Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Recombinant human soluble domain of CD39L3 and ticagrelor: cardioprotective effects in experimental myocardial infarction.

BACKGROUND AND AIMS: The ecto-nucleoside triphosphate diphosphohydrolases of the CD39 family degrade ATP and ADP into AMP, which is converted into adenosine by the extracellular CD73/ecto-5-nucleotidase. This pathway has been explored in antithrombotic treatments but little in myocardial protection. We have investigated whether the administration of solCD39L3 (AZD3366) confers additional cardioprotection to that of ticagrelor alone in a pre-clinical model of myocardial infarction (MI). METHODS: Ticagrelor-treated pigs underwent balloon-induced MI (90 min) and, before reperfusion, received intravenously either vehicle, 1 mg/kg AZD3366 or 3 mg/kg AZD3366. All animals received ticagrelor twice daily for 42 days. A non-treated MI group was run as a control. Serial cardiac magnetic resonance (baseline, Day 3 and Day 42 post-MI), light transmittance aggregometry, bleeding time, and histological and molecular analyses were performed. RESULTS: Ticagrelor reduced oedema formation and infarct size at Day 3 post-MI vs. controls. A 3 mg/kg AZD3366 provided an additional 45% reduction in oedema and infarct size compared with ticagrelor and a 70% reduction vs. controls (P < .05). At Day 42, infarct size declined in all ticagrelor-administered pigs, particularly in 3 mg/kg AZD3366-treated pigs (P < .05). Left ventricular ejection fraction was diminished at Day 3 in placebo pigs and worsened at Day 42, whereas it remained unaltered in ticagrelor ± AZD3366-administered animals. Pigs administered with 3 mg/kg AZD3366 displayed higher left ventricular ejection fraction upon dobutamine stress at Day 3 and minimal dysfunctional segmental contraction at Day 42 (χ2P < .05 vs. all). Cardiac and systemic molecular readouts supported these benefits. Interestingly, AZD3366 abolished ADP-induced light transmittance aggregometry without affecting bleeding time. CONCLUSIONS: Infusion of AZD3366 on top of ticagrelor leads to enhanced cardioprotection compared with ticagrelor alone.

Myeloperoxidase Inhibition Reverses Biomarker Profiles Associated With Clinical Outcomes in HFpEF.

BACKGROUND: Systemic microvascular dysfunction and inflammation are postulated to play a pathophysiologic role in heart failure with preserved ejection fraction (HFpEF). OBJECTIVES: This study aimed to identify biomarker profiles associated with clinical outcomes in HFpEF and investigate how inhibition of the neutrophil-derived reactive oxygen species-producing enzyme, myeloperoxidase, affects these biomarkers. METHODS: Using supervised principal component analyses, the investigators assessed the associations between baseline plasma proteomic Olink biomarkers and clinical outcomes in 3 independent observational HFpEF cohorts (n = 86, n = 216, and n = 242). These profiles were then compared with the biomarker profiles discriminating patients treated with active drug vs placebo in SATELLITE (Safety and Tolerability Study of AZD4831 in Patients With Heart Failure), a double-blind randomized 3-month trial evaluating safety and tolerability of the myeloperoxidase inhibitor AZD4831 in HFpEF (n = 41). Pathophysiological pathways were inferred from the biomarker profiles by interrogation of the Ingenuity Knowledge Database. RESULTS: TNF-R1, TRAIL-R2, GDF15, U-PAR, and ADM were the top individual biomarkers associated with heart failure hospitalization or death, and FABP4, HGF, RARRES2, CSTB, and FGF23 were associated with lower functional capacity and poorer quality of life. AZD4831 downregulated many markers (most significantly CDCP1, PRELP, CX3CL1, LIFR, VSIG2). There was remarkable consistency among pathways associated with clinical outcomes in the observational HFpEF cohorts, the top canonical pathways being associated with tumor microenvironments, wound healing signaling, and cardiac hypertrophy signaling. These pathways were predicted to be downregulated in AZD4831 relative to placebo-treated patients. CONCLUSIONS: Biomarker pathways that were most strongly associated with clinical outcomes were also the ones reduced by AZD4831. These results support the further investigation of myeloperoxidase inhibition in HFpEF.

