In JoVE (2)

Other Publications (56)

Articles by Arne Hansen in JoVE

 JoVE Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine

JoVE 2605

Other articles by Arne Hansen on PubMed

Immunglobulin Repertoire Analysis Provides New Insights into the Immunopathogenesis of Sjögren's Syndrome

Autoimmunity Reviews. May, 2002  |  Pubmed ID: 12849004

This review focuses on the use of immunglobulin (Ig) variable region genes by B cells from patients with primary Sjögren's syndrome (pSS) and the biologic insights that this provides. Comparison of the Ig repertoire from the blood and parotid gland of pSS patients with that of normal donors suggests that there are typical disturbances of B cell homeostasis with depletion of memory B cells from the peripheral blood and accumulation/retention of these antigen-experienced B cells in the inflamed tissue. Although there are clonally expanded B cells in the parotid gland, generalized abnormalities in the B cell repertoire are also found in pSS patients. The vast majority of the current data indicate that there is no major molecular abnormality in generating the IgV chain repertoire in patients with pSS. In contrast, disordered selection leads to considerable differences in the V(L) gene usage and V(H) CDR3 length of the B cell Ig repertoire in pSS patients. The nature of the influences that lead to disordered selection in pSS remains to be determined, but should provide important clues to the etiology of this autoimmune inflammatory disorder.

Dose-reduced Conditioning Regimen Followed by Allogeneic Stem Cell Transplantation in Patients with Myelofibrosis with Myeloid Metaplasia

British Journal of Haematology. Dec, 2002  |  Pubmed ID: 12437657

Three patients with myelofibrosis received allogeneic stem cell transplantation after a dose-reduced conditioning regimen of busulphan (8 mg/kg), fludarabine (180 mg/m2) and antithymocyte globulin (4 x 10 mg/kg). The median age at transplantation was 51 years (range 44-58). All patients engrafted with a leucocyte count > 1.0 x 10(9)/l after a median of 18 d (range 16-20). Grade II acute skin graft-versus-host disease (GvHD) occurred in one patient. One limited and one extensive chronic GvHD was observed. All patients achieved complete haematological remission. In one patient the fibrosis resolved completely 180 d post transplant. All patients are alive 126, 466 and 764 d after transplantation.

Chronic Lymphocytic Leukemia Preceded by Cold Agglutinin Disease: Intraclonal Immunoglobulin Light-chain Diversity in V(H)4-34 Expressing Single Leukemic B Cells

Blood. Nov, 2002  |  Pubmed ID: 12384446

Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription-polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, the IgV(kappa) genes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgV(H) genes were invariant. Thus, the light-chain-restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population.

Diminished Peripheral Blood Memory B Cells and Accumulation of Memory B Cells in the Salivary Glands of Patients with Sjögren's Syndrome

Arthritis and Rheumatism. Aug, 2002  |  Pubmed ID: 12209521

To delineate the mechanism of the abnormalities in B cell biology found in patients with primary Sjögren's syndrome (SS).

Analysis of Immunoglobulin Light Chain Rearrangements in the Salivary Gland and Blood of a Patient with Sjögren's Syndrome

Arthritis Research. 2002  |  Pubmed ID: 12106503

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.

Peracetic Acid-ethanol Treatment of Allogeneic Avital Bone Tissue Transplants--a Reliable Sterilization Method

Annals of Transplantation : Quarterly of the Polish Transplantation Society. 2003  |  Pubmed ID: 14626574

Based on the European Standard EN 1040, the validation guidelines of the German Federal Institute for Drugs and Medical Devices and CPMP guidelines we tested the antimicrobial effectiveness of a peracetic acid-ethanol sterilization procedure (PES) in allogenic avital bone transplants.

New Concepts in the Pathogenesis of Sjögren Syndrome: Many Questions, Fewer Answers

Current Opinion in Rheumatology. Sep, 2003  |  Pubmed ID: 12960481

Although a modified European-American consensus classification of Sjögren syndrome has been introduced during the last year, the etiopathogenesis of this disease characterized by chronic lymphocytic inflammation, impaired function, and, finally, destruction of the salivary and lacrimal glands as well as systemic manifestations remains to be elucidated. Recent insights into the pathogenesis of Sjögren syndrome resulting from immunogenetic, hormonal, and epidemiologic evaluations as well as animal and in vitro studies are highlighted by this review. Evidence confirms that lymphocytic disturbances, including ectopic germinal center formation and aberrations of cellular signaling play a significant role in Sjögren syndrome. Although some of these features are unique to Sjögren syndrome, others are also found in a number of systemic autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. The underlying cause of Sjögren syndrome remains largely enigmatic. However, distinct characteristics may provide the basis for the classification of the disease entities. Finally, an enhanced risk of lymphomagenesis is a well-known hallmark of primary Sjögren syndrome, indicating the central role of derangement of lymphocyte regulation. As demonstrated by the introduction of the new targeted therapeutic approaches in rheumatoid arthritis, solid insights into the pathogenesis of Sjögren syndrome may pave the way toward new therapeutic approaches.

Autoantibodies in Normals--the Value of Predicting Rheumatoid Arthritis

Arthritis Research & Therapy. 2004  |  Pubmed ID: 15535841

Abnormalities in Peripheral B Cell Memory of Patients with Primary Sjögren's Syndrome

Arthritis and Rheumatism. Jun, 2004  |  Pubmed ID: 15188366

To delineate disturbances in peripheral B cell memory in primary Sjögren's syndrome (SS).

Cryopreserved Human B Cells As an Alternative Source for Single Cell MRNA Analysis

Cell and Tissue Banking. 2005  |  Pubmed ID: 16308769

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

Immunopathogenesis of Primary Sjögren's Syndrome: Implications for Disease Management and Therapy

Current Opinion in Rheumatology. Sep, 2005  |  Pubmed ID: 16093833

Recent studies have broadened our understanding of the etiopathogenesis and immunopathology of primary Sjögren's syndrome. This review highlights recent advances in understanding the underlying mechanisms of the disease as well as their implications for clinical handling and therapeutic options.

Dysregulation of Chemokine Receptor Expression and Function by B Cells of Patients with Primary Sjögren's Syndrome

Arthritis and Rheumatism. Jul, 2005  |  Pubmed ID: 15986367

To assess whether abnormal chemokine receptor expression and/or abnormal responsiveness to the cognate ligands might underlie some of the disturbances in B cell homeostasis characteristic of primary Sjögren's syndrome (SS).

A Method for the Determination of the Residual Chloroform in Defatted Cancellous Bone Transplants

Cell and Tissue Banking. 2005  |  Pubmed ID: 15735904

The removal of fat from cancellous bone tissue promotes the clinical healing of the transplant and improves the penetration of chemical sterilisation media into the tissue. Treatment using chloroform/methanol (2:1, 2 h) is frequently used as a defatting procedure. Eight rinses with methanol followed by two rinses with aqua ad iniectabilia (20 min each, with ultrasonic effect) ensure depletion in the level of chloroform from defatted cancellous bone to a concentration below 25 ppm (limit value). For the necessary routine quality checks on the production process, a gas chromatography method has been developed that determines the level of chloroform in cancellous bone, for which the detection limit is 0.003 ppm.

Generation of Migratory Antigen-specific Plasma Blasts and Mobilization of Resident Plasma Cells in a Secondary Immune Response

Blood. Feb, 2005  |  Pubmed ID: 15507523

Maintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19(+)/CD27(high)/intracellular immunoglobulin G(high) (IgG(high))/HLA-DR(high)/CD38(high)/CD20(-)/CD95(+) tetanus toxin-specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19(+)/CD27(high)/intracellular IgG(high)/HLA-DR(low)/CD38(+)/CD20(-)/CD95(+) cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.

