136 results found
Normahani P, Boyle JJ, Cave L, et al., 2022, Peripheral blood mononuclear cell gene expression and cytokine profiling in patients with intermittent claudication who exhibit exercise induced acute renal injury., PLoS One, Vol: 17, Pages: 1-15, ISSN: 1932-6203
BACKGROUND: Intermittent claudication (IC) is a common manifestation of peripheral arterial disease. Some patients with IC experience a rise in Urinary N-acetyl-β-D-Glucosaminidase (NAG)/ Creatinine (Cr) ratio, a marker of renal injury, following exercise. In this study, we aim to investigate whether peripheral blood mononuclear cells (PBMC) from patients with IC who exhibit a rise in urinary NAG/ Cr ratio following exercise exhibit differential IL-10/ IL-12 ratio and gene expression compared to those who do not have a rise in NAG/ Cr ratio. METHODS: We conducted a single center observational cohort study of patients diagnosed with IC. Blood and urine samples were collected at rest and following a standardised treadmill exercise protocol. For comparative analysis patients were separated into those with any rise in NAG/Cr ratio (Group 1) and those with no rise in NAG/Cr ratio (Group 2) post exercise. Isolated PBMC from pre- and post-exercise blood samples were analysed using flow cytometry. PBMC were also cultured for 20 hours to perform further analysis of IL-10 and IL-12 cytokine levels. RNA-sequencing analysis was performed to identify differentially expressed genes between the groups. RESULTS: 20 patients were recruited (Group 1, n = 8; Group 2, n = 12). We observed a significantly higher IL-10/IL-12 ratio in cell supernatant from participants in Group 1, as compared to Group 2, on exercise at 20 hours incubation; 47.24 (IQR 9.70-65.83) vs 6.13 (4.88-12.24), p = 0.04. 328 genes were significantly differentially expressed between Group 1 and 2. The modulated genes had signatures encompassing hypoxia, metabolic adaptation to starvation, inflammatory activation, renal protection, and oxidative stress. DISCUSSION: Our results suggest that some patients with IC have an altered immune status making them 'vulnerable' to systemic inflammation and renal injury following exercise. We have identified a panel of genes which are differentially expressed in this group of
Walter ERH, Cooper SM, Boyle JJ, et al., 2021, Enzyme-activated probes in optical imaging: a focus on atherosclerosis, DALTON TRANSACTIONS, Vol: 50, Pages: 14486-14497, ISSN: 1477-9226
Walter E, Ge Y, Mason J, et al., 2021, A coumarin-porphyrin FRET break-apart probe for heme oxygenase-1, Journal of the American Chemical Society, Vol: 143, Pages: 6460-6469, ISSN: 0002-7863
Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin–porphyrin FRET break-apart probe to detect HO-1 activity, Fe–L1. We designed Fe–L1 to “break-apart” upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC–MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe–L2, we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.
Boyle J, Seneviratne A, Cave L, et al., 2021, Metformin directly suppresses atherosclerosis in normoglycemic mice via haematopoietic Adenosine Monophosphate-Activated Protein Kinase (AMPK), Cardiovascular Research, Vol: 117, Pages: 1295-1308, ISSN: 0008-6363
AimsAtherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque hemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and ATF1. The anti-diabetic drug metformin may also activate AMPK-dependent signalling.HypothesisMetformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, and thereby suppresses atherogenesis.Methods and ResultsNormoglycemic Ldlr-/- hyperlipidemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (p < 5x10−11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK (ANOVA, p < 0.03). Metformin at a clinically relevant concentration (10μM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1 and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin induced lesional macrophage expression of p-AMPK, p-ATF1 and downstream M2-like protective effects.ConclusionMetformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidemic mice via haematopoietic AMPK.Translational perspectiveThe work shows that oral antidiabetic drug metformin may suppress atherosclerotic lesion development via hematopoietic AMPK at clinically relevant concentrations, rather than via a hypoglycemic effect. Activating Transcription Factor 1 (ATF1) may mediate induction of key atheroprotective genes
Forte E, Panahi M, Baxan N, et al., 2021, Type 2 MI induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart, Journal of Cellular and Molecular Medicine, Vol: 25, Pages: 229-243, ISSN: 1582-1838
Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol hydrochloride is used for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis. To assess whether isoproterenol-induced cardiomyocyte necrosis triggers an adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.
Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require deeper understanding of the healthy heart’s molecular processes. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavor. Here, using state-of-the-art analyses of large-scale single-cell and nuclei transcriptomes, we characterise six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes, and fibroblasts, revealing distinct atrial and ventricular subsets with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment we identify cardiac resident macrophages with inflammatory and protective transcriptional signatures. Further, inference of cell-cell interactions highlight different macrophage-fibroblast-cardiomyocyte networks between atria and ventricles that are distinct from skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a healthy reference for future studies.
Boyle J, Long NJ, Walter ERH, et al., 2020, COMPOUNDS FOR THE DETECTION OF HEME OXYGENASE 1 (HO-1), AND METHODS AND USES INVOLVING THE SAME, 2017871.1
The present invention relates to compounds for the detection of heme oxygenase 1 (HO-1), in particular porphyrin, chlorin, bacteriochlorin or isobacteriochlorin compounds having a tetrapyrrole or reduced tetrapyrrole backbone and a fluorophore. Such compounds can be used in the detection of HO-1 in vivo, ex vivo and in vitro, and can also be used in methods of diagnosis and as research reagents.
Seneviratne A, Han Y, Wong E, et al., 2020, Hematoma resolution in vivo is directed by Activating Transcription Factor 1, Circulation Research, Vol: 127, Pages: 928-944, ISSN: 0009-7330
Rationale: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an adenosine monophosphate activated protein kinase / activating transcription factor 1 (AMPK/ATF1) pathway that directs Mhem macrophages through coregulation of heme oxygenase 1 (HMOX1, HO-1) and lipid homeostasis genes.Objective: We asked whether this pathway had an in vivo role in mice.Methods and Results: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow derived macrophages (mBMM), heme induced HO-1, lipid regulatory genes including LXR, the growth factor IGF1, and the splenic red pulp macrophage gene Spic. This response was lost in mBMM from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between day 8 and day 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-) (n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent.Conclusions: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution.
Evans RJ, Lavin B, Phinikaridou A, et al., 2020, Targeted molecular iron oxide contrast agents for imaging atherosclerotic plaque, Nanotheranostics, Vol: 4, Pages: 184-194, ISSN: 2206-7418
Overview: Cardiovascular disease remains a leading cause of death worldwide, with vulnerable plaque rupture the underlying cause of many heart attacks and strokes. Much research is focused on identifying an imaging biomarker to differentiate stable and vulnerable plaque. Magnetic Resonance Imaging (MRI) is a non-ionising and non-invasive imaging modality with excellent soft tissue contrast. However, MRI has relatively low sensitivity (micromolar) for contrast agent detection compared to nuclear imaging techniques. There is also an increasing emphasis on developing MRI probes that are not based on gadolinium chelates because of increasing concerns over associated systemic toxicity and deposits1. To address the sensitivity and safety concerns of gadolinium this project focused on the development of a high relaxivity probe based on superparamagnetic iron oxide nanoparticles for the imaging of atherosclerotic plaque with MRI. With development, this may facilitate differentiating stable and vulnerable plaque in vivo.Aim: To develop a range of MRI contrast agents based on superparamagnetic iron oxide nanoparticles (SPIONs), and test them in a murine model of advanced atherosclerosis.Methods: Nanoparticles of four core sizes were synthesised by thermal decomposition and coated with poly(maleicanhydride-alt-1-octadecene) (PMAO), poly(ethyleneimine) (PEI) or alendronate, then characterised for core size, hydrodynamic size, surface potential and relaxivity. On the basis of these results, one candidate was selected for further studies. In vivo studies using 10 nm PMAO-coated SPIONs were performed in ApoE-/- mice fed a western diet and instrumented with a perivascular cuff on the left carotid artery. Control ApoE-/- mice were fed a normal chow diet and were not instrumented. Mice were scanned on a 3T MR scanner (Philips Achieva) with the novel SPION contrast agent, and an elastin-targeted gadolinium agent that was shown previously to enable visualisation of plaque burden. Histo
Hultman K, Edsfeldt A, Björkbacka H, et al., 2019, Cartilage oligomeric matrix protein associates with a vulnerable plaque phenotype in human atherosclerotic plaques, Stroke, Vol: 50, ISSN: 0039-2499
Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.
