Gudrun Brandes

Tissue engineering of heart valves – human endothelial cell seeding of detergent acellularized porcine valves

OBJECTIVE Tissue engineering of heart valves represents a new experimental concept to improve current modes of therapy in valvular heart disease. Drawbacks of glutaraldehyde fixed tissue valves or mechanical valves include the short durability or the need for life-long anticoagulation, respectively. Both have in common the inability to grow, which makes valvular heart disease especially problematic in children. The aim of this study was to develop a new methodology for a tissue engineered heart valve combining human cells and a xenogenic acellularized matrix. METHODS Porcine aortic valves were acellularized by deterging cell extraction using Triton without tanning. Endothelial cells were isolated in parallel from human saphenous veins and expanded in vitro. Specimens of the surface of the acellular matrix were seeded with endothelial cells. Analysis of acellularity was performed by light microscopy and scanning electron microscopy. Cell viability following seeding was assayed by fluorescence staining of viable cells. RESULTS The acellularization procedure resulted in an almost complete removal of the original cells while the 3D matrix was loosened at interfibrillar zones. However the 3D arrangement of the matrix fibers was grossly maintained. The porcine matrix could be seeded with in vitro expanded human endothelial cells and was maintained in culture for up to 3 days to document the formation of confluent cultures. CONCLUSIONS Porcine aortic valves can be almost completely acellularized by a non-tanning detergent extraction procedure. The xenogenic matrix was reseeded with human endothelial cells. This approach may eventually lead to the engineering of tissue heart valves repopulated with the patients own autologous cells.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3′ untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Argyrin A Reveals a Critical Role for the Tumor Suppressor Protein p27kip1 in Mediating Antitumor Activities in Response to Proteasome Inhibition

A reduction in the cellular levels of the cyclin kinase inhibitor p27(kip1) is frequently found in many human cancers and correlates directly with patient prognosis. In this work, we identify argyrin A, a cyclical peptide derived from the myxobacterium Archangium gephyra, as a potent antitumoral drug. All antitumoral activities of argyrin A depend on the prevention of p27(kip1) destruction, as loss of p27(kip1) expression confers resistance to this compound. We find that argyrin A exerts its effects through a potent inhibition of the proteasome. By comparing the cellular responses exerted by argyrin A with siRNA-mediated knockdown of proteasomal subunits, we find that the biological effects of proteasome inhibition per se depend on the expression of p27(kip1).

Preclinical Testing of Tissue-Engineered Heart Valves Re-Endothelialized Under Simulated Physiological Conditions

Background— The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. Methods and Results— PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-α-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-α-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. Conclusion— In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

Effect of Clathrin Assembly Lymphoid Myeloid Leukemia Protein Depletion on Clathrin Coat Formation

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron‐specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the α‐appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino‐terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round‐coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin‐coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective: In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20‐mm sciatic nerve defect in rats. Materials and methods: We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results: Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S‐100 and neurofilament indicated existence of Schwann cells and axonal re‐growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell‐free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion: We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re‐growth in a long‐distance model for peripheral nerve regeneration.

Orthotopic replacement of the aortic valve with decellularized allograft in a sheep model

Tissue engineered (TE) allografts have been successfully applied in pulmonary circulation. The behavior of TE valves based on decellularized scaffolds in systemic circulation remains unexplored. We investigated the function, histological changes, potential of in-vivo re-endothelialization of decellularized aortic valve allografts in orthotopic position in sheep. Ovine aortic valve conduits (n=12) were decellularized with detergents and implanted as an aortic root in lambs (35-45kg). For controls, fresh native ovine aortic valve conduits (n=6) were implanted. The valves were explanted at 3 and 9 months. In the experimental group, the valves exhibited trivial regurgitation and normal morphology with no signs of graft dilatation, degeneration or rejection. In some animals (n=2), we documented minimal calcification in the area of arterial anastomosis and in one, microthrombi formation on the leaflet surface. The luminal sides of the grafts were partially covered with an endothelial cell monolayer, neovasculogenesis was observed at the adventitial side. The valves in the control group appeared thickened, shrunken with marked calcification/degeneration signs, and advanced valve insufficiency. Detergent decellularized aortic valve allografts satisfy the higher requirements of the systemic circulation in sheep. As valve conduits become repopulated by endothelial and interstitial cells, they may re-gain the potential for growth.

Phagocytosis of O4+ axonal fragments in vitro by p75− neonatal rat olfactory ensheathing cells

Olfactory ensheathing cells (OECs) have gained wide interest because of their unique regeneration‐promoting capacity. However, despite their frequent use in regeneration studies, the characterization of the cells has remained fragmentary. In the present study, we analyzed freshly dissociated neonatal rat OECs at the light and electron microscopic level and studied their fate in vitro using a novel two‐step labeling protocol based on antibody internalization. We report the identification and characterization of two distinct OEC populations in situ and in primary cell suspensions that differed in number, p75 NGF receptor expression, and O4 immunoreactivity. The major OEC population in primary cells suspensions did not express p75 but stained positive for the glycolipid O4 (p75−/O4+). During culturing, these cells upregulated p75 expression and lost O4 immunoreactivity. Conversely, the minor OEC population consisted of p75+/O4− OECs that maintained p75 expression in vitro. Interestingly, ultrastructural analysis revealed not only that O4 immunoreactivity of p75− OECs was, in fact, due to O4+ axonal fragments adhering to the cell surface but also that p75− OECs rapidly phagocytosed these fragments in vitro. Taken together, the identification of two distinct OEC populations in the neonatal olfactory bulb that converge into single p75+ phenotype in vitro is reported. The observation that upregulation of p75 receptor expression in vitro was only apparent in those OECs closely associated with O4+ axonal processes may suggest that axonal signalling in vivo negatively regulates p75 receptor expression. The strong phagocytic activity of OECs in vitro may reflect one important aspect of their physiological function.

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep.

Background Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep. Methodology/Principal Findings The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation. Conclusions/Significance This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

Clostridium difficile Toxins A and B Directly Stimulate Human Mast Cells

ABSTRACT Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D2 and E2. The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.