miR-210/NF-κB axis: A new direction for regulating cadmium-induced pig artery inflammatory injury.

Cadmium (Cd) is a toxic metal pollutant that still exists in the environment. The microRNA (miRNA) is a type of noncoding RNA that plays an important role in gene posttranscriptional regulation and disease development. Although the toxic effects of Cd have been extensively studied, studies on the mechanism of Cd from the perspective of miRNA are still limited. So, we established a Cd-exposure pig model, which confirmed that Cd exposure would cause pig artery damage. The miR-210 with the most reduced expression and the nuclear factor kappa B (NF-κB) that had a targeting relationship with miR-210 were screened. The effect of miR-210/NF-κB on the artery damage induced by Cd exposure was investigated by acridine orange/ethidium bromide staining, reactive oxygen species (ROS) staining, quantitative PCR, and western blotting. The results showed that miR-210 inhibitor, pcDNA-NF-κB could induce ROS overproduction in pig hip artery endothelial cells, thus inducing Th1/Th2 imbalance and necroptosis, leading to increased inflammation, while small interfering RNA-NF-κB played a mitigating role. In conclusion, Cd can induce artery necroptosis and Th1/Th2 imbalance by regulating the miR-210/NF-κB axis, so as to lead to artery inflammatory damage. In this study, we explored the way in which Cd exposure causes artery damage in pig, providing a new perspective on the regulatory damage of miR-210/NF-κB axis.

Arterial wall inflammation assessed by 18F-FDG-PET/CT is higher in individuals with Type 1 diabetes and associated with circulating inflammatory proteins.

AIMS: The article investigates whether chronic hyperglycaemia in Type 1 diabetes (T1D) is associated with a proinflammatory immune signature and with arterial wall inflammation, driving the development of atherosclerosis. METHODS AND RESULTS: Patients with T1D (n = 41), and healthy age-, sex-, and body mass index-matched controls (n = 20) were recruited. Arterial wall inflammation and haematopoietic activity were measured with 2'-deoxy-2'-(18F)-fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography. In addition, flow cytometry of circulating leucocytes was performed as well as targeted proteomics to measure circulating inflammatory markers. 18F-FDG uptake in the wall of the abdominal aorta, carotid arteries, and iliac arteries was higher in T1D compared with that in the healthy controls. Also, 18F-FDG uptake in the bone marrow and spleen was higher in patients with T1D. CCR2 and CD36 expressions on circulating monocytes were higher in patients with T1D, as well as several circulating inflammatory proteins. In addition, several circulating inflammatory markers (osteoprotegerin, transforming growth factor-alpha, CX3CL1, and colony-stimulating factor-1) displayed a positive correlation with FDG uptake. Within T1D, no differences were found between people with a high and low HbA1c. CONCLUSION: These findings strengthen the concept that chronic hyperglycaemia in T1D induces inflammatory changes that fuel arterial wall inflammation leading to atherosclerosis. The degree of hyperglycaemia appears to play a minor role in driving this inflammatory response in patients with T1D.

Histologic and Immunohistochemical Evaluation of Infiltrating Inflammatory Cells in Kawasaki Disease Arteritis Lesions.

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology which predominantly affects medium- and small-sized muscular arteries. Histopathologic studies of KD vasculitis lesions have demonstrated characteristic T cell infiltration and an abundance of CD8 T cells; however, the contribution of cytotoxic lymphocytes to KD vasculitis lesions has not been identified. Here, we histopathologically and immunohistochemically examined infiltrating inflammatory cells, particularly cytotoxic protein-positive cells, such as granzyme B cells and TIA-1 cells, in KD vasculitis lesions. Three autopsy specimens with acute-phase KD were observed and contained 24 vasculitis lesions affecting medium-sized muscular arteries, excluding pulmonary arteries. Infiltrating neutrophils in vasculitis lesions were evaluated by hematoxylin and eosin staining, and monocytes/macrophages and lymphocytes were evaluated by immunohistochemistry. The predominant cells were CD163 monocytes/macrophages and CD3 T cells. CD8 T cells, granzyme B cells, and TIA-1 cells were also observed, but CD56 natural killer cells were rare. To the best of our knowledge, the current study is the first histopathologic report confirming the infiltration of inflammatory cells with cytotoxic proteins in vasculitis lesions in patients with KD. Cytotoxic T cells may play a role in the development of vasculitis lesions in KD patients.

