MOMMENTUM-CVD

TEAM
Roger Foo, Chester Drum, Jiang Jianming, Roshni Singaraja, Mark Chan, Koh Cho Yeow, Matthew Ackers-Johnson, Wong Lee Lee, Vitaly A. Sorokin, Wang JiongWei, Arthur Mark Richards, Wang Peipei

SCOPE
Molecular Discovery will expand our strength in epigenetics and biomarkers encompassing-omics technologies, including non-coding RNA studies, genomics, peptide immunoassay and proteomic screening platforms to discover and validate new molecular targets with marker and therapeutic potential.

Platforms supported by Core 1 include:

Genomic / epigenetic discovery
Analysis of DNA, chromatin and RNA from multiple sources in both clinical and experimental pre-clinical settings. DNA sequencing via a custom-built NGS inherited cardiac gene panel (applied to SG10K and ATTRACT genomes). For RNA and epigenomes we deploy RNAseq, single-cell transcriptomics, assessment of histone modifications, chromatin interactomes, HiC, and perform transcription factor ChIPseq. Data are stored in computing clusters at GIS and the National Super Computer Centre. Capabilities through this Centre contribute strategically to developing bioinformatics expertise among clinician-scientists on the NUHS campus within and beyond CVRI.

Circulating biomarker discovery and assay development
Technologies include, immunoassay, PCR and mass spectrometry. Since 2009, CVRI has bio-banked samples from annotated clinical cohorts (>20,000 participants recruited nationwide) spanning coronary disease, heart failure, atrial fibrillation, and valve disease – totalling >200,000 individual aliquots of DNA, PBMCs, EDTA, heparin and citrate anticoagulated plasma. The department of cardiothoracic and vascular surgery tissue repository has 16,820 samples of aorta, peripheral artery, veins, myocardium and cardiac valves. Methods include standard ELISA, multiplex bead-based assay, FRET-based assay and proximity-extension assay platforms. Equipment includes automated immunoanalysers (Abbott Architect i2000SR and Roche Cobas e411 analysers), Bio-PlexÒ Multi-Suspension Array System, ELLA (“Next Generation” ELISAs on single or multiplex microfluidic cartridges) and the EnspireÒ Multi-Mode Plate Reader with access to the BioMark™ System (Fluidigm). The Richards’ group develops in-house immunoassays via targeted cloning of epitope-directed monoclonal antibodies characterized (isotyping, surface plasmon resonance-based affinity, epitope mapping and mass spectroscopy) and validated for ELISA, Western blotting and immunohistochemistry.

The Mass Spectrometry Laboratory
Runs 3 highly sensitive Agilent 6495 triple-quadrupole (QqQ) MS machines and the Agilent Ultivo QqQ MS. MS-targeted metabolomics profiling methods detect over 400 endogenous small molecule metabolites including (i) a comprehensive panel of oxidative stress markers, (ii) components of multiple metabolic pathways (iii) NAD metabolome/electron transport chain, (iv) amino acids (v) drugs and (vi) deoxyguanosine (DNA damage) and 8-Hydroxyguanine (RNA damage).

Team

Mark Chan, Ronald Lee Chi Hang, Lim Shir Lynn, Vitaly A. Sorokin, Ling Lieng Hsi, Chester Drum, Arthur Mark Richards, Andrew Mark Choong Tze Liang

Scope

Clinical Science will underpin and expand our clinical links facilitating the acquisition of biological samples and accelerating our participation in early (Phase 2) clinical trials.

Clinical Trails

Clinical Trials infrastructure (Clinical Trial Coordinators, space, Ethics, documentation) is overseen by Dr Lim Shir Lynn with input from clinical investigators. The Cardiovascular Clinical Trials Unit employs 12 full-time CRCs supporting investigator-initiated and industry-supported studies. The unit has a physical space and an electronic platform for secure data storage.

Cardiovascular Imaging Core Laboratory

The Cardiovascular Imaging Core Laboratory (CICL; Assoc Prof Ling Lieng Hsi, Dr Lynette Teo) at NUHCS is Singapore’s core echocardiography and vascular imaging laboratory providing state-of-the-art cardiac ultrasound and vascular phenotyping (atherosclerosis, regional, arterial stiffness, vascular reactivity, skin autofluoresence for advanced glycation end-products) in 3 dedicated imaging suites. Data are archived on a GE-based server system (pending an upgrade to the ViewPoint platform). CICL serves as the core ultrasound laboratory for both NUHCS CG and the Collaborative CG held with our partner heart centre, NHCS.

Complementing CICL, our access to 4 ultrafast CT and 6 MRI machines, MR-PET and MR-SPECT in NUH/NUS facilities, positions us well for high throughput in imaging cohort studies and clinical trials.

Computational fluid dynamics

Vascular Computational fluid dynamics overseen by Dr Andrew Choong.

