The sLOX-1 protein has been previously demonstrated to be elevated in STEMI and to originate from the coronary circulation; and was hypothesised to be released by atherosclerotic plaque during rupture. Here, we assessed the utility of sLOX-1 as a biomarker to discriminate between STEMI and STEMI "mimics", such as Takotsubo cardiomyopathy and myopericarditis. Serum sLOX-1 values were obtained from patients enrolled in the BioHEART biobank who had invasive angiography for the provisional diagnosis of STEMI. The cohort consisted of 106 patients having an acute coronary syndrome (ACS, inclusive of both STEMI and non-STEMI), 29 patients with Takotsubo cardiomyopathy, and 16 patients with myopericarditis. The sLOX-1 levels seen with acute myocarditis were significantly lower than in Takotsubo cardiomyopathy (1602.0+/-1155.7 pg/mL vs 3166.6+/-2027.8 pg/mL, p=0.007) and in ACS (1602.0+/-1155.7 pg/mL vs 3003.1+/-2610.6 pg/mL, p<0.001). sLOX-1 levels were not useful in discriminating between Takotsubo cardiomyopathy and ACS (3166.6+/-2027.8 pg/mL vs 3003.1+/-2610.6 pg/mL, p=0.756). In this suspected STEMI cohort, serum sLOX-1 levels were nearly 2-fold lower in acute myopericarditis; a cutoff of 1875 pg/mL gave a sensitivity of 69%, specificity of 60%, PPV of 17% and NPV of 94% for the diagnosis of myopericarditis. Future studies should be undertaken in larger myopericarditis cohorts to determine if this could be developed into a clinically reliable "rule-out" test to avoid invasive angiography in this patient group.

Abstract Background Current methods for the early detection of coronary artery disease (CAD) are limited, particularly in those without traditional risk factors. CAD remains a significant cause of morbidity and mortality worldwide, despite advances in diagnostics, treatment and prevention. There is a major unmet need for novel, prognostic blood-based CAD biomarkers, applied to diagnostic strategies or trial endpoints, to accelerate the drug development pipeline and improve clinical trial efficiency. Peripheral blood mononuclear cells (PBMCs) are easily accessible and may serve as a potential cell-derived biomarker or a tool in candidate selection in drug discovery. Purpose This study aims to determine if IL-1β secretion from patient-derived monocytes could differ between patients with calcified atherosclerotic CAD compared with those with no calcified disease and serve as a tool in drug screening. Methods PBMCs were isolated from a cohort of 41 BioHEART participants (ACTRN12618001322224) undergoing clinically indicated CT coronary angiography. We compared 23 participants with CAD, as defined by a coronary artery calcium score (CACS) >0 Agatston units (AU) with 18 participants with no calcified CAD, defined as CACS=0 AU. Monocyte enrichment from PBMCs was achieved through a brief period of in vitro incubation, in which monocytes adhered to the culture plate, while non-monocyte cells were removed by washing. Monocytes were split into 3 conditions; incubated with either (a) 0.01% DMSO in PBS (basal, unstimulated), or (b) stimulated with 1 mM BzATP,or (c) following a 30-minute pre-treatment with a novel P2X7 receptor antagonist, PKT100 (1 μM in PBS 0.01% DMSO), stimulated with BzATP (with PKT100 maintained during the stimulation period). The concentration of IL-1β in supernatant was determined by colormetric ELISA detection. Results We demonstrated that monocytes derived from participants with calcified CAD have a significant elevation in IL-1β secretion following ATP stimulation, when compared to baseline (p<0.0001). While baseline concentration of IL-1β did not differ between control and CAD groups, ATP stimulation of monocytes resulted in a 3.40-fold higher IL-1β concentration in the CAD group compared with the control group (p<0.0001). ATP stimulation of the CAD group in the presence of PKT100 markedly reduced the monocyte IL-1β secretion to near baseline concentrations (p<0.0001). Conclusions Here, we have shown for the first time that PBMCs isolated from participants with CAD secrete higher concentrations of IL-1β upon ATP stimulation compared with non-CAD individuals. This response was markedly reduced after treatment with PKT100, confirming a role for the P2X7 receptor in modulating the downstream IL-1β response to ATP signalling. Further studies are required to understand whether a P2X7 receptor antagonist could be a potential treatment of inflammation associated with CAD.ATP stimulation of CAD patient monocytes

Introduction: Considerable efforts targeting standard modifiable cardiovascular risk factors (SMuRFs) have contributed to significant improvements in age-adjusted mortality for coronary artery disease (CAD). However, the development of atherosclerotic CAD and subsequent myocardial infarction (MI) in the absence of SMuRFs remains a significant unmet clinical need. Between 10-27% of first presentation STEMI patients are “SMuRFless”, with higher 30-day mortality than patients with at least one SMuRF. Addressing this group requires early CAD identification by novel methods, independent of traditional risk scores. Here we examine the burden of CAD in individuals considered low risk by traditional scores.

Abstract Background Exposure to air pollution is known to have both acute and chronic adverse effects on the cardiovascular system, but the pathophysiology of cardiac complications associated with air pollutants remains poorly understood; however, utilizing high-throughput omic studies can provide a better understanding of involved biomarkers, which may ultimately help identify, prevent, and discover drugs for high-risk populations. Purpose Utilising the targeted metabolomics in the BioHEART Discovery cohort we set to identify metabolites implicated in cardiovascular diseases in relation to acute exposure to air pollution. Method We included 1002 clinically stable study participants who were referred for a clinically indicated CT coronary angiogram (CTCA) between November 2015 and March 2020. Coronary artery disease (CAD) burden was assessed using coronary artery calcium scores (CACS) and the Gensini scores. We performed targeted metabolomic analysis of hydrophilic metabolites in positive and negative ionisation using Liquid chromatography–mass spectrometry (LC-MS). Acute air pollution exposure was defined as the daily average and maximum climatic (temperature and humidity) and particulate matter levels on the day of and up to 3 days prior to enrollment, based on the patient's postcode. Using K-means clustering, we grouped the population based on their overall maximum PM2.5 exposure. We also used PCA and sPLS-DA to cluster and perform variable importance selection. Results PCA demonstrated good separation between the high vs normal PM2.5 exposure groups in PC1-PC2 dimensions, with scores of 29.5% and 8.5% respectively [Figure 1]. All of the top five metabolites detected by sPLS-DA, including Trans-4-Hydroxyproline and Serine, demonstrated down-regulation in the high exposure cohort.Trans-4-Hydroxyproline is involved in the urea cycle and arginine and proline metabolism, while Serine is a marker of glycine, serine, and threonine metabolism. Previous studies [1] have shown that these metabolites have an inverse correlation with carotid-femoral pulse-wave velocity (PWV), an indirect measure of arterial stiffness and an indication of increased risk of all-cause mortality and cardiac morbidity [2]. Conclusion Here we have demonstrated that acute exposure to PM2.5 can potentially result in a shift in metabolimic signature that is associated with vascular dysfunction.PCA clusters demonstrating a good separaComparing Trans-4-HYP and Serine

STEMI is underdiagnosed in females, and they are less likely to receive evidence-based management despite growing evidence identifying these discrepancies. We conducted a retrospective cohort study of sex differences in demographics, risk factors, in-hospital mortality and pharmacotherapies on discharge among all STEMI patients presenting to a single tertiary centre in NSW from 2017–2021. The electronic medical records of 1,175 sequential patients (262 females [22%]) presenting with STEMI were reviewed. A significant difference in age at presentation was found between males and females (64+/-13 vs 73+/-14 years, p<0.001). A significantly higher proportion of females had hypertension (64% vs 46%, p<0.001), but lower rates of smoking history (31% vs 47%, p<0.001) and family history of coronary artery disease (13% vs 21%, p=0.02). After adjusting for age there was no significant difference in in-hospital mortality between males and females (p=0.59). Female-specific cardiovascular risk factors including hysterectomy and hormone-replacement therapy were identified in 6.4% of female STEMI patients; the rates of specific diagnoses were lower than expected given their population prevalence, suggesting that these conditions may not have been inquired about. Women were less likely to be prescribed DAPT (85% vs 95%, p<0.001), statins (88% vs 96%, p<0.001) and ACEi/ARBs (60% vs 75%, p<0.001) on discharge. Females that presented with STEMI were older, had fewer traditional risk factors and were less likely to be discharged on evidence-based pharmacotherapy. Further research should explore why these sex differences persist despite increasing awareness of these discrepancies.

sLOX-1 has been previously demonstrated to be elevated in STEMI and in patients with stable angina. We hypothesised that sLOX-1 would be detectably elevated in patients with early atherosclerosis, potentially having utility as a blood-based biomarker for early diagnosis of CAD.