Boyle J, Seneviratne A, Han Y, et al., 2019, VERTEBRATE HEMATOMA RESOLUTION IS DIRECTED BY ACTIVATING TRANSCRIPTION FACTOR 1 (ATF1) AND ADENOSINE-MONOPHOSPHATE-ACTIVATED-PROTEIN-KINASE (AMPK), 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E246-E246, ISSN: 0021-9150
Boyle J, Seneviratne A, Tsao A, et al., 2019, SMARCA4 REDIRECTS BINDING OF MACROPHAGE ACTIVATING TRANSCRIPTION FACTOR 1 (ATF1) FROM GENES FOR INFLAMMATION RESOLUTION TO GENES FOR ERYTHROCYTE RESOLUTION, 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E78-E78, ISSN: 0021-9150
Boyle J, Seneviratne A, Hyde G, et al., 2019, METFORMIN DIRECTLY SUPPRESSES ATHEROSCLEROSIS IN NORMOGLYCEMIC MICE VIA HAEMATOPOIETIC ADENOSINE MONOPHOSPHATE-ACTIVATED PROTEIN KINASE (AMPK), 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E45-E46, ISSN: 0021-9150
Kalna V, Yang Y, Peghaire C, et al., 2019, The transcription factor ERG regulates super-enhancers associated with an endothelial-specific gene expression program, Circulation Research, Vol: 124, Pages: 1337-1349, ISSN: 0009-7330
Rationale:The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers).Objective:To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers.Methods and Results:Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-assoc
Alrashed F, Calay D, Lang M, et al., 2018, Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling, Scientific Reports, Vol: 8, ISSN: 2045-2322
Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000 ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1β-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs.
Khamis RY, Hughes AD, Caga-Anan M, et al., 2016, High serum immunoglobulin G and M levels predict freedom from adverse cardiovascular events in hypertension: a nested case-control substudy of the Anglo-Scandinavian Cardiac Outcomes Trial, EBioMedicine, Vol: 9, Pages: 372-380, ISSN: 2352-3964
Aims: We aimed to determine whether the levels of total serum IgM and IgG, together with specific antibodies against malondialdehyde-conjugated low-density lipoprotein (MDA-LDL),can improve cardiovascular risk discrimination.Methods and Results: The Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) randomized 9098 patients in the UK and Ireland into the Blood Pressure-Lowering Arm. 485 patients that had cardiovascular (CV) events over 5.5 years were age and sex matched with 1367 controls. Higher baseline total serum IgG, and to a lesser extent IgM, were associated with decreased risk of CV events (IgG odds ratio (OR) per one standard deviation (SD) 0.80[95% confidence interval, CI 0.72,0.89], p<0.0001; IgM 0.83[0.75,0.93], p=0.001), and particularly events due to coronary heart disease (CHD) (IgG OR 0.66 (0.57,0.76); p<0.0001, IgM OR 0.81 (0.71,0.93); p=0.002). The association persisted after adjustment for a basic model with variables in the Framingham Risk Score (FRS) as well as following inclusion of C-reactive protein (CRP) and N-terminal pro-B-type natriuretic peptide (NtProBNP). IgG and IgM antibodies against MDA-LDL were also associated with CV events but their significance was lost following adjustment for total serum IgG and IgM respectively. The area under the receiver operator curve for CV events was improved from the basic risk model when adding in total serum IgG, and there was improvement in continuous and categorical net reclassification(17.6% and 7.5% respectively) as well as in the integrated discrimination index.Conclusion: High total serum IgG levels are an independent predictor of freedom from adverse cardiovascular events, particularly those attributed to CHD, in patients with hypertension.
Lindsay D, Ismajli M, Bucknall R, et al., 2016, Simultaneous presentation of IgG4-related chronic peri-aortitis and coeliac disease in a patient with Marfan's Syndrome, Rheumatology, Vol: 55, Pages: 1141-1143, ISSN: 1462-0332
IgG4 related chronic peri-aortitis can present relatively early and without aortic calcification in Marfan’s disease.
