Cathelicidin Antimicrobial Peptide Levels in Atherosclerosis and Myocardial Infarction in Mice and Human.

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.

Decreased circulating CTRP3 levels in acute and chronic cardiovascular patients.

C1q/TNF-related protein 3 (CTRP3) represents an adipokine with various metabolic and immune-regulatory functions. While circulating CTRP3 has been proposed as a potential biomarker for cardiovascular disease (CVD), current data on CTRP3 regarding coronary artery disease (CAD) remains partially contradictory. This study aimed to investigate CTRP3 levels in chronic and acute settings such as chronic coronary syndrome (CCS) and acute coronary syndrome (ACS). A total of 206 patients were classified into three groups: CCS (n = 64), ACS having a first acute event (ACS-1, n = 75), and ACS having a recurrent acute event (ACS-2, n = 67). The control group consisted of 49 healthy individuals. ELISA measurement in peripheral blood revealed decreased CTRP3 levels in all patient groups (p < 0.001) without significant differences between the groups. This effect was exclusively observed in male patients. Females generally exhibited significantly higher CTRP3 plasma levels than males. ROC curve analysis in male patients revealed a valuable predictive potency of plasma CTRP3 in order to identify CAD patients, with a proposed cut-off value of 51.25 ng/mL. The sensitivity and specificity of prediction by CTRP3 were congruent for the subgroups of CCS, ACS-1, and ACS-2 patients. Regulation of circulating CTRP3 levels in murine models of cardiovascular pathophysiology was found to be partly opposite to the clinical findings, with male mice exhibiting higher circulating CTRP3 levels than females. We conclude that circulating CTRP3 levels are decreased in both male CCS and ACS patients. Therefore, CTRP3 might be useful as a biomarker for CAD but not for distinguishing an acute from a chronic setting. KEY MESSAGES: CTRP3 levels were found to be decreased in both male CCS and ACS patients compared to healthy controls. Plasma CTRP3 has a valuable predictive potency in order to identify CAD patients among men and is therefore proposed as a biomarker for CAD but not for distinguishing between acute and chronic settings. Regulation of circulating CTRP3 levels in murine models of cardiovascular pathophysiology was found to be partly opposite to the clinical findings in men.

Cellular and Systemic Effects of Micro- and Nanoplastics in Mammals-What We Know So Far.

Microplastics (MP) and nanoplastics (NP) are accumulating more and more in our environment and have been frequently detected in water and soil, but also in a variety of mainly marine organisms. Polymers such as polyethylene, polypropylene, and polystyrene are those most commonly found. Once in the environment, MP/NP are carriers for many other substances, which often convey toxic effects. Even though intuitively it is thought that ingesting MP/NP cannot be healthy, little is known about their effects on mammalian cells and organisms so far. To better understand the potential hazards of MP/NP on humans and to offer an overview of the already associated pathological effects, we conducted a comprehensive literature review on cellular effects, as well as experimental animal studies on MP/NP in mammals.

Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study.

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve. Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold's biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.

Case Report: Arterial Wall Inflammation in Atherosclerotic Cardiovascular Disease is Reduced by Olamkicept (sgp130Fc).

Inflammation is a strong driver of atherosclerotic cardiovascular disease (ASCVD). There is a large unmet need for therapies that prevent or reduce excessive inflammation while avoiding systemic immunosuppression. We showed previously that selective inhibition of pro-inflammatory interleukin-6 (IL-6) trans-signalling by the fusion protein olamkicept (sgp130Fc) prevented and reduced experimental murine atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr -/-) mice on a high-fat, high-cholesterol diet independently of low-density lipoprotein (LDL) cholesterol metabolism. Therefore, we allowed compassionate use of olamkicept (600 mg intravenously biweekly for 10 weeks) in a patient with very-high-risk ASCVD. Despite optimal LDL cholesterol under maximum tolerated lipid-lowering treatment, the patient had a remaining very high risk for future cardiovascular events related to significant arterial wall inflammation with lipoprotein (a) [Lp(a)]-cholesterol as the main contributor. 18Fluorodeoxyglucose positron emission tomography/computed tomography (18FDG PET/CT) measurements were performed before and after the treatment period. Olamkicept reduced arterial wall inflammation in this patient without interfering with lipoprotein metabolism. No clinical or laboratory side effects were observed during or after treatment with olamkicept. Our findings in this patient matched the results from our mechanistic study in Ldlr -/- mice, which were extended by additional analyses on vascular inflammation. Olamkicept may be a promising option for treating ASCVD independently of LDL cholesterol metabolism. A Phase II trial of olamkicept in ASCVD is currently being prepared.

