PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-9 (9)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Increased afterload induces pathological cardiac hypertrophy: a new in vitro model 
Basic Research in Cardiology  2012;107(6):307.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-012-0307-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00395-012-0307-z
PMCID: PMC3505530  PMID: 23099820
Afterload enhancement; Cardiac hypertrophy; Cardiac metabolism; Cardiac tissue engineering; Endothelin receptor antagonist; Fibrosis
2.  Complex Interactions between Human Myoblasts and the Surrounding 3D Fibrin-Based Matrix 
PLoS ONE  2012;7(4):e36173.
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.
doi:10.1371/journal.pone.0036173
PMCID: PMC3338613  PMID: 22558372
3.  Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology 
PLoS ONE  2011;6(10):e26397.
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.
doi:10.1371/journal.pone.0026397
PMCID: PMC3197640  PMID: 22028871
4.  Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus 
Arthritis Research & Therapy  2010;12(6):R204.
Introduction
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 CD27negative 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.
Methods
Epratuzumab binding specificity and the surface expression of adhesion molecules (CD62L, β7 integrin and β1 integrin) after culture with epratuzumab was studied on B-cell subsets of SLE patients by flow cytometry. In addition, in vitro transwell migration assays were performed to analyze the effects of epratuzumab on migration towards different chemokines such as CXCL12, CXCL13 or to CXCR3 ligands, and to assess the functional consequences of altered adhesion molecule expression.
Results
Epratuzumab binding was considerably higher on B-cells relative to other cell types assessed. No binding of epratuzumab was observed on T-cells, while weak non-specific binding of epratuzumab on monocytes was noted. On B-cells, binding of epratuzumab was particularly enhanced on CD27negative B-cells compared to CD27positive B-cells, primarily related to a higher expression of CD22 on CD27negative B-cells. Moreover, epratuzumab binding led to a decrease in the cell surface expression of CD62L and β7 integrin, while the expression of β1 integrin was enhanced. The effects on the pattern of adhesion molecule expression observed with epratuzumab were principally confined to a fraction of the CD27negative B-cell subpopulation and were associated with enhanced spontaneous migration of B-cells. Furthermore, epratuzumab also enhanced the migration of CD27negative B-cells towards the chemokine CXCL12.
Conclusions
The current data suggest that epratuzumab has effects on the expression of the adhesion molecules CD62L, β7 integrin and β1 integrin as well as on migration towards CXCL12, primarily of CD27negative B-cells. Therefore, induced changes in migration appear to be part of the mechanism of action of epratuzumab and are consistent with the observation that CD27negative B-cells were found to be preferentially reduced in the peripheral blood under treatment.
doi:10.1186/ar3179
PMCID: PMC3046510  PMID: 21050432
5.  B cells in Sjögren's syndrome: indications for disturbed selection and differentiation in ectopic lymphoid tissue 
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 cμ 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.
doi:10.1186/ar2210
PMCID: PMC2206371  PMID: 17697366
6.  Developments in lupus 2006 
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.
doi:10.1186/ar2183
PMCID: PMC2206361  PMID: 17634152
8.  Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome 
Arthritis Research  2002;4(4):R4.
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 immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin 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 (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related VL chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ 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 VL chain genes caused by selection and clonal expansion of B cells expressing particular VL genes. In addition, the data document an accumulation of B cells bearing mutated VL 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.
doi:10.1186/ar423
PMCID: PMC125296  PMID: 12106503
B cells; parotid gland; Sjögren's syndrome; somatic mutation; V light chain genes
9.  Perturbations in the impact of mutational activity on Vλ genes in systemic lupus erythematosus 
Arthritis Research  2001;3(6):368-374.
To assess the impact of somatic hypermutation and selective influences on the Vλ light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of mutations were analyzed in individual CD19+ B cells from a patient with previously undiagnosed SLE. The mutational frequency of nonproductive and productive rearrangements in the SLE patient was greater (3.1 × 10-2 vs 3.4 × 10-2, respectively) than that in normal B cells (1.2 × 10-2 vs 2.0 × 10-2, both P < 0.001). The frequencies of mutated rearrangements in both the nonproductive and productive repertoires were significantly higher in the patient with SLE than in normal subjects. Notably, there were no differences in the ratio of replacement to silent (R/S) mutations in the productive and nonproductive repertoires of the SLE patient, whereas the R/S ratio in the framework regions of productive rearrangements of normal subjects was reduced, consistent with active elimination of replacement mutations in this region. The pattern of mutations was abnormal in the SLE patient, with a significant increase in the frequency of G mutations in both the productive and nonproductive repertoires. As in normal subjects, however, mutations were found frequently in specific nucleotide motifs, the RGYW/WRCY sequences, accounting for 34% (nonproductive) and 46% (productive) of all mutations. These data are most consistent with the conclusion that in this SLE patient, the mutational activity was markedly greater than in normal subjects and exhibited some abnormal features. In addition, there was decreased subsequent positive or negative selection of mutations. The enhanced and abnormal mutational activity along with disturbances in selection may play a role in the emergence of autoreactivity in this patient with SLE.
PMCID: PMC64848  PMID: 11714391
autoimmunity; B cells; SLE; somatic hypermutation; V genes

Results 1-9 (9)