and have firmly demonstrated a critical role of MR as a key pathophysiologic variable in cardiac hypertrophy, transition to heart failure, adipose inflammation, and atherosclerosis. Finally, the central nervous system activation of MR in permeable regions of the blood-brain barrier may play a role in peripheral inflammation. Key Message: Ongoing clinical trials will help clarify the role of MR blockade in conditions, such as atherosclerosis and chronic kidney disease.

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages.

Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.

"Eat me" imaging and therapy.

Clearance of apoptotic debris is a vital role of the innate immune system. Drawing upon principles of apoptotic clearance, convenient delivery vehicles including intrinsic anti-inflammatory characteristics and specificity to immune cells can be engineered to aid in drug delivery. In this article, we examine the use of phosphatidylserine (PtdSer), the well-known "eat-me" signal, in nanoparticle-based therapeutics making them highly desirable "meals" for phagocytic immune cells. Use of PtdSer facilitates engulfment of nanoparticles allowing for imaging and therapy in various pathologies and may result in immunomodulation. Furthermore, we discuss the targeting of the macrophages and other cells at sites of inflammation in disease. A thorough understanding of the immunobiology of "eat-me" signals is requisite for the successful application of "eat-me"-bearing materials in biomedical applications.

Hybrid nanoparticles improve targeting to inflammatory macrophages through phagocytic signals.

Macrophages are innate immune cells with great phenotypic plasticity, which allows them to regulate an array of physiological processes such as host defense, tissue repair, and lipid/lipoprotein metabolism. In this proof-of-principle study, we report that macrophages of the M1 inflammatory phenotype can be selectively targeted by model hybrid lipid-latex (LiLa) nanoparticles bearing phagocytic signals. We demonstrate a simple and robust route to fabricate nanoparticles and then show their efficacy through imaging and drug delivery in inflammatory disease models of atherosclerosis and obesity. Self-assembled LiLa nanoparticles can be modified with a variety of hydrophobic entities such as drug cargos, signaling lipids, and imaging reporters resulting in sub-100nm nanoparticles with low polydispersities. The optimized theranostic LiLa formulation with gadolinium, fluorescein and "eat-me" phagocytic signals (Gd-FITC-LiLa) a) demonstrates high relaxivity that improves magnetic resonance imaging (MRI) sensitivity, b) encapsulates hydrophobic drugs at up to 60% by weight, and c) selectively targets inflammatory M1 macrophages concomitant with controlled release of the payload of anti-inflammatory drug. The mechanism and kinetics of the payload discharge appeared to be phospholipase A2 activity-dependent, as determined by means of intracellular Förster resonance energy transfer (FRET). In vivo, LiLa targets M1 macrophages in a mouse model of atherosclerosis, allowing noninvasive imaging of atherosclerotic plaque by MRI. In the context of obesity, LiLa particles were selectively deposited to M1 macrophages within inflamed adipose tissue, as demonstrated by single-photon intravital imaging in mice. Collectively, our results suggest that phagocytic signals can preferentially target inflammatory macrophages in experimental models of atherosclerosis and obesity, thus opening the possibility of future clinical applications that diagnose/treat these conditions. Tunable LiLa nanoparticles reported here can serve as a model theranostic platform with application in various types of imaging of the diseases such as cardiovascular disorders, obesity, and cancer where macrophages play a pathogenic role.

T-cell costimulation protects obesity-induced adipose inflammation and insulin resistance.