Extracellular Ribosomal RNA Acts Synergistically with Toll-like Receptor 2 Agonists to Promote Inflammation.

Self-extracellular RNA (eRNA), which is released under pathological conditions from damaged tissue, has recently been identified as a new alarmin and synergistic agent together with toll-like receptor (TLR)2 ligands to induce proinflammatory activities of immune cells. In this study, a detailed investigation of these interactions is reported. The macrophage cell line J774 A.1 or C57 BL/6 J wild-type mice were treated with 18S rRNA and different TLR2 agonists. Gene and protein expression of tumor necrosis factor (Tnf)-α; interleukin (Il)-1ß, Il-6; or monocyte chemoattractant protein (Mcp)-1 were analyzed and furthermore in vitro binding studies to TLR2 were performed. The TLR2/TLR6-agonist Pam2 CSK4 (Pam2) together with 18S rRNA significantly increased the mRNA expression of inflammatory genes and the release of TNF-α from macrophages in a TLR2- and nuclear factor kappa B (NF-κB)-dependent manner. The injection of 18S rRNA/Pam2 into mice increased the cytokine levels of TNF-α, IL-6, and MCP-1 in the peritoneal lavage. Mechanistically, 18S rRNA built complexes with Pam2 and thus enhanced the affinity of Pam2 to TLR2. These results indicate that the alarmin eRNA, mainly consisting of rRNA, sensitizes TLR2 to enhance the innate immune response under pathological conditions. Thus, rRNA might serve as a new target for the treatments of bacterial and viral infections.

Monocyte subpopulation profiling indicates CDK6-derived cell differentiation and identifies subpopulation-specific miRNA expression sets in acute and stable coronary artery disease.

Coronary artery disease (CAD) is a long-lasting inflammatory disease characterized by monocyte migration into the vessel wall leading to clinical events like myocardial infarction (MI). However, the role of monocyte subsets, especially their miRNA-driven differentiation in this scenario is still in its infancy. Here, we characterized monocyte subsets in controls and disease phenotypes of CAD and MI patients using flow cytometry and miRNA and mRNA expression profiling using RNA sequencing. We observed major differences in the miRNA profiles between the classical (CD14++CD16-) and nonclassical (CD14+CD16++) monocyte subsets irrespective of the disease phenotype suggesting the Cyclin-dependent Kinase 6 (CDK6) to be an important player in monocyte maturation. Between control and MI patients, we found a set of miRNAs to be differentially expressed in the nonclassical monocytes and targeting CCND2 (Cyclin D2) that is able to enhance myocardial repair. Interestingly, miRNAs as miR-125b playing a role in vascular calcification were differentially expressed in the classical subset in patients suffering from CAD and not MI in comparison to control samples. In conclusion, our study describes specific peculiarities of monocyte subset miRNA expression in control and diseased samples and provides basis to further functional analysis and to identify new cardiovascular disease treatment targets.

Polystyrene microplastic particles induce endothelial activation.

Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the µm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 µm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.

Anti-Inflammatory Effects of C1q/Tumor Necrosis Factor-Related Protein 3 (CTRP3) in Endothelial Cells.

