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.

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.

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Annexin A5 (annexin V, anchorin CII) represents the prototype member of the large annexin family, characterized by its ability to interact with phospholipids in a calcium-dependent manner and to form calcium-specific ion channels. Despite intense biochemical analysis, the in vivo expression and function of this annexin during mouse development, still remains unclear. Immunohistochemistry, in situ hybridization and reporter gene expression were used to define expression of annexin A5 during early mouse development. First, annexin A5 expression is associated with the developing vascular system. Later, expression is detected within the notochord and found in parallel to the differentiation of cartilage and bone. Therefore, expression of the Anxa5 gene may represent a novel marker characterizing cell lineages involved in the development of the vascular as well as the skeletal system.

The immediate-early 1 and 2 gene locus of human cytomegalovirus (HCMV) that encodes trans-activator proteins with effects on both homologous and heterologous promoters is expressed under control of a complex enhancer/promoter regulatory region. This enhancer contains four types of repetitive sequence elements with 