ß-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis.

Trabecular bone formation is the last step in endochondral ossification. This remodeling process of cartilage into bone involves blood vessel invasion and removal of hypertrophic chondrocytes (HTCs) by chondroclasts and osteoclasts. Periosteal- and chondrocyte-derived osteoprogenitors utilize the leftover mineralized HTC matrix as a scaffold for primary spongiosa formation. Here, we show genetically that ß-catenin (encoded by Ctnnb1), a key component of the canonical Wnt pathway, orchestrates this remodeling process at multiple levels. Conditional inactivation or stabilization of ß-catenin in HTCs by a Col10a1-Cre line locally modulated osteoclastogenesis by altering the Rankl:Opg ratio in HTCs. Lack of ß-catenin resulted in a severe decrease of trabecular bone in the embryonic long bones. Gain of ß-catenin activity interfered with removal of late HTCs and bone marrow formation, leading to a continuous mineralized hypertrophic core in the embryo and resulting in an osteopetrotic-like phenotype in adult mice. Furthermore, ß-catenin activity in late HTCs is required for chondrocyte-derived osteoblastogenesis at the chondro-osseous junction. The latter contributes to the severe trabecular bone phenotype in mutants lacking ß-catenin activity in HTCs.

Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity.

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.

Synergistic control of mesenchymal stem cell differentiation by nanoscale surface geometry and immobilized growth factors on TiO2 nanotubes.

The aim of this study is to elucidate whether combined environmental signals provided by nanoscale topography and by growth factors control cell behavior of mesenchymal stem cells (MSCs) in a synergistic or simply additive manner. Chondrogenic and osteogenic differentiation of MSCs is studied on vertically aligned TiO(2) nanotubes of size 15 and 100 nm with and without immobilized bone morphogenetic protein-2 (BMP-2). Although BMP-2 coating stimulates both chondrogenic and osteogenic differentiation of MSCs, the response strongly depends on the surface nanoscale geometry of the BMP-2-coated nanotubes. Chondrogenic differentiation is strongly supported on 100 nm BMP-2-coated nanotubes, but not on 15 nm nanotubes, which induce spreading and de-differentiation of chondrocytes. A similar response is observed with primary chondrocytes, which maintain their chondrogenic phenotype on BMP-2-coated 100 nm nanotubes, but de-differentiate on 15 nm nanotubes. In contrast, osteogenic differentiation is greatly enhanced on 15 nm but not on 100 nm BMP-2-coated nanotubes as shown previously. Furthermore, covalent immobilization of BMP-2 rescues MSCs from apoptosis occurring on uncoated 100 nm TiO(2) nanotube surfaces. Thus, combined signals provided by BMP-2 immobilized to a defined lateral nanoscale spacing geometry seem to contain environmental cues that are able to modulate a lineage-specific decision of MSC differentiation and cell survival in a synergistic manner.

Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue.

OBJECTIVE: To investigate the effect of chondromodulin 1 on the phenotype of osteochondral progenitor cells in cartilage repair tissue. METHODS: Self-complementary adeno-associated virus (AAV) vectors carrying chondromodulin 1 complementary DNA (AAV-Chm-1) were applied to cartilage lesions in the knee joints of miniature pigs that were treated by the microfracture technique. Alternatively, isolated porcine osteochondral progenitor cells were infected with AAV-Chm-1 or with AAV-GFP control vectors ex vivo prior to being transplanted into cartilage lesions in which the subchondral bone plate was left intact. The quality of the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods. The effects of chondromodulin 1 overexpression were also analyzed by angiogenesis assays and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: AAV-Chm-1-infected cells efficiently produced chondromodulin 1, which had strong antiangiogenic effects, as verified by the inhibition of tube formation of endothelial cells. Gene expression analyses in vitro revealed the cell cycle inhibitor p21WAF1/Cip1 as one target up-regulated by AAV-Chm-1. Direct application of AAV-Chm-1 vectors into microfractured porcine cartilage lesions stimulated chondrogenic differentiation of ingrowing progenitor cells, but significantly inhibited terminal chondrocyte hypertrophy, the invasion of vessel structures, and excessive endochondral ossification, which were otherwise observed in untreated lesions. Indirect gene transfer, with infection of porcine osteochondral progenitor cells by AAV-Chm-1 ex vivo, also supported chondrogenic differentiation of these transplanted cells. AAV-Chm-1-infected cells maintained a chondrocyte-like phenotype and formed a hyaline-like matrix that was superior to that formed by uninfected or AAV-GFP-infected cells. CONCLUSION: Our findings indicate that the antiangiogenic factor chondromodulin 1 stabilizes the chondrocyte phenotype by supporting chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral ossification.

Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer.

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA-microarray, real-time-quantitative-PCR and immunohistochemistry. DNA-microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I-III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real-time-quantitative-PCR confirmed the results of the DNA-microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA-microarry and real-time-quantitative-PCR results on the protein level. Finally, these findings correspond with the surgeon's clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis.

Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Here we report that Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone. Coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt factors show specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Moreover, Wif-1 was able to block Wnt3a-mediated activation of the canonical Wnt signalling pathway. Consequently, Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells. These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage biology.

Correction of Vertebral Bone Development in Ectodysplasin A1-Deficient Mice by Prenatal Treatment With a Replacement Protein.

X-linked hypohidrotic ectodermal dysplasia with the cardinal symptoms hypodontia, hypotrichosis and hypohidrosis is caused by a genetic deficiency of ectodysplasin A1 (EDA1). Prenatal EDA1 replacement can rescue the development of skin appendages and teeth. Tabby mice, a natural animal model of EDA1 deficiency, additionally feature a striking kink of the tail, the cause of which has remained unclear. We studied the origin of this phenomenon and its response to prenatal therapy. Alterations in the distal spine could be noticed soon after birth, and kinks were present in all Tabby mice by the age of 4 months. Although their vertebral bones frequently had a disorganized epiphyseal zone possibly predisposing to fractures, cortical bone density was only reduced in vertebrae of older Tabby mice and even increased in their tibiae. Different availability of osteoclasts in the spine, which may affect bone density, was ruled out by osteoclast staining. The absence of hair follicles, a well-known niche of epidermal stem cells, and much lower bromodeoxyuridine uptake in the tail skin of 9-day-old Tabby mice rather suggest the kink being due to a skin proliferation defect that prevents the skin from growing as fast as the skeleton, so that caudal vertebrae may be squeezed and bent by a lack of skin. Early postnatal treatment with EDA1 leading to delayed hair follicle formation attenuated the kink, but did not prevent it. Tabby mice born after prenatal administration of EDA1, however, showed normal tail skin proliferation, no signs of kinking and, interestingly, a normalized vertebral bone density. Thus, our data prove the causal relationship between EDA1 deficiency and kinky tails and indicate that hair follicles are required for murine tail skin to grow fast enough. Disturbed bone development appears to be partially pre-determined in utero and can be counteracted by timely EDA1 replacement, pointing to a role of EDA1 also in osteogenesis.

Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix.

The ultimate goal in the design of biomimetic materials for use in tissue engineering as permanent or resorbable tissue implants is to generate biocompatible scaffolds with appropriate biomechanical and chemical properties to allow the adhesion, ingrowth, and survival of cells. Recent efforts have therefore focused on the construction and modification of biomimetic surfaces targeted to support tissue-specific cell functions including adhesion, growth, differentiation, motility, and the expression of tissue-specific genes. Four decades of extensive research on the structure and biological influence of the extracellular matrix (ECM) on cell behavior and cell fate have shown that three types of information from the ECM are relevant for the design of biomimetic surfaces: (1) physical properties (elasticity, stiffness, resilience of the cellular environment), (2) specific chemical signals from peptide epitopes contained in a wide variety of extracellular matrix molecules, and (3) the nanoscale topography of microenvironmental adhesive sites. Initial physical and chemical approaches aimed at improving the adhesiveness of biomaterial surfaces by sandblasting, particle coating, or etching have been supplemented by attempts to increase the bioactivity of biomaterials by coating them with ECM macromolecules, such as fibronectin, elastin, laminin, and collagens, or their integrin-binding epitopes including RGD, YIGSR, and GFOGER. Recently, the development of new nanotechnologies such as photo- or electron-beam nanolithography, polymer demixing, nano-imprinting, compression molding, or the generation of TiO(2) nanotubes of defined diameters (15-200 nm), has opened up the possibility of constructing biomimetic surfaces with a defined nanopattern, eliciting tissue-specific cellular responses by stimulating integrin clustering. This development has provided new input into the design of novel biomaterials. The new technologies allowing the construction of a geometrically defined microenvironment for cells at the nanoscale should facilitate the investigation of nanotopography-dependent mechanisms of integrin-mediated cell signaling.

Narrow window in nanoscale dependent activation of endothelial cell growth and differentiation on TiO2 nanotube surfaces.

Critical features of biomimetic materials used for vascular grafts and stents are surface structure and chemical features of the implant material supporting adhesion, proliferation, and differentiation of endothelial cells and smooth muscle cells, the major cell types of blood vessels. Recently, experimental evidence from several laboratories have indicated a strong stimulation of cellular activities on vertically aligned TiO(2) nanotube surfaces in comparison to amorphous TiO(2) surfaces. Conflicting reports exist, however, concerning the nanoscale dimension, and the role of the chemistry and crystallinity of the nanotubes in eliciting cell responses. Here we demonstrate that 15 nm nanotubes provide a substantially stronger stimulation of differentiation of mesenchymal cells to endothelial cells and smooth muscle cells than 70-100 nm nanotubes, while high rates of apoptosis were seen on 100 nm nanotubes. Also endothelial cell adhesion, proliferation, and motility were several-fold higher on 15 nm than on 100 nm nanotubes. Furthermore, our data indicate a clear dominance of the nanoscale geometry on endothelial cell behavior over surface chemistry and crystallinity of the TiO(2) nanotube surface. These findings indicate that fine-tuning of TiO(2) surfaces at nanoscale will be an essential parameter in optimizing endothelial cell and smooth muscle cell responses to vascular implants.

Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages of chondrocyte differentiation.

Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor-like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function.

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts.

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.

Molecular dynamics simulations on human fibulin-4 mutants D203A and E126K reveal conformational changes in EGF domains potentially responsible for enhanced protease lability and impaired extracellular matrix assembly.

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca2+ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-ß-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca2+ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca2+ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes.

Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter.

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183-194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.

Distinct acidic clusters and hydrophobic residues in the alternative splice domains X1 and X2 of alpha7 integrins define specificity for laminin isoforms.

The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.

Improved attachment of mesenchymal stem cells on super-hydrophobic TiO2 nanotubes.

Self-organized layers of vertically orientated TiO(2) nanotubes providing defined diameters ranging from 15 up to 100nm were grown on titanium by anodic oxidation. These TiO(2) nanotube layers show super-hydrophilic behavior. After coating TiO(2) nanotube layers with a self-assembled monolayer (octadecylphosphonic acid) they showed a diameter-dependent wetting behavior ranging from hydrophobic (108+/-2 degrees ) up to super-hydrophobic (167+/-2 degrees ). Cell adhesion, spreading and growth of mesenchymal stem cells on the unmodified and modified nanotube layers were investigated and compared. We show that cell adhesion and proliferation are strongly affected in the super-hydrophobic range. Adsorption of extracellular matrix proteins as fibronectin, type I collagen and laminin, as well as bovine serum albumin, on the coated and uncoated surfaces showed a strong influence on wetting behavior and dependence on tube diameter.

The role of annexin A5 in the modulation of the immune response against dying and dead cells.

Annexins are characterized by the ability to bind phospholipids of membranes in the presence of Ca2+. Annexin A5 represents a typical member of this protein family and is a natural occurring highly specific ligand for phosphatidylserine (PS). The exposure of PS is one major "eat me" signal for phagocytes of apoptotic and necrotic cells. Apoptotic cells are normally cleared via an anti-inflammatory pathway. In contrast, the uptake and removal of necrotic cells normally involves inflammation and an immune response. Interestingly, the lack of endogenous annexin A5 also leads to a reduced inflammatory potential of necrotic cells. Annexin A5 may interfere in vivo with the immunosuppressive effects of apoptotic cells since it preferentially binds PS with high affinity and inhibits apoptotic cell uptake by macrophages. In this review we focus on how defects in the clearance process can lead to chronic autoimmunity. Furthermore, the role of annexin A5 as important adjuvant for apoptotic cell-based tumour vaccines is discussed. The mechanism of how the immunogenicity of apoptotic cells can be restored by blocking their PS-dependent clearance is outlined in detail. Taken together, annexin A5 is an important modulator of the immune response against PS-exposing particles like apoptotic cells, necrotic cells, and certain viruses.

Modulation of the immune system by dying cells and the phosphatidylserine-ligand annexin A5.

Apoptotic cell death and the efficient clearance of dying cells are essential mechanisms to control tissue homeostasis and to eliminate potential autoantigens. Numerous alterations on the surfaces of dying cells define a highly characteristic membrane signature and enable an unequivocal distinction from vital cells. This way, phagocytosis is initiated and signalling events induced which minimize inflammatory reactions. Therefore, the use of proteins interfering with the clearance process may open up new vistas to improve immunization strategies and may help to understand the mechanisms of autoimmune diseases.

Annexin A5 is not essential for skeletal development.

Annexins are highly conserved proteins that are characterized by their ability to interact with phospholipids in a calcium-dependent manner. Although diverse functions have been ascribed to annexins based on in vitro analyses, their in vivo functions still remain unclear. The intensively studied annexin A5 has been identified by its effects on blood coagulation, and subsequently, its function as a calcium-specific ion channel was described. In vitro experiments and expression studies suggested a potential role of annexin A5 during calcification processes in vivo, especially in endochondral ossification. To gain insights into the relevance of annexin A5 in this process, we generated an annexin A5-deficient mouse mutant. Mice lacking annexin A5 are viable, are fertile, and reveal no significant alterations in the biochemical parameters characteristic for metabolic or functional defects. Neither the development of skeletal elements nor the in vitro calcification properties of isolated chondrocytes is significantly impaired by the absence of annexin A5. Therefore, annexin A5 is dispensable for the formation and maintenance of skeletal elements in the mouse and may possibly be pointing to a compensatory effect of other members from the annexin family due to their high functional and structural similarity.