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Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

Faithfully recapitulating human physiology “in a dish” from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.

Aim Late outgrowth endothelial cells (EOC) are strong contenders to be the true circulating endothelial progenitor cells since they are capable of clonogenic expansion, exhibit a mature endothelial phenotype, and contribute to angiogenesis in vivo . These cells may play a crucial role in the process of vascular repair, but whether they are able to accumulate at sites of vascular damage in vivo is not clear. We hypothesise that EOC, delivered locally or systemically, accumulate at, and incorporate into, a site of arterial injury. Methods Experimental groups comprised systemic administration of the glucose analogue radiotracer 18 F-Flurodeoxyglucose (FDG) (Sys-Free) or FDG-labelled EOC (Sys-EOC), or local administration of free FDG (Local-Free) or FDG-labelled EOC (Local-EOC). EOC were isolated from peripheral blood from patients with coronary heart disease (n=3). Left femoral artery injury was achieved in male Sprague Dawley rats (300–350g) under general anaesthesia by inserting micro-renathane tubing via the popliteal artery. EOC were labelled with FDG (25 MBq/ml, 30 min, 37°C) and 1 million cells were administered either locally into the femoral artery or systemically via the tail vein (0.3–1.3 MBq, n=3 per group). Following injection of radiolabelled cells or free FDG, rats underwent dynamic PET scanning over 4 hours (Mediso nanoPET/CT scanner, Hungary). AuroVist and Fenestra (MediLumine Inc, Canada) were used as computed tomography vascular contrast agents. Images were analysed with PMOD software (PMOD, Switzerland) and standardised uptake values were calculated. Results FDG radioactivity was successfully visualised by micro-PET/CT. The activity was distributed in the bladder, kidneys, heart, brain, lungs, spleen and liver (descending order). Radioactivity in the lungs was significantly higher (80 and 120 min) following systemic EOC administration compared with the other three groups (two-way ANOVA with Bonferroni post-test, p Conclusion Preliminary analysis shows that EOC are able to target sites of vascular injury following systemic and local administration. These observations suggest that late outgrowth endothelial cells have the potential to contribute to vascular repair and regeneration.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such, hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However, for their full potential to be realized, both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however, they suffer from some major limitations including lack of definition, xenobiotic nature, batch-to-batch variation, and labor-intensive production. Therefore, hESC culture definition is essential if hESC lines, and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state, for over 30 passages using MT and SP. Additionally, we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture, contributing to the standardization of hESC in vitro models and ultimately their application.

s / Atherosclerosis 241 (2015) e32ee71 e44 Methods: Type 1 diabetes was induced to apolipoprotein E deficient mice (male, 8-week old) by streptozotocin injection. Diabetic animals were treated with either the HSP90 inhibitor 17-(dimethylaminoethylamino)17-demetoxygeldanamycin (DMAG, 2-4 mg/kg body weight/day, i.p., every second day, 10 weeks, n1⁄46 per group) or vehicle (n1⁄49). We analysed aortic and renal lesions (Oil-Red-O/hematoxylin and periodic acid-Schiff staining), cellularity (immunohistochemistry of leukocytes and a-smooth muscle actin), fibrotic content (picrosirius red staining), gene expression (real-time quantitative PCR) and NF-kB activity (Southwestern in situ). Results: DMAG treatment reduced the size and the extension of atherosclerotic lesions, without affecting hyperglycemia and lipid profile. It also induced a more stable phenotype of plaque by reducing the amount of lipid, infiltrating leukocytes (macrophages and T-cells) and inflammatory markers (CCL2, CCL5, TNFa and NF-kB), while increasing smooth muscle cell and collagen content. DMAG treatment also improved renal function and reduced renal lesions associated to diabetes, such as mesangial expansion, leukocyte infiltration, inflammation and fibrosis (TGFb, collagen and fibronectin). Conclusions: HSP90 inhibition by DMAG restrains the progression of vascular and renal and damage in experimental diabetes. EAS-0519. IDENTIFICATION OF THE ORIGIN OF CIRCULATING ENDOTHELIAL PROGENITOR CELLS T. Fujisawa , M. Brittan , O. Tura-Ceide , A. Hunter , A. Mitchell , A. Vesey , E.M. Skinner , C. Medine , S. Gallogly , P.W.F. Hadoke , C. Keith , A. Sproul , H. Roddie , G. McQuaker , I. Wilmut , N.L. Mills . BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom; Department of Pulmonary Medicine, University of Barcelona, Barcelona, Spain; Cytogenetics, Western General Hospital, Edinburgh, United Kingdom; Department of Haematology, Western General Hospital, Edinburgh, United Kingdom; Bone Marrow Transplant Unit, Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 6 Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom Aim: The discovery of circulating endothelial progenitor cells (EPCs) has stimulated clinical interest in the potential for these cells to treat patients with cardiovascular diseases. The precise origin of the circulating EPCs is unknown and has hampered the development of effective therapies. We hypothesize that circulating EPCs do not originate in the bone marrow, but reside in a niche within the vascular endothelial lining. Methods: Peripheral blood was used to isolate late endothelial outgrowth cells (EOCs) and early outgrowth cells (CFU-Hill) from patients with allogeneic sex-mismatched bone marrow transplants. Vascular endothelial cells were grown from vascular biopsy with J-shape wire from vein. The origin of cells were analysed by FISH against X and Y chromosome as well as multiplex short tandem repeats (STRs) analysis. Flow cytomerty was used for phenotypic analysis. Results: EOCs and endothelial cells obtained from vascular biopsies were of recipient genotype by FISH as well as STRs analysis. These cells had typical endothelial morphology and cell surface marker expression by flow cytometry analysis, while CFU-Hill were haematopoietic cells, did not express endothelial markers and had a donor genotype. Conclusion: Preliminary data suggests true circulating endothelial progenitor cells, capable of forming mature endothelial cells, are derived from the vasculature. These observations challenge the concept of a bone marrow derived endothelial progenitor cell. EAS-0539. ANGIOTENSIN-(1-7) AND ANGIOTENSIN-(1-9) INHIBIT VASCULAR SMOOTH MUSCLE CELL GROWTH AND MIGRATION IN VITRO AND VASCULAR REMODELLING IN VIVO C.A. McKinney , S. Kennedy , G.S. Baillie , G. Milligan , S.A. Nicklin . 1 Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom; 2 Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, United Kingdom Background: Vascular smooth muscle cell(VSMC) proliferation and migration underlies the pathogenesis of atherosclerosis, vein graft failure and in-stent restenosis. Angiotensin II(AngII), acting via the AT1R, is integral in these processes. AngII is inhibited by the counter-regulatory axis of the renin angiotensin system, which is centered around the actions of angiotensin-converting-enzyme-2 and the production of Ang(1-7) and Ang-(1-9), which act via the Mas receptor and AT2R, respectively. Here we investigated the role of Ang-(1-9) and Ang-(1-7) in primary human VSMC migration and proliferation, and in a mouse model of vascular injury. Methods: Migration was assessed via scratch assay and proliferation using the MTS-assay. Vascular injury was induced in vivo via wire injury to the left carotid artery. Ang-(1-7) and Ang-(1-9) were delivered via osmotic minipump and vascular remodeling quantified at 28 days postinjury. Results: VSMCmigration and proliferationwas inhibited by Ang-(1-9) and Ang-(1-7); these effects were selectively blocked by the pharmacological antagonists PD123,319 and A779, respectively, suggesting Ang-(1-9) acts via the AT2R and Ang-(1-7) via Mas. In vivowire injury of the mouse carotid artery induced significant neointimal formation(NI) at 28 days post-injury; this was attenuated by Ang-(1-7) and Ang-(1-9) via Mas and the AT2R, respectively. Conclusion: These data demonstrate that Ang-(1-7) and Ang-(1-9) inhibit VSMC proliferation and migration in vitro and neointimal formation in vivo via the AT2R and Mas receptor, respectively. Here we demonstrate for the first time a direct biological effect of Ang-(1-9) within the vasculature. These findings highlight the potential of Ang-(1-9) and Ang-(1-7) as therapeutic agents in vascular injury. EAS-0553. ROLE OF PERIODONTAL PATHOGENS IN INDUCTION OF ATHEROSCLEROSIS IN LDLR-/MICE M. Easwaran , S. Chukkapalli , M. Rivera-Kweh , I. Velsko , D. Zheng , A. Lucas , L. Kesavalu . 1 Periodontology, University of Florida, Gainesville, USA; Medicine, University of Florida, Gainesville, USA Aim: Periodontal disease (PD) is a polymicrobial chronic inflammatory disease. A distinct pathogenic consortium is found in the subgingival plaque includes Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum. Atherosclerosis has been linked to PD. We have examined whether oral periodontal pathogens can infect vascular tissues and induce atherosclerotic plaque formation in LDLR-/mice. Methods: LDLR-/mice (Group I) were orally infected with early colonizer F.nucleatum for the first 12 weeks followed by late colonizers P.gingivalis/ T.denticola/T.forsythia for the remaining 12 weeks. Group II mice were sham-infected. After 8 infection periods (4 days a week every 3rd week for 24 weeks), the mice were euthanized. Periodontal disease parameters like bone loss, antibody levels and oral plaque samples were analyzed. Heart, aorta, spleen, liver, lungs and kidney were evaluated for systemic infection and aorta was examined for atherosclerosis. Results: P.gingivalis, T.denticola, T.forsythia and F.nucleatum genomic DNA were detected in oral plaque samples demonstrating infection of the mice oral cavities. Infected mice serum contain significant levels (p

In the present study, it is shown that mice heterozygous for wt1 develop glomerular sclerosis and the nature and time course of events leading to the glomerular scarring are determined. Wt1-heterozygous (wt1het) mice and their wild-type littermates were closely monitored from birth and plasma levels of urea, creatinine, and albumin were compared with histological data and clinical features. One of the first indications of nephropathy in the wt1het mouse was the development of proteinuria, accompanied by progressive elevation of the plasma levels of urea and creatinine. Subsequently, the mice developed albuminuria, which correlated with thickening of the glomerular basement membrane and fusion of the podocyte foot processes. Glomerulosclerosis was a relatively late event, accompanied by severe albuminuria and loss of WT1, nephrin, CD2AP, and alpha-actinin-4.

