Simon Kennedy

Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4 ROLES FOR CD14, LPS-BINDING PROTEIN, AND MD2 AS TARGETS FOR SPECIFICITY OF INHIBITION

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-α production, IκBα degradation, p38 MAPK phosphorylation, and NF-κB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from ∼30 μm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation.

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

RAGE, vascular tone and vascular disease

Evidence provided by both clinical and pre-clinical studies regarding a central involvement of the receptor for advanced glycation endproducts (RAGE) in vascular disease continues to mount. RAGE is upregulated as a consequence of activation of the ubiquitous pro-inflammatory transcription factor NF-kappaB which is activated in response to diverse inflammatory stimuli including hyperglycaemia, oxidised low density lipoprotein (oxLDL) and reduced shear stress. RAGE may maintain and amplify inflammatory responses in the vasculature if ligand for the receptor is present. RAGE binding by circulating advanced glycation endproducts (AGEs) or S100 protein released by activated leukocytes results in the generation of reactive oxygen species (ROS) and further activation of NF-kappaB. This leads to upregulation of adhesion molecules for circulating monocytes as well as further upregulation of RAGE itself. In addition, these ROS may scavenge and reduce bioavailability of the labile vasodilator nitric oxide (NO), reducing its anti-inflammatory effects and possibly compromising control of vascular tone directly. In addition to atherosclerosis and vascular diseases associated with diabetes, recent data from studies in transgenic mice overexpressing the RAGE ligand S100A4/MTS1 suggest a role for RAGE in the pathogenesis of pulmonary arterial hypertension (PAH). RAGE antagonism also prevents proliferation and migration of pulmonary arterial smooth muscle cells in response to 5-HT, suggesting that S100-RAGE signalling may be of key importance in pulmonary vascular homeostasis and/or disease. Further study of the role of RAGE in inflammation seems likely to yield, not only promising therapeutics but key insights into the pathophysiology of vascular disease as well.

AMPK and vasculoprotection.

AMP-activated protein kinase (AMPK) is proposed to be a key regulator of cellular and organismal metabolism and has reported vasculoprotective effects. In addition, many therapeutic agents used in the treatment of diabetes and atherosclerosis such as metformin, thiazolidinediones and statins may exert their vasculoprotective effects through activation of AMPK. Activation of AMPK has a number of potentially beneficial anti-atherosclerotic effects including reducing adhesion of inflammatory cells to the blood vessel endothelium, reducing lipid accumulation and the proliferation of inflammatory cells caused by oxidised lipids, stimulation of gene expression responsible for cellular antioxidant defenses and stimulation of enzymes responsible for nitric oxide formation. In humans and animals the AMPK cascade triggers vascular protective mechanisms that have been shown to reduce myocardial ischaemic injury and mutations in AMPK can cause familial hypertrophic cardiomyopathy. Taken together, these data suggest that activation and function of AMPK contributes to cardiovascular health. In this review we propose to focus on the vasculoprotective effects of AMPK, the evidence for AMPK activation with currently used therapeutic agents and the potential for agents which specifically activate AMPK as a treatment for vascular disease.

Reducing In-Stent Restenosis: Therapeutic Manipulation of miRNA in Vascular Remodeling and Inflammation

Background Drug-eluting stents reduce the incidence of in-stent restenosis, but they result in delayed arterial healing and are associated with a chronic inflammatory response and hypersensitivity reactions. Identifying novel interventions to enhance wound healing and reduce the inflammatory response may improve long-term clinical outcomes. Micro–ribonucleic acids (miRNAs) are noncoding small ribonucleic acids that play a prominent role in the initiation and resolution of inflammation after vascular injury. Objectives This study sought to identify miRNA regulation and function after implantation of bare-metal and drug-eluting stents. Methods Pig, mouse, and in vitro models were used to investigate the role of miRNA in in-stent restenosis. Results We documented a subset of inflammatory miRNAs activated after stenting in pigs, including the miR-21 stem loop miRNAs. Genetic ablation of the miR-21 stem loop attenuated neointimal formation in mice post-stenting. This occurred via enhanced levels of anti-inflammatory M2 macrophages coupled with an impaired sensitivity of smooth muscle cells to respond to vascular activation. Conclusions MiR-21 plays a prominent role in promoting vascular inflammation and remodeling after stent injury. MiRNA-mediated modulation of the inflammatory response post-stenting may have therapeutic potential to accelerate wound healing and enhance the clinical efficacy of stenting.

Perivascular fat, AMP-activated protein kinase and vascular diseases

Perivascular adipose tissue (PVAT) is an active endocrine and paracrine organ that modulates vascular function, with implications for the pathophysiology of cardiovascular disease (CVD). Adipocytes and stromal cells contained within PVAT produce mediators (adipokines, cytokines, reactive oxygen species and gaseous compounds) with a range of paracrine effects modulating vascular smooth muscle cell contraction, proliferation and migration. However, the modulatory effect of PVAT on the vascular system in diseases, such as obesity, hypertension and atherosclerosis, remains poorly characterized. AMP‐activated protein kinase (AMPK) regulates adipocyte metabolism, adipose biology and vascular function, and hence may be a potential therapeutic target for metabolic disorders such as type 2 diabetes mellitus (T2DM) and the vascular complications associated with obesity and T2DM. The role of AMPK in PVAT or the actions of PVAT have yet to be established, however. Activation of AMPK by pharmacological agents, such as metformin and thiazolidinediones, may modulate the activity of PVAT surrounding blood vessels and thereby contribute to their beneficial effect in cardiometabolic diseases. This review will provide a current perspective on how PVAT may influence vascular function via AMPK. We will also attempt to demonstrate how modulating AMPK activity using pharmacological agents could be exploited therapeutically to treat cardiometabolic diseases.

Multiphoton Microscopy for 3-Dimensional Imaging of Lymphocyte Recruitment Into Apolipoprotein-E–Deficient Mouse Carotid Artery

Two recent elegant studies have shown that in apolipoprotein-E–deficient mice, the lamina adventitia is a major site of arterial wall inflammation associated with lymphocyte infiltration into atherosclerotic arteries and with formation of adventitial lymphoid-like tissues.1,2 These results suggest that lymphocyte responses in the lamina adventitia may play a crucial role in atherosclerosis development.1,2 However, in both studies, the detection of immune cells within the artery was limited to conventional analysis systems (flow cytometry, immunohistochemistry), which require the removal, disruption, and processing of tissue at defined time points to give a “snapshot” of the immune response to vascular damage in vivo. For evaluation of atherosclerotic processes and their relationship to immune activation, imaging of 3-dimensional structures in intact vascular tissues and functional aspects of the diseased artery (eg, details of the interactions of immune cell with vessel wall structures) are required. A potential solution to this problem is offered by the development of multiphoton laser-scanning microscopy,3 with the ability to penetrate directly into tissues at sufficient depths to image tissues in situ in the absence of significant phototoxicity. By moving the focal point axially, …

miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation

Aims The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation. Methods and results We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation. Conclusion This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

Activation of mouse protease-activated receptor-2 induces lymphocyte adhesion and generation of reactive oxygen species

Protease‐activated receptor‐2 (PAR‐2) is expressed on lymphocytes and endothelial cells, and plays a significant role in inflammatory reactions. Since leukocyte‐endothelial cell interaction and reactive oxygen species (ROS) generation are hallmarks of the development of inflammation, the effects of PAR‐2 activation by trypsin on lymphocyte adhesion and ROS generation was examined utilising PAR‐2 wild type and knockout (PAR‐2−/−) mice.

Targeting sphingosine-1-phosphate signalling for cardioprotection.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid generated by the sphingosine kinase (SK1 or SK2)-catalysed phosphorylation of sphingosine. Plasma S1P is carried in high-density lipoprotein (HDL) or bound to albumin and is reported to arise from activated platelets and erythrocytes. In addition, extracellular SK1 released from vascular endothelial cells may also contribute to plasma S1P levels. S1P exerts its effects through a family of five high affinity S1P-specific G protein-coupled receptors (GPCRs), S1P(1-5). Various S1P receptors are present in the cardiovascular system, including cardiac tissue. Additionally, intracellular S1P may have a second messenger action. Since S1P is recognised as a survival factor in many tissues, there has been much interest in S1P as a cardioprotective agent. Recent evidence indicates that S1P can pre-condition and post-condition the heart and that the cardioprotective effect of HDL may be because of its S1P content. In addition, evidence is emerging that the cardioprotective effects of cannabinoids and S1P may be linked.