The C1q/TNF-related protein 3 (CTRP3) represents a pleiotropic adipokine reciprocally associated with obesity and type 2 diabetes mellitus and exhibits anti-inflammatory properties in relation to lipopolysaccharides (LPS)-mediated effects in adipocytes, as well as monocytes/macrophages. Here, we focused on the influence of CTRP3 on LPS-mediated effects in endothelial cells in order to expand the understanding of a possible anti-inflammatory function of CTRP3 in a setting of endotoxemia. An organ- and tissue-specific expression analysis by real-time PCR revealed a considerable Ctrp3 expression in various adipose tissue compartments; however, higher levels were detected in the aorta and in abundantly perfused tissues (bone marrow and the thyroid gland). We observed a robust Ctrp3 expression in primary endothelial cells and a transient upregulation in murine endothelial (MyEND) cells by LPS (50 ng/mL). In MyEND cells, CTRP3 inhibited the LPS-induced expression of interleukin (Il)-6 and the tumor necrosis factor (Tnf)-α, and suppressed the LPS-dependent expression of the major endothelial adhesion molecules Vcam-1 and Icam-1. The LPS-induced adhesion of monocytic cells to an endothelial monolayer was antagonized by CTRP3. In C57BL/6J mice with an LPS-induced systemic inflammation, exogenous CTRP3 did not affect circulating levels of TNF-α, ICAM-1, and VCAM-1. In conclusion, we characterized CTRP3 beyond its function as an adipokine in a setting of vascular inflammation. CTRP3 inhibited LPS-induced endothelial expression of adhesion molecules and monocyte cell adhesion, indicating an important vascular anti-inflammatory role for CTRP3 in endotoxemia.

Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis.

Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldlr-/-Nod1/2-/-) which were subsequently analyzed regarding experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition. Compared to Ldlr-/- mice, Ldlr-/-Nod1/2-/- mice showed reduced plasma lipids and increased hepatic expression of the scavenger receptor LDL receptor-related protein 1 after feeding a high-fat diet for 12 weeks. Furthermore, intestinal cholesterol and its bacterial degradation product coprostanol were elevated in Ldlr-/-Nod1/2-/- mice, correlating with the increased abundance of Eubacterium coprostanoligenes as assessed by 3rd generation sequencing of the gut microbiota. Atherosclerotic plaques of Ldlr-/-Nod1/2-/- mice exhibited less lipid deposition and macrophage accumulation. Moreover, macrophages from Ldlr-/-Nod1/2-/- mice showed higher expression of the cholesterol efflux transporters Abca1 and Abcg1 and accordingly reduced foam cell formation. Deficiency of Nod1 and Nod2 led to reduced plaque lipid deposition and inflammatory cell infiltration in atherosclerotic plaques. This might be explained by diminished plasma lipid levels and foam cell formation due to altered expression of key regulators of the hepatic cholesterol pathway as well as differential intestinal cholesterol metabolism and microbiota composition.

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

The Lipopeptide MALP-2 Promotes Collateral Growth.

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth.

Heparan Sulfate-Editing Extracellular Sulfatases Enhance VEGF Bioavailability for Ischemic Heart Repair.