We investigated the thrombin generation and fibrin generation profiles of patients with cardiovascular risk factors using the overall haemostatic potential (OHP) and calibrated automated thrombin (CAT) assays.

Inflammation is now a well-established component of the pathophysiology of coronary artery disease (CAD), but it is unknown whether atherosclerosis is associated with a distinct circulating immune cell profile. Mass cytometry time-of-flight (CYTOF) is a new precision technology which can be used to assess leukocyte populations comprehensively. To determine if patients with calcified and non-calcified (soft) coronary plaque have distinct circulating immune cell profiles when compared to healthy controls, and to assess whether this could be used to detect sub-clinical CAD. Patients referred for a CT coronary angiogram were recruited; blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated. Imaging data was analysed using a modified Gensini scoring system which incorporated plaque composition, with higher weighting given to soft plaque. The modified Gensini scores were then used to further segregate into calcified-predominant and soft-predominant disease groups. CYTOF analysis was performed on the PBMCs, with groups as outlined in Table 1. Age was significantly higher in the CAD+ group, but all other demographic features and risk factors did not differ between groups. Patients with predominantly calcified disease showed an increase in memory CD8 T cells (p=0.004), an increase in CD 39+ CD4 T cells (p=0.028), and a decrease in naïve CD8 T cells (p=0.005), which suggests an accumulated memory response in more quiescent disease. Patients with predominantly soft-plaque disease have higher pro-inflammatory monocyte populations (p=0.013) and proliferative CD4 T cell populations (p=0.011), suggesting acute innate and adaptive responses to biologically active plaque. This pilot study has shown that further study should be pursued into the utility of CYTOF to identify sub-clinical CAD through differences in peripheral circulating immune cell profiles. Figure 1 Type of funding source: Public Institution(s). Main funding source(s): National Health and Medical Research Council of Australia, Heart Research Australia

The progression of coronary artery disease (CAD) is known to involve plaque micro-ruptures with associated thrombosis, but it is unknown whether subclinical disorders of coagulation predispose patients to early atherosclerosis. We employed two global assays of coagulation in patients with and without CAD to determine if hypercoagulation was a novel risk factor for atherosclerosis development. This study utilised the BioHEART-CT biobank, a cohort of patients recruited when having a clinically indicated CT coronary angiogram (CTCA). Imaging data was analysed using a modified Gensini segmental scoring system to determine disease severity and plaque composition. Platelet poor plasma samples were assessed by calibrated automated thrombin (CAT) and overall haemostatic potential (OHP) assays on a total of 206 patients which were 50% female, had an average age of 62.9+/- 9.9 years, and had no significant differences in major cardiovascular risk factor distribution or medication use between the sexes. In patients who had soft plaque (SPS>0), significant differences in the results of the CAT assay (prolonged time to peak: p=0.016, prolonged thrombin tail: p=0.015) and OHP assay (increased OHP: p=0.027, higher OD max: p=0.038) were detected. Subgroup analysis demonstrated that these differences were apparent in males, but not in females. The OHP and CAT assays demonstrate a detectable disparity in coagulation in male patients with soft plaque, identifying imbalances in thrombin formation and fibrin generation that may represent a unique gender-specific biomarker for biologically active atherosclerotic plaque.

The P2X7 receptor is an ATP-gated ion channel mediating transactivation of the NLRP3 inflammasome and release of IL-1ß. It plays an important, yet relatively unexplored role in cardiovascular disease. We investigated the immunophenotypic distribution of the P2X7R in patients with atherosclerosis and correlation with CTCA measures of plaque burden, in addition to effects of pharmacological P2X7R blockade in ex-vivo STEMI monocytes, and in a mouse model of unstable plaque. P2X7R expression in an NK cell-subpopulation, characterised by CD56hiCD16- cell-surface markers was strongly associated with calcium score (p=0.0015), calcium centile (p=0.008), and Gensini (p<0.05) in multivariate regression analyses. PKT100 attenuated IL-1ß secretion in STEMI monocytes (53.8±9.4pg/ml to 13.9±2.4pg/ml; p<0.0001) and inhibited ATP-driven increases in caspase-1 function measured by fluorescent assay (12.51±2.2 to 7.44±1.9,n=12; p<0.01). Six weeks after commencement of high fat diet, a carotid-artery tandem-stenosis was introduced in 12-week-old apolipoprotein-E-deficient mice treated with PKT100 or DMSO-control by implantable osmotic mini-pumps and euthanised 7 weeks later. Aortic arch/carotids were harvested; plaque burden, measured by Oil-Red-O staining was significantly reduced in PKT100 mice (26.5±3.0%,n=6) versus DMSO (40.6±2.4%,n=5;p=0.009). Compared to controls, PKT100-treated mice expressed a more stable plaque phenotype, demonstrating decreased necrotic core size (25±3% vs 37±3,n=5; p<0.05), increased fibrous cap thickness, and non-significantly decreased plaque rupture rate (80% vs 33%,n=5; p=0.07). P2X7R levels in CD56hiCD16- NK cells may be a useful biomarker for CVD screening. The inhibition of ATP-induced inflammasome activation in monocytes from STEMI patients and in-vivo plaque stabilising effects of our novel P2X7R antagonist PKT100 has tremendous translational therapeutic potential in CVD.

Circulating endothelial progenitor cells (EPCs) play a role in vascular regeneration and can be cultured from human peripheral blood mononuclear cells (PBMCs). We hypothesise that EPCs provide a model of endothelial dysfunction in patients with coronary artery disease and sought to characterise the factors that impact their spontaneous growth and their redox signalling profiles. PBMCs were purified from patient blood samples from the BioHEART-CT biobank1 and were cultured in appropriate medium. Following the emergence of EPCs the cells were expanded, and protein lysate was collected to determine expression of redox signalling proteins by immunoblotting. Demographic and clinical information was recorded during recruitment, and CT coronary angiograms were analysed using Gensini scores to determine the burden of disease. Spontaneous growth of EPCs occurred in 164 of the 835 patients (19.6%), and the only demographic or clinical feature that was associated with EPC growth was having a BMI ≤30 (p<0.017). Coronary artery calcium scores did not predict EPC growth (p=0.90). Biochemically, NADPH oxidase-2 protein expression was found to be higher in EPCs of patients with CAD compared to those without (p=0.02), but there were no significant differences in other redox proteins assessed. In EPC angiogenesis assays, male patients with CAD were found to have significantly increased migration compared to those without CAD (p=0.024). EPCs show differential redox signalling and migration in association with CAD, but no difference in spontaneous growth rates was detected.

A positive stress test in a patient with no significant obstructive coronary artery disease (CAD) has previously been considered a “false” positive, but the underlying microvascular dysfunction driving it is now recognised as prognostically important. We aim to assess demographics and clinical factors in this patient group to determine how they might be better identified and treated. We reviewed 1498 participants in the BioHEART-CT biobank and identified 98 patients who had been referred for CT coronary angiogram (CTCA) following a positive exercise stress test. CTCAs were analysed, and the group split into microvascular disease (no obstructive lesion >50%, n=71) and macrovascular disease (n=28). The groups had no significant differences in terms of age, gender or BMI. The macrovascular group had a borderline higher rate of patients with greater than 10 pack years of smoking history (25.0% vs 9.9%, p=0.052), but otherwise rates of hypertension, hyperlipidaemia, type 2 diabetes and family history of ischaemic heart disease were similar. The was a higher rate of ACE inhibitor use in the macrovascular group (60.7% vs 25.4%, p=0.001), likely reflecting a more significant degree of hypertension, but usage of anticoagulants, aspirin, statins and beta-blockers was similar. Coronary microvascular dysfunction has similar risk factors to macrovascular dysfunction, limiting the utility of risk factor profiling as a method of early diagnosis. Biomarkers should be sought by multi-omics approaches that correlate to microvascular disease, as patients with reassuringly non-obstructive CTCAs often do not have aggressive risk factor management.

An association between type 2 immunity and coronary artery atherosclerosis has recently been described. Ixodes holocyclus (Australian paralysis tick) bites can result in IgE sensitization to the mammalian oligosaccharide galactose-α-1,3-galactose (α-Gal) which in turn is associated with mammalian meat anaphylaxis. A previous angiography-based study identified that IgE sensitization to α-Gal is associated with coronary artery disease plaque burden.

Aims: The J-CTO score is an easily calculated clinical tool which can predict the success of CTO PCI. However, it is unknown whether this score remains relevant in the context of the modern techniques used in contemporary CTO PCI. Methods & Results: We reviewed patients undergoing CTO PCI at our centre from April 2010 to February 2019. A total of 301 patients were included. The mean age was 69.3 years (±10.3) with 16.6% females and a mean BMI of 28.5 kg/m2 (±5.6). 28.6% of patients had a previous coronary artery bypass graft, with 7% of CTOs being within a stented vessel. The right coronary artery (RCA) was the commonest target vessel, accounting for 53.8% of cases, followed by the left circumflex artery (25.2%) and left anterior descending artery (19.3%). The remainder comprised of vein grafts or a protected left main stem (1.7%). The mean J-CTO score was 1.9, with a median score of 2. The overall procedural success rate was 72.1%. A higher J-CTO score was associated with greater utilisation of the retrograde approach (p < 0.01) although not dissection re-entry approach (p = 0.18). A higher J-CTO score was associated with a lower rate of procedural success (p < 0.0001). In multi-variate analysis, after correcting for age, gender and BMI, the J-CTO score remained predictive of procedural success (p < 0.0001). Conclusions: The J-CTO score remains a valuable tool to predict procedural success in patients undergoing contemporary CTO PCI. Routine calculation of the J-CTO score remains useful to identify challenging cases which may require additional planning or proctoring.

Aims: The coronary collateral circulation has numerous benefits during CTO PCI, including visualisation of the distal vessel, as a conduit to cross the distal cap and maintaining perfusion to the occluded vessel. The degree of collateralisation is not included in current predictive scoring tools. We sought to determine whether the degree of collateralisation was associated with CTO PCI outcomes. Methods & Results: We reviewed patients undergoing CTO PCI at our centre from April 2010 to February 2019. After excluding patients with only bridging collaterals or a bypass graft as the major collateral vessel, 275 patients were assessed. 94 patients (34.2%) had an extensive collateral formation as defined as Rentrop grade 3, whilst 65.8% had less developed collaterals. Those with well-developed collaterals were more likely to be females (26.6% vs 12.2%, p < 0.01) than those without, although there were similar ages in both cohorts (69.0 vs 69.1, p = 0.94). The presence of well-developed collaterals was associated with a higher rate of procedural success (92.5% vs 62.4%, p < 0.0001). When an antegrade approach was the only strategy attempted (n = 193), well developed collaterals remained predictive of success (95.5% vs 65.1%, p < 0.0001). When a retrograde approach was initially trialled (n = 45), well developed collaterals had a trend towards higher CTO PCI success rate (94.4% vs 77.8%, p = 0.10). Conclusions: The degree of collateralisation is predictive of CTO PCI being successful, even in those undergoing an anterograde revascularisation strategy. Collateral circulation grading should be considered in the development of scoring tools aiming to predict CTO PCI outcomes.

We present the case of a 36-year-old man who presented with acute-onset dyspnoea and palpitations while playing badminton. The patient initially reported having Shprintzen–Goldberg syndrome, which has associations with valve prolapse and aortic root dilatation. His manifestations were of cleft palate, craniofacial otological abnormalities, and hypoparathyroidism. He denied any cardiac involvement or symptoms prior. A bedside transthoracic echocardiogram showed a dilated aortic root but no dissection flap or regurgitation into the left ventricle. There was an echodense mass on the tricuspid valve associated with severe tricuspid regurgitation and signs of right heart failure. Empirical antibiotics were initiated for potential infective endocarditis, although inflammatory markers were low and blood cultures later returned negative results. A ventilation-perfusion scan was negative for pulmonary embolism.

AIMS/HYPOTHESIS:Adult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets.METHODS:Gene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined.RESULTS:We identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling.CONCLUSIONS/INTERPRETATION:Our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.