B-cell Lymphoproliferation in Chronic Inflammatory Rheumatic Diseases

Nature Clinical Practice. Rheumatology. Oct, 2007  |  Pubmed ID: 17906611

Patients with chronic inflammatory rheumatic diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and especially primary Sjögren's syndrome (SS), are at higher risk than the general population of developing B-cell non-Hodgkin lymphoma (NHL). Analyses of the association between various lymphoma subtypes and specific disease entities suggest that this association might be mediated by disease-specific mechanisms, as well as by mechanisms unique to lymphoma subtype. These specific associations can provide important information about abnormal B-cell stimulation in these conditions. Patients with primary SS, SLE and RA are at high risk of developing diffuse large B-cell lymphomas, a group of high-grade NHLs with remarkable heterogeneity. Patients with primary SS are at particularly high risk of developing marginal-zone B-cell lymphomas. The risk factors of lymphoma development in primary SS seem to be closely related to the underlying mechanisms of abnormal stimulation and/or impaired censoring mechanisms of B cells. In patients with RA and SLE, more intense disease activity and/or long-lasting disease might be indications of a higher risk of lymphoma development. This Review will focus on the risk of lymphoma, common and disease-specific mechanisms of B-cell lymphoma development, and on the clinical consequences of lymphoma in patients with inflammatory rheumatic diseases.

B Cells in Sjögren's Syndrome: Indications for Disturbed Selection and Differentiation in Ectopic Lymphoid Tissue

Arthritis Research & Therapy. 2007  |  Pubmed ID: 17697366

Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathological features. A hallmark of pSS is B-cell hyperactivity as manifested by the production of autoantibodies, hypergammaglobulinemia, formation of ectopic lymphoid structures within the inflamed tissues, and enhanced risk of B-cell lymphoma. Changes in the distribution of peripheral B-cell subsets and differences in post-recombination processes of immunoglobulin variable region (IgV) gene usage are also characteristic features of pSS. Comparison of B cells from the peripheral blood and salivary glands of patients with pSS with regard to their expression of the chemokine receptors CXCR4 and CXCR5, and their migratory capacity towards the corresponding ligands, CXCL12 and CXCL13, provide a mechanism for the prominent accumulation of CXCR4+ CXCR5+ memory B cells in the inflamed glands. Glandular B cells expressing distinct features of IgV light and heavy chain rearrangements, (re)circulating B cells with increased mutations of cmu transcripts in both CD27- and CD27+ memory B-cell subsets, and enhanced frequencies of individual peripheral B cells containing IgV heavy chain transcripts of multiple isotypes indicate disordered selection and incomplete differentiation processes of B cells in the inflamed tissues in pSS. This may possibly be related to a lack of appropriate censoring mechanisms or different B-cell activation pathways within the ectopic lymphoid structures of the inflamed tissues. These findings add to our understanding of the pathogenesis of this autoimmune inflammatory disorder and may result in new therapeutic approaches.

Developments in Lupus 2006

Arthritis Research & Therapy. 2007  |  Pubmed ID: 17634152

Published reports in 2006 on systemic lupus erythematosus are reviewed with regard to preclinical and clinical studies on disturbances of the immune system including co-stimulation, cytokines and recent insights into new therapeutic approaches. Increasing knowledge of components of the innate immune system, such as certain receptors (Toll-like receptors, Fc receptors and complement receptors) and cytokines as well as immune cells (dendritic cells, plasmacytoid cells and neutrophils) supports their immunopathogenic relevance and enhance our understanding of the pathogenic complexity of lupus. Although it remains to be shown which of those could be targets for therapy or biomarkers, lymphocyte-directed therapy is currently under promising clinical investigation.

Using Brain Slice Cultures of Mouse Brain to Assess the Effect of Growth Factors on Differentiation of Bone Marrow Derived Stem Cells

Ideggyógyászati Szemle. Mar, 2007  |  Pubmed ID: 17451052

Bone marrow derived stem cells (BMDSCs) have been reported to form neurons and supportive cells in the brain. We describe a technique that combines the simplicity of in vitro studies with many of the advantages of in vivo experiments. We cultured mouse brain slices, deposited GFP-tagged BMDSCs evenly distributed on their surfaces, and then added test factors to the culture medium. Addition of both SDF-1 and EGF resulted in morphological changes of BMDSC and in the induction of islet-1, a marker of neuroepithelial progenitors. We conclude that organotypic tissue culture (OTC) may allow us to detect the effects of exogenous factors on the differentiation of BMDSCs (or any other type of stem cells) in an environment that may resemble the CNS after brain injury. Once such factors have been identified they could be evaluated for tissue regeneration in more complex, whole animal models.

Sensitive and Specific Method for Detecting G Protein-coupled Receptor MRNAs

Nature Methods. Jan, 2007  |  Pubmed ID: 17115035

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.