RATIONALE: Mechanistic insight into the inflammatory response after acute myocardial infarction may inform new molecularly targeted treatment strategies to prevent chronic heart failure. OBJECTIVE: We identified the sulfatase SULF2 in an in silico secretome analysis in bone marrow cells from patients with acute myocardial infarction and detected increased sulfatase activity in myocardial autopsy samples. SULF2 (Sulf2 in mice) and its isoform SULF1 (Sulf1) act as endosulfatases removing 6-O-sulfate groups from heparan sulfate (HS) in the extracellular space, thus eliminating docking sites for HS-binding proteins. We hypothesized that the Sulfs have a role in tissue repair after myocardial infarction. METHODS AND RESULTS: Both Sulfs were dynamically upregulated after coronary artery ligation in mice, attaining peak expression and activity levels during the first week after injury. Sulf2 was expressed by monocytes and macrophages, Sulf1 by endothelial cells and fibroblasts. Infarct border zone capillarization was impaired, scar size increased, and cardiac dysfunction more pronounced in mice with a genetic deletion of either Sulf1 or Sulf2. Studies in bone marrow-chimeric Sulf-deficient mice and Sulf-deficient cardiac endothelial cells established that inflammatory cell-derived Sulf2 and endothelial cell-autonomous Sulf1 promote angiogenesis. Mechanistically, both Sulfs reduced HS sulfation in the infarcted myocardium, thereby diminishing Vegfa (vascular endothelial growth factor A) interaction with HS. Along this line, both Sulfs rendered infarcted mouse heart explants responsive to the angiogenic effects of HS-binding Vegfa164 but did not modulate the angiogenic effects of non-HS-binding Vegfa120. Treating wild-type mice systemically with the small molecule HS-antagonist surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide, 1 mg/kg/day) for 7 days after myocardial infarction released Vegfa from HS, enhanced infarct border-zone capillarization, and exerted sustained beneficial effects on cardiac function and survival. CONCLUSIONS: These findings establish HS-editing Sulfs as critical inducers of postinfarction angiogenesis and identify HS sulfation as a therapeutic target for ischemic tissue repair.

Elevated expression of the metalloproteinase ADAM8 associates with vascular diseases in mice and humans.

BACKGROUND AND AIMS: Members of the family of a disintegrin and metalloproteinases (ADAMs) and their substrates have been previously shown to modulate the inflammatory response in cardiac diseases, but studies investigating the relevance of ADAM8 are still rare. Our aim is to provide evidence for the inflammatory dysregulation of ADAM8 in vascular diseases and its association with disease severity. METHODS: Western-type diet fed Apoe-/- and Ldlr-/- mice and artery ligation served as murine model for atherosclerosis and myocardial infarction, respectively. Human bypass grafts were used to study the association with coronary artery disease (CAD), with the simplified acute physiology score II (SAPS II) as a measure of postoperative organ dysfunction. Human primary vascular and blood cells were analyzed under basal and inflammatory conditions. mRNA levels were determined by RT-qPCR, ADAM8 protein levels by ELISA, immunohistochemistry or flow cytometry. RESULTS: ADAM8/ADAM8 expression is associated with atherosclerosis and CAD such as myocardial infarction in both mice and humans, especially in endothelial cells and leukocytes. We observed a strong in vivo and in vitro correlation of ADAM8 with the vascular disease markers VCAM-1, ICAM-1, TNF, IL-6, and CCL-2. Serum analysis revealed a significant elevation of soluble ADAM8 serum levels correlating with soluble CXCL16 levels and SAPS II. CONCLUSIONS: We demonstrate a general association of ADAM8 with cardiovascular diseases in mice and humans predominantly acting in endothelial cells and leukocytes. The correlation with postoperative organ dysfunctions in CAD patients highlights the value of further studies investigating the specific function of ADAM8 in cardiovascular diseases.

Suppressor of Cytokine Signaling 1 is Involved in Gene Regulation Which Controls the Survival of Ly6Clow Monocytes in Mice.

BACKGROUND/AIMS: Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6Chigh) and to a lesser extent on patrolling monocytes (Ly6Clow). Our previous study revealed an accumulation of Ly6Chigh monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6Chigh and Ly6Clow monocytes. METHODS: In monocyte subsets from SOCS-1competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA). RESULTS: mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators. CONCLUSION: Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6Clow monocytes.

Variety matters: Diverse functions of monocyte subtypes in vascular inflammation and atherogenesis.

