The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

c-kit+ cells minimally contribute cardiomyocytes to the heart.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display.

BACKGROUND: Extracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed "cloaking" to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration. RESULTS: We demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins. CONCLUSIONS: EV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology.

Cardiac and systemic rejuvenation after cardiosphere-derived cell therapy in senescent rats.

AIM: The aim is to assess the effects of CDCs on heart structure, function, gene expression, and systemic parameters in aged rats. Diastolic dysfunction is characteristic of aged hearts. Cardiosphere-derived cell (CDC) therapy has exhibited several favourable effects on heart structure and function in humans and in preclinical models; however, the effects of CDCs on aging have not been evaluated. METHODS AND RESULTS: We compared intra-cardiac injections of neonatal rat CDCs to vehicle (phosphate-buffered saline, PBS) in 21.8 ± 1.6 month-old rats (mean ± standard deviation; n = 23 total). Ten rats 4.1 ± 1.5 months of age comprised a young reference group. Blood, echocardiographic, haemodynamic and treadmill stress tests were performed at baseline in all animals, and 1 month after treatment in old animals. Histology and the transcriptome were assessed after terminal phenotyping. For in vitro studies, human heart progenitors from older donors, or cardiomyocytes from aged rats were exposed to human CDCs or exosomes secreted by CDCs (CDC-XO) from paediatric donors. Transcriptomic analysis revealed that CDCs, but not PBS, recapitulated a youthful pattern of gene expression in the hearts of old animals (85.5% of genes differentially expressed, P < 0.05). Telomeres in heart cells were longer in CDC-transplanted animals (P < 0.0001 vs. PBS). Cardiosphere-derived cells attenuated hypertrophy by echo (P < 0.01); histology confirmed decreases in cardiomyocyte area (P < 0.0001) and myocardial fibrosis (P < 0.05) vs. PBS. Cardiosphere-derived cell injection improved diastolic dysfunction [lower E/A (P < 0.01), E/E' (P = 0.05), end-diastolic pressure-volume relationship (P < 0.05) compared with baseline), and lowered serum brain natriuretic peptide (both P < 0.05 vs. PBS). In CDC-transplanted old rats, exercise capacity increased ∼20% (P < 0.05 vs. baseline), body weight decreased ∼30% less (P = 0.05 vs. PBS) and hair regrowth after shaving was more robust (P < 0.05 vs. PBS). Serum biomarkers of inflammation (IL-10, IL-1b, and IL-6) improved in the CDC group (P < 0.05 for each, all vs. PBS). Young CDCs secrete exosomes which increase telomerase activity, elongate telomere length, and reduce the number of senescent human heart cells in culture. CONCLUSION: Young CDCs rejuvenate old animals as gauged by cardiac gene expression, heart function, exercise capacity, and systemic biomarkers.

Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction.

Aims: Naturally secreted nanovesicles known as exosomes are required for the regenerative effects of cardiosphere-derived cells (CDCs), and exosomes mimic the benefits of CDCs in rodents. Nevertheless, exosomes have not been studied in a translationally realistic large-animal model. We sought to optimize delivery and assess the efficacy of CDC-secreted exosomes in pig models of acute (AMI) and convalescent myocardial infarction (CMI). Methods and Results: In AMI, pigs received human CDC exosomes (or vehicle) by intracoronary (IC) or open-chest intramyocardial (IM) delivery 30 min after reperfusion. No-reflow area and infarct size (IS) were assessed histologically at 48 h. Intracoronary exosomes were ineffective, but IM exosomes decreased IS from 80 ± 5% to 61 ± 12% (P= 0.001) and preserved left ventricular ejection fraction (LVEF). In a randomized placebo-controlled study of CMI, pigs 4 weeks post-myocardial infarction (MI) underwent percutaneous IM delivery of vehicle (n = 6) or CDC exosomes (n = 6). Magnetic resonance imaging (MRI) performed before and 1 month after treatment revealed that exosomes (but not vehicle) preserved LV volumes and LVEF (−0.1 ± 2.2% vs. −5.4 ± 3.6%, P= 0.01) while decreasing scar size. Histologically, exosomes decreased LV collagen content and cardiomyocyte hypertrophy while increasing vessel density. Conclusion: Cardiosphere-derived cell exosomes delivered IM decrease scarring, halt adverse remodelling and improve LVEF in porcine AMI and CMI. While conceptually attractive as cell-free therapeutic agents for myocardial infarction, exosomes have the disadvantage that IM delivery is necessary.