A key pathophysiologic role for activated T-cells in mediating adipose inflammation and insulin resistance (IR) has been recently postulated. However, mechanisms underlying their activation are poorly understood. In this study, we demonstrated a previously unrecognized homeostatic role for the costimulatory B7 molecules (CD80 and CD86) in preventing adipose inflammation. Instead of promoting inflammation, which was found in many other disease conditions, B7 costimulation reduced adipose inflammation by maintaining regulatory T-cell (Treg) numbers in adipose tissue. In both humans and mice, expression of CD80 and CD86 was negatively correlated with the degree of IR and adipose tissue macrophage infiltration. Decreased B7 expression in obesity appeared to directly impair Treg proliferation and function that lead to excessive proinflammatory macrophages and the development of IR. CD80/CD86 double knockout (B7 KO) mice had enhanced adipose macrophage inflammation and IR under both high-fat and normal diet conditions, accompanied by reduced Treg development and proliferation. Adoptive transfer of Tregs reversed IR and adipose inflammation in B7 KO mice. Our results suggest an essential role for B7 in maintaining Tregs and adipose homeostasis and may have important implications for therapies that target costimulation in type 2 diabetes.

CXCR3 modulates obesity-induced visceral adipose inflammation and systemic insulin resistance.

OBJECTIVE: Chemokine (C-X-C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regulation of immune cell trafficking and activation. Increased CXCR3 expression in the visceral adipose of obese humans and mice was observed. A pathophysiologic role for CXCR3 in diet-induced obesity (DIO) was hypothesized. METHODS: Wild-type (WT) C57B/L6J and chemokine receptor 3 knockout (CXCR3(-/-) ) mice were fed a high-fat diet (HFD) for 20 weeks followed by assessment of glucose metabolism and visceral adipose tissue (VAT) inflammation. RESULTS: CXCR3(-/-) mice exhibited lower fasting glucose and improved glucose tolerance compared with WT-HFD mice, despite similar body mass. HFD-induced VAT innate and adaptive immune cell infiltration, including immature myeloid cells (CD11b(+) F4/80(lo) Ly6C(+) ), were markedly ameliorated in CXCR3(-/-) mice. In vitro IBIDI and in vivo migration assays demonstrated no CXCR3-mediated effect on macrophage or monocyte migration, respectively. CXCR3(-/-) macrophages, however, had a blunted response to interferon-γ, a TH 1 cytokine that induces macrophage activation. CONCLUSIONS: A previously unreported role for CXCR3 in the development of HFD-induced insulin resistance (IR) and VAT macrophage infiltration in mice was demonstrated. Our results support pharmaceutical targeting of the CXCR3 receptor as a potential treatment for obesity/IR.

Aliskiren effect on plaque progression in established atherosclerosis using high resolution 3D MRI (ALPINE): a double-blind placebo-controlled trial.

BACKGROUND: The renin-angiotensin system is well recognized as a mediator of pathophysiological events in atherosclerosis. The benefits of renin inhibition in atherosclerosis, especially when used in combination with angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEIs/ARBs) are currently not known. We hypothesized that treatment with the renin inhibitor aliskiren in patients with established cardiovascular disease will prevent the progression of atherosclerosis as determined by high-resolution magnetic resonance imaging (MRI) measurements of arterial wall volume in the thoracic and abdominal aortas of high-risk patients with preexisting cardiovascular disease. METHODS AND RESULTS: This was a single-center, randomized, double-blind, placebo-controlled trial in patients with established cardiovascular disease. After a 2-week single-blind placebo phase, patients were randomized to receive either placebo (n=37, mean ± SD age 64.5 ± 8.9 years, 3 women) or 150 mg of aliskiren (n=34, mean ± SD age 63.9 ± 11.5 years, 9 women). Treatment dose was escalated to 300 mg at 2 weeks and maintained during the remainder of the study. Patients underwent dark-blood, 3-dimensional MRI assessment of atherosclerotic plaque in the thoracic and abdominal segments at baseline and on study completion or termination (up to 36 weeks of drug or matching placebo). Aliskiren use resulted in significant progression of aortic wall volume (normalized total wall volume 5.31 ± 6.57 vs 0.15 ± 4.39 mm(3), P=0.03, and percentage wall volume 3.37 ± 2.96% vs 0.97 ± 2.02%, P=0.04) compared with placebo. In a subgroup analysis of subjects receiving ACEI/ARB therapy, atherosclerosis progression was observed only in the aliskiren group, not in the placebo group. CONCLUSIONS: MRI quantification of atheroma plaque burden demonstrated that aliskiren use in patients with preexisting cardiovascular disease resulted in an unexpected increase in aortic atherosclerosis compared with placebo. Although preliminary, these results may have implications for the use of renin inhibition as a therapeutic strategy in patients with cardiovascular disease, especially in those receiving ACEI/ARB therapy. CLINICAL TRIAL REGISTRATION: URL: http://ClinicalTrials.gov Unique identifier: NCT01417104.

