Andrew Jefferson

Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression

Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate-cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.

Anti-Inflammatory Effects of Nicotinic Acid in Human Monocytes Are Mediated by GPR109A Dependent Mechanisms

Objective—Nicotinic acid (NA) treatment has been associated with benefits in atherosclerosis that are usually attributed to effects on plasma lipoproteins. The NA receptor GPR109A is expressed in monocytes and macrophages, suggesting a possible additional role for NA in modulating function of these immune cells. We hypothesize that NA has the potential to act directly on monocytes to alter mediators of inflammation that may contribute to its antiatherogenic effects in vivo. Methods and Results—In human monocytes activated by Toll-like receptor (TLR)-4 agonist lipopolysaccharide, NA reduced secretion of proinflammatory mediators: TNF-&agr; (by 49.2±4.5%); interleukin-6 (by 56.2±2.8%), and monocyte chemoattractant protein-1 (by 43.2±3.1%) (P<0.01). In TLR2 agonist, heat-killed Listeria monocytogenes-activated human monocytes, NA reduced secretion of TNF-&agr; (by 48.6±7.1%), interleukin-6 (by 60.9±1.6%), and monocyte chemoattractant protein-1 (by 59.3±5.3%) (P<0.01; n=7). Knockdown of GPR109A by siRNA resulted in a loss of this anti-inflammatory effect in THP-1 monocytes. However, inhibition of prostaglandin D2 receptor by MK0524 or COX2 by NS398 did not alter the anti-inflammatory effects of NA observed in activated human monocytes. Preincubation of THP-1 monocytes with NA 0.1 mmol/L reduced phosphorylated IKK&bgr; by 42±2% (P<0.001) IKB-&agr; by 54±14% (P<0.01). Accumulation of nuclear p65 NF-&kgr;B in response to lipopolysaccharide treatment was also profoundly inhibited, by 89±1.3% (n=4; P<0.01). NA potently inhibited monocyte adhesion to activated HUVEC, and VCAM, mediated by the integrin, very late antigen 4. Monocyte chemotaxis was also significantly reduced (by 45.7±1.2%; P<0.001). Conclusion—NA displays a range of effects that are lipoprotein-independent and potentially antiatherogenic. These effects are mediated by GPR109A and are independent of prostaglandin pathways. They suggest a rationale for treatment with NA that is not dependent on levels of plasma cholesterol and possible applications beyond the treatment of dyslipidemia.

A leukocyte-mimetic magnetic resonance imaging contrast agent homes rapidly to activated endothelium and tracks with atherosclerotic lesion macrophage content.

Objective—Endothelial cell activation is an important mediator of monocyte recruitment to sites of vascular inflammation. We hypothesized that high-affinity dual-ligand microparticles of iron oxide (MPIO), targeted to P-selectin and vascular cell adhesion molecule-1 (PV-MPIO), would identify activated endothelial cells during atherosclerosis progression. Methods and Results—In vivo magnetic resonance imaging in apolipoprotein E-deficient mice showed rapid binding of PV-MPIO to the aortic root, which was maximal 30 minutes post-MPIO injection and maintained at 60 minutes. Minimal binding was observed for control IgG-MPIO. Intensely low magnetic resonance signal areas, corresponding to PV-MPIO binding, were detected early (14 weeks), during foam cell formation. Contrast effects increased at 20 weeks during fibrofatty lesion development (P<0.05), but reduced by 30 weeks (P<0.01). Across all lesion severities, magnetic resonance imaging contrast effects correlated with lesion macrophage area quantified by immunohistochemistry (R=0.53; P<0.01). Near-infrared fluorescently labeled PV-MPIO were shown, by flow cytometry, to bind only activated endothelial cells and not to macrophages. Using en face immunofluorescence, we further demonstrate selective PV-MPIO accumulation at atherosclerosis-susceptible sites, with minimal binding to atherosclerosis-spared regions. Conclusion—This high-affinity leukocyte-mimetic magnetic resonance imaging agent reveals endothelial activation. PV-MPIO demonstrate exceptionally rapid in vivo steady state accumulation, providing conspicuous magnetic resonance contrast effects that can be objectively quantified. In atherosclerosis progression, PV-MPIO tracked closely with the burden and distribution of plaque macrophages, not merely plaque size. On a biocompatible platform, this approach has potential for quantitative magnetic resonance imaging of inflammatory disease activity.