Cellular Sources and Regional Variations in the Expression of the Neuroinflammatory Marker Translocator Protein (TSPO) in the Normal Brain.

The inducible expression of the mitochondrial translocator protein 18 kDa (TSPO) by activated microglia is a prominent, regular feature of acute and chronic-progressive brain pathology. This expression is also the rationale for the continual development of new TSPO binding molecules for the diagnosis of "neuroinflammation" by molecular imaging. However, there is in the normal brain an ill-defined, low-level constitutive expression of TSPO. Taking advantage of healthy TSPO knockout mouse brain tissue to validate TSPO antibody specificity, this study uses immunohistochemistry to determine the regional distribution and cellular sources of TSPO in the normal mouse brain. Fluorescence microscopy revealed punctate TSPO immunostaining in vascular endothelial cells throughout the brain. In the olfactory nerve layers and glomeruli of the olfactory bulb, choroid plexus and ependymal layers, we confirm constitutive TSPO expression levels similar to peripheral organs, while some low TSPO expression is present in regions of known neurogenesis, as well as cerebellar Purkinje cells. The distributed-sparse expression of TSPO in endothelial mitochondria throughout the normal brain can be expected to give rise to a low baseline signal in TSPO molecular imaging studies. Finally, our study emphasises the need for valid and methodologically robust verification of the selectivity of TSPO ligands through the use of TSPO knockout tissues.

Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.

Small heat shock protein HSPB1 regulates growth of embryonic zebrafish craniofacial muscles.

The small heat shock protein HspB1 (Hsp27) is abundantly expressed in embryonic muscle tissues of a wide variety of vertebrate species. However, the functional significance of this expression pattern is not well established. In the present study, we observed specific, high level expression of HspB1 protein and an HspB1 gene reporter in developing craniofacial muscles of the zebrafish, Danio rerio, and examined the consequences of reducing HspB1 expression to the development and growth of these muscles. Quantitative morphometric analyses revealed a reduction in the cross-sectional area of myofibers in embryos expressing reduced HspB1 levels by as much as 47% compared to controls. In contrast, we detected no differences in the number of myofibrils or associated nuclei, nor the number, size or development of chondrocytes in surrounding tissues. We also did not detect changes to the overall organization of sarcomeres or myofibrils in embryos expressing reduced levels of HspB1. Together our results reveal a critical role for HspB1 in the growth of myofibrils and provide new insight into the mechanism underlying its developmental function.

Cytotoxicity of phosphoramidate, bis-amidate and cycloSal prodrug metabolites against tumour and normal cells.

Phosphonate and phosphate prodrugs are integral to enhancing drug permeability, but the potential toxicity of their metabolites requires careful consideration. This study evaluates the impact of widely used phosphoramidate, bis-amidate, and cycloSal phosph(on)ate prodrug metabolites on BxPC3 pancreatic cancer cells, GL261-Luc glioblastoma cells, and primary cultured mouse astrocytes. 1-Naphthol and 2-naphthol demonstrated the greatest toxicity. Notably, 2-naphthol exhibited an ED50 of 21 µM on BxPC3 cells, surpassing 1-naphthol with an ED50 of 82 µM. Real-time xCELLigence experiments revealed notable activity for both metabolites at a low concentration of 16 µM. On primary cultured mouse astrocyte cells, all prodrugs exhibited reduced viability at 128 to 256 µM after only 4 hours of exposure. A cell-type-dependent sensitivity to phosph(on)ate prodrug metabolites was evident, with normal cells showing greater susceptibility than corresponding tumour cells. The results suggest it is essential to consider the potential cytotoxicity of phosph(on)ate prodrugs in the drug design and evaluation process.