Activated Memory B Cell Subsets Correlate with Disease Activity in Systemic Lupus Erythematosus: Delineation by Expression of CD27, IgD, and CD95

Arthritis and Rheumatism. Jun, 2008  |  Pubmed ID: 18512812

Analysis of peripheral B cell subsets in patients with systemic lupus erythematosus (SLE) has provided evidence of specific alterations, such as an expansion of CD27++ plasma cells/blasts and transitional B cells. However, memory B cells in lupus have not been thoroughly investigated, and only recently a CD27- memory B cell subset was identified in the peripheral blood of lupus patients. Focusing on CD27- B cells, this study aimed to identify abnormalities in peripheral B cell subsets in patients with SLE.

Role of the Spleen in Peripheral Memory B-cell Homeostasis in Patients with Autoimmune Thrombocytopenia Purpura

Clinical Immunology (Orlando, Fla.). Feb, 2009  |  Pubmed ID: 18977183

The effect of splenectomy on circulating memory B cells in autoimmune thrombocytopenia purpura (AITP) patients has not yet been addressed. We therefore analyzed the distribution and phenotypic characteristics of B-cell subsets in non-splenectomized and splenectomized AITP patients and controls, as well as CD95 expression after B cell activation. Decreased frequencies of memory B cells in splenectomized individuals were observed, with a rapid decline of CD27+IgD+ and a slower decrease of CD27+IgD- and CD27-/IgD- cells. Similar results were noted following splenectomy in healthy donors (HD). CD95+ B cells were substantially increased in all subsets in patients with active AITP, indicating their enhanced activation status. After splenectomy, the percentage of CD95+ B cells were further increased in the CD27+IgD- post-switch memory population in AITP, but not in HD. CD95+CD27+ memory B cells largely reside in the region in the human spleen analogous to the murine marginal zone. Thus, the spleen plays a fundamental role in controlling peripheral memory B cell homeostasis in both AITP and HD and regulates activated CD95+ B cells in patients with AITP.

MicroRNA-199b Targets the Nuclear Kinase Dyrk1a in an Auto-amplification Loop Promoting Calcineurin/NFAT Signalling

Nature Cell Biology. Dec, 2010  |  Pubmed ID: 21102440

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure.

Epratuzumab Targeting of CD22 Affects Adhesion Molecule Expression and Migration of B-cells in Systemic Lupus Erythematosus

Arthritis Research & Therapy. 2010  |  Pubmed ID: 21050432

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27(negative) B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.

Myomasp/LRRC39, a Heart- and Muscle-specific Protein, is a Novel Component of the Sarcomeric M-band and is Involved in Stretch Sensing

Circulation Research. Nov, 2010  |  Pubmed ID: 20847312

The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear.

What Do We Know About Memory B Cells in Primary Sjögren's Syndrome?

Autoimmunity Reviews. Jul, 2010  |  Pubmed ID: 20452465

Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögrens Syndrome (pSS) and its malignant complication, B cell lymphoma. Recent studies on B cells in pSS add to our understanding of the distinct memory B cell subsets in pSS. Reduction of peripheral memory CD27(+) B cells, most strikingly of the CD27(+)IgM(+) subset, may indicate a lack of appropriate censoring mechanisms and incomplete differentiation processes within the ectopic lymphoid tissues in pSS. This ectopically formed lymphoid tissue might protect autoreactive memory B cells from deletion by physiological check-points and, thereby, may contribute to the perpetuation of the disease as well as to an enhanced lymphoma risk. Thus, B cells may be potential targets of direct or indirect treatment in pSS.

Development of a Drug Screening Platform Based on Engineered Heart Tissue

Circulation Research. Jul, 2010  |  Pubmed ID: 20448218

Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. Objective: Here, we present a new miniaturized and automated method based on engineered heart tissue (EHT).

Human Engineered Heart Tissue As a Versatile Tool in Basic Research and Preclinical Toxicology

PloS One. 2011  |  Pubmed ID: 22028871

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.

EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI): Development of a Consensus Patient Index for Primary Sjogren's Syndrome

Annals of the Rheumatic Diseases. Jun, 2011  |  Pubmed ID: 21345815

To develop a score for assessment of patients' symptoms in primary Sjögren's syndrome (SS): the EULAR SS Patient Reported Index (ESSPRI).