Autocrine function of aldehyde dehydrogenase 1 as a determinant of diet- and sex-specific differences in visceral adiposity.

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1(-/-) mice, but not males, were resistant to high-fat (HF) diet-induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1(-/-) mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet-induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

A potential role for dendritic cell/macrophage-expressing DPP4 in obesity-induced visceral inflammation.

Dipeptidyl peptidase-4 (DDP4) inhibitors target the enzymatic degradation of incretin peptides and represent a major advance in the treatment of type 2 diabetes. DPP4 has a number of nonenzymatic functions that involve its interaction with adenosine deaminase (ADA) and other extracellular matrix proteins. Here, we assessed the nonenzymatic role of DPP4 in regulating dendritic cell (DC)/macrophage-mediated adipose inflammation in obesity. Both obese humans and rodents demonstrated increased levels of DPP4 expression in DC/macrophage cell populations from visceral adipose tissue (VAT). The DPP4 expression increased during monocyte differentiation to DC/macrophages and with lipopolysaccharide (LPS)-induced activation of DC/macrophages. The DPP4 colocalized with membrane-bound ADA on human DCs and enhanced the ability of the latter to stimulate T-cell proliferation. The DPP4 interaction with ADA in human DC/macrophages was competitively inhibited by the addition of exogenous soluble DPP4. Knockdown of DPP4 in human DCs, but not pharmacologic inhibition of their enzymatic function, significantly attenuated the ability to activate T cells without influencing its capacity to secrete proinflammatory cytokines. The nonenzymatic function of DPP4 on DC may play a role in potentiation of inflammation in obesity by interacting with ADA. These findings suggest a novel role for the paracrine regulation of inflammation in adipose tissue by DPP4.

Effects of a novel pharmacologic inhibitor of myeloperoxidase in a mouse atherosclerosis model.

Inflammation and oxidative stress play fundamental roles in the pathogenesis of atherosclerosis. Myeloperoxidase has been extensively implicated as a key mediator of inflammatory and redox-dependent processes in atherosclerosis. However, the effect of synthetic myeloperoxidase inhibitors on atherosclerosis has been insufficiently studied. In this study, ApoE(-/-) mice were randomized to low- and high-dose INV-315 groups for 16 weeks on high-fat diet. INV-315 resulted in reduced plaque burden and improved endothelial function in response to acetylcholine. These effects occurred without adverse events or changes in body weight or blood pressure. INV-315 treatment resulted in a decrease in iNOS gene expression, superoxide production and nitrotyrosine content in the aorta. Circulating IL-6 and inflammatory CD11b(+)/Ly6G(low)/7/4(hi) monocytes were significantly decreased in response to INV-315 treatment. Acute pretreatment with INV-315 blocked TNFα-mediated leukocyte adhesion in cremasteric venules and inhibited myeloperoxidase activity. Cholesterol efflux was significantly increased by high-dose INV-315 via ex-vivo reverse cholesterol transport assays. Our results suggest that myeloperoxidase inhibition may exert anti-atherosclerotic effects via inhibition of oxidative stress and enhancement of cholesterol efflux. These findings demonstrate a role for pharmacologic modulation of myeloperoxidase in atherosclerosis.