Cardiosleep Laboratory

PI: Prof Ronald Lee
The cardiosleep lab runs trials including (1) CRESCENT – multicentre, randomized comparison of CPAP vs mandibular advancement device (MAD) (2) POACH – determining the CV effects of OSA in atrial fibrillation (3) SAGIC-CVD – multi-national propensity scored trial to evaluate the impact of CPAP therapy on BP in the excessive sleepiness phenotype, (4) MANTA -a collaboration with University of Sydney trialling a novel MAD in atrial fibrillation (5) Belun Ring study- a collaboration with a commercial start-up to validate a novel sleep monitoring device (6) Drug-induced sleep endoscopy (DISE) – a collaboration with otolaryngologists to characterize anatomical patterns of upper airway obstruction using drug-induced sleep endoscopy.

Team

Theodoros Kofidis, Roshni Rebecca Singaraja, Roger Foo, Wong Lee Lee, Mark Chan, Koh Cho Yeow, Matthew Ackers-Johnson, Wang Jiongwei, Arthur Mark Richards, Wang Pei Pei

Scope 

Preclinical Modelling will support the array of in vitro, ex vivo and in vivo cell and animal models underpinning our basic research efforts including the following platform.

Capabilities

The in vitro modelling platform for (1) primary vascular tissues (Singaraja), (2) iPS-derived human vascular tissues (Ackers-Johnson) and (3) 3D-organoid models of vessels (Sorokin) supports to validate selected targets. Dissociated vascular tissue will be used to acquire primary patient-specific VSMCs and Endothelial cells to create organoids. PBMC-derived M0 macrophages will be added into 3D organoids to elaborate vascular tissue-immune cell interactions relevant to atherogenesis. PBMC-derived iPS cells are generated for comparison of blood-acquired vs tissue-originated organoids. iPSC-derived organoids will provide patient-derived 3D organoids for downstream target validation.

• In vivo mouse models of atherosclerosis include high-fat diet-fed Ldlr-/- and Apoe-/- and AAV-Pcsk9 gain-of-function models. We also have a rabbit atherosclerosis model to better mimic human atherosclerosis (Koh). Target-specific knockout or transgenic mouse models can be generated as required. 

• PAD model platform – models of atherosclerosis (Apoe-/- mice subjected to carotid-wire injury) and aortic atherosclerosis (Ldlr-/- mice fed western-type diet) for testing novel PAD targets will be established. Diabetic mice (C57BL6) are generated by high-fat feeding. These mouse models will be compounded by (1) stable femoral artery plaques using wire injury or partial ligation of the femoral artery; (2) unstable femoral artery plaques, using the Tandem Stenosis approach and (3) hind-limb femoral artery ischaemia models. These are good models of acute lower limb ischaemia. To model chronic onset PAD in humans, we will utilize the Ameroid constrictor to occlude the femoral artery over weeks (Singaraja).

• For cardiac studies we will maintain our range of small animal models (conducted under oversight by Wang P, Wang J-W, Jiang, Foo, Koh and Ackers-Johnson) of myocardial infarction, ischaemia-reperfusion, thoracic aortic constriction, renal dysfunction/cardiac fibrosis (5/6ths nephrectomy).

• Large animal models (porcine) overseen by Richards and Kofidis – of myocardial ischaemia, haemodynamic overload, heart failure with preserved or reduced ejection fraction and novel cardiac implants in pigs.

• Pre-clinical imaging including cardiac ultrasound (operated by several CVRI investigators) and cardiac magnetic resonance as supported by Comparative Medicine.

• MicroRNA laboratory – (Wang P) Mechanistic investigation of miRs by mimics and antagomirs for influence upon myocardial response (inflammation, fibrosis, function) to injury.

• Human ex vivo We will also use an ex vivo model of thrombosis for testing of efficacy of antithrombotic therapeutics (THETA study). Human participants’ antecubital veins will be directly connected to extracorporeal perfusion chambers containing strips of endothelium-denuded pig aorta, mimicking vascular damage. Molecules under investigation will be infused with assessment of impact upon thrombus formation and inflammatory activation. 
• Target delivery platform consisting of (1) vascular and/or heart-specific nanoparticle delivery systems (Wang JW), and (2) vascular and/or heart-specific viral delivery systems (Jiang J). Sharpening our current AAV tools to be specific for vascular and/or heart tissues will be useful to assess target therapeutic potential.
• Single cell sequencing (Ackers-Johnson) is established for cardiac cell types in health and disease allowing prediction of cell-cell interaction pathways. Primary culture and human iPSC-derived multicellular co-culture models enable interrogation of molecular mechanisms of cardiac disease, including identified candidates and SG rare genetic variants.

Team

Choi Hyungwon and Chan Siew Pang

Scope

Cardioinformatics, which will provide both analytical services and development of original analytic methods, has universal applicability to the data management and analytic requirements of all our research efforts.

• Biostatistics training for research staff in CVRI- from basic data management to advanced statistical analysis methods for clinical trials (Chan SP).
• Advanced Statistical Methods for omics-scale data analysis (Choi H-W).
• Single cell-based bulk-seq analysis. Novel statistical approaches for incorporating single-cell RNA-sequencing data into the differential gene expression analysis of bulk tissue data.
• Advanced omics-based biomarker discovery. Prototype modelling methods to infer a latent variable model for circulating marker data and organ-specific gene expression data in collaboration with multiple groups in NUSmed, including CVRI. The “site-of-origin” latent variables for organ systems / tissues are inferred from circulating marker data.
• Develop bioinformatics tools for proteomics, metabolomics and lipidomics.