Cardiovascular Side Effects of Cancer Therapies: a Position Statement from the Heart Failure Association of the European Society of Cardiology

European Journal of Heart Failure. Jan, 2011  |  Pubmed ID: 21169385

The reductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti-cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under-represent older patients and those with significant co-morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre-clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross-disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.

Succinate Reverses In-vitro Platelet Inhibition by Acetylsalicylic Acid and P2Y Receptor Antagonists

Platelets. 2012  |  Pubmed ID: 21736422

High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000 µM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100 µM succinate. Similarly, succinate completely (at 1000 µM) or partially (at 100 µM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P expression was used as a different readout for platelet activation. In summary, extracellular succinate may have a co-stimulatory role in platelet aggregation and, by (partially) antagonizing the effects of platelet inhibitors, may contribute to the inter-individual variability frequently observed in platelet function testing.

Increased Afterload Induces Pathological Cardiac Hypertrophy: a New in Vitro Model

Basic Research in Cardiology. Nov, 2012  |  Pubmed ID: 23099820

Increased afterload results in 'pathological' cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.

Physiological Aspects of Cardiac Tissue Engineering

American Journal of Physiology. Heart and Circulatory Physiology. Jul, 2012  |  Pubmed ID: 22582087

Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic research and predictive toxicology applications. Since the first description of engineered heart tissue 15 years ago, major development steps were directed toward these three goals. Technical innovations led to improved three-dimensional cardiac tissue structure and near physiological contractile force development. Automation and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were tested for cardiac repair and were associated with functional improvements in rats. Whether these approaches can be simply transferred to larger animals or the human patients remains to be tested. The availability of an unrestricted human cardiac myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue engineering techniques with their strengths and limitations and possible future applications.

Complex Interactions Between Human Myoblasts and the Surrounding 3D Fibrin-based Matrix

PloS One. 2012  |  Pubmed ID: 22558372

Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5 ± 0.8 to 7.4 ± 4.7 kPa during proliferation (p<0.05) and reached 12.2 ± 6.0 kPa during differentiation (p<0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.

Evidence for FHL1 As a Novel Disease Gene for Isolated Hypertrophic Cardiomyopathy

Human Molecular Genetics. Jul, 2012  |  Pubmed ID: 22523091

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.

Aberrant ZNF423 Impedes B Cell Differentiation and is Linked to Adverse Outcome of ETV6-RUNX1 Negative B Precursor Acute Lymphoblastic Leukemia

The Journal of Experimental Medicine. Oct, 2013  |  Pubmed ID: 24081948

Differentiation arrest is a hallmark of acute leukemia. Genomic alterations in B cell differentiation factors such as PAX5, IKZF1, and EBF-1 have been identified in more than half of all cases of childhood B precursor acute lymphoblastic leukemia (ALL). Here, we describe a perturbed epigenetic and transcriptional regulation of ZNF423 in ALL as a novel mechanism interfering with B cell differentiation. Hypomethylation of ZNF423 regulatory sequences and BMP2 signaling result in transactivation of ZNF423α and a novel ZNF423β-isoform encoding a nucleosome remodeling and histone deacetylase complex-interacting domain. Aberrant ZNF423 inhibits the transactivation of EBF-1 target genes and leads to B cell maturation arrest in vivo. Importantly, ZNF423 expression is associated with poor outcome of ETV6-RUNX1-negative B precursor ALL patients. Our work demonstrates that ALL is more than a genetic disease and that epigenetics may uncover novel mechanisms of disease with prognostic implications.

Contractile Abnormalities and Altered Drug Response in Engineered Heart Tissue from Mybpc3-targeted Knock-in Mice

Journal of Molecular and Cellular Cardiology. Oct, 2013  |  Pubmed ID: 23896226

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.

