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Schafer CM, Martin-Almedina S, Kurylowicz K, et al., 2023, Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge., bioRxiv
BACKGROUND: During infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNFα or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5(PAC)Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek , a gene target of ERG previously implicated in maintaining pulmonary vascular stability during infl
Khamis R, Hartley A, Caga-Anan M, et al., 2022, A monoclonal autoantibody against a cryptic epitope on tissue-adherent low-density lipoprotein for molecular imaging in atherosclerosis, JACC: Cardiovascular Imaging, Vol: 15, ISSN: 1876-7591
Objectives We aimed to generate and characterize IgG monoclonal autoantibodies in atherosclerosis for targeting of novel molecular determinants.Background Antibody-based constructs for molecular imaging and therapeutic delivery provide promising opportunities for the diagnosis and treatment of atherosclerosis.Methods We created hybridomas from an unimmunized LDL receptor-deficient (Ldlr-/-) mouse and selected an IgG2b isotype autoantibody, LO9, for further characterization.Results LO9 reacted well with native LDL bound to immobilized matrix components, and less well to oxidized LDL. LO9 binding to immobilized native LDL was not neutralized by fluid-phase native LDL, indicating an adhesion-dependent epitope. We localized the epitope to a 20 amino-acid peptide sequence (P5) in the globular amino-terminus of apolipoprotein B. LO9 reacted with antigen in mouse atherosclerosis and in both human stable and ruptured coronary atherosclerosis. Furthermore, in vivo near-infrared fluorescence molecular tomographic imaging and ex vivo confocal microscopy showed that intravenously injected LO9 localized beneath endothelium of the aortic arch in Ldlr-/- mice, in the vicinity of macrophages.Conclusions We believe LO9 is the first example of an IgG autoantibody that reacts with a native LDL epitope revealed by adherence to tissue matrix. Antibodies against adherent native LDL have potential as molecular targeting agents for imaging of and therapeutic delivery to atherosclerosis.
Kaura A, Hartley A, Panoulas V, et al., 2022, Mortality risk prediction of high-sensitivity C-reactive protein in suspected acute coronary syndrome: a cohort study, PLoS Medicine, Vol: 19, ISSN: 1549-1277
BACKGROUND: There is limited evidence on the use of high-sensitivity C-reactive protein (hsCRP) as a biomarker for selecting patients for advanced cardiovascular (CV) therapies in the modern era. The prognostic value of mildly elevated hsCRP beyond troponin in a large real-world cohort of unselected patients presenting with suspected acute coronary syndrome (ACS) is unknown. We evaluated whether a mildly elevated hsCRP (up to 15 mg/L) was associated with mortality risk, beyond troponin level, in patients with suspected ACS. METHODS AND FINDINGS: We conducted a retrospective cohort study based on the National Institute for Health Research Health Informatics Collaborative data of 257,948 patients with suspected ACS who had a troponin measured at 5 cardiac centres in the United Kingdom between 2010 and 2017. Patients were divided into 4 hsCRP groups (<2, 2 to 4.9, 5 to 9.9, and 10 to 15 mg/L). The main outcome measure was mortality within 3 years of index presentation. The association between hsCRP levels and all-cause mortality was assessed using multivariable Cox regression analysis adjusted for age, sex, haemoglobin, white cell count (WCC), platelet count, creatinine, and troponin. Following the exclusion criteria, there were 102,337 patients included in the analysis (hsCRP <2 mg/L (n = 38,390), 2 to 4.9 mg/L (n = 27,397), 5 to 9.9 mg/L (n = 26,957), and 10 to 15 mg/L (n = 9,593)). On multivariable Cox regression analysis, there was a positive and graded relationship between hsCRP level and mortality at baseline, which remained at 3 years (hazard ratio (HR) (95% CI) of 1.32 (1.18 to 1.48) for those with hsCRP 2.0 to 4.9 mg/L and 1.40 (1.26 to 1.57) and 2.00 (1.75 to 2.28) for those with hsCRP 5 to 9.9 mg/L and 10 to 15 mg/L, respectively. This relationship was independent of troponin in all suspected ACS patients and was further verified in those who were confirmed to have an ACS diagnosis by clinical coding. The main limitation of our study is that we did not