Khamis RY, Woollard KJ, Hyde GD, et al., 2016, Near Infrared Fluorescence (NIRF) Molecular Imaging of Oxidized LDL with an Autoantibody in Experimental Atherosclerosis, Scientific Reports, Vol: 6, ISSN: 2045-2322
We aimed to develop a quantitative antibody-based near infrared fluorescence (NIRF) approachfor the imaging of oxidized LDL in atherosclerosis. LO1, a well- characterized monoclonalautoantibody that reacts with malondialdehyde-conjugated LDL, was labeled with a NIRF dye toyield LO1-750. LO1-750 specifically identified necrotic core in ex vivo human coronary lesions.Injection of LO1-750 into high fat (HF) fed atherosclerotic Ldlr-/-mice led to specific focallocalization within the aortic arch and its branches, as detected by fluorescence moleculartomography (FMT) combined with micro-computed tomography (CT). Ex vivo confocalmicroscopy confirmed LO1-750 subendothelial localization of LO1-750 at sites ofatherosclerosis, in the vicinity of macrophages. When compared with a NIRF reporter of MMPactivity (MMPSense-645-FAST), both probes produced statistically significant increases inNIRF signal in the Ldlr-/- model in relation to duration of HF diet. When withdrawing the HFdiet, the reduction in oxLDL accumulation, as demonstrated with LO1-750, was less markedthan the effect seen on MMP activity. In the rabbit, in vivo injected LO1-750 localization wassuccessfully imaged ex vivo in aortic lesions with a customised intra-arterial NIRF detectioncatheter. A partially humanized chimeric LO1-Fab-Cys localized similarly to the parentantibody in murine atheroma showing promise for future translation.
Thornton CC, Al-Rashed F, Calay D, et al., 2016, Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation, Annals of the Rheumatic Diseases, Vol: 75, Pages: 439-448, ISSN: 0003-4967
Aims Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes.Methods/results In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression.Conclusions These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against
Wei K, Diaz-Trelles R, Liu Q, et al., 2015, Developmental origin of age-related coronary artery disease, CARDIOVASCULAR RESEARCH, Vol: 107, Pages: 287-294, ISSN: 0008-6363
Mylroie H, Dumont O, Bauer A, et al., 2015, PKC epsilon-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis, CARDIOVASCULAR RESEARCH, Vol: 106, Pages: 509-519, ISSN: 0008-6363
Amini N, Boyle JJ, Moers B, et al., 2014, Requirement of JNK1 for endothelial cell injury in atherogenesis, Atherosclerosis, Vol: 235, Pages: 613-618, ISSN: 1879-1484
ObjectiveThe c-Jun N-terminal kinase (JNK) family regulates fundamental physiological processes including apoptosis and metabolism. Although JNK2 is known to promote foam cell formation during atherosclerosis, the potential role of JNK1 is uncertain. We examined the potential influence of JNK1 and its negative regulator, MAP kinase phosphatase-1 (MKP-1), on endothelial cell (EC) injury and early lesion formation using hypercholesterolemic LDLR−/− mice.Methods and resultsTo assess the function of JNK1 in early atherogenesis, we measured EC apoptosis and lesion formation in LDLR−/− or LDLR−/−/JNK1−/− mice exposed to a high fat diet for 6 weeks. En face staining using antibodies that recognise active, cleaved caspase-3 (apoptosis) or using Sudan IV (lipid deposition) revealed that genetic deletion of JNK1 reduced EC apoptosis and lesion formation in hypercholesterolemic mice. By contrast, although EC apoptosis was enhanced in LDLR−/−/MKP-1−/− mice compared to LDLR−/− mice, lesion formation was unaltered.ConclusionWe conclude that JNK1 is required for EC apoptosis and lipid deposition during early atherogenesis. Thus pharmacological inhibitors of JNK may reduce atherosclerosis by preventing EC injury as well as by influencing foam cell formation.
Khamis RY, Woollard KJ, Hyde GD, et al., 2014, DEVELOPMENT OF WHOLE BODY AND INTRAVASCULAR NEAR-INFRARED OPTICAL MOLECULAR IMAGING OF MARKERS OF PLAQUE VULNERABLITY IN ATHEROSCLEROSIS, Annual Conference of the British-Cardiovascular-Society
Hamdulay SS, Wang B, Calay D, et al., 2014, Synergistic therapeutic vascular cytoprotection against complement- mediated injury induced via a PKC alpha-, AMPK-, and CREB- dependent pathway, Journal of Immunology, Vol: 192, Pages: 4316-4327, ISSN: 1550-6606
Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.
