Tissue vascularization is requisite to successful cell‐based therapies, biomaterial design and implant integration. Thus, known problems in ossointegration of avascular implants in connection with the generation of bone tissue reflect arrays of general problems of socio‐economic relevance existing in reparative medicine still waiting for to be solved. For this purpose, morphogenesis and remodeling of endothelial angio‐architectures in tissue and in vitro by isolated non‐mitogenic angio‐morphogens [angiotropins] are considered in terms of their structure, function and action mechanisms. Extracellular angiotropins are secreted by activated leukocytes/monocytes/macrophages. They are a family of cytokines with morphogen bioactivity selectively directed to endothelial cells. Their structure was deciphered as metalloregulated copper‐ribonucleoproteins [CuRNP ribokines]. They are built up of angiotropin‐related S100‐EF‐hand protein [ARP] and highly modified and edited 5'end‐phosphorylated RNA [ARNA], complexed together by copper ions. Oxidant‐sensitive ARNA and their precursors represent novel types in a RNA world: They are the first isolated and sequenced forms of extracellular RNA [eRNA], may act as cytokine and bioaptamer, contain isoguanosine [crotonoside] as modified nucleoside and show up copper as RNA‐structuring transition metal ion. By metalloregulated bioaptamer functions, ARNA impart novel biofunctions to RAGE‐binding S100‐EF‐hand proteins. Angiotropin morphogens were shown suitable for neointiation and remodeling of blood vessel patterns in different, adult, embryonal and artificial tissues. These neovascular patterns manifest regulated hemodynamics for preventing tissue necrosis, supporting tissue functions and promoting wound healing. As evaluated in skin and muscle vascularization, the neovascular patterns are integrated into homeostatic control mechanisms of tissue. Thus, the morphogens show up beneficial perspectives and are suggested useful tools for further investigations in angiotherapy, engineering of blood vessel patterns in tissues and biocompatible artificial organs as well as in the preparation of novel implants with morphogen‐coated surfaces.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo-and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used 1-4 , no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay 5,6 , (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye 7-9 , (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM 10-12 , (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice 13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM 14 .In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study. ProtocolAll equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and ...

Bioassays for cellular differentiation and tissue morphogenesis were used to design methods for isolation of bioactive redox‐ and metalloregulated nucleic acids and copper ion complexes with proteins from extracellular, circulating, wound, and supernatant fluids of cultured cells. In extracellular biospheres, diversities of nucleic acids were found to be secreted by cells upon activation. They may reflect nucleic acid biolibraries with molecular imprints of cellular history. After removal of protein components, eRNA prototypes exuded by activated cells were sequenced. They are small, endogenous, highly modified and edited, redox‐ and metalloregulated 5′‐end phosphorylated extracellular eRNA (∼2–200 bases) with cellular, enzymic, and bioaptamer functions. Fenton‐type OH* radical redox reactions may form modified nucleotides in RNA as wobbles eRNA per se, or as copper ion‐complex with protein (e.g., S100A12‐EF‐hand protein, angiotropin‐related protein, calgranulin‐C, hippocampal neurite differentiation factor) are shown to be bioactive in vivo and in vitro as cytokines (ribokines) and as nonmitogenic angiomorphogens for endothelial cell differentiation in the formation of organoid supracellular capillary structures. As bioaptamers, copper ion‐structured eRNA imparts novel biofunctions to proteins that they do not have on their own. The origin of extracellular RNA and intermediate precursors (up to 500 bases) was traced to intracellular parent nucleic acids. Intermediate precursors with and without partial homology were found. This suggests that bioaptamers are not directly retranslatable gene products. Metalloregulated eRNA bioaptamer function was investigated by domains (e.g. 5′…CUG…3′ hairpin loop) for folding, bioactivity, and binding of protein with copper, calcium, and alkali metal ion affinity. Vice versa, metalloregulated nucleic acid‐binding domains (K3H, R3H) in proteins were identified. Interaction of protein and eRNA docking potentials were visualized by 3D‐rapid prototyping of accurate molecular image models based on crystallographic or NMR data. For S100A12‐homologous proteins, receptor‐ and metalloregulated RNA chaperone‐shaped protein assemblies were investigated. They suggest insight into signaling cascades as to how eRNA transmits its cytokine (ribokine) bioinformation from the extracellular RNA biosphere into cells. Proteomics of the extracellular RNA biosphere demonstrate the presence of nucleic acid‐binding domain homologies in defense‐, aging‐, and disease‐associated neuronal and other proteins as targets for RNA orphans. By structural relationships found to transmissible processes, proteinaceous transfer (“infectivity”) and feedback of bioinformation beyond the central dogma of molecular biology are considered in terms of metalloregulated RNA bioaptamer function, nucleic acid‐binding domains, and protein conformation.

No abstract

No abstract