Heterozygous LmnadelK32 Mice Develop Dilated Cardiomyopathy Through a Combined Pathomechanism of Haploinsufficiency and Peptide Toxicity

Human Molecular Genetics. Aug, 2013  |  Pubmed ID: 23575224

Dilated cardiomyopathy (DCM) associates left ventricular (LV) dilatation and systolic dysfunction and is a major cause of heart failure and cardiac transplantation. LMNA gene encodes lamins A/C, proteins of the nuclear envelope. LMNA mutations cause DCM with conduction and/or rhythm defects. The pathomechanisms linking mutations to DCM remain to be elucidated. We investigated the phenotype and associated pathomechanisms of heterozygous Lmna(ΔK32/+) (Het) knock-in mice, which carry a human mutation. Het mice developed a cardiac-specific phenotype. Two phases, with two different pathomechanisms, could be observed that lead to the development of cardiac dysfunction, DCM and death between 35 and 70 weeks of age. In young Het hearts, there was a clear reduction in lamin A/C level, mainly due to the degradation of toxic ΔK32-lamin. As a side effect, lamin A/C haploinsufficiency probably triggers the cardiac remodelling. In older hearts, when DCM has developed, the lamin A/C level was normalized and associated with increased toxic ΔK32-lamin expression. Crossing our mice with the Ub(G76V)-GFP ubiquitin-proteasome system (UPS) reporter mice revealed a heart-specific UPS impairment in Het. While UPS impairment itself has a clear deleterious effect on engineered heart tissue's force of contraction, it also leads to the nuclear aggregation of viral-mediated expression of ΔK32-lamin. In conclusion, Het mice are the first knock-in Lmna model with cardiac-specific phenotype at the heterozygous state. Altogether, our data provide evidence that Het cardiomyocytes have to deal with major dilemma: mutant lamin A/C degradation or normalization of lamin level to fight the deleterious effect of lamin haploinsufficiency, both leading to DCM.

Impact of ANKRD1 Mutations Associated with Hypertrophic Cardiomyopathy on Contraction Parameters of Engineered Heart Tissue

Basic Research in Cardiology. May, 2013  |  Pubmed ID: 23572067

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M-ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired.

Effects of Proarrhythmic Drugs on Relaxation Time and Beating Pattern in Rat Engineered Heart Tissue

Basic Research in Cardiology. 2014  |  Pubmed ID: 25209140

The assessment of proarrhythmic risks of drugs remains challenging. To evaluate the suitability of rat engineered heart tissue (EHT) for detecting proarrhythmic effects. We monitored drug effects on spontaneous contractile activity and, in selected cases, on action potentials (sharp microelectrode) and Ca2+ transients (Fura-2) and contraction under electrical pacing. The Ito-blocker inhibitor 4-aminopyridine increased action potential duration and T2 and caused aftercontractions, which were abolished by inhibitors of ryanodine receptors (RyR2; JTV-519) or sodium calcium exchanger (NCX; SEA0400). 77 Drugs were then tested at 1-10-100× free therapeutic plasma concentrations (FTPC): Inhibitors of IKr, IKs, Ito, antiarrhythmics (8), drugs withdrawn from market for torsades des pointes arrhythmias (TdP, 5), drugs with measurable (7) or isolated TdP incidence (13), drugs considered safe (14), 28 new chemical entities (NCE). Inhibitors of IKr or IKs had no effect alone, but substantially prolonged relaxation time (T2) when combined at high concentration. 15/33 drugs associated with TdP and 6/14 drugs considered non-torsadogenic (cibenzoline, diltiazem, ebastine, ketoconazole, moxifloxacin, and phenytoin) induced concentration-dependent T2 prolongations (10-100× FTPC). Bepridil, desipramine, imipramine, thioridazine, and erythromycin induced irregular beating. Three NCE prolonged T2, one reduced force. Drugs inhibiting repolarization prolong relaxation in rat EHTs and cause aftercontractions involving RyR2 and NCX. Insensitivity to IKr inhibitors makes rat EHTs unsuitable as general proarrhythmia screen, but favors detection of effects on Ito, IKs + Ito or IKs + IKr. Screening a large panel of drugs suggests that effects on these currents, in addition to IKr, are more common than anticipated.

Generation of Strip-format Fibrin-based Engineered Heart Tissue (EHT)

Methods in Molecular Biology (Clifton, N.J.). 2014  |  Pubmed ID: 25070332

This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately ¼ of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

Functional Improvement and Maturation of Rat and Human Engineered Heart Tissue by Chronic Electrical Stimulation

Journal of Molecular and Cellular Cardiology. Sep, 2014  |  Pubmed ID: 24852842

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

Automated Analysis of Contractile Force and Ca2+ Transients in Engineered Heart Tissue

American Journal of Physiology. Heart and Circulatory Physiology. May, 2014  |  Pubmed ID: 24585781

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).