Nicotinic acid receptor GPR109A is down-regulated in human macrophage-derived foam cells.

Nicotinic acid (NA) regresses atherosclerosis in human imaging studies and reduces atherosclerosis in mice, mediated by myeloid cells, independent of lipoproteins. Since GPR109A is expressed by human monocytes, we hypothesized that NA may drive cholesterol efflux from foam cells. In THP-1 cells NA suppressed LPS-induced mRNA transcription of MCP-1 by 76.6±12.2% (P<0.01) and TNFα by 56.1±11.5% (P<0.01), yet restored LPS-induced suppression of PPARγ transcription by 536.5±46.4% (P<0.001) and its downstream effector CD36 by 116.8±19.8% (P<0.01). Whilst direct PPARγ-agonism promoted cholesterol efflux from THP-1 derived foam cells by 37.7±3.1% (P<0.01) and stimulated transcription of LXRα by 87.9±9.5% (P<0.001) and ABCG1 by 101.2±15.5% (P<0.01), NA showed no effect in foam cells on either cholesterol efflux or key RCT genes transcription. Upon foam cell induction, NA lost its effect on PPARγ and cAMP pathways, since its receptor, GPR109A, was down-regulated by foam cell transformation. This observation was confirmed in explanted human carotid plaques. In conclusion, despite NA’s anti-inflammatory effect on human macrophages, it has no effect on foam cells in reverse cholesterol transport; due to GPR109A down-regulation.

Exogenous Microparticles of Iron Oxide Bind to Activated Endothelial Cells but, Unlike Monocytes, Do Not Trigger an Endothelial Response

Targeting particles to sites of inflammation is of considerable interest for applications relating to molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands (e.g. antibodies, peptides and oligosaccharides) to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question has been whether the binding of imaging particles to these mediators induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. Here, we address these questions by looking for changes in endothelial cells following binding of contrast agent. Specifically, we have quantified calcium flux, rearrangement of the actin cytoskeleton, production of reactive oxygen species (ROS), apoptosis and potential secondary changes, such as changes in gene and protein expression follow binding events to primary endothelial cells in vitro. Although leukocytes induced changes to endothelial cell function, we did not see any significant changes to endothelial calcium flux, cytoskeletal organisation, production of ROS or induction of apoptosis in response to antibody-MPIO binding. Furthermore, there were no changes to gene expression monitored via real-time RT-PCR or presentation of protein on the cell surface measured using flow cytometry. Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.

Melanoma Inhibition by Anthocyanins Is Associated with the Reduction of Oxidative Stress Biomarkers and Changes in Mitochondrial Membrane Potential

AbstractAnthocyanins are water soluble pigments which have been proved to exhibit health benefits. Several studies have investigated their effects on several types of cancer, but little attention has been given to melanoma. The phytochemical content of nine different berry samples was assessed by liquid chromatography followed by electrospray ionization mass spectrometry (LC-ESI+-MS). Twenty-six anthocyanins were identified, after a previous C18 Sep-pak clean-up procedure. Chokeberry and red grape anthocyanins rich extracts (C-ARE and RG-ARE) were selected to be tested on normal and melanoma cell lines, due to their different chemical pattern. C-ARE composition consists of cyanidin aglycone glycosylated with different sugars; while RG-ARE contains glucosylated derivatives of five different aglycones. Both C-ARE and RG-ARE anthocyanins reduced proliferation, increased oxidative stress biomarkers and diminished mitochondrial membrane potential in melanoma cells, having no negative influence on normal cells. A synergistic response may be attributed to the five different aglycones present in RG-ARE, which proved to exert greater effects on melanoma cells than the mixture of cyanidin derivatives with different sugars (C-ARE). In conclusion, C-ARE and RG-ARE anthocyanins may inhibit melanoma cell proliferation and increase the level of oxidative stress, with opposite effect on normal cells. Therefore, anthocyanins might be recommended as active ingredients for cosmetic and nutraceutical industry. Graphical Abstractᅟ

NICOTINIC ACID RECEPTOR GPR109A IS DOWN-REGULATED IN HUMAN MACROPHAGE-DERIVED FOAM CELLS

Introduction Nicotinic acid (NA) has been known to exert favourable effects on plasma lipoproteins. It regresses atherosclerosis in human imaging studies and reduces atherosclerosis in mice, mediated by its receptor GPR109a on myeloid cells, independent of its lipoprotein effect. Since GPR109a is expressed by human monocytes, we hypothesized that NA may drive cholesterol efflux from human macrophage-derived foam cells. Methods THP-1 cells were induced into foam cells by acetylated LDL. After treatment with NA, GW1929 (a PPARγ agonist) and vehicle controls, cholesterol efflux was assessed using HDL3 and apo-AI as cholesterol acceptors. qRT-PCR was performed using primers for genes responsible for inflammation and reverse cholesterol transport (RCT). ELISAs for PPARγ activity and cAMP response were performed to investigate NAs putative cellular mechanisms of action. Fluorescence immunohistochemistry on ex-vivo human carotid atherosclerotic plaques was carried out against CD68, adipophilin (a foam cells marker), as well as GPR109a. Immediately adjacent sections were stained with Oil-red-O to visualized lipid content and distribution. Results In basal THP-1 cells, NA suppressed LPS-induced mRNA transcription of MCP-1 by 76.6±12.2% (P<0.01) and TNF-α by 56.1±11.5% (P<0.01), yet restored LPS-induced suppression of PPARγ transcription by 536.5±46.4% (P<0.001) and its downstream effector CD36 by 116.8±19.8% (P<0.01). Whilst direct PPARγ-agonism promoted cholesterol efflux from THP-1 derived foam cells by 37.7±3.1% (P<0.01) and stimulated transcription of LXRα by 87.9±9.5% (P<0.001) and ABCG1 by 101.2±15.5% (P<0.01), NA showed no effect in foam cells on either cholesterol efflux or key RCT genes transcription. NA was found to activate PPARγ pathway and suppress cAMP response in basal macrophages; but these effects were lost in foam cells, since its receptor, GPR109a, was down-regulated by foam cell transformation. This observation was confirmed in explanted human carotid plaques. Plaque CD68 positive macrophages were found clustered at the interface between the acellular lipid pool (LP) and the overlying fibrous cap. GPR109a co-expression was observed in CD68-positive cells outside the LP, whereas CD68-positive cells within the LP, which are highly likely to represent macrophage-derived foam cells, do not co-express GPR109a. This co-expression finding was further confirmed using antibody raised against a specific lipid-droplet associated protein, adipophilin. Conclusions This study shows that despite NAs anti-inflammatory effect on human macrophages, it does not appear to enhance cholesterol efflux in foam cells, which is at least partially ascribed to GPR109a down-regulation upon foam cell transformation. This implies that direct NA-promotion of cholesterol efflux from foam cells may not contribute to atherosclerosis regression, which has been demonstrated with this drug. Figure 1 Figure 2

Myocardial infarction causes inflammation and leukocyte recruitment at remote sites in the myocardium and in the renal glomerulus

Background Acute myocardial infarction (AMI) results in both systemic inflammation and recruitment of leukocytes to injured myocardium. Additionally, myocardium remote to the infarct zone, also becomes inflamed and is associated with adverse LV remodelling. Renal ischaemic syndromes have been associated with remote organ inflammation and impaired function. We therefore tested the hypothesis thatAMI results in acute inflammation at remote sites which may contribute to organ dysfunction. Methods Female C57BL/6J mice underwent AMI by surgical coronary artery ligation or sham procedure. At 24 hours, mice underwent transthoracic echocardiography prior to sacrifice (n=8/group). The inflammatory response in peripheral blood, injured and remote myocardium, and kidneys was studied. Results At 24 hours, in comparison to sham operated mice, AMI resulted in increased circulating neutrophils and monocytes (P<0.001). Using quantitative RT-PCR, mRNA for inflammatory mediators increased in infarcted myocardium; IL6 by 11±0.3-fold (P<0.01), TNF-α by 32.9±2.3-fold (P<0.01), IL1RN by 5.4±0.2-fold (P<0.05), IL1R2 by 4.3±0.9-fold (P<0.05) and in remote myocardium; IL6 by 5.0±0.7-fold (P<0.05), TNF-α by 15.2±4.2-fold (P<0.05)). VCAM-1 mRNA was significantly increased in both infarcted and remote myocardium. In kidneys of AMI mice, VCAM-1 protein was increased by 2.6±0.1 fold (P<0.01) (Figure 1) and immunofluorescence revealed localisation of VCAM-1 to the same glomerular cells expressing PECAM-1 indicating endothelial expression of VCAM-1 (Figure 2, blue: DAPI, red: PECAM-1, green: VCAM-1). This was associated with leukocyte infiltration (P<0.01) and increased inflammatory mRNA expression; IL6 by 2.9±0.1-fold (P<0.001), TNF-α by 7.4±1.8-fold (P<0.01), IL1RN by 3.9±0.2-fold (P<0.01) and IL1R2 by 14.6±4.9-fold (P<0.01). AMI did not affect plasma creatinine. Conclusion (1) AMI induces a local inflammatory response, characterised by infiltration of leukocytes and increased expression of mRNA for inflammatory cytokines; (2) cytokines are upregulated in myocardium that is remote from the site of ischaemic injury; (3) AMI is associated with systemic inflammation, reflected in increased peripheral blood neutrophils and monocytes; (4) AMI is also associated with remote organ inflammation evidenced by (i) increased expression of mRNA for inflammatory cytokines, (ii) marked upregulation of VCAM-1 in renal glomeruli and (iii) by the recruitment and infiltration of leukocytes in the kidney. Figure 1 Figure 2

213 EXOGENOUS MICROPARTICLES OF IRON OXIDE BIND TO ACTIVATED ENDOTHELIAL CELLS BUT, UNLIKE MONOCYTES, DO NOT TRIGGER AN ENDOTHELIAL RESPONSE

Introduction Targeting particles to sites of inflammation is of considerable interest for molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question is whether the binding of imaging particles induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. We address this by looking for changes in human aortic endothelial cells (HAEC) following binding of contrast agent. Methods 1 µm MPIO dual-targeted to E-selectin and VCAM were prepared by labelling with specific antibodies, and cellular responses to binding events assessed. Calcium flux was monitored using the calcium indicator Fluo-4 and fluorescent microscopy for analysis. Production of reactive oxygen species was investigated using a nitroblue tetrazolium blue (NBT) assay. Real-time RT-PCR was used to assess gene expression of several endothelial markers of inflammation. Antibody staining of cell surface proteins and flow cytometry analysis monitored protein levels presented by HAEC. Results Binding of E+V-MPIO did not cause calcium flux in HAEC; normalised fluorescence intensity of Fluo-4 dropped to 0.81 (SEM ±0.02) of baseline 60 seconds post MPIO binding and to 0.64 (SEM±0.02) with vehicle only, whilst a significant increase in Fluo-4 fluorescence to 1.96 (SEM±0.48) was seen following THP-1 binding indicating endothelial calcium flux post-leukocyte binding (P<0.001). Assessing cells for formazan precipitate revealed minimal production of ROS in cells incubated with E+V-MPIO and ICAM-1-MPIO, and negative controls (0.3% ±0.3, 3.6% ±1.2% and 0% ±0%) (P≤0.001 all comparisons). Real-time RT-PCR revealed no significant changes to gene expression when antibody-MPIO was bound (P≥0.05 all comparisons); mRNA levels in arbitrary units for E-selectin were 0.93 (SEM ±0.01) compared with 0.93 (SEM ±0.006); VCAM-1 were 0.74 (SEM ±0.13) and 0.79 (SEM ±0.04); ICAM-1 were 0.68 (SEM ±0.25) and 0.6 (SEM ±0.26) for cells with E+V-MPIO bound versus controls respectively. In concordance with gene expression data, there was no change to the cell surface presentation of E-selectin in HAEC with MPIO bound compared with controls; geometric mean fluorescence intensities were 125.5 (SEM±7.5) and 157.5 (SEM±1.5) (P=0.053). Conclusions Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.