Elisabeth Ehler

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF 164 and VEGF 188

Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells

In polarized epithelial cells measles virus (MV) is predominantly released at the apical cell surface, irrespective of the sorting of its two envelope glycoproteins F and H. It has been reported previously that the viral matrix (M) protein modulates the fusogenic capacity of the viral envelope glycoproteins. Here, extant MV mutants and chimeras were used to determine the role of M protein in the transport of viral glycoproteins and release of progeny virions in polarized epithelial CaCo2 cells. In the absence of M, envelope glycoproteins are sorted to the basolateral surface, suggesting that they possess intrinsic basolateral sorting signals. However, interactions of M with the glycoprotein cytoplasmic tails allow M–glycoprotein co‐segregation to the apical surface, suggesting a vectorial function of M to retarget the glycoproteins for apical virion release. Whereas this may allow virus airway shedding, the intrinsic sorting of the glycoproteins to the basolateral surface may account for systemic host infection by allowing efficient cell–cell fusion.

Mass Production of Embryoid Bodies in Microbeads

Abstract: Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell‐derived, differentiated cells are of increasing importance as a potential source for non‐proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula‐like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Progressive cardiac hypertrophy and dysfunction in atrial natriuretic peptide receptor (GC-A) deficient mice

Objective: To investigate how permanent inhibition of guanylyl cyclase A receptor (GC-A) affects cardiac function. Methods: Hearts of GC-A−/− and corresponding wild type mice (GC-A+/+) were characterised by histological, western blotting, and northern blotting analyses. Cardiac function was evaluated in isolated, working heart preparations. Results: At 4 months of age, GC-A−/− mice had global cardiac hypertrophy (about a 40% increase in cardiac weight) without interstitial fibrosis. Examination of heart function found a significant delay in the time of relaxation; all other parameters of cardiac contractility were similar to those in wild type mice. At 12 months, the hypertrophic changes were much more severe (about a 61% increase in cardiac weight), together with a shift in cardiac gene expression (enhanced concentrations of atrial natriuretic peptide (3.8-fold), B type natriuretic peptide (2-fold), β myosin heavy chain (1.6-fold) and α skeletal actin (1.7-fold) mRNA), increased expression of cytoskeletal tubulin and desmin (by 29.6% and 25.6%, respectively), and pronounced interstitial fibrosis. These changes were associated with significantly impaired cardiac contractility (+dP/dt decreased by about 10%) and relaxation (−dP/dt decreased by 21%), as well as depressed contractile responses to pressure load (all p < 0.05). Conclusions: Chronic hypertension in GC-A−/− mice is associated with progressive cardiac changes—namely, initially compensated cardiomyocyte hypertrophy, which is complicated by interstitial fibrosis and impaired cardiac contractility at later stages.

Measles Virus Spreads in Rat Hippocampal Neurons by Cell-to-Cell Contact and in a Polarized Fashion

ABSTRACT Measles virus (MV) can infect the central nervous system and, in rare cases, causes subacute sclerosing panencephalitis, characterized by a progressive degeneration of neurons. The route of MV transmission in neurons was investigated in cultured rat hippocampal slices by using MV expressing green fluorescent protein. MV infected hippocampal neurons and spread unidirectionally, in a retrograde manner, from CA1 to CA3 pyramidal cells and from there to the dentate gyrus. Spreading of infection depended on cell-to-cell contact and occurred without any detectable release of infectious particles. The role of the viral proteins in the retrograde MV transmission was determined by investigating their sorting in infected pyramidal cells. MV glycoproteins, the fusion protein (F) and hemagglutinin (H), the matrix protein (M), and the phosphoprotein (P), which is part of the viral ribonucleoprotein complex, were all sorted to the dendrites. While M, P, and H proteins remained more intracellular, the F protein localized to prominent, spine-type domains at the surface of infected cells. The detected localization of MV proteins suggests that local microfusion events may be mediated by the F protein at sites of synaptic contacts and is consistent with a mechanism of retrograde transmission of MV infection.

Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure

During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O(2) in serum- and glucose-free medium) for 5 h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30 min, strongly inhibited after 5 h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.

Identification, characterization, and expression of a novel α‐tropomyosin isoform in cardiac tissues in developing chicken

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non‐muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM‐4 type. This novel α‐Tm tropomyosin isoform, designated as α‐Tm‐2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (α‐Tm‐1) was also expressed in embryonic hearts along with the striated muscle isoform of TM‐4. In adult heart, TM‐4 was expressed, however, expression of both α‐Tm‐1 and α‐Tm‐2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of α‐Tm‐2 in embryonic and adult skeletal muscle, however, the α‐Tm‐1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the α‐TM gene, i.e., α‐smooth muscle tropomyosin (α‐Sm), α‐Fibroblast‐1 (α‐F1), and α‐Fibroblast‐2 (α‐F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of α‐Tm‐1 and α‐Tm‐2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the α‐TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

Design and Characterization of Cardiomyocyte-Derived Microtissues

Classical mammalian cell culture focuses on growing established cell lines as two-dimensional monolayers or suspension cultures. However, in such cultivation conditions mammalian primary cells may loose key morphological characteristics and specialized functions. By contrast, three-dimensional (3D) cell culture may preserve specific function and cell phenotypes since the 3D arrangement of cells favors (i) in vivo-like cell shape, (ii) intercellular crosstalk as well as (iii) development of a complex extracellular matrix. Thus, 3D cultivation may create unique microenvironments required for key processes including differentiation, proliferation, apoptosis and angiogenesis. We have refined the hanging drop technology to produce fully functional and beating myocardial microtissues that maintain the differentiated morphology typical for cardiomyocytes. Myocardial microtissues derived from primary mouse and rat cardiomyocytes as well as mixed populations, reflecting the cell type composition of rodent hearts, were characterized for muscle-specific cell morphologies, extracellular matrix components as well as VEGF (vascular endothelial growth factor) production. Novel findings included (i) a linear correlation between cell number and microtissue size, (ii) intensive cell-cell contacts, (iii) maintenance/development of cardiomyocyte-specific cell structures, (iv) development of an extracellular matrix, and (v) size-dependent VEGF production of microtissues which is expected to increase vascularization and tissue integration following implantation. We also established human HIV-1-based transduction of cardiomyocytes that revealed increased specific productivity of microtissues compared to isogenic populations cultivated as monolayers.