[68Ga]Ga-Pentixafor for PET Imaging of Vascular Expression of CXCR-4 as a Marker of Arterial Inflammation in HIV-Infected Patients: A Comparison with 18F[FDG] PET Imaging.

People living with human immunodeficiency virus (PLHIV) have excess risk of atherosclerotic cardiovascular disease (ASCVD). Arterial inflammation is the hallmark of atherogenesis and its complications. In this study we aimed to perform a head-to-head comparison of fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG PET/CT) and Gallium-68 pentixafor positron emission tomography/computed tomography [68Ga]Ga-pentixafor PET/CT for quantification of arterial inflammation in PLHIV. We prospectively recruited human immunodeficiency virus (HIV)-infected patients to undergo [18F]FDG PET/CT and [68Ga]Ga-pentixafor PET/CT within two weeks of each other. We quantified the levels of arterial tracer uptake on both scans using maximum standardized uptake value (SUVmax) and target-background ratio. We used Bland and Altman plots to measure the level of agreement between tracer quantification parameters obtained on both scans. A total of 12 patients were included with a mean age of 44.67 ± 7.62 years. The mean duration of HIV infection and mean CD+ T-cell count of the study population were 71.08 ± 37 months and 522.17 ± 260.33 cells/µL, respectively. We found a high level of agreement in the quantification variables obtained using [18F]FDG PET and [68Ga]Ga-pentixafor PET. There is a good level of agreement in the arterial tracer quantification variables obtained using [18F]FDG PET/CT and [68Ga]Ga-pentixafor PET/CT in PLHIV. This suggests that [68Ga]Ga-pentixafor may be applied in the place of [18F]FDG PET/CT for the quantification of arterial inflammation.

FDG PET/CT for evaluating systemic arterial inflammation induced by anthracycline-based chemotherapy of Hodgkin lymphoma: A retrospective cohort study.

To evaluate arterial fluorodeoxyglucose (FDG) uptake as a marker of arterial inflammation in multiple vascular beds in patients treated with anthracycline-based chemotherapy for Hodgkin lymphoma (HL).We used maximum standardized uptake value (SUVmax) and target-to-background ratio (TBR) to quantify arterial FDG uptake in the carotid artery, ascending aorta, abdominal aorta, and femoral artery obtained on positron emission tomography/computed tomography (PET/CT) imaging performed at baseline before chemotherapy and after completion of chemotherapy in patients with HL treated with an anthracycline-containing regimen. We compared the SUVmax and TBR obtained at baseline with that obtained post-chemotherapy for each arterial bed to evaluate the effect of anthracycline-based chemotherapy. We evaluated the effect of cardiovascular risk factors such as human immunodeficiency virus (HIV) infection, smoking, hypertension, and diabetes on the changes in SUVmax and TBR seen in the different arterial beds after anthracycline-based chemotherapy.Fifty-two patients were included with a mean age of 34.56 ±â€Š10.19 years. There were 33 males, and 18 patients were HIV-infected. The mean interval between completion of chemotherapy and follow-up flourine-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) scan was 65 weeks. We found no significant difference in arterial FDG uptake measured by SUVmax and TBR in all arterial beds between the pre- and post-chemotherapy FDG PET/CT. There was no significant impact of HIV infection, smoking, and hypertension on the changes in arterial FDG uptake following treatment with anthracycline-based chemotherapy.In patients with HL who were treated with anthracycline-based chemotherapy, we found no significant increase in arterial inflammation measured by FDG PET/CT after an average follow-up period of about 65 weeks since completion of chemotherapy.

l-Arginine prevents inflammatory and pro-calcific differentiation of interstitial aortic valve cells.

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.

Dectin-2-induced CCL2 production in tissue-resident macrophages ignites cardiac arteritis.

Environmental triggers, including those from pathogens, are thought to play an important role in triggering autoimmune diseases, such as vasculitis, in genetically susceptible individuals. The mechanism by which activation of the innate immune system contributes to vessel-specific autoimmunity in vasculitis is not known. Systemic administration of Candida albicans water-soluble extract (CAWS) induces vasculitis in the aortic root and coronary arteries of mice that mimics human Kawasaki disease. We found that Dectin-2 signaling in macrophages resident in the aortic root of the heart induced early CCL2 production and the initial recruitment of CCR2+ inflammatory monocytes (iMo) into the aortic root and coronary arteries. iMo differentiated into monocyte-derived dendritic cells (Mo-DC) in the vessel wall and were induced to release IL-1ß in a Dectin-2-Syk-NLRP3 inflammasome dependent pathway. IL-1ß then activated cardiac endothelial cells to express CXCL1 and CCL2 and adhesion molecules that induced neutrophil and further iMo recruitment and accumulation in the aortic root and coronary arteries. Our findings demonstrate that Dectin-2-mediated induction of CCL2 production by macrophages resident in the aortic root and coronary arteries initiates vascular inflammation in a model of Kawasaki disease, suggesting an important role for the innate immune system in initiating vasculitis.

Stage-dependent differential effects of interleukin-1 isoforms on experimental atherosclerosis.

AIMS: Targeting interleukin-1 (IL-1) represents a novel therapeutic approach to atherosclerosis. CANTOS demonstrated the benefits of IL-1ß neutralization in patients post-myocardial infarction with residual inflammatory risk. Yet, some mouse data have shown a prominent role of IL-1α rather than IL-1ß in atherosclerosis, or even a deleterious effect of IL-1 on outward arterial remodelling in atherosclerosis-susceptible mice. To shed light on these disparate results, this study investigated the effect of neutralizing IL-1α or/and IL-1ß isoforms starting either early in atherogenesis or later in ApoE-/- mice with established atheroma. METHODS AND RESULTS: The neutralization of IL-1α or of both IL-1 isoforms impaired outward remodelling during early atherogenesis as assessed by micro-computed tomographic and histologic assessment. In contrast, the neutralization of IL-1ß did not impair outward remodelling either during early atherogenesis or in mice with established lesions. Interleukin-1ß inhibition promoted a slant of blood monocytes towards a less inflammatory state during atherogenesis, reduced the size of established atheromata, and increased plasma levels of IL-10 without limiting outward remodelling of brachiocephalic arteries. CONCLUSION: This study established a pivotal role for IL-1α in the remodelling of arteries during early experimental atherogenesis, whereas IL-1ß drives inflammation during atherogenesis and the evolution of advanced atheroma in mice.

The suppressive effect of the three-herb extract mixture on vascular and liver inflammation in atherogenic diet with high fructose-fed mice.

CONTEXT: Cynanchum wilfordii (Maximowicz) Hemsley (Apocynaceae), Arctium lappa L. var. rubescens Frivald (Asteraceae) and Dioscorea opposite Thunb (Dioscoreaceae) root extracts have been widely used as an alternative for intervening obesity. OBJECTIVES: The synergistic effect of three-herb mixture of C. wilfordii, A. lappa and D. opposita was determined on aortic and liver inflammatory responses. MATERIALS AND METHODS: CWE, ALE and DOE were prepared from the root of C. wilfordii, A. lappa and D. opposite by 70% ethanol extraction, respectively. CADE was prepared using a powder mixture of 2 CWE:1 ALE:1 DOE. C57BL/6 mice were fed an atherogenic diet combined with 10% fructose (ATHFR) in the presence of 200 mg/kg/day CWE, ALE, DOE or CADE for 8 weeks (each group, n = 6). Biochemical profiles, protein expression of vascular cell adhesion molecule-1 (VCAM-1) on the aorta and liver were determined. RESULTS: CADE could significantly suppress the protein expression of VCAM-1 in both the aorta and liver (80% reduction) compared to ATHFR-fed mice. Impairment of liver function was significantly ameliorated by CADE supplement, as determined by GOT (60% reduction) and GPT (51% reduction) levels. CONCLUSIONS: CADE should be considered when developing medications to suppress the vascular and liver inflammatory responses for individuals who are either non-responsive or resistant to lipid-lowering drugs.

CCR2 expression on circulating monocytes is associated with arterial wall inflammation assessed by 18F-FDG PET/CT in patients at risk for cardiovascular disease.