Endothelin-1 increases expression and activity of arginase 2 via ETB receptors and is co-expressed with arginase 2 in human atherosclerotic plaques.

BACKGROUND AND AIMS: Endothelin-1 (ET-1) and arginase are both suggested to be involved in the inflammatory processes and development of endothelial dysfunction in atherosclerosis. However, information regarding the roles of ET-1 and arginase, as well as the interactions between the two in human atherosclerosis, is scarce. We investigated the expression of ET-1 and its receptors, ETA and ETB, as well as arginase in human carotid atherosclerotic plaques and determined the functional interactions between ET-1 and arginase in endothelial cells and THP-1-derived macrophages. METHODS: Carotid plaques and blood samples were retreived from patients undergoing surgery for symptomatic or asymptomatic carotid stenosis. Plaque gene and protein expression was determined and related to clinical characteristics. Functional interactions between ET-1 and arginase were investigated in endothelial cells and THP-1 cells. RESULTS: Expression of ET-1 and ETB receptors was increased in plaques from patients with symptomatic carotid artery disease. ET-1 was co-localized with arginase 1 and arginase 2 in the necrotic core, together with macrophage markers CD163 and CD68. Arginase 2, ET-1 and ETB receptors were expressed in endothelial cells as well as in smooth muscle cells in the fibrous cap. ET-1 increased arginase 2 mRNA expression and arginase activity in endothelial cells and arginase activity in macrophages. Moreover, ET-1 stimulated formation of reactive oxygen species (ROS) in THP-1-derived macrophages via an arginase-dependent mechanism. CONCLUSIONS: This is the first study that demonstrates co-localization of ET-1 and arginase 2 in human atherosclerotic plaques. ET-1 stimulated arginase 2 expression and activity in endothelial cells, as well as arginase activity and ROS formation in macrophages via an arginase-dependent mechanism. These results indicate an important interaction between the ET pathway and arginase in human atherosclerotic plaques.

High-sensitivity C-reactive protein and the risk of chronic kidney disease progression or acute kidney injury in post-myocardial infarction patients.

BACKGROUND: Persistent, low-grade inflammation likely participates in the pathophysiology of both atherosclerosis and kidney disease. Although high-sensitivity C-reactive protein (hsCRP) predicts future cardiovascular risk in patients with chronic kidney disease (CKD), it is unknown whether hsCRP levels predict adverse renal outcomes in patients with cardiovascular disease. METHODS: We studied all myocardial infarction (MI) survivors undergoing hsCRP testing >30 days after their MI during routine health care in Stockholm, Sweden (2006-2011), with available information on estimated glomerular filtration rate (eGFR). HsCRP tests measured during hospitalization/emergency room visits, followed by antibiotics or indicative of acute illness, were excluded, together with patients with ongoing/recent cancer, chronic infections, or immunosuppression. Inflammation was defined over a 3-month baseline window. Study outcomes were CKD progression (composite of doubling plasma creatinine, renal replacement therapy, or renal death) and acute kidney injury (AKI, acute creatinine peaks according to Kidney Disease: Improving Global Outcomes criteria). Multivariable Cox regression was used to adjust for age, sex, eGFR, hemoglobin, time since MI, comorbidities, undertaken procedures, and medications. RESULTS: A total of 12,905 patients (62% men, mean age 73 years and 3 years since MI) were included, of whom 35% had an eGFR<60 mL/min/1.73 m2. The mean (SD) hsCRP was 3.0 (4.4) mg/L. Baseline hsCRP levels were increasingly higher across lower eGFR categories. During a median follow-up of 3.2 years, 1,019 CKD progressions and 1,481 AKI events were recorded. Patients with hsCRP ≥2 mg/L were at higher risk of both CKD progression (adjusted hazard ratio 1.42; 95% CI 1.21-1.66) and AKI (1.29; 1.13-1.47) compared to those with hsCRP <2 mg/L. This association persisted across single CKD severity stages and after further hsCRP categorization into 4 groups (≤1, 1-3, 3-10, >10 mg/L). Results were robust across subgroups of patients and after exclusion of events occurring during the first 6-12 months. CONCLUSIONS: In post-MI patients undergoing routine health care, elevated hsCRP was associated with subsequent risk of AKI and progression of CKD, irrespective of baseline kidney function.

hsCRP Level and the Risk of Death or Recurrent Cardiovascular Events in Patients With Myocardial Infarction: a Healthcare-Based Study.

Background Beyond the controlled setting of trials, scarce information exists on the burden, predictors, and outcomes associated with elevated hs CRP (high-sensitivity C-reactive protein) in "real-world" patients with myocardial infarction ( MI ). Methods and Results We included all-coming MI survivors undergoing hs CRP testing >30 days after an MI during routine health care in Stockholm, Sweden (2006-2011). hs CRP tests measured during hospitalization/emergency department visits, followed by antibiotics or indicative of acute illness, were excluded, together with patients with ongoing/recent cancer, chronic infections, or immunosuppression. Inflammation was defined over a 3-month baseline window and associated with subsequent death and major adverse cardiovascular events (composite of MI, ischemic stroke, or cardiovascular death). Included were 17 464 patients (63% men; mean age, 72.6 years) with a median hs CRP level of 2.2 (interquartile range, 1.0-6.0) mg/L and a median of 2.2 (interquartile range, 0.8-4.9) years since their MI . Most (66%) had hs CRP ≥2 mg/L, and 40% had hs CRP >3 mg/L. Lower hemoglobin, lower estimated glomerular filtration rate, and comorbidities (eg, heart failure, peripheral vascular disease, stroke, atrial fibrillation, diabetes mellitus, and rheumatoid diseases) were associated with higher odds of hs CRP ≥2 mg/L. Conversely, previous percutaneous coronary intervention, ongoing renin-angiotensin blockade, and statins were associated with lower hs CRP ≥2 mg/L odds. Patients with hs CRP ≥2 mg/L were at higher risk of major adverse cardiovascular events (n=3900; adjusted hazard ratio, 1.28; 95% CI, 1.18-1.38) and death (n=4138; adjusted hazard ratio, 1.42; 95% CI, 1.31-1.53). Results were robust across subgroups of patients and after exclusion of events occurring during the first 6 to 12 months. On a continuous scale, the association between hs CRP and outcomes was linear until hs CRP >5 mg/L, plateauing thereafter. Conclusions Most patients with MI exhibit elevated hs CRP levels. Besides identifying populations at high-inflammatory risk, this study extends the prognostic validity of this biomarker from trial evidence to real-world healthcare settings.

Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe(-/-) mice.

Discovery of novel biomarkers for atherosclerosis is important to aid in early diagnosis of pre-symptomatic patients at high risk of cardiovascular events. The aim of the present study was therefore to identify potential biomarkers in circulating cells reflecting atherosclerotic lesion progression in the vessel wall. We performed gene arrays on circulating leucocytes from atherosclerosis prone Apoe(-/-) mice with increasing ages, using C57BL/6 mice as healthy controls. We identified fatty acid binding protein 4 (FABP4) mRNA to be augmented in mice with established disease compared with young Apoe(-/-) or controls. Interestingly, the transcript FABP4 correlated significantly with lesion size, further supporting a disease associated increase. In addition, validation of our finding on protein level showed augmented FABP4 in circulating leucocytes whereas, importantly, no change could be observed in plasma. Immunofluorescence analysis demonstrated FABP4 to be present mainly in circulating neutrophils and to some extent in monocytes. Moreover, FABP4-positive neutrophils and macrophages could be identified in the subintimal space in the plaque. Using human circulating leucocytes, we confirmed the presence of FABP4 protein in neutrophils and monocytes. In conclusion, we have showed that cellular levels of FABP4 in circulating leucocytes associate with lesion development in the experimental Apoe(-/-) model. The increased expression is primarily localized to neutrophils, but also in monocytes. We have identified FABP4 in leucocytes as a potential and easy accessible biomarker of atherosclerosis which could be of future clinical relevance.

Identification of the BCAR1-CFDP1-TMEM170A locus as a determinant of carotid intima-media thickness and coronary artery disease risk.

BACKGROUND: Carotid intima-media thickness (cIMT) is a widely accepted marker of subclinical atherosclerosis. To date, large-scale investigations of genetic determinants of cIMT are sparse. METHODS AND RESULTS: To identify cIMT-associated genes and genetic variants, a discovery analysis using the Illumina 200K CardioMetabochip was conducted in 3430 subjects with detailed ultrasonographic determinations of cIMT from the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study. Segment-specific IMT measurements of common carotid, bifurcation, and internal carotid arteries, and composite IMT variables considering the whole carotid tree (IMT(mean), IMT(max), and IMT(mean-max)), were analyzed. A replication stage investigating 42 single-nucleotide polymorphisms for association with common carotid IMT was undertaken in 5 independent European cohorts (total n=11,590). A locus on chromosome 16 (lead single-nucleotide polymorphism rs4888378, intronic in CFDP1) was associated with cIMT at significance levels passing multiple testing correction at both stages (array-wide significant discovery P=6.75 × 10(-7) for IMT(max); replication P=7.24×10(-6) for common cIMT; adjustments for sex, age, and population substructure where applicable; minor allele frequency 0.43 and 0.41, respectively). The protective minor allele was associated with lower carotid plaque score in a replication cohort (P=0.04, n=2120) and lower coronary artery disease risk in 2 case-control studies of subjects with European ancestry (odds ratio [95% confidence interval] 0.83 [0.77-0.90], P=6.53 × 10(-6), n=13 591; and 0.95 [0.92-0.98], P=1.83 × 10(-4), n=82 297, respectively). Queries of human biobank data sets revealed associations of rs4888378 with nearby gene expression in vascular tissues (n=126-138). CONCLUSIONS: This study identified rs4888378 in the BCAR1-CFDP1-TMEM170A locus as a novel genetic determinant of cIMT and coronary artery disease risk in individuals of European descent.

High plasma adiponectin concentration is associated with all-cause mortality in patients with carotid atherosclerosis.

BACKGROUND: The role of adiponectin as a risk factor for mortality and recurrent ischemic cardiovascular events in patients with carotid artery disease is unknown. METHODS: Consecutive patients (n = 292) undergoing carotid endarterectomy for symptomatic and asymptomatic carotid stenosis were included in the study. Mortality and cardiovascular ischemic events were recorded during a median follow-up of 5.2 years. Baseline plasma concentrations of adiponectin were measured. Cox regression models stratified for gender were used for estimation of risk of events. RESULTS: Fifty-two patients died and 73 had an ischemic event (ischemic stroke, n = 52 and/or MI, n = 28) during follow-up. In univariate analyses, adiponectin was associated with mortality, hazard ratio (HR) per standard deviation (SD) increase of adiponectin, 1.46 (95% confidence interval [CI], 1.14-1.86). In multivariate analysis age, type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), plasma interleukin-6 (IL-6) and plasma adiponectin (HR per SD increase of adiponectin, 1.73 [95% CI, 1.29-2.32]) were independently associated with mortality. T2DM, CHD, fibrinogen, contralateral carotid artery stenosis, systolic blood pressure, symptomatic carotid artery stenosis were independently associated with ischemic events, whereas plasma adiponectin was not (HR per SD increase of adiponectin, 0.96 [95% CI, 0.75-1.23]). CONCLUSIONS: High plasma adiponectin concentration is associated with mortality in patients with established atherosclerosis undergoing surgery for carotid artery stenosis. Further studies to determine the role for adiponectin as a biomarker are warranted.

Cysteinyl leukotriene signaling aggravates myocardial hypoxia in experimental atherosclerotic heart disease.

BACKGROUND: Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe(-/-) mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia. METHODS AND PRINCIPAL FINDINGS: Myocardial biopsies from Apoe(-/-) mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C(4) synthase (LTC(4)S) and CysLT1 expression in the myocardium of Apoe(-/-) mice. Acute bouts of hypoxia further induced LTC(4)S expression, increased LTC(4)S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe(-/-) mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC(4)S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling. CONCLUSION: Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.

Prediction of ischemic events on the basis of transcriptomic and genomic profiling in patients undergoing carotid endarterectomy.

Classic risk factors, including age, smoking, serum cholesterol, diabetes and blood pressure, constitute the basis of present risk prediction models but fail to identify all individuals at risk. The objective of this study was to investigate if genomic and transcriptional patterns improve prediction of ischemic events in patients with established carotid artery disease. Genotype and gene expression profiles were obtained from carotid plaque tissue (n = 126) and peripheral blood mononuclear cells (n = 97) of patients undergoing carotid endarterectomy. Patients were followed for an average of 44 months, and 25 ischemic events occurred (18 ischemic strokes and 7 myocardial infarctions). Blinded leave-one-out cross-validation on Cox regression coefficients was used to assign gene expression-based risk scores to each patient. When compared with classic risk factors, addition of carotid plaque gene expression-based risk score improved the prediction of future ischemic events from an area under the curve (AUC) of 0.66 to an AUC of 0.79. The inclusion of gene expression risk score from peripheral blood mononuclear cells or from 25 established myocardial infarction risk single nucleotide polymorphisms only exhibited marginal effects on the prediction of ischemic events. Prediction of ischemic events is improved by inclusion of gene expression profiling from carotid endarterectomy tissue compared with prediction on the basis of classic risk markers alone in patients with atherosclerosis. The method may be developed to identify subjects at very high risk of ischemic events.

Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve patients with thoracic aortic dilatation: the ASAP study.

Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.

Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm.

Leukotrienes (LTs) are arachidonic acid-derived lipid mediators involved in the pathogenesis and progression of diverse inflammatory disorders. The cysteinyl-leukotrienes LTC(4), LTD(4), and LTE(4) are important mediators of asthma, and LTB(4) has recently been implicated in atherosclerosis. Here we report that mRNA levels for the three key enzymes/proteins in the biosynthesis of cysteinyl-leukotrienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTC(4) synthase (LTC(4)S), are significantly increased in the wall of human abdominal aortic aneurysms (AAAs). In contrast, mRNA levels of LTA(4) hydrolase, the enzyme responsible for the biosynthesis of LTB(4), are not increased. Immunohistochemical staining of AAA wall revealed focal expression of 5-LO, FLAP, and LTC(4)S proteins in the media and adventitia, localized in areas rich in inflammatory cells, including macrophages, neutrophils, and mast cells. Human AAA wall tissue converts arachidonic acid and the unstable epoxide LTA(4) into significant amounts of cysteinyl-leukotrienes and to a lesser extent LTB(4). Furthermore, challenge of AAA wall tissue with exogenous LTD(4) increases the release of matrix metalloproteinase (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by montelukast blocks this effect. The increased expression of LTC(4)S, together with the predominant formation of cysteinyl-leukotrienes and effects on MMPs production, suggests a mechanism by which LTs may promote matrix degradation in the AAA wall and identify the components of the cysteinyl-leukotriene pathway as potential targets for prevention and treatment of AAA.

Sustained inflammation due to nuclear factor-kappa B activation in irradiated human arteries.

OBJECTIVES: The aim of this study was to investigate gene expression networks related to cardiovascular disease in radiated human arteries. BACKGROUND: Recent epidemiological studies have shown that radiotherapy is associated with cardiovascular disease years after treatment. However, the molecular mechanisms underlying late effects of radiation are poorly described. METHODS: Arterial biopsies from radiated and nonradiated human conduit arteries, from the same patient, were simultaneously harvested during microvascular free tissue transfer for cancer-reconstruction in 13 patients, 4 to 500 weeks from radiation treatment. Radiated and nonradiated arteries were compared, with Affymetrix (Santa Clara, California) microarrays on a subset of the material to generate candidate genes. A Taqman (Applied Biosystems, Foster City, California) low-density array of 45 selected genes was designed for analysis of the whole material. RESULTS: Thirteen genes were synchronously expressed in all patients (p = 0.0015), including CCL8, CCL3, CXCL2, DUSP5, FGFR2, HMOX1, HOXA9, IL-6, MMP-1, PTX3, RDH10, SOD2, and TNFAIP3. A majority of differentially regulated genes related to the nuclear factor-kappa B (NF-kappaB) signaling pathway and were dysregulated even years after radiation. The NF-kappaB activation was confirmed by immunohistochemistry and immunofluorescence. CONCLUSIONS: In the present study, we found sustained inflammation due to NF-kappaB activation in human radiated arteries. The results are supported by previous in vitro findings suggesting that deoxyribonucleic acid injury, after radiation, activates NF-kappaB. We also suggest that HOXA9 might be involved in the regulation of NF-kappaB activation. The observed sustained inflammatory response can explain cardiovascular disease years after radiation.

Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants.

BACKGROUND: Several genome-wide association studies have recently linked a group of single nucleotide polymorphisms in the 9p21 region with cardiovascular disease. The molecular mechanisms of this link are not fully understood. We investigated five different expression microarray datasets in order to determine if the genotype had effect on expression of any gene transcript in aorta, mammary artery, carotid plaque and lymphoblastoid cells. METHODOLOGY/PRINCIPAL FINDINGS: After multiple testing correction, no genes were found to have relation to the rs2891168 risk genotype, either on a genome-wide scale or on a regional (8 MB) scale. The neighbouring ANRIL gene was found to have eight novel transcript variants not previously known from literature and these varied by tissue type. We therefore performed a detailed probe-level analysis and found small stretches of significant relation to genotype but no consistent associations. In all investigated tissues we found an inverse correlation between ANRIL and the MTAP gene and a positive correlation between ANRIL and CDKN2A and CDKN2B. CONCLUSIONS/SIGNIFICANCE: Investigation of relation of the risk genotype to gene expression is complicated by the transcript complexity of the locus. With our investigation of a range of relevant tissue we wish to underscore the need for careful attention to the complexity of the alternative splicing issues in the region and its implications to the design of future gene expression studies.

YKL-40 a new biomarker in patients with acute coronary syndrome or stable coronary artery disease.

BACKGROUND: YKL-40 is involved in remodelling and angiogenesis in non-cardiac inflammatory diseases. Aim was to quantitate plasma YKL-40 in patients with ST-elevation myocardial infarction (STEMI) or stable chronic coronary artery disease (CAD), and YKL-40 gene activation in human myocardium. METHODS AND RESULTS: We included 73 patients: I) 20 patients with STEMI; II) 28 patients with stable CAD; III) 15 CAD patients referred for coronary by-pass surgery. YKL-40 mRNA expression was measured in myocardium subtended by stenotic or occluded arteries and areas with no apparent disease; and IV) 10 age-matched healthy controls. Plasma YKL-40 was significantly increased in patients with STEMI (88 microg/l, median) and CAD (66 microg/l) compared to controls (16 microg/l, p<0.01 for both). Plasma YKL-40 correlated with CRP at baseline in STEMI (r=0.53, p=0.02) and CAD patients (r=0.41, p=0.031).YKL-40 gene expression was similar in ischemic and non-ischemic myocardium. CONCLUSIONS: Plasma YKL-40 was significantly increased in patients with STEMI and stable CAD. Further studies will define the role of YKL-40 as a clinically useful marker for myocardial ischemia, remodelling and maybe prognosis.

Reduced perlecan expression and accumulation in human carotid atherosclerotic lesions.

Heparan sulfate in the extracellular matrix of the artery wall has been proposed to possess anti-atherogenic properties by interfering with lipoprotein retention, suppression of inflammation, and inhibition of smooth muscle cell growth. Previously, the amount of heparan sulfate in atherosclerotic lesions from humans and animals has been shown to be reduced but the identity or identities of the heparan sulfate molecules being down regulated in this disease are not known. In this study, atherosclerotic lesions were retrieved from 44 patients undergoing surgery for symptomatic carotid stenosis. Normal iliac arteries from organ donors were used as controls. Analysis of the specimens by gene microarray showed a selective reduction in perlecan gene expression, whereas, expression of the other heparan sulfate proteoglycans in the artery wall, agrin and collagen XVIII, remained unchanged. Expression of the large chondroitin sulfate proteoglycan, versican, also remained unchanged. Real-time PCR confirmed the decrease in perlecan gene expression and the unchanged expression of versican. The findings were supported by immunohistochemical analysis demonstrating a reduced accumulation of both perlecan core protein and heparan sulfate in carotid lesions. The study demonstrates a reduction of perlecan mRNA-expression and protein deposition in human atherosclerosis, which in part explains the low levels of heparan sulfate in this disease.

Induction of neutrophil gelatinase-associated lipocalin in vascular injury via activation of nuclear factor-kappaB.

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.