Hartley A, Pradeep M, Van den Berg V, et al., 2022, Depletion of homeostatic antibodies against malondialdehyde-modified low-density lipoprotein correlates with adverse events in major vascular surgery, Antioxidants, Vol: 11, ISSN: 2076-3921
We aimed to investigate if major vascular surgery induces LDL oxidation, and whether circulating antibodies against malondialdehyde-modified LDL (MDA-LDL) alter dynamically in this setting. We also questioned relationships between these biomarkers and post-operative cardiovascular events. Major surgery can induce an oxidative stress response. However, the role of the humoral immune system in clearance of oxidized LDL following such an insult is unknown. Plasma samples were obtained from a prospective cohort of 131 patients undergoing major non-cardiac vascular surgery, with samples obtained preoperatively and at 24- and 72 h postoperatively. Enzyme-linked immunoassays were developed to assess MDA-LDL-related antibodies and complexes. Adverse events were myocardial infarction (primary outcome), and a composite of unstable angina, stroke and all-cause mortality (secondary outcome). MDA-LDL significantly increased at 24 h post-operatively (p < 0.0001). Conversely, levels of IgG and IgM anti-MDA-LDL, as well as IgG/IgM-MDA-LDL complexes and total IgG/IgM, were significantly lower at 24 h (each p < 0.0001). A smaller decrease in IgG anti-MDA-LDL related to combined clinical adverse events in a post hoc analysis, withstanding adjustment for age, sex, and total IgG (OR 0.13, 95% CI [0.03–0.5], p < 0.001; p value for trend <0.001). Major vascular surgery resulted in an increase in plasma MDA-LDL, in parallel with a decrease in antibody/complex levels, likely due to antibody binding and subsequent removal from the circulation. Our study provides novel insight into the role of the immune system during the oxidative stress of major surgery, and suggests a homeostatic clearance role for IgG antibodies, with greater reduction relating to downstream adverse events.
Hartley A, Khamis R, Al-Lamee R, et al., 2021, The placebo-controlled effect of percutaneous coronary intervention on exercise induced changes in anti- malondialdehyde-LDL antibody levels in stable coronary artery disease: a substudy of the ORBITA Trial, Frontiers in Cardiovascular Medicine, Vol: 8, ISSN: 2297-055X
Aim: Malondialdehyde-modified low-density lipoprotein (MDA-LDL) forms a significantcomponent of oxidized LDL. The effects of exercise on levels of MDA-LDL and antiMDA-LDL antibodies are not well understood. Furthermore, it is not known whetherthese can be modified in patients with coronary artery disease by percutaneouscoronary intervention (PCI).Methods: The Objective Randomised Blinded Investigation with optimal medicalTherapy of Angioplasty in stable angina (ORBITA) trial was the first blinded, multicentre randomised trial of PCI versus placebo procedure for angina relief. Serumsamples were available at four time-points: pre-randomisation pre- (P1) and post- (P2)exercise and post-randomisation (six-weeks following the PCI or placebo procedure),pre- (P3) and post- (P4) exercise. ELISAs were performed using laboratory-developedassays for MDA-LDL (adjusted for Apolipoprotein B) and anti-MDA-LDL antibodies.Results: 196 of the 200 patients (age 66.1 [SD 8.99] years, 28% female) with severesingle vessel coronary artery disease suitable for PCI enrolled in the ORBITA trial hadblood available for analysis. With exercise at pre-randomisation (P2 - P1) there wasno significant change in adjusted MDA-LDL (-0.001, 95% CI -0.004 to 0.001; p=0.287);however, IgG and IgM anti-MDA-LDL significantly declined (-0.022, 95% CI -0.029 to-0.014, p<0.0001; -0.016, 95% CI -0.024 to -0.008, p=0.0002, respectively). PCI didnot have a significant impact on either the pre-exercise values (P3 controlling for P1)of MDA-LDL (p=0.102), IgG (p=0.444) or IgM anti-MDA-LDL(p=0.909). Nor did PCIimpact the exercise induced changes in these markers (P4 controlling for P1, P2, andP3) for MDA-LDL (p=0.605), IgG (p=0.725) or IgM anti-MDA-LDL (p=0.171). Prerandomisation ischaemia on stress echo did not impact these interactions.Conclusions: Exercise results in an acute reduction in anti-oxLDL antibodies inpatients with severe single vessel coronary disease, possibly indicating an inductionin homoeostatic
