Britta Blumenthal

Polyurethane Scaffolds Seeded With Genetically Engineered Skeletal Myoblasts: A Promising Tool to Regenerate Myocardial Function

In animal models, intramyocardial injection of primary skeletal myoblasts is supposed to promote tissue regeneration and to improve cardiac function after myocardial infarction. The usage of genetically engineered myoblasts overexpressing the paracrine factors involved in tissue repair is believed to enhance these effects. However, cell therapy via injection is always accompanied by a high death rate of the injected cells. Here, we describe the construction of a growth factor-producing myoblast-seeded scaffold to overcome this limitation. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were seeded on polyurethane (PU) scaffolds (Artelon) and transfected with DNA of VEGF-A, HGF, SDF-1, or Akt1 using the lipid-based Metafectene Pro method. Overexpression was verified by ELISA, RT-PCR (VEGF-A, HGF, and SDF-1) and Western blot analysis (Akt1). The seeded scaffolds were transplanted onto damaged myocardium of Lewis rats 2 weeks after myocardial infarction. Six weeks later, their therapeutic potential in vivo was analyzed by measurement of infarction size and capillary density. Primary rat skeletal myoblasts seeded on PU scaffolds were efficiently transfected, achieving transfection rates of 20%. In vitro, we noted a significant increase in expression of VEGF-A, HGF, SDF-1, and Akt1 after transfection. In vivo, transplantation of growth factor-producing myoblast-seeded scaffolds resulted in enhanced angiogenesis (VEGF-A, HGF, and Akt1) or a reduced infarction zone (SDF-1 and Akt1) in the ischemically damaged myocardium. In summary, we constructed a growth factor-producing myoblast-seeded scaffold which combines the beneficial potential of stem cell transplantation with the promising effects of gene-therapeutic approaches. Because this matrix also allows us to circumvent previous cell application drawbacks, it may represent a promising tool for tissue regeneration and the re-establishment of cardiac function after myocardial infarction.

The Cytotoxic Necrotizing Factors from Yersinia pseudotuberculosis and from Escherichia coli Bind to Different Cellular Receptors but Take the Same Route to the Cytosol

ABSTRACT The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNFY of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNFY exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNFY does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNFY into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNFY, suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schägger and von Jagow 1991); (Schägger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Cleavage of Escherichia coli cytotoxic necrotizing factor 1 is required for full biologic activity.

ABSTRACT Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by pathogenic Escherichia coli strains. CNF1 constitutively activates small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. The toxin is taken up into mammalian cells by receptor-mediated endocytosis and is delivered from late endosomes into the cytosol. Here, we show that an approximately 55-kDa fragment of CNF1, which contains the catalytic domain and an additional part of the toxin, is present in the cytosol. The processing of this fragment requires an acidic pH and insertion of the toxin into the endosomal membrane. We define the cleavage site region as the region located between amino acids 532 and 544 of CNF1. The data provide insight into the complex mechanism of uptake of bacterial toxins into mammalian cells.

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.

Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy

The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.

Hepatocyte growth factor-transfected skeletal myoblasts to limit the development of postinfarction heart failure.

Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF-overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold-based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU-HGF-SkM) or the infection solution was intramyocardially injected (group: Inj-HGF-SkM). Two groups (Inj-SkM and PU-SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control (n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU-HGF-SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB-Ked, pressure half time [PHT], Tau). In group Inj-HGF-SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU-HGF-SkM than in sham and Inj-SkM group. The infarction size was not affected by the therapy. Transplanting HGF-transfected myoblasts after MI can limit the development of ventricular dysfunction. Scaffold-based therapy in combination with gene therapy accelerates this capacity. This hemodynamic amelioration is accompanied by neovascularization, but not by smaller infarction sizes.

Functional regeneration of ischemic myocardium by transplanted cells overexpressing stromal cell-derived factor-1 (SDF-1): intramyocardial injection versus scaffold-based application §

OBJECTIVE Stromal cell-derived factor-1 (SDF-1) is a potent chemotaxin. Increased SDF-1 levels can be found in ischemic myocardium and might protect against ischemia-reperfusion injury. We hypothesized that transplantation of stem cells overexpressing SDF-1 might improve cardiac function after myocardial infarction (MI). We compared intramyocardial injection with a scaffold-based application of SDF-1-transfected cells. METHODS Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huSDF-1 and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. Two weeks after myocardial infarction, seeded scaffolds were implanted epicardially into rats (group: PU-SDF-1-SkM) or the injection solution was applied intramyocardially (Inj-SDF-1-SkM). Additional groups were treated with non-transfected myoblasts either by injection (Inj-SkM) or by scaffold-based application (PU-SkM) or received a sham operation (Sham). Before this intervention and 6 weeks later, hemodynamic parameters were measured. Infarction size and neovascularization were assessed by histology at study end. RESULTS In sham animals, we detected a clear decrease in systolic function from intervention to study end. In group Inj-SkM and PU-SkM, all hemodynamic parameters that were assessed remained unchanged during observation time. Systolic function as measured by dP/dt(max) and SB-Emax was significantly improved in groups Inj-SDF-1-SkM and PU-SDF-1-SkM at study end without a difference between the two SDF-1 groups. Diastolic function measured by post-interventional dP/dt(min) was also increased in group Inj-SDF-1-SkM but not in PU-SDF-1-SkM. Histological analysis revealed a reduced infarction size in all treatment groups at study end but enhanced neovascularization was not observable. CONCLUSIONS Transplantation of myoblasts overexpressing SDF-1 improves cardiac function after MI. The restoration of hemodynamic parameters is accompanied by a reduction in infarction size. This reverse remodeling capacity is independent of a scaffold-based application of the SDF-1-transfected cells.

Scaffold-based transplantation of vascular endothelial growth factor-overexpressing stem cells leads to neovascularization in ischemic myocardium but did not show a functional regenerative effect.

The transplantation of skeletal myoblasts (SkM) might improve cardiac function after myocardial infarction via paracrine action. We used scaffold-based cell transfer by using vascular endothelial growth factor (VEGF)-overexpressing myoblasts. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were transfected with pCINeo-VEGF121 and seeded on polyurethane (PU) scaffolds. The seeded scaffolds were epicardially implanted in rats 2 weeks after myocardial infarction (group: PU-VEGF-SkM). Before this intervention and 6 weeks later, pressure/volume loops were analyzed followed by histology. Additional study groups (n = 10 per group) were injected with VEGF-overexpressing myoblasts (Inj-VEGF-SkM) or unmodified myoblasts (Inj-SkM) or underwent a sham operation. Overexpression of VEGF was verified in vitro. The transplantation of growth factor producing myoblast-seeded scaffolds resulted in enhanced angiogenesis of ischemically damaged myocardium in vivo. However, the infarction size was not reduced. In group Inj-SkM, hemodynamics remained unchanged. Systolic function as measured by dP/dt(max) was not significantly altered in PU-VEGF-SkM (preinterventionally 2,156 ± 1,222 mmHg vs. postinterventionally 2,134 ± 850 mmHg). Other systolic function and diastolic function parameters as measured by dP/dt(min), tau, and pressure half-time were not restored in groups PU-VEGF-SkM and Inj-VEGF-SkM either. Transplantation of VEGF-overexpressing skeletal myoblasts leads to neovascularization in infarcted hearts. No functional myocardial recovery was observed. Scaffold-based transfer of genetically-modified cells remains a useful tool to study paracrine stem cell action.

Detection of phosphorylated T and B cell antigen receptor species by Phos-tag SDS- and Blue Native-PAGE

Detection of phospho-proteins and differently phosphorylated forms of the same protein are important in understanding cell behaviour. One novel method is Phos-tag SDS-PAGE. A dinuclear Mn(2+) complex that binds to phosphate groups (the Phos-tag) is covalently attached to the polyacrylamide gel matrix. Thus, phosphorylated proteins are retarded in their migration and can be distinguished from their non-phosphorylated counterparts. We applied Phos-tag SDS-PAGE to the analysis of the zeta, CD3epsilon and CD3delta subunits of the T cell antigen receptor (TCR-CD3). Pervanadate stimulation generated six different phospho-zeta and each two different CD3epsilon and CD3delta forms. This corresponds to the phosphorylatable tyrosines on their cytoplasmic tails. The phosphorylation pattern was compatible with random phosphorylation events. Further, we showed that the Phos-tag technology can be applied to Blue Native (BN)-PAGE. This extends the applicability to the analysis of native protein complexes. Upon pervanadate stimulation the TCR-CD3 complex was predominantly detected as two distinct phospho-complexes. In contrast, the B cell antigen receptor (BCR) appeared as one phospho-form. Thus, Phos-tag BN-PAGE is useful for the analysis of different phosphorylation states of multiprotein complexes.