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Heinrich A.,University of Gottingen | Heinrich A.,Albert Ludwigs University of Freiburg | der Heyde A.S.V.,University of Gottingen | Boer U.,University of Gottingen | And 4 more authors.
Cellular Signalling | Year: 2013

Lithium salts are important drugs to treat bipolar disorder. Previous work showed that lithium by enforcing the interaction between the transcription factor CREB and its coactivator CRTC1 enhanced cAMP-stimulated CREB-dependent gene transcription. Both CREB and CRTC have been implicated in neuronal adaptation, which might underlie lithium's therapeutic action. In the present study the mechanisms of lithium action on cAMP-induced CREB-dependent gene transcription were further elucidated. Transient transfection assays revealed that all three CRTC isoforms conferred lithium responsiveness to CREB whereas their intrinsic transcriptional activities remained unchanged by lithium, suggesting a conformational change of CREB or CRTC by lithium. In in vitro protein-protein interaction assays lithium enhanced the interaction between CREB and both coactivators CRTC and CBP. Furthermore, lithium enforced the oligomerization of CRTC, a prerequisite for CREB interaction. For further evaluation it was investigated whether lithium competes with magnesium, which coordinates the conformation of the CREB basic region leucine zipper (bZip). Mutational analysis of the magnesium coordinating lysine-290 within the bZip, in vitro and intracellular interaction assays and luciferase reporter-gene assays revealed that the effect of lithium on the CREB-CRTC interaction or on the transcriptional activity, respectively, was not affected by the mutation, thus excluding a magnesium-lithium competition. However, the CREB-CRTC interaction was strongly increased in lysine-290-mutants thereby extending the CRTC-CREB interaction domain. Taken together the results exclude a competition between lithium and magnesium at the bZip, but suggest that lithium by enforcing the CRTC-oligomer formation and the interaction of CREB-CBP-CRTC enhances cAMP-induced CREB-dependent gene transcription. © 2012 Elsevier Inc.

Boer U.,GMP Model Laboratory for Tissue Engineering | Boer U.,Hannover Medical School | Hurtado-Aguilar L.G.,AME Institute of Applied Medical Engineering | Klingenberg M.,GMP Model Laboratory for Tissue Engineering | And 8 more authors.
Annals of Biomedical Engineering | Year: 2015

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts. © 2015 Biomedical Engineering Society

Boer U.,GMP Model Laboratory for Tissue Engineering | Boer U.,Hannover Medical School | Spengler C.,GMP Model Laboratory for Tissue Engineering | Jonigk D.,Hannover Medical School | And 11 more authors.
Tissue Engineering - Part A | Year: 2013

Decellularized equine carotid arteries (dEAC) are potential alternatives to alloplastic vascular grafts although there are certain limitations in biocompatibility and immunogenicity. Here, dEAC were coated with the matricellular protein CCN1 and evaluated in vitro for its cytotoxic and angiogenic effects and in vivo for cellular repopulation, local biocompatibility, neovascularization, and immunogenicity in a sheep model. CCN1 coating resulted in nontoxic matrices not compromising viability of L929 fibroblasts and endothelial cells (ECs) assessed by WST-8 assay. Functionality of CCN1 was maintained as it induced typical changes in fibroblast morphology and MMP3 secretion. For in vivo testing, dEAC±CCN1 (n=3 each) and polytetrafluoroethylene (PTFE) protheses serving as controls (n=6) were implanted as cervical arteriovenous shunts. After 14 weeks, grafts were harvested and evaluated immunohistologically. PTFE grafts showed a patency rate of only 33% and lacked cellular repopulation. Both groups of bioartificial grafts were completely patent and repopulated with ECs and smooth muscle cells (SMCs). However, whereas dEAC contained only patch-like aggregates of SMCs and a partial luminal lining with ECs, CCN1-coated grafts showed multiple layers of SMCs and a complete endothelialization. Likewise, CCN1 coating reduced leukocyte infiltration and fibrosis and supported neovascularization. In addition, in a three-dimensional assay, CCN1 coating increased vascular tube formation in apposition to the matrix 1.6-fold. Graft-specific serum antibodies were increased by CCN1 up to 6 weeks after implantation (0.89±0.03 vs. 1.08±0.04), but were significantly reduced after 14 weeks (0.85±0.04 vs. 0.69±0.02). Likewise, restimulated lymphocyte proliferation was significantly lower after 14 weeks (1.78±0.09 vs. 1.32±0.09-fold of unstimulated). Thus, CCN1 coating of biological scaffolds improves local biocompatibility and accelerates scaffold remodeling by enhancing cellular repopulation and immunologic tolerance, making it a promising tool for generation of bioartificial vascular prostheses. © 2013, Mary Ann Liebert, Inc.

Boer U.,GMP Model Laboratory for Tissue Engineering | Boer U.,Hannover Medical School | Lohrenz A.,GMP Model Laboratory for Tissue Engineering | Klingenberg M.,GMP Model Laboratory for Tissue Engineering | And 6 more authors.
Biomaterials | Year: 2011

Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices were homogenized and extracted by high detergent concentrations westernblot analyses revealed cytosolic and cytosceleton proteins like GAPDH and smooth muscle actin which were depleted to 4.1 ± 1.9% and 13.8 ± 0.55%, resp. Also putative immunogenic MHC I complexes and the alpha-Gal epitop were reduced to only 14.8 ± 1.2% and 15.1 ± 2.05%. Mass spectrometry of matrix extracts identified 306 proteins belonging to cytosol, organelles, nucleus and cell membrane. Moreover, aqueous matrix extracts evoked a pronounced antibody formation when administered in mice and thus display high immunogenic potential. Our data indicate that an established decellularization protocol which results in acellular matrices evaluated by low DNA content reduces but not eliminates cellular components which may contribute to its immunogenic potential in vivo. © 2011 Elsevier Ltd.

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