Hartley A, Khamis R, 2021, A novel immunoassay for malondialdehyde-conjugated low-density lipoprotein measures dynamic changes in the blood of patients undergoing coronary artery bypass graft surgery, Antioxidants, Vol: 10, Pages: 1-14, ISSN: 2076-3921
Oxidized low-density lipoproteins play an important role in tissue pathology. In this study, we report a sensitive novel enzyme-linked immunosorbent assay for the detection of malondial-dehyde-modified low-density lipoprotein (MDA-LDL), a key component of oxidized LDL. The assay is capable of measuring a variable presence of MDA-LDL within human plasma and serum. We demonstrate the robust nature of the assay on samples stored for over 20 months, as well as high inter-operator reproducibility (r=0.74, p <0.0001). The assay was capable of detecting dynamic changes in patient blood samples after coronary artery bypass graft surgery, indicating synthesis or release of MDA-LDL with the oxidative stress of surgery, followed by homeostatic clearance. This robust, sensitive and specific assay for circulating MDA-LDL will serve as a valuable trans-lational tool for the improved detection of oxidative forms of LDL in response to a range of physiological or pathological stimuli, with potential clinical applicability.
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
Haskard DO, 2021, Natural IgM antibodies help fend off thrombosis, BLOOD, Vol: 137, Pages: 1280-1281, ISSN: 0006-4971
Mehta P, Haskard DO, Laffan MA, et al., 2021, Thromboses and COVID-19: reducing inflammation in addition to thromboprophylaxis, LANCET RHEUMATOLOGY, Vol: 3, Pages: E171-E172, ISSN: 2665-9913
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Khan TZ, Haskard D, Hartley A, et al., 2021, Oxidised LDL and Anti-Oxidised LDL Antibodies Are Reduced by Lipoprotein Apheresis in a Randomised Controlled Trial on Patients with Refractory Angina and Elevated Lipoprotein(a), Antioxidants, Vol: 10, ISSN: 2076-3921
Aims: An abundance of epidemiological evidence demonstrates that elevated lipoprotein(a) (Lp(a)) represents a significant contributing risk factor towards the development of cardiovascular disease. In particular, raised Lp(a) may play a mechanistic role in patients with refractory angina. Studies have also shown a correlation between oxidised LDL (oxLDL) levels and atherosclerotic burden as well as rates of cardiovascular events. Antibodies against oxLDL (anti-oxLDL) are involved in the removal of oxLDL. Lipoprotein apheresis (LA), which removes lipoproteins using extra-corporeal processes, is an established means of reducing Lp(a), and thereby reduces cardiovascular events. The aim of this study was to investigate the effect of LA on oxLDL and anti-oxLDL levels amongst those with refractory angina in the context of raised Lp(a). Methods: We performed a sub-study within a randomised controlled crossover trial involving 20 patients with refractory angina and raised Lp(a) > 500 mg/L, comparing the effect of three months of blinded weekly LA or sham, followed by crossover to the opposite study arm. We utilized enzyme-linked immunosorbent assays (ELISA) to quantify oxLDL and IgG/ IgM anti-oxLDL antibody levels at baseline and following three months of active LA or sham sessions. Results: Following three months of LA, there was a 30% reduction in oxLDL from 0.37 ± 0.06 to 0.26 ± 0.04 with a mean drop of −0.11 units (U) (95% CI −0.13, −0.09) compared to no significant change with sham therapy (p < 0.0001 between treatment arms). LA also led to a 22% reduction in levels of IgG and IgM anti-oxLDL, again with no significant change demonstrated during sham (p = 0.0036 and p = 0.012, respectively, between treatment arms). Conclusion: Amongst patients with refractory angina in the context of elevated Lp(a), LA significantly lowers levels of oxLDL and anti-oxLDL antibodies, representing potential mechanisms by which LA yields symptomatic and
Rahman MS, Haskard DO, Frost G, et al., 2020, Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses, European-Society-of-Cardiology (ESC) Congress, Publisher: OXFORD UNIV PRESS, Pages: 3622-3622, ISSN: 0195-668X
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.
van den Berg VJ, Vroegindewey MM, Kardys I, et al., 2019, Anti-oxidized LDL antibodies and coronary artery disease: a systematic review, Antioxidants (Basel), Vol: 8, ISSN: 2076-3921
Antibodies to oxidized LDL (oxLDL) may be associated with improved outcomes in cardiovascular disease. However, analysis is restricted by heterogenous study design and endpoints. Our objective was to conduct a comprehensive systematic review assessing anti-oxLDL antibodies in relation to coronary artery disease (CAD). Through a systematic literature search, we identified all studies assessing the relationship of either, IgG or IgM ox-LDL/ copper-oxLDL/ malondialdehyde-LDL, with coronary atherosclerosis or cardiovascular events in populations with, and without, established CAD. Systematic review best practices were adhered to and study quality was assessed. An initial electronic database search identified 2059 records, which was subsequently followed by abstract and full-text review. Finally, we included 18 studies with over 1811 patients with CAD. The studies varied according to populations studied, conventional cardiovascular risk factors and interventional modalities used to assess CAD. IgM anti-oxLDL antibodies were found to indicate protection from more severe CAD and possibly cardiovascular events, whilst the relationship with IgG is more complex and difficult to elucidate, with studies reporting divergent results. In this systematic review, there is evidence that suggests a relationship between anti-oxLDL antibodies and CAD, especially for the IgM subclass. However, further studies, with well-characterized prospective cohorts, will be important to clarify these associations.
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, 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
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
Makmur EL, Myers SH, Hanns L, et al., 2019, Comparing the clinical profile of adults and children with Behçet's syndrome in the UK., Clinical and Experimental Rheumatology, ISSN: 0392-856X
OBJECTIVES: Behçet's syndrome (BS) is a rare multi-system inflammatory disorder. Clinical phenotypic variance across geographical regions is recognised but UK BS patients' variance by age groups and gender has not been studied. This study compares the clinical features of adult and juvenile onset Behçet's Syndrome (JBS) in a UK population. METHODS: Two clinical databases of BS patients were compared. The JBS database was collected at the Great Ormond Street Hospital for Children, London (n=46). The adult database was collected at the Hammersmith Hospital, London (n=560). RESULTS: Oro-genital aphthosis had high prevalence in both the JBS and the adult cohort (oral: 97.8% vs. 96.6%, genital: 73.9% vs. 75.7%). The JBS cohort was more likely to have gastrointestinal involvement (21.7% vs. 4.5%, p<0.001) and arthritis (21.7% vs. 9.6%, p=0.021) compared to adults. The JBS cohort was less likely to have eye involvement (4.3% vs. 37%, p<0.001), skin (21.7% vs. 55.4%, p<0.001) and vascular involvement (6.5% vs. 17.5% p=0.063). JBS females had a higher rate of genital aphthosis than JBS males (87.5% vs. 59.1%, p=0.044). Adult females had higher rates of genital (85.2% vs. 64.5%, p<0.001) and oral (99.0% vs. 93.8%, p=0.001) aphthosis than adult males. Adult males were more likely to have ophthalmological (44.9% vs. 30.3%, p<0.001) and vascular (23.0% vs. 12.8%, p=0.002) manifestations than adult females. CONCLUSIONS: UK JBS patients displayed less ocular and skin manifestations compared to the adult BS patients. This information will aid clinicians in diagnosing BS in UK adult and paediatric populations.
Kalesh K, Lukauskas S, Borg AJ, et al., 2019, An integrated chemical proteomics approach for quantitative profiling of intracellular ADP-Ribosylation, Scientific Reports, Vol: 9, Pages: 1-12, ISSN: 2045-2322
ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells. Although similar labelling profiles were observed via fluorescence imaging for 2YnAd and 6YnAd, a previously reported clickable NAD+ precursor, quantitative mass spectrometry analysis of the two probes in MDA-MB-231 breast cancer cells revealed a significant increase in protein coverage of the 2YnAd probe. To facilitate global enrichment of ADP-ribosylated proteins, we developed a dual metabolic labelling approach that involves simultaneous treatment of live cells with both 2YnAd and 6YnAd. By combining this dual metabolic labelling strategy with highly sensitive tandem mass tag (TMT) isobaric mass spectrometry and hierarchical Bayesian analysis, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib.
