Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels

Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secrete and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options

Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome

Introduction: Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS. Materials and methods: We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies. Results: The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS “criteria” antibody against CL (P = 0.764). Conclusion: We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS

Global comparative transcriptome analysis of cartilage formation in vivo

<p>Abstract</p> <p>Background</p> <p>During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of <it>in vivo </it>gene expression.</p> <p>Results</p> <p>Tibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for <it>Agc1</it>, <it>Bmp8a</it>, <it>Col2a1</it>, <it>Fgfr4</it>, <it>Foxa3</it>, <it>Gdf5</it>, <it>Klf2</it>, <it>Klf4</it>, <it>Lepre1</it>, <it>Ncad</it>, <it>Sox11</it>, and <it>Trpv4</it>. Further, independent validation of the microarray data was achieved by <it>in situ </it>hybridization to analyse the expression of <it>Lepre1</it>, <it>Pcdh8</it>, <it>Sox11</it>, and <it>Trpv4 </it>from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several <it>Bmp, Gdf, Wnt, Sox and Fox </it>family members.</p> <p>Conclusion</p> <p>These data represent the first global gene expression profiling analysis of chondrogenic tissues during <it>in vivo </it>development. They identify genes for further study on their functional roles in chondrogenesis, and provide a comprehensive and important resource for future studies on cartilage development and disease.</p

Loss of maternal annexin A5 increases the likelihood of placental platelet thrombosis and foetal loss

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.Peer reviewe

MXene-Integrated Silk Fibroin-Based Self-Assembly-Driven 3D-Printed Theragenerative Scaffolds for Remotely Photothermal Anti-Osteosarcoma Ablation and Bone Regeneration

Aiming to address the bone regeneration and cancer therapy functionalities in one single material, in this study, we developed a dual-functional theragenerative three-dimensional (3D) aerogel-based composite scaffold from hybridization of photo-cross-linked silk fibroin (SF) biopolymer with MXene (Ti3C2) two-dimensional (2D) nanosheets. To fabricate the scaffold, we first develop a dual-cross-linked SF-based aerogel scaffold through 3D printing and photo-cross-linking of the self-assembly-driven methacrylate-modified SF (SF-MA) gel with controlled pore size, macroscopic geometry, and mechanical stability. In the next step, to endow a remotely controlled photothermal antiosteosarcoma ablation function to fabricated aerogel scaffold, MXene 2D nanosheets with strong near-infrared (NIR) photon absorption properties were integrated into the 3D-printed scaffolds. While 3D-printed MXene-modified dual-cross-linked SF composite scaffolds can mediate the in vitro growth and proliferation of preosteoblastic cell lines, they also endow a strong photothermal effect upon remote irradiation with NIR laser but also significantly stimulate bone mineral deposition on the scaffold surface. Additionally, besides the local release of the anticancer model drug, the generated heat (45-53 ?) mediated the photothermal ablation of cancer cells. The developed aerogel-based composites and chosen therapeutic techniques are thought to render a significant breakthrough in biomaterials' future clinical applications

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against ß2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS. The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages

Effects of long-term immobilisation on endomysium of the soleus muscle in humans

Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-tofibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness

Effects of long‐term immobilisation on endomysium of the soleus muscle in humans

New findings: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. Abstract: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness