New models of atherosclerosis and multi-drug therapeutic interventions.

MOTIVATION: Atherosclerosis is amongst the leading causes of death globally. However, it is challenging to study in vivo or in vitro and no detailed, openly-available computational models exist. Clinical studies hint that pharmaceutical therapy may be possible. Here, we develop the first detailed, computational model of atherosclerosis and use it to develop multi-drug therapeutic hypotheses. RESULTS: We assembled a network describing atheroma development from the literature. Maps and mathematical models were produced using the Systems Biology Graphical Notation and Systems Biology Markup Language, respectively. The model was constrained against clinical and laboratory data. We identified five drugs that together potentially reverse advanced atheroma formation. AVAILABILITY AND IMPLEMENTATION: The map is available in the Supplementary Material in SBGN-ML format. The model is available in the Supplementary Material and from BioModels, a repository of SBML models, containing CellDesigner markup. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exploring the Involvement of NLRP3 and IL-1ß in Osteoarthritis of the Hand: Results from a Pilot Study.

Hand osteoarthritis (HOA) includes different subsets; a particular and uncommon form is erosive HOA (EHOA). Interleukin- (IL-) 1ß plays a crucial role in the pathogenesis of osteoarthritis (OA); it is synthesized as an inactive precursor which requires the intervention of a cytosolic multiprotein complex, named inflammasome, for its activation. The aim of this study was to investigate the involvement of IL-1ß and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in patients with EHOA and nonerosive HOA (NEHOA) compared to healthy controls. In particular, we evaluated the gene expression of IL-1ß and NLRP3, the serum levels of IL-1ß, IL-6, IL-17, and tumor necrosis factor- (TNF-) α, and the protein levels of IL-1ß and NLRP3. We also assessed the relationships between IL-1ß and NLRP3 and clinical, laboratory, and radiological findings. Fifty-four patients with HOA (25 EHOA and 29 NEHOA) and 20 healthy subjects were included in the study. Peripheral blood mononuclear cell (PBMC) gene and protein expressions of IL-1ß and NLRP3 were quantified by quantitative real-time PCR and western blot. IL-1ß, IL-6, IL-17, and TNF-α serum levels were determined by ELISA. IL-1ß gene expression was significantly reduced (p = 0.0208) in EHOA compared to healthy controls. NLRP3 protein levels were significantly increased in the NEHOA group versus the control (p = 0.0063) and EHOA groups (p = 0.0038). IL-1ß serum levels were not significantly different across the groups; IL-6, IL-17, and TNF-α were not detectable in any sample. IL-1ß concentrations were negatively correlated with the Kellgren-Lawrence score in the whole population (r = -0.446; p = 0.0008) and in NEHOA (r = -0.608; p = 0.004), while IL-1ß gene expression was positively correlated with the number of joint swellings in the EHOA group (r = 0.512; p = 0.011). Taken together, our results, showing poorly detectable IL-1ß concentrations and minimal inflammasome activity in the PBMCs of HOA patients, suggest a low grade of systemic inflammation in HOA. This evidence does not preclude a possible involvement of these factors at the local level.

An exploratory analysis investigating blood protein biomarkers to augment ECG diagnosis of ACS.

BACKGROUND: Acute Coronary Syndrome (ACS) is currently diagnosed using a 12­lead Electrocardiogram (ECG). Our recent work however has shown that interpretation of the 12­lead ECG is complex and that clinicians can be sub-optimal in their interpretation. Additionally, ECG does not always identify acute total occlusions in certain patients. PURPOSE: The aim of the present study was to undertake an exploratory analysis to compare protein expression profiles of ACS patients that may in the future augment ECG diagnosis. METHODS: Patients were recruited consecutively at the cardiac catheterization laboratory at Altnagelvin Hospital over a period of 6 months. A low risk control group was recruited by advertisement. Blood samples were analysed using the multiplex proximity extension assays by OLINK proteomics. Support vector machine (SVM) learning was used as a classifier to distinguish between patient groups on training data. The ST segment elevation level was extracted from each ECG for a subset of patients and combined with the protein markers. Quadratic SVM (QSVM) learning was then used as a classifier to distinguish STEMI from NSTEMI on training and test data. RESULTS: Of the 344 participants recruited, 77 were initially diagnosed with NSTEMI, 7 with STEMI, and 81 were low risk controls. The other participants were those diagnosed with angina (stable and unstable) or non-cardiac patients. Of the 368 proteins analysed, 20 proteins together could significantly differentiate between patients with ACS and patients with stable angina (ROC-AUC = 0.96). Six proteins discriminated significantly between the stable angina and the low risk control groups (ROC-AUC = 1.0). Additionally, 16 proteins together perfectly discriminated between the STEMI and NSTEMI patients (ROC-AUC = 1). ECG comparisons with the protein biomarker data for a subset of patients (STEMI n = 6 and NSTEMI n = 6), demonstrated that 21 features (20 proteins + ST elevation) resulted in the highest classification accuracy 91.7% (ROC-AUC = 0.94). The 20 proteins without the ST elevation feature gave an accuracy of 80.6% (ROC-AUC 0.91), while the ST elevation feature without the protein biomarkers resulted in an accuracy of 69.3% (ROC-AUC = 0.81). CONCLUSIONS: This preliminary study identifies panels of proteins that should be further explored in prospective studies as potential biomarkers that may augment ECG diagnosis and stratification of ACS. This work also highlights the importance for future studies to be designed to allow a comparison of blood biomarkers not only with ECG's but also with cardio angiograms.

Living the PCSK9 adventure: from the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs.

A decade after our discovery of the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in cholesterol metabolism through the identification of the first mutations leading to hypercholesterolemia, PCSK9 has become one of the most promising targets in cholesterol and cardiovascular diseases. This challenging work in the genetics of hypercholesterolemia paved the way for a plethora of studies around the world allowing the characterization of PCSK9, its expression, its impact on reducing the abundance of LDL receptor, and the identification of loss-of-function mutations in hypocholesterolemia. We highlight the different steps of this adventure and review the published clinical trials especially those with the anti-PCSK9 antibodies evolocumab (AMG 145) and alirocumab (SAR236553/REGN727), which are in phase III trials. The promising results in lowering LDL cholesterol levels raise hope that the PCSK9 adventure will lead, after the large and long-term ongoing phase III studies evaluating efficacy and safety, to a new anticholesterol pharmacological class.

Senescence Biomarkers CKAP4 and PTX3 Stratify Severe Kidney Disease Patients.

INTRODUCTION: Cellular senescence is the irreversible growth arrest subsequent to oncogenic mutations, DNA damage, or metabolic insult. Senescence is associated with ageing and chronic age associated diseases such as cardiovascular disease and diabetes. The involvement of cellular senescence in acute kidney injury (AKI) and chronic kidney disease (CKD) is not fully understood. However, recent studies suggest that such patients have a higher-than-normal level of cellular senescence and accelerated ageing. METHODS: This study aimed to discover key biomarkers of senescence in AKI and CKD patients compared to other chronic ageing diseases in controls using OLINK proteomics. RESULTS: We show that senescence proteins CKAP4 (p-value < 0.0001) and PTX3 (p-value < 0.0001) are upregulated in AKI and CKD patients compared with controls with chronic diseases, suggesting the proteins may play a role in overall kidney disease development. CONCLUSIONS: CKAP4 was found to be differentially expressed in both AKI and CKD when compared to UHCs; hence, this biomarker could be a prognostic senescence biomarker of both AKI and CKD.

Multiomics unveils extracellular vesicle-driven mechanisms of endothelial communication in human carotid atherosclerosis.

Background: Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV cargos were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions, and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-miRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV- vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.

Biliverdin Reductase B Is a Plasma Biomarker for Intraplaque Hemorrhage and a Predictor of Ischemic Stroke in Patients with Symptomatic Carotid Atherosclerosis.

BACKGROUND: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. OBJECTIVE: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. METHODS: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study (n = 177, 5 year follow-up) with and without IPH as indicated by MRI. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). RESULTS: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092-2.344]; p = 0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs. 1752 ± 366.84 MFI; p = 0.004). CONCLUSIONS: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability.

TACE/ADAM17 substrates associate with ACS (Ep-CAM, HB-EGF) and follow-up MACE (TNFR1 and TNFR2).