Hartley A, Dorian H, Khamis RYJ, 2019, Oxidized LDL and anti-oxidized LDL antibodies in atherosclerosis - novel insights and future directions in diagnosis and therapy, Trends in Cardiovascular Medicine, Vol: 29, Pages: 22-26, ISSN: 1050-1738
We provide an up-to-date overview of current topics surrounding oxidized low-density lipoprotein (oxLDL) and its related antibodies in the quest to better identify the individuals at risk of cardiovascular disease and atherosclerotic plaques with unfavorable characteristics. We discuss the potential of oxLDL and anti-oxLDL antibodies as serum biomarkers of cardiovascular disease and emerging studies examining the targeting of arterial oxLDL for imaging and therapeutic delivery.
van den Berg VJ, Haskard DO, Fedorowski A, et al., 2018, IgM anti-malondialdehyde low density lipoprotein antibody levels indicate coronary heart disease and necrotic core characteristics in the Nordic Diltiazem (NORDIL) study and the Integrated Imaging and Biomarker Study 3 (IBIS-3)., EBioMedicine, Vol: 36, Pages: 63-72, ISSN: 2352-3964
BACKGROUND: Certain immunoglobulins (Ig) are proposed to have protective functions in atherosclerosis. OBJECTIVES: We tested whether serum levels of IgG and IgM autoantibodies against malondialdehyde low density lipoprotein (MDA-LDL) are associated with clinical coronary heart disease (CHD) and unfavorable plaque characteristics. METHODS: NORDIL was a prospective study investigating adverse cardiovascular outcomes in hypertensive patients. IBIS-3 analyzed lesions in a non-culprit coronary artery with <50% stenosis using radiofrequency intravascular ultrasound (RF-IVUS) and near-infrared spectroscopy (NIRS). Imaging was repeated after a median of 386 days on rosuvastatin. Associations of antibodies with incident CHD and imaging parameters were assessed in the two sub-studies respectively. FINDINGS: From 10,881 NORDIL patients, 87 had serum sampled at baseline and developed CHD over 4.5 years, matched to 227 controls. Higher titers of IgM anti-MDA-LDL had a protective effect on adverse outcomes, with odds ratio 0.29 (0.11, 0.76; p = 0.012; p = 0.016 for trend). Therefore, the effect was explored at the lesional level in IBIS-3. 143 patients had blood samples and RF-IVUS measurements available, and NIRS was performed in 90 of these. At baseline, IgM anti-MDA-LDL levels had a strong independent inverse relationship with lesional necrotic core volume (p = 0.027) and percentage of plaque occupied by necrotic core (p = 0.011), as well as lipid core burden index (p = 0.024) in the worst 4 mm segment. INTERPRETATION: Our study supports the hypothesis that lower circulating levels of IgM anti-MDA-LDL are associated with clinical CHD development, and for the first time relates these findings to atherosclerotic plaque characteristics that are linked to vulnerability.
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.
Hartley A, Haskard D, Khamis R, 2018, Markers of Apoptosis predict cardiovascular outcomes and point to 'response to injury' as a common pathway leading to diabetes and cardiovascular events, EBioMedicine, Vol: 28, Pages: 19-20, ISSN: 2352-3964
Metabolic stress, chronic vascular injury and cellular apoptosis are considered as instrumentally connected in the pathogenesis of atherosclerosis and diabetes (Gistera and Hansson, 2017). The work from Mattisson et al. (Mattisson et al., 2017) sheds light on this potentially important pathway linking cell-mediated death and cardiovascular outcomes. The study spans translational science, from describing an elegant laboratory technique using Fas ligand to induce apoptosis in peripheral blood mononuclear cells and demonstrating release of TNF receptor 1 (TNFR-1), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR-2) and Fas, to studying the cell death receptor plasma levels in a large well-characterized prospective population.