Neutron Capture Enhances Dose and Reduces Cancer Cell Viability in and out of Beam During Helium and Carbon Ion Therapy.

PURPOSE: Neutron capture enhanced particle therapy (NCEPT) is a proposed augmentation of charged particle therapy that exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with 2 different targeted neutron capture agents (NCAs). METHODS AND MATERIALS: Human glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a polymethyl methacrylate phantom either laterally adjacent to or within a 100 × 100 × 60 mm spread out Bragg peak (SOBP). The effect of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in 6 independent experiments. Neutron fluence within the phantom was characterized by quantifying the neutron activation of gold foil. RESULTS: Cells placed inside the treatment volume reached 10% survival by 2 Gy of carbon or 2 to 3 Gy of helium in the presence of NCAs compared with 5 Gy of carbon and 7 Gy of helium with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of carbon or helium (to the treatment volume), compared with no detectable effect on cells without NCA. The mean thermal neutron fluence at the center of the SOBP was approximately 2.2 × 109 n/cm2/Gy (relative biological effectiveness) for the carbon beam and 5.8 × 109 n/cm2/Gy (relative biological effectiveness) for the helium beam and gradually decreased in all directions. CONCLUSIONS: The addition of NCAs to cancer cells during carbon and helium beam irradiation has a measurable effect on cell survival and growth in vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumors, by targeting this dose to microinfiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumor tissue within the treatment volume, with a reduction in radiation doses to off-target tissue.

Green fluorescent protein alters the transcriptional regulation of human mitochondrial genes after gamma irradiation.

Green fluorescent proteins (GFP), extensively used as reporters in biological and imaging studies, are assumed to be mostly biologically inert. Here, we test the assumption in regard to the transcriptional regulation of 18 mitochondrially encoded genes in GFP expressing human T-cell line (JURKAT cells) exposed to gamma radiation. Using quantitative polymerase chain reaction, we demonstrate that wild type and GFP expressing JURKAT cells have different baseline mitochondrial transcript expression (10 out of the 18 tested genes) and after a single dose of radiation (100 Gy) show a significantly different transcriptional regulation of their mitochondrial genes. While in wild type cells, ten of the tested genes are up-regulated in response to radiation exposure, GFP expressing cells show less transcriptional regulation with a small down-regulation in five genes. Our results indicate that the presence of GFP in the cytoplasm can alter the cellular response to ionizing radiation.

Reprogramming of iron metabolism confers ferroptosis resistance in ECM-detached cells.

Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. Although alterations in membrane lipid content are observed during ECM detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM detachment because of changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of ferritin sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy toward ECM-detached cells.

Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth.

Newts have the extraordinary ability to fully regenerate lost or damaged cardiac, neural and retinal tissues, and even amputated limbs. In contrast, mammals lack these broad regenerative capabilities. While the molecular basis of newts' regenerative ability is the subject of active study, the underlying paracrine signaling factors involved remain largely uncharacterized. Extracellular vesicles (EVs) play an important role in cell-to-cell communication via EV cargo-mediated regulation of gene expression patterns within the recipient cells. Here, we report that newt myogenic precursor (A1) cells secrete EVs (A1EVs) that contain messenger RNAs associated with early embryonic development, neuronal differentiation, and cell survival. Exposure of rat primary superior cervical ganglion (SCG) neurons to A1EVs increased neurite outgrowth, facilitated by increases in mitochondrial respiration. Canonical pathway analysis pinpointed activation of NGF/ERK5 signaling in SCG neurons exposed to A1EV, which was validated experimentally. Thus, newt EVs drive neurite growth and complexity in mammalian primary neurons.

Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence.

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

Intravascularly infused extracellular matrix as a biomaterial for targeting and treating inflamed tissues.

Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO).

Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment.

Control of Neuroinflammation through Radiation-Induced Microglial Changes.

Microglia, the innate immune cells of the central nervous system, play a pivotal role in the modulation of neuroinflammation. Neuroinflammation has been implicated in many diseases of the CNS, including Alzheimer's disease and Parkinson's disease. It is well documented that microglial activation, initiated by a variety of stressors, can trigger a potentially destructive neuroinflammatory response via the release of pro-inflammatory molecules, and reactive oxygen and nitrogen species. However, the potential anti-inflammatory and neuroprotective effects that microglia are also thought to exhibit have been under-investigated. The application of ionising radiation at different doses and dose schedules may reveal novel methods for the control of microglial response to stressors, potentially highlighting avenues for treatment of neuroinflammation associated CNS disorders, such as Alzheimer's disease and Parkinson's disease. There remains a need to characterise the response of microglia to radiation, particularly low dose ionising radiation.

Mitochondrial Translocator Protein (TSPO) Expression in the Brain After Whole Body Gamma Irradiation.

The brain's early response to low dose ionizing radiation, as may be encountered during diagnostic procedures and space exploration, is not yet fully characterized. In the brain parenchyma, the mitochondrial translocator protein (TSPO) is constitutively expressed at low levels by endothelial cells, and can therefore be used to assess the integrity of the brain's vasculature. At the same time, the inducible expression of TSPO in activated microglia, the brain's intrinsic immune cells, is a regularly observed early indicator of subtle or incipient brain pathology. Here, we explored the use of TSPO as a biomarker of brain tissue injury following whole body irradiation. Post-radiation responses were measured in C57BL/6 wild type (Tspo +/+) and TSPO knockout (Tspo -/-) mice 48 h after single whole body gamma irradiations with low doses 0, 0.01, and 0.1 Gy and a high dose of 2 Gy. Additionally, post-radiation responses of primary microglial cell cultures were measured at 1, 4, 24, and 48 h at an irradiation dose range of 0 Gy-2 Gy. TSPO mRNA and protein expression in the brain showed a decreased trend after 0.01 Gy relative to sham-irradiated controls, but remained unchanged after higher doses. Immunohistochemistry confirmed subtle decreases in TSPO expression after 0.01 Gy in vascular endothelial cells of the hippocampal region and in ependymal cells, with no detectable changes following higher doses. Cytokine concentrations in plasma after whole body irradiation showed differential changes in IL-6 and IL-10 with some variations between Tspo-/- and Tspo +/+ animals. The in vitro measurements of TSPO in primary microglial cell cultures showed a significant reduction 1 h after low dose irradiation (0.01 Gy). In summary, acute low and high doses of gamma irradiation up to 2 Gy reduced TSPO expression in the brain's vascular compartment without de novo induction of TSPO expression in parenchymal microglia, while TSPO expression in directly irradiated, isolated, and thus highly activated microglia, too, was reduced after low dose irradiation. The potential link between TSPO, its role in mitochondrial energy metabolism and the selective radiation sensitivity, notably of cells with constitutive TSPO expression such as vascular endothelial cells, merits further exploration.

Microgravity × Radiation: A Space Mechanobiology Approach Toward Cardiovascular Function and Disease.

In recent years, there has been an increasing interest in space exploration, supported by the accelerated technological advancements in the field. This has led to a new potential environment that humans could be exposed to in the very near future, and therefore an increasing request to evaluate the impact this may have on our body, including health risks associated with this endeavor. A critical component in regulating the human pathophysiology is represented by the cardiovascular system, which may be heavily affected in these extreme environments of microgravity and radiation. This mini review aims to identify the impact of microgravity and radiation on the cardiovascular system. Being able to understand the effect that comes with deep space explorations, including that of microgravity and space radiation, may also allow us to get a deeper understanding of the heart and ultimately our own basic physiological processes. This information may unlock new factors to consider with space exploration whilst simultaneously increasing our knowledge of the cardiovascular system and potentially associated diseases.