Background and aims: TACE/ADAM17 is a membrane bound metalloprotease, which cleaves substrates involved in immune and inflammatory responses and plays a role in coronary artery disease (CAD). We measured TACE and its substrates in CAD patients to identify potential biomarkers within this molecular pathway with potential for acute coronary syndrome (ACS) and major adverse cardiovascular events (MACE) prediction. Methods: Blood samples were obtained from consecutive patients (n = 229) with coronary angiographic evidence of CAD admitted with ACS or electively. MACE were recorded after a median 3-year follow-up. Controls (n = 115) had a <10% CAD risk as per the HeartSCORE. TACE and TIMP3 protein and mRNA levels were measured by ELISA and RT-qPCR respectively. TACE substrates were measured using a multiplex proximity extension assay. Results: TACE mRNA and cell protein levels (p < 0.01) and TACE substrates LDLR (p = 0.006), TRANCE (p = 0.045), LAG-3 (p < 0.001) and ACE2 (p < 0.001) plasma levels were significantly higher in CAD patients versus controls. TACE inhibitor TIMP3 mRNA levels were significantly lower in CAD patients and tended to be lower in the ACS population (p < 0.05). TACE substrates TNFR1 (OR:3.237,CI:1.514-6.923,p = 0.002), HB-EGF (OR:0.484,CI:0.288-0.813,p = 0.006) and Ep-CAM (OR:0.555,CI:0.327-0.829,p = 0.004) accurately classified ACS patients with HB-EGF and Ep-CAM levels being lower compared to electively admitted patients. TNFR1 (OR:2.317,CI:1.377-3.898,p = 0.002) and TNFR2 (OR:1.902,CI:1.072-3.373,p = 0.028) were significantly higher on admission in those patients who developed MACE within 3 years. Conclusions: We demonstrate a possible role of TACE substrates LAG-3, HB-EGF and Ep-CAM in atherosclerotic plaque development and stability. We also underline the importance of measuring TNFR1 and TNFR2 earlier than previously appreciated for MACE prediction. We report an important role of TIMP3 in regulating TACE levels.

Therapeutic potential of the Proprotein Convertase Subtilisin/Kexin family in vascular disease.

Proprotein convertase subtilisin/kexins (PCSKs) constitute a family of nine related proteases: PCSK1-7, MBTPS1, and PCSK9. Apart from PCSK9, little is known about PCSKs in cardiovascular disease. Here, we aimed to investigate the expression landscape and druggability potential of the entire PCSK family for CVD. We applied an integrative approach, combining genetic, transcriptomic and proteomic data from three vascular biobanks comprising carotid atherosclerosis, thoracic and abdominal aneurysms, with patient clinical parameters and immunohistochemistry of vascular biopsies. Apart from PCSK4, all PCSK family members lie in genetic regions containing variants associated with human cardiovascular traits. Transcriptomic analyses revealed that FURIN, PCSK5, MBTPS1 were downregulated, while PCSK6/7 were upregulated in plaques vs. control arteries. In abdominal aneurysms, FURIN, PCSK5, PCSK7, MBTPS1 were downregulated, while PCSK6 was enriched in diseased media. In thoracic aneurysms, only FURIN was significantly upregulated. Network analyses of the upstream and downstream pathways related to PCSKs were performed on the omics data from vascular biopsies, revealing mechanistic relationships between this protein family and disease. Cell type correlation analyses and immunohistochemistry showed that PCSK transcripts and protein levels parallel each other, except for PCSK9 where transcript was not detected, while protein was abundant in vascular biopsies. Correlations to clinical parameters revealed a positive association between FURIN plaque levels and serum LDL, while PCSK6 was negatively associated with Hb. PCSK5/6/7 were all positively associated with adverse cardiovascular events. Our results show that PCSK6 is abundant in plaques and abdominal aneurysms, while FURIN upregulation is characteristic for thoracic aneurysms. PCSK9 protein, but not the transcript, was present in vascular lesions, suggesting its accumulation from circulation. Integrating our results lead to the development of a novel 'molecular' 5D framework. Here, we conducted the first integrative study of the proprotein convertase family in this context. Our results using this translational pipeline, revealed primarily PCSK6, followed by PCSK5, PCSK7 and FURIN, as proprotein convertases with the highest novel therapeutic potential.

Targeting the NLRP3 Inflammasome in Glaucoma.

Glaucoma is a group of optic neuropathies characterised by the degeneration of retinal ganglion cells, resulting in damage to the optic nerve head (ONH) and loss of vision in one or both eyes. Increased intraocular pressure (IOP) is one of the major aetiological risk factors in glaucoma, and is currently the only modifiable risk factor. However, 30-40% of glaucoma patients do not present with elevated IOP and still proceed to lose vision. The pathophysiology of glaucoma is therefore not completely understood, and there is a need for the development of IOP-independent neuroprotective therapies to preserve vision. Neuroinflammation has been shown to play a key role in glaucoma and, specifically, the NLRP3 inflammasome, a key driver of inflammation, has recently been implicated. The NLRP3 inflammasome is expressed in the eye and its activation is reported in pre-clinical studies of glaucoma. Activation of the NLRP3 inflammasome results in IL-1ß processing. This pro inflammatory cytokine is elevated in the blood of glaucoma patients and is believed to drive neurotoxic inflammation, resulting in axon degeneration and the death of retinal ganglion cells (RGCs). This review discusses glaucoma as an inflammatory disease and evaluates targeting the NLRP3 inflammasome as a therapeutic strategy. A hypothetical mechanism for the action of the NLRP3 inflammasome in glaucoma is presented.

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD.