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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.

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G > A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1–45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems., Highlights • iPS-CM were generated from a Brugada Syndrome patient who carries an SNV in SCN5A. • Patient-specific iPS-CM show a loss of function of the sodium current. • Use of iPS-CM uncovers changes in INa properties not apparent in tsA201 cells.

Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of “hepatocytes”; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.

Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. Aims: In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. Methods: hESC-derived HLCs were injected into the spleen of acutely injured NODscidIL-2Rγnull mice and analysed at various time points post-transplantation up to 3 months. Results: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. Conclusions: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases.

The spatial distribution of the target (t-) SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two-and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.

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.

Objective: Following chronic liver injury or when hepatocyte proliferation is impaired, ductular reactions (DR) containing hepatic progenitor cells (HPC) appear in the periportal regions and can regenerate the liver parenchyma. HPCs exist in a niche composed of myofibroblasts, macrophages and laminin matrix. Galectin-3 (Gal-3) is a β-galactoside-binding lectin that binds to laminin and is expressed in injured liver in mice and humans.Design: We examined the role of Gal-3 in HPC activation. HPC activation was studied following dietary induced hepatocellular (choline-deficient ethionine-supplemented diet) and billiary (3,5-diethoxycarbonyl-1,4-dihydrocollidine supplemented diet) injury in wild type and Gal-3(-/-) mice.Results: HPC proliferation was significantly reduced in Gal-3(-/-) mice. Gal-3(-/-) mice failed to form a HPC niche, with reduced laminin formation. HPCs isolated from wild type mice secrete Gal-3 which enhanced adhesion and proliferation of HPCs on laminin in an undifferentiated form. These effects were attenuated in Gal3(-/-) HPCs and in wild type HPCs treated with the Gal-3 inhibitor lactose. Gal-3(-/-) HPCs in vitro showed increased hepatocyte function and prematurely upregulated both biliary and hepatocyte differentiation markers and regulated cell cycle genes leading to arrest in G0/G1.Conclusions: We conclude that Gal-3 is required for the undifferentiated expansion of HPCs in their niche in injured liver.

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.

The human liver is a vital organ within the body and plays a major role in normal homeostasis. The “work horse” of the liver, termed the “hepatocyte,” is estimated to make up approximately 70–80% of the liver’s mass. Therefore, the study of hepatocyte biology has an important role to play in medicine and the drug discovery process. At present the routine use of human primary hepatocytes is limited due to poor supply and their loss of function upon isolation. Therefore, additional and renewable sources of hepatocytes are being sought. Rodent hepatocytes have been utilised for many years, and although informative, they possess significant limitations and do not accurately extrapolate to human liver. To overcome the issue of cell viability, several groups have tried to generate immortalised hepatocytes; however, the derivative cells exhibit dramatic decreases in function and karyotypic instability over prolonged culture. It has therefore been necessary to find an alternative source of hepatocytes and efficient methods for deriving hepatic endoderm from stem cells in vitro. We have employed human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to derive human hepatic endoderm (HE). hESCs and iPSCs represent scalable and highly efficient resources with which to generate human HE in vitro, and hESC-derived HE will be the focus of this chapter.

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.

Accurate prediction of human drug toxicity is a vital part of the drug discovery process. However, the safety evaluation process is hindered by the availability and quality of primary human liver models with which to study drug toxicity. In an attempt to overcome this limitation, research has focused on deriving human hepatocytes from a number of sources, including progenitors from fetal and adult liver, human cell lines derived from liver tumours, immortalized human hepatocytes and pluripotent stem cells. The major hurdles in developing scalable and high-fidelity human hepatocytes from hepatic cell lines and fetal and adult progenitors have been limited organ availability, homogeneous cell purification, short-term cell culture, and the rapid loss of hepatocyte phenotype and function in culture. Therefore it has been necessary to find alternative sources of human hepatocytes which circumvent these issues. The research in our group has focused on generating human hepatic endoderm from the scalable pluripotent stem cell populations, human embryonic stem cells and induced pluripotent stem cells. We have developed efficient and scalable models of human hepatocyte differentiation from these cell populations. Moreover, stem-cell-derived hepatic endoderm displays many of the functional attributes of primary human hepatocytes. Our research is now focused on developing defined culture systems and improving cell culture microenvironments in order to improve our understanding of the mechanisms regulating human liver development. This will in turn facilitate the generation of broad-range functioning hepatic endoderm in vitro. By taking these approaches, we believe that it will be possible to improve the predictive nature of our in vitro models, revolutionizing the manner in which industry measures human drug toxicity and having an impact on drug attrition.

The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

The emergence of regenerative medicine has led to significant advances in the identification and understanding of human stem cells and adult progenitor cells. Both cell populations exhibit plasticity and theoretically offer a potential source of somatic cells in large numbers. Such a resource has an important role to play in the understanding of human development, in modeling human disease and drug toxicity, and in the generation of somatic cells in large numbers for cell-based therapies. Presently, liver transplantation is the only effective treatment for end-stage liver disease. Although this procedure can be carried out with high levels of success, the routine transplant of livers is severely limited by organ donor availability. As a result, attention has focused on the ability to restore liver mass and function by alternative approaches ranging from the bioartificial device to transplantation of human hepatocytes. In this review we will focus on the generation of human hepatic endoderm from different stem/progenitor cell populations with a view to its utility in regenerative medicine.

Eukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE complex in neurons and neuroendocrine cells, is regulated directly by munc18-1, its cognate Sec1p/munc18 (SM) protein. SM proteins show remarkable structural conservation throughout evolution, indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and its mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore, these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1.

Recent developments in cellular imaging now permit the minimally invasive study of protein interactions in living cells. These advances are of enormous interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Up until recently, all protein interactions had to be determined in vitro using biochemical approaches. This biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with spatial resolution. More recent developments in TCSPC imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. Here, we discuss some findings we have made along the way which may be useful for physiologists to consider. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

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.

Summary The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4α. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4α in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types.

Human stem cells are scalable cell populations capable of cellular differentiation. This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells. Such an approach has the potential to improve our understanding of human biology and treating disease. In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases. In recent years, efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own. In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology. (C) 2012 Baishideng. All rights reserved.