Cardiac Tissue Engineering: State of the Art

Circulation Research. Jan, 2014  |  Pubmed ID: 24436431

The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade. Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing.

In Vitro Perfusion of Engineered Heart Tissue Through Endothelialized Channels

Tissue Engineering. Part A. Feb, 2014  |  Pubmed ID: 24156346

In engineered heart tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitro perfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm³). Thin dry alginate fibers (17×0.04×0.04 mm) were embedded into the cell-fibrin-thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the tissue. Perfusion rapidly increased it to 10%-12% in the immediate vicinity of the microchannel. Continuous perfusion (20 μL/h, for 3 weeks) of the tubular lumina (100-500 μm) via hollow posts of the silicone rack increased mean dystrophin-positive cardiomyocyte density (36%±6% vs. 10%±3% of total cell number) and cross sectional area (73±2 vs. 48±1 μm²) in the central part of the tissue compared to nonperfused EHTs. The channels were populated by endothelial cells present in the reconstitution cell mix. In conclusion, we developed a novel approach to generate small tubular structures suitable for perfusion of spontaneously contracting and force-generating EHTs and showed that prolonged perfusion improved cardiac tissue structure.

Functional MicroRNA Library Screening Identifies the Hypoxamir MiR-24 As a Potent Regulator of Smooth Muscle Cell Proliferation and Vascularization

Antioxidants & Redox Signaling. Sep, 2014  |  Pubmed ID: 24063572

Smooth muscle cells (SMCs) are key components within the vasculature. Dependent on the stimulus, SMC can either be in a proliferative (synthetic) or differentiated state. Alterations of SMC phenotype also appear in several disease settings, further contributing to disease progression.

Development of Long Noncoding RNA-Based Strategies to Modulate Tissue Vascularization

Journal of the American College of Cardiology. Nov, 2015  |  Pubmed ID: 26516004

Long noncoding ribonucleic acids (lncRNAs) are a subclass of regulatory noncoding ribonucleic acids for which expression and function in human endothelial cells and angiogenic processes is not well studied.

Immunobiology of Fibrin-based Engineered Heart Tissue

Stem Cells Translational Medicine. Jun, 2015  |  Pubmed ID: 25947338

Different tissue-engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin-based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3- and CD68-positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme-linked immunospot and immunofluorescence experiments revealed matrix-directed TH1-based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase-positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin-based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected.

Deciphering the MicroRNA Signature of Pathological Cardiac Hypertrophy by Engineered Heart Tissue- and Sequencing-technology

Journal of Molecular and Cellular Cardiology. Apr, 2015  |  Pubmed ID: 25633833

Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on engineered heart tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.

Towards a Tissue-Engineered Contractile Fontan-Conduit: The Fate of Cardiac Myocytes in the Subpulmonary Circulation

PloS One. 2016  |  Pubmed ID: 27875570

The long-term outcome of patients with single ventricles improved over time, but remains poor compared to other congenital heart lesions with biventricular circulation. Main cause for this unfavourable outcome is the unphysiological hemodynamic of the Fontan circulation, such as subnormal systemic cardiac output and increased systemic-venous pressure. To overcome this limitation, we are developing the concept of a contractile extracardiac Fontan-tunnel. In this study, we evaluated the survival and structural development of a tissue-engineered conduit under in vivo conditions. Engineered heart tissue was generated from ventricular heart cells of neonatal Wistar rats, fibrinogen and thrombin. Engineered heart tissues started beating around day 8 in vitro and remained contractile in vivo throughout the experiment. After culture for 14 days constructs were implanted around the right superior vena cava of Wistar rats (n = 12). Animals were euthanized after 7, 14, 28 and 56 days postoperatively. Hematoxylin and eosin staining showed cardiomyocytes arranged in thick bundles within the engineered heart tissue-conduit. Immunostaining of sarcomeric actin, alpha-actin and connexin 43 revealed a well -developed cardiac myocyte structure. Magnetic resonance imaging (d14, n = 3) revealed no constriction or stenosis of the superior vena cava by the constructs. Engineered heart tissues survive and contract for extended periods after implantation around the superior vena cava of rats. Generation of larger constructs is warranted to evaluate functional benefits of a contractile Fontan-conduit.

Cardiac Repair in Guinea Pigs with Human Engineered Heart Tissue from Induced Pluripotent Stem Cells

Science Translational Medicine. Nov, 2016  |  Pubmed ID: 27807283

Myocardial injury results in a loss of contractile tissue mass that, in the absence of efficient regeneration, is essentially irreversible. Transplantation of human pluripotent stem cell-derived cardiomyocytes has beneficial but variable effects. We created human engineered heart tissue (hEHT) strips from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived endothelial cells. The hEHTs were transplanted onto large defects (22% of the left ventricular wall, 35% decline in left ventricular function) of guinea pig hearts 7 days after cryoinjury, and the results were compared with those obtained with human endothelial cell patches (hEETs) or cell-free patches. Twenty-eight days after transplantation, the hearts repaired with hEHT strips exhibited, within the scar, human heart muscle grafts, which had remuscularized 12% of the infarct area. These grafts showed cardiomyocyte proliferation, vascularization, and evidence for electrical coupling to the intact heart tissue in a subset of engrafted hearts. hEHT strips improved left ventricular function by 31% compared to that before implantation, whereas the hEET or cell-free patches had no effect. Together, our study demonstrates that three-dimensional human heart muscle constructs can repair the injured heart.

Ca(2+)-Currents in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Effects of Two Different Culture Conditions

Frontiers in Pharmacology. 2016  |  Pubmed ID: 27672365

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca(2+)-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca(2+)-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.

Human Engineered Heart Tissue: Analysis of Contractile Force

Stem Cell Reports. Jul, 2016  |  Pubmed ID: 27211213

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

PloS One. 2016  |  Pubmed ID: 26840448

Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism.

Spontaneous Formation of Extensive Vessel-Like Structures in Murine Engineered Heart Tissue

Tissue Engineering. Part A. Feb, 2016  |  Pubmed ID: 26763667

Engineered heart tissue (EHT) from primary heart cells contains endothelial cells (ECs), but the extent to which ECs organize into vessel-like structures or even functional vessels remains unknown and is difficult to study by conventional methods. In this study, we generated fibrin-based mini-EHTs from a transgenic mouse line (Cdh5-CreERT2 × Rosa26-LacZ), in which ECs were specifically and inducibly labeled by applying tamoxifen (EC(iLacZ)). EHTs were generated from an unpurified cell mix of newborn mouse hearts and were cultured under standard serum-containing conditions. Cre expression in 15-day-old EHTs was induced by addition of o-hydroxytamoxifen to the culture medium for 48 h, and ECs were visualized by X-gal staining. EC(iLacZ) EHTs showed a dense X-gal-positive vessel-like network with distinct tubular structures. Immunofluorescence revealed that ECs were mainly associated with cardiomyocytes within the EHT. EC(iLacZ) EHT developed spontaneous and regular contractility with forces up to 0.1 mN. Coherent contractility and the presence of an extensive vessel-like network were both dependent on the presence of animal sera in the culture medium. Contractile EC(iLacZ) EHTs successfully served as grafts in implantation studies onto the hearts of immunodeficient mice. Four weeks after implantation, EHTs showed X-gal-positive lumen-forming vessel structures connected to the host myocardium circulation as they contained erythrocytes on a regular basis. Taken together, genetic labeling of ECs revealed the extensive formation of vessel-like structures in EHTs in vitro. The EC(iLacZ) EHT model could help simultaneously study biological effects of compounds on cardiomyocyte function and tissue vascularization.

Human Engineered Heart Tissue As a Model System for Drug Testing

Advanced Drug Delivery Reviews. Jan, 2016  |  Pubmed ID: 26026976

Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered heart tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.

Thymosin β4 Improves Differentiation and Vascularization of EHTs

Stem Cells International. 2017  |  Pubmed ID: 28191018

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin β4 (Tβ4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of Tβ4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that Tβ4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tβ4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tβ4 promotes vascularization and contractility in EHTs, highlighting Tβ4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes.

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