Wan X, Huo Y, Johns M, et al., 2014, HEME AND METFORMIN COORDINATE HUMAN AND MURINE MACROPHAGE HEME OXYGENASE 1 EXPRESSION WITH FOAM CELL RESISTANCE PARTLY VIA ADENOSINE MONOPHOSPHATE KINASE AND ACTIVATING TRANSCRIPTION FACTOR 1 (AMPK-ATF1), Autumn Meeting of the British-Atherosclerosis-Society (BAS), Publisher: ELSEVIER IRELAND LTD, Pages: E4-E4, ISSN: 0021-9150
Wan X, Huo Y, Johns M, et al., 2013, 5 '-AMP-Activated Protein Kinase-Activating Transcription Factor 1 Cascade Modulates Human Monocyte-Derived Macrophages to Atheroprotective Functions in Response to Heme or Metformin, ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY, Vol: 33, Pages: 2470-2480, ISSN: 1079-5642
Landis RC, Philippidis P, Domin J, et al., 2013, Haptoglobin Genotype-Dependent Anti-Inflammatory Signaling in CD163(+) Macrophages., International Journal of Inflammation, Vol: 2013, ISSN: 2042-0099
Intraplaque hemorrhage causes adaptive remodelling of macrophages towards a protective phenotype specialized towards handling iron and lipid overload, denoted Mhem. The Mhem phenotype expresses elevated levels of hemoglobin (Hb) scavenger receptor, CD163, capable of endocytosing pro-oxidant free Hb complexed to acute phase protein haptoglobin (Hp). It is notable that individuals homozygous for the Hp 2 allele (a poorer antioxidant) are at increased risk of cardiovascular disease compared to the Hp 1 allele. In this study, we examined whether scavenging of polymorphic Hp:Hb complexes differentially generated downstream anti-inflammatory signals in cultured human macrophages culminating in interleukin (IL)-10 secretion. We describe an anti-inflammatory signalling pathway involving phosphatidylinositol-3-kinase activation upstream of Akt phosphorylation (pSer473Akt) and IL-10 secretion. The pathway is mediated specifically through CD163 and is blocked by anti-CD163 antibody or phagocytosis inhibitor. However, levels of pSer473Akt and IL-10 were significantly diminished when scavenging polymorphic Hp2-2:Hb complexes compared to Hp1-1:Hb complexes (P < 0.05). Impaired anti-inflammatory macrophage signaling through a CD163/pAkt/IL-10 axis may thus represent a possible Hp2-2 disease mechanism in atherosclerosis.
Haskard DO, Boyle JJ, Evans PC, et al., 2013, Cytoprotective Signaling and Gene Expression in Endothelial Cells and MacrophagesLessons for Atherosclerosis, MICROCIRCULATION, Vol: 20, Pages: 203-216, ISSN: 1073-9688
Dumont O, Mylroie H, Bauer A, et al., 2012, Protein kinase C epsilon activity induces anti-inflammatory and anti-apoptotic genes via an ERK1/2-and NF-kappa B-dependent pathway to enhance vascular protection, BIOCHEMICAL JOURNAL, Vol: 447, Pages: 193-204, ISSN: 0264-6021
Boyle JJ, 2012, Heme and haemoglobin direct macrophage Mhem phenotype and counter foam cell formation in areas of intraplaque haemorrhage., Curr Opin Lipidol, Vol: 23, Pages: 453-461
PURPOSE OF REVIEW: Several studies have recently shown that haemoglobin drives a novel macrophage subset that is protected from foam cell formation. RECENT FINDINGS: In a previously overlooked area, two centres have independently shown that heme and haemoglobin drive an atheroprotective macrophage subset. We compare and contrast the approaches and findings of the laboratories and discuss some of the underlying biology and implications, concentrating on the aspects of lipidological relevance. SUMMARY: Treatments based on direct heme-mimetics or other agonists of this pathway have enormous potential for linked antioxidant protection via heme oxygenase 1 and reduced foam cell formation via liver X receptor, a potent combination for treating atherosclerosis.
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