Introduction It was previously believed that the heart was a terminally differentiated organ, with limited or no regenerative capacity. However, over the last decade compelling evidence suggests that the heart does undergo regeneration with a limited population of resident progenitor cells able to replenish cardiomyocytes lost due to injury throughout adult life. The therapeutic manipulation of resident cardiac progenitor cells could transform outcomes in patients with heart failure if these cells can be harnessed for myocardial regeneration. In recent years, the search for cardiac progenitor candidates has gathered momentum, and several populations of resident cardiac progenitor cells have now been isolated in rat, mouse, dog, porcine and human hearts using a variety of stem cell markers. With a variety of cardiac progenitor populations now described it is important that we fully understand the properties and differentiation potential of these progenitor populations. Methods We describe the isolation of c-kit+ potential cardiac progenitor cells from atrial appendages of patients undergoing coronary artery bypass or valve replacement surgery (n=6) and from human fetal hearts (n=6). C-kit+ magnetic activated cell sorting (MACS) was used to isolate c-kit+ cells that were expanded in culture. Immunostaining and fluorescent activated cell sorting (FACS) were used to assess the expression of a variety of markers. Cardiomyocyte and endothelial differentiation techniques were used to assess the differentiation potential the cardiac progenitors. Single-cell gene expression techniques also allowed us to detect variations among cells in seemingly homogenous populations. Results These populations were CD45-, CD34-, but positive for CD146, CD105, KDR and the early cardiac transcription factors Mef2c, GATA4, Nkx2.5 and Isl1. The c-kit+ populations were able to differentiate in vitro to cardiomyocytes and endothelial cells. To improve our understanding of the dynamic networks of transcriptional regulation in cardiac progenitor cells both fetal and adult c-kit+ cardiac progenitors were used as a tool for gene profiling to identify key genetic differences between the populations. This allowed us to detectoverlapping and non-overlapping expression patterns and thus provide novel insights into the complex networks of transcriptional regulation in cardiac progenitor cells. Conclusion Understanding the transcriptional regulation of cardiac progenitor cells will identify novel approaches for cellular reprogramming and permit us to develop robust methods for isolating and expanding these cells for clinical application in the treatment of patients with heart failure.

Objective The ability to derive cardiomyocytes from human induced pluripotent stem (iPS) cells provides a novel approach to study inherited cardiac channelopathies. Fibroblasts from patients can be reprogrammed to iPS cells and differentiated into cardiomyocytes that carry the patient9s exact genetic background. Although this technique has enormous potential for modeling inherited channelopathies the derived cardiomyocytes have not been fully characterised and compared to adult and foetal cells. Knowledge about their maturity will be critical if assessments of cellular function are to provide insight into disease processes that manifest in the adult. Methods Healthy control human iPS cells were differentiated into cardiomyocytes using an unguided differentiation protocol. Embryoid bodies from undifferentiated iPS cells were cultured in suspension for 4 days before being plated onto 0.1% gelatin. Spontaneously contracting areas were disaggregated and single cardiomyocytes were characterised using immunocytochemistry, TEM, electrophysiology and Ca 2+ imaging. Cells were stained for Troponin T, Troponin I and α-actinin. Ca 2+ release into the cytoplasm was detected by fluo-4 and fluorescence intensity was traced before and after Tetrodotoxin (TTX) treatment. Sodium channel activity was measured in a low sodium buffer using perforated patch-clamping. Ultrastructure of cells was analysed with transmission electron microscopy while the presence of t-tubules was investigated using the fluorescent dye Di-8-ANEPPS. Cardiac cells derived from iPS cells were then compared to primary cardiomyocytes isolated from human foetal and adult cardiac tissue. Results iPS derived cardiomyocytes express a wide range of cardiac markers as well as Tetrodotoxin resistant voltage activated sodium channels that inactivate and recover from inactivation and exhibit action potential triggered Ca 2+ -induced-Ca 2+ -release. Spontaneous contraction and lack of t-tubules were observed in derived cardiomyocytes and immature human foetal cardiomyocytes. Conclusion iPS cells give rise to mostly immature cardiomyocytes, that express typical cardiac proteins and have functional cardiac sodium channels. This differentiation system may be utilised to investigate patients with and without known genetic mutations to provide a better understanding of the pathophysiology of inherited cardiac channelopathies.