AIMS: Circulating monocytes infiltrate the plaque and differentiate into macrophages, contributing to an inflammatory environment which is associated with higher risk of cardiovascular events. Although the pivotal role of circulating monocytes in plaque inflammation has been firmly established, the search continues to identify specific monocyte subsets that may be especially atherogenic. Therefore, we evaluated the relation between monocyte phenotype, particularly surface receptor expression, and arterial wall inflammation in patients at increased cardiovascular risk. METHODS AND RESULTS: We performed a multivariate linear regression analysis in 79 patients at increased cardiovascular risk who had both an 18F-fluorodeoxyglucose positron emission tomography/computed tomography to assess arterial wall inflammation and extensive monocyte characterization (using flow cytometry). We found that CCR2, a monocyte chemokine receptor essential for transmigration, significantly correlates with arterial wall inflammation. This relationship was independent of traditional cardiovascular risk factors and statin use (ß = 0.429, P = 0.015). We found no relation between arterial wall inflammation and monocyte count or monocyte subsets, namely CD14+CD16-, CD14+CD16+, CD14+CD16 ++, CCR5+, CD18+, CD11b+, or CD11c+ monocytes. CONCLUSION: Monocyte CCR2 expression is associated with arterial wall inflammation in patients at increased cardiovascular risk. Our data warrant further studies to assess if inhibition of CCR2 may attenuate atherosclerotic plaque inflammation.

Lymphocytic thrombophilic arteritis: A distinct inflammatory type I interferon and C5b-9 mediated subcutaneous endovasculitis.

BACKGROUND: Lymphocytic thrombophilic arteritis is a recently recognized subcuticular larger vessel vasculitis characterized by striking vascular luminal thrombosis. METHODS: The clinical features, histopathology and phenotypic profile of ten patients with lymphocytic thrombophilic arteritis were explored in an attempt to better define the entity from a clinical and pathophysiologic perspective. RESULTS: The patients were all female (mean age of 43) presenting with generally asymptomatic lower and upper extremity hyperpigmented macules. A consistent picture diagnostic of a connective tissue disease syndrome was not seen. The disease was not progressive although it was typically persistent. The morphology was characterized by a temporally heterogeneous subcutaneous arteritis targeting the endothelium and intima with changes ranging from incipient intimal expansion by hyaluronic acid to concentric intimal fibrin deposition to one of an end stage acellular intraluminal obliterative fibrous arteriopathy. The infiltrate was predominated by lymphocytes and histiocytes. The intimal elastic lamina was intact in most cases. All tested cases showed intimal and endothelial C5b-9 deposition, an upregulated type I interferon microenvironment and marked upregulation of the inducible interferon gamma 16 protein. CONCLUSIONS: Lymphocytic thrombophilic arteritis is a unique form of C5b-9 mediated arteritic endotheliopathy where the brunt of the changes involves the endothelium and intima and that is morphologically distinct from the transmural arteritis of benign cutaneous polyarteritis nodosa.

Allograft Inflammatory Factor-1 Links T-Cell Activation, Interferon Response, and Macrophage Activation in Chronic Kawasaki Disease Arteritis.

BACKGROUND: Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. METHODS: Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. RESULTS: Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. CONCLUSIONS: Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.

Oligodendrocyte death, neuroinflammation, and the effects of minocycline in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

Purpose: Optic nerve (ON) damage following nonarteritic anterior ischemic optic neuropathy (NAION) and its models is associated with neurodegenerative inflammation. Minocycline is a tetracycline derivative antibiotic believed to exert a neuroprotective effect by selective alteration and activation of the neuroinflammatory response. We evaluated minocycline's post-induction ability to modify early and late post-ischemic inflammatory responses and its retinal ganglion cell (RGC)-neuroprotective ability. Methods: We used the rodent NAION (rNAION) model in male Sprague-Dawley rats. Animals received either vehicle or minocycline (33 mg/kg) daily intraperitoneally for 28 days. Early (3 days) ON-cytokine responses were evaluated, and oligodendrocyte death was temporally evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Cellular inflammation was evaluated with immunohistochemistry, and RGC preservation was compared with stereology of Brn3a-positive cells in flat mounted retinas. Results: Post-rNAION, oligodendrocytes exhibit a delayed pattern of apoptosis extending over a month, with extrinsic monocyte infiltration occurring only in the primary rNAION lesion and progressive distal microglial activation. Post-induction minocycline failed to improve retinal ganglion cell survival compared with the vehicle treated (893.14 vs. 920.72; p>0.9). Cytokine analysis of the rNAION lesion 3 days post-induction revealed that minocycline exert general inflammatory suppression without selective upregulation of cytokines associated with the proposed alternative or neuroprotective M2 inflammatory pathway. Conclusions: The pattern of cytokine release, extended temporal window of oligodendrocyte death, and progressive microglial activation suggests that selective neuroimmunomodulation, rather than general inflammatory suppression, may be required for effective repair strategies in ischemic optic neuropathies.

Perinatal Arterial Ischemic Stroke Is Associated to Materno-Fetal Immune Activation and Intracranial Arteritis.

The medium-size intra-cranial arteries arising from the carotid bifurcation are prone to perinatal arterial ischemic strokes (PAIS). PAIS' physiopathology needs to be better understood to develop preventive and therapeutic interventions that are currently missing. We hypothesized that materno-fetal inflammation leads to a vasculitis affecting selectively the carotidian tree and promoting a focal thrombosis and subsequent stroke. Dams were injected with saline or lipopolysaccharide (LPS) from Escherichia coli. A prothrombotic stress was applied on LPS-exposed vs. saline (S)-exposed middle cerebral arteries (MCA). Immunolabeling detected the inflammatory markers of interest. In S-exposed newborn pups, a constitutive higher density of macrophages combined to higher expressions of tumor necrosis factor-α (TNF-α), and interleukin 1ß (IL-1ß) was observed within the wall of intra- vs. extra-cranial cervicocephalic arteries. LPS-induced maternal and placental inflammatory responses mediated by IL-1ß, TNF-α and monocyte chemotactic protein 1 (MCP-1) were associated with: (i) increased density of pro-inflammatory macrophages (M1 phenotype); and (ii) pro-inflammatory orientation of the IL-1 system (IL-1ß/IL-1 receptor antagonist (IL-1Ra) ratio) within the wall of LPS-, vs. S-exposed, intra-cranial arteries susceptible to PAIS. LPS plus photothrombosis, but not sole photothrombosis, triggered ischemic strokes and subsequent motor impairments. Based on these preclinical results, the combination of pro-thrombotic stress and selective intra-cranial arteritis arising from end gestational maternal immune activation seem to play a role in the pathophysiology of human PAIS.

Resistin Enhances Inflammatory Cytokine Production in Coronary Artery Tissues by Activating the NF-κB Signaling.

Purpose. Kawasaki disease (KD) is a systemic vasculitis and is a leading cause of coronary artery lesions (CALs) in childhood. Our previous study has shown higher levels of serum Resistin in KD patients with coronary aneurysm. This study aimed at examining the association of Resistin with inflammatory cytokine expression in mouse model of coronary arteritis and determining the potential mechanisms. Methods. C57BL/6 mice were injected with Lactobacillus cell wall extract (LCWE) to induce coronary arteritis. The relative levels of Resistin, TNF-α, IL-1ß, and MMP-9 expression and inflammatory infiltrates in the coronary arteries were determined longitudinally by quantitative RT-PCR, ELISA, and histology. The effect of TLR4 and NF-κB activation on Resistin-induced TNF-α and IL-1ß expression in human coronary artery endothelium cells (HCAECs) was examined by quantitative RT-PCR. Results. Higher levels of Resistin, TNF-α, IL-1ß, and MMP-9 expression were associated with the degrees of inflammatory infiltrates in the coronary artery walls of the LCWE-injected mice. Resistin enhanced TNF-α and IL-1ß expression in HCAECs at 18 or 24 hours after stimulation. Pretreatment with anti-TLR4 attenuated Resistin-enhanced IL-1ß, but not TNF-α, expression and pretreatment with parthenolide or QNZ demolished Resistin-enhanced TNF-α expression in HACECs. Pretreatment with parthenolide, but not QNZ, blocked Resistin-enhanced IL-1ß expression in HCAECs. Conclusion. Resistin may enhance inflammation by cross-talking with TLR4/NF-κB signaling during the development of coronary arteritis in mice.

GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo.