Effect of co-exposure to nickel and particulate matter on insulin resistance and mitochondrial dysfunction in a mouse model.

BACKGROUND: It has been well recognized that toxicity of fine ambient air particulate matter (PM(2.5)) may depend on its chemical constituents, including components such as soluble metals that may theoretically exert distinctive effects. We have recently demonstrated an important effect of PM(2.5) on metabolic function. Since transition metals, such as nickel (Ni), represent an important component of exposure in certain environments, and may significantly influence the toxicity of inhalational exposure, we investigated the effects of Ni as a variable component of ambient PM(2.5) exposure. METHODS: Male ApoE knockout mice were exposed to filtered air (FA), fine-sized nickel sulfate particles alone (Ni) at 0.44 µg/m(3), concentrated ambient air PM(2.5) (CAPs) at a mean of 70 µg/m(3), or CAPs+Ni in Tuxedo, NY, 6 hours/day, 5 days/week, for 3 months. RESULTS: Exposure to Ni, irrespective of co-exposure to CAPs, resulted in body weight gain, while exposure to CAPs+Ni significantly enhanced fasting glucose and worsened insulin resistance measures (HOMA-IR), when compared with exposure to CAPs alone. CAPs+Ni exposure induced a significant decrease in phosphorylation of AMP-activated protein kinase (AMPK) α. Exposure to Ni or CAPs+Ni significantly induced microcirculatory dysfunction and increased monocytic cell infiltration into lung and adipose, and decreased uncoupling protein 1 expression at gene and protein levels and several brown adipocyte-specific genes in adipose tissue. CONCLUSIONS: Ni exposure has effects on metabolic and inflammatory parameters that are comparable to that of CAPs. Additionally, Ni synergistically exacerbates CAPs-induced adverse effects on some of, but not all of, these parameters, that may be mediated via the AMPK signaling pathway. These findings have important implications for inhaled transition metal toxicity that may exert synergistic effects with other PM(2.5) components.

In vivo targeting of inflammation-associated myeloid-related protein 8/14 via gadolinium immunonanoparticles.

OBJECTIVE: Myeloid-related protein (Mrp) 8/14 complex (is a highly expressed extracellularly secreted protein, implicated in atherosclerosis. In this study, we evaluated the feasibility of targeting Mrp in vivo through synthetic immuno-nanoprobes. METHODS AND RESULTS: Anti-Mrp-14 and nonspecific IgG-conjugated gadolinium nanoprobes (aMrp-) were synthesized and characterized. Pharmacokinetics and vascular targeting via MRI of the formulations were assessed in vivo in high fat-fed apolipoprotein E deficient (ApoE(-/-)), ApoE(-/-)/Mrp14(-/-) (double knockout) and chow-fed wild-type (C57BL/6) mice. Bone marrow-derived myeloid progenitor cells were isolated from both ApoE(-/-) and double knockout mice, differentiated to macrophages, and were treated with LPS, with or without Mrp8, Mrp14, or Mrp8/14; conditioned media was used for in vitro studies. Mrp-activated cells secreted significant amounts of proinflammatory cytokines, which was abolished by pretreatment with aMrp-NP. We show in vitro that aMrp-NP binds endothelial cells previously treated with conditioned media containing Mrp8/14. MRI following intravenous delivery of aMrp-NP revealed prolonged and substantial delineation of plaque in ApoE(-/-) but not double knockout or wild-type animals. Nonspecific IgG-conjugated gadolinium nanoprobe-injected animals in all groups did not show vessel wall enhancement. Flow-cytometric analysis of aortic digesta revealed that aMrp-NP present in Ly-6G(+), CD11b(+), CD11c(+), and CD31(+) cells in ApoE(-/-) but not in double knockout animals. CONCLUSIONS: Targeted imaging with aMrp-NP demonstrates enhancement of plaque with binding to inflammatory cells and reduction in inflammation. This strategy has promise as a theranostic approach for atherosclerosis.