Pandey S, Haskard DO, Khamis R, 2017, Developing a strategy for interventional molecular imaging of oxidized low density lipoprotein in atherosclerosis. An auto-commentary., Molecular Imaging, Vol: 16, ISSN: 1536-0121
The identification of vulnerable coronary artery atherosclerotic plaques offers the prospect of either localized or systematic therapeutic targeting in order to prevent myocardial infarction. Molecular imaging of atherosclerosis adds to morphological imaging by focusing on the immunobiology hidden in and behind the endothelium and therefore may be able to improve the identification of prospective culprit lesions. Our focus has been on identifying arterial accumulation of oxidized low density lipoprotein (oxLDL) by exploiting advances in knowledge of vascular pathobiology. Here, we reflect on our work developing Near Infra-Red Fluorescence (NIRF) imaging of oxLDL using LO1, a monoclonal autoantibody generated in our laboratory. We detail progress to date and discuss our vision on taking the work through the early translational pipeline towards a multi-targeted approach in imaging rupture prone atherosclerotic plaques. Ultimately, molecular imaging of coronary arteries should be able to assess the regional risk that is specific to a lesion, which can then be used in concert with global risk factors to personalize the therapeutic strategy for patients in a way that goes beyond generalized population-based therapies.
Alrashed F, Calay D, Thornton CC, et al., 2017, SELECTIVE ACTIVATION OF AN AMPK-CREB-NRF2-DEPENDENT PATHWAY BY CELECOXIB INDUCES VASCULOPROTECTIVE GENES AND MITIGATES AGAINST CARDIOVASCULAR RISK, Annual European Congress of Rheumatology, Publisher: BMJ PUBLISHING GROUP, Pages: 219-219, ISSN: 0003-4967
Boyle JJ, Seneviratne A, Carling D, et al., 2017, Hematoma Resolution In Vivo is Mediated by AMP-Activated Kinase (AMPK) and Activating Transcription Factor 1 (ATF1) Via Coregulation of Tissue Homeostatic Genes, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 29-29, ISSN: 1018-1172
Boyle JJ, Seneviratne AA, Haskard DO, et al., 2017, Oral Metformin Profoundly Suppresses Atherosclerotic Lesion Development In Vivo Independently of Glucose Lowering in a Mild Hyperlipidemic Model Via AMPK, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 8-9, ISSN: 1018-1172
Boyle JJ, Seneviratne A, Haskard DO, et al., 2017, AMP-Activated Protein Kinase-Mediated Chromatin Remodelling Redirects Activating Transcription Factor 1 From Cyclic-AMP Response Genes to Heme-Response Genes, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 29-29, ISSN: 1018-1172
Khan E, Ambrose N, Ahnstrom J, et al., 2016, A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet’s Syndrome, Clinical and Experimental Rheumatology, Vol: 6, ISSN: 1593-098X
Thrombosis is common in Behçet’s Syndrome (BS), and there is a need for better biomarkers for risk assessment. As microparticles expressing Tissue Factor (TF) can contribute to thrombosis in preclinical models, we investigated whether plasma microparticles expressing Tissue Factor (TF) are increased in BS. We compared blood plasma from 72 healthy controls with that from 88 BS patients (21 with a history of thrombosis (Th+) and 67 without (Th−). Using flow cytometry, we found that the total plasma MP numbers were increased in BS compared to HC, as were MPs expressing TF and Tissue Factor Pathway Inhibitor (TFPI) (all p < 0.0001). Amongst BS patients, the Th+ group had increased total and TF positive MP numbers (both p ≤ 0.0002) compared to the Th- group, but had a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI positive to TF positive MP counts (TFPI/TF) was significantly lower in Th+ versus Th− BS patients (p = 0.0002), and no patient with a TFPI/TF MP ratio >0.7 had a history of clinical thrombosis. We conclude that TF-expressing MP are increased in BS and that an imbalance between microparticulate TF and TFPI may predispose to thrombosis.
Khan E, Ambrose N, Ahnstrom J, et al., 2016, A LOW BALANCE BETWEEN MICROPARTICLES EXPRESSING TISSUE FACTOR PATHWAY INHIBITOR AND TISSUE FACTOR IS ASSOCIATED WITH THROMBOSIS IN BEHCET'S SYNDROME, Publisher: CLINICAL & EXPER RHEUMATOLOGY, Pages: S143-S143, ISSN: 0392-856X
Serbanovic-Canic J, de Luca A, Warboys C, et al., 2016, Zebrafish model for functional screening of flow-responsive genes, Arteriosclerosis Thrombosis and Vascular Biology, Vol: 36, ISSN: 1524-4636
OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.
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