UNLABELLED: (18)F-FDG accumulates in glycolytically active tissues and is known to concentrate in tissues that are rich in activated macrophages. In this study, we tested the hypotheses that human granulocyte-macrophage colony-stimulating factor (GM-CSF), a clinically used cytokine, increases macrophage glycolysis and deoxyglucose uptake in vitro and acutely enhances (18)F-FDG uptake within inflamed tissues such as atherosclerotic plaques in vivo. METHODS: In vitro experiments were conducted on human macrophages whereby inflammatory activation and uptake of radiolabeled 2-deoxyglucose was assessed before and after GM-CSF exposure. In vivo studies were performed on mice and New Zealand White rabbits to assess the effect of GM-CSF on (18)F-FDG uptake in normal versus inflamed arteries, using PET. RESULTS: Incubation of human macrophages with GM-CSF resulted in increased glycolysis and increased 2-deoxyglucose uptake (P < 0.05). This effect was attenuated by neutralizing antibodies against tumor necrosis factor-α or after silencing or inhibition of 6-phosphofructo-2-kinase. In vivo, in mice and in rabbits, intravenous GM-CSF administration resulted in a 70% and 73% increase (P < 0.01 for both), respectively, in arterial (18)F-FDG uptake in atherosclerotic animals but not in nonatherosclerotic controls. Histopathologic analysis demonstrated a significant correlation between in vivo (18)F-FDG uptake and macrophage staining (R = 0.75, P < 0.01). CONCLUSION: GM-CSF substantially augments glycolytic flux in vitro (via a mechanism dependent on ubiquitous type 6-phosphofructo-2-kinase and tumor necrosis factor-α) and increases (18)F-FDG uptake within inflamed atheroma in vivo. These findings demonstrate that GM-CSF can be used to enhance detection of inflammation. Further studies should explore the role of GM-CSF stimulation to enhance the detection of inflammatory foci in other disease states.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

AIM: The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. METHODS: Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. RESULTS: Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. CONCLUSION: AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity.

Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common causes of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase (COX)-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A(2) were determined in human endothelial cells stimulated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). Moreover, Gper-deficient (Gper(-/-)) and WT mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, contractions to acetylcholine-stimulated endothelial vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by twofold. Ovariectomy also augmented prostanoid-dependent contractions by twofold in WT mice but had no additional effect in Gper(-/-) mice. These contractions were blocked by the COX inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor l-N(G)-nitroarginine methyl ester. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to treat increased prostanoid-dependent vasomotor tone or vascular disease in postmenopausal women.

Identification of Pathogenic Cardiac CD11c+ Macrophages in Nod1-Mediated Acute Coronary Arteritis.

OBJECTIVE: Nod1 is an intracellular pattern recognition receptor for bacterial peptidoglycan fragments. We previously reported that a synthetic Nod1 ligand, FK565, induced acute coronary arteritis in mice similar to that of Kawasaki disease. However, the molecular mechanisms underlying this characteristic inflammation have remained elusive. APPROACH AND RESULTS: We found that CD11c(+)MHC class II(+) cells accumulated in the heart of FK565-treated mice before arteritis development. Morphological features and gene expression signatures of the cardiac CD11c(+)MHC class II(+) cells suggested that this population is closely related to macrophages, and thus, we designated them cardiac CD11c(+) macrophages. Nod1 in nonhematopoietic cells, rather than hematopoietic cells, was required for the increase of cardiac CD11c(+) macrophages and arteritis development. Among nonhematopoietic cells, cardiac endothelial cells produced a large amount of chemokines in response to FK565. Endothelial cell-specific blockade of Nod1 signaling suppressed FK565-induced expression of these chemokines, accumulation of cardiac CD11c(+) macrophages, and subsequent coronary arteritis development. We also found that CCR2(+)Ly6C(hi) inflammatory monocytes in peripheral blood supplied precursors of cardiac CD11c(+) macrophages. CCR2-deficient mice or pertussis toxin-treated mice exhibited decreased numbers of cardiac CD11c(+) macrophages and reduced arteritis. CONCLUSIONS: These results suggest that Ly6C(hi) monocytes are recruited to FK565-activated endothelial cells to generate cardiac CD11c(+) macrophages, which play a pivotal role in the pathogenesis of acute coronary arteritis.