Monocytes are important mediators of the innate immunity by recognizing and attacking especially bacterial pathogens but also play crucial roles in various inflammatory diseases, including vascular inflammation and atherosclerosis. Maturation, differentiation and function of monocytes have been intensively explored for a long time in innumerable experimental and clinical studies. Monocytes do not represent a uniform cell type but could be further subdivided into subpopulations with distinct features and functions. Those subpopulations have been identified in experimental mouse models as well as in humans, albeit distinguished by different cell surface markers. While Ly6C is used for subpopulation differentiation in mice, corresponding human subsets are differentiated by CD14 and CD16. In this review, we specifically focused on new experimental insights from recent years mainly in regard to murine monocyte subpopulations and their roles in vascular inflammation und atherogenesis.

Anti-tumor necrosis factor-α therapy increases plaque burden in a mouse model of experimental atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is critically fueled by vascular inflammation through oxidized lipids and inflammatory cytokines such as tumor necrosis factor (TNF)-α. Genetic disruption of Tnf-α reduces atherosclerosis in experimental mouse models. However, less is known about the therapeutic potential of Tnf-α blockage by pharmacological inhibitors such as monoclonal antibodies, which are already approved for several inflammatory disorders in patients. Therefore, we investigated the effect of pharmacological TNF-α inhibition on plaque development in experimental atherosclerosis. RESULTS: 10 week old male Ldlr-/- mice were divided into 4 groups (n = 7-10) and fed a high fat, high cholesterol diet for 6 and 12 weeks. Simultaneously, the mouse-specific anti-Tnf-α monoclonal antibody CNTO5048 (CNT) or a control IgG was administered. RESULTS: CNT reduced circulating inflammatory markers without affecting body weight and glucose metabolism. Unexpectedly, CNT treatment increased plasma triglyceride levels and pro-atherogenic very-low-density lipoprotein (VLDL) cholesterol as well as plaque burden in the thoracoabdominal aorta and in the aortic root. In addition, we observed decreased smooth muscle cell content in the lesions and a trend towards reduced collagen deposition upon Tnf-α inhibition. Furthermore, inflammatory gene expression in the aortic arch was increased following Tnf-α inhibitor treatment. CONCLUSIONS: Although up to 12-week pharmacological inhibition of TNF-α in Ldlr-/- mice diminishes systemic inflammation, experimental plaque burden and vascular inflammatory gene expression are increased, while markers of plaque stability decrease. These observations may be explained by the development of a pro-atherogenic plasma lipid profile.

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

Differential Expression of MicroRNAs in Endarterectomy Specimens Taken from Patients with Asymptomatic and Symptomatic Carotid Plaques.

OBJECTIVE: Stroke and transient ischemic attacks are considered as clinical manifestations of atherosclerotic disease due to on-going vascular inflammation and finally atherothrombosis of the carotid arteries. MicroRNAs (miRNA/miR) are known to be involved in vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of selected miRNAs in endarterectomy specimen from carotid arteries that were taken from patients with asymptomatic and symptomatic atherosclerotic plaques. METHODS AND RESULTS: 11 miRNAs were selected and their expression was analyzed using real-time RT-PCR. Therefore, samples were divided into three different groups. On the one hand we investigated the expression patterns from patients in asymptomatic (n = 14) and symptomatic (n = 10) plaques; on the other hand we took samples from normal configurated internal mammary arteries (n = 15). Out of these 11 targets we identified some miRNAs, which were up- or down-regulated in either one of the two groups. Interestingly, the expression of two miRNAs was significantly different between asymptomatic and symptomatic samples, namely miR-21 (P<0.01) and miR-143 (P<0.05). CONCLUSION: In the present study, we identified miRNA subtypes which showed different expression in endarterectomy specimen from patients with asymptomatic and symptomatic plaques, suggesting that these miRNAs correlated with advanced vascular inflammation and plaque stability. They may represent new therapeutic targets for vascular proliferative diseases such as atherosclerosis.

Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion.

OBJECTIVE: It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab-which is approved for several inflammatory disorders-on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions. METHODS AND RESULTS: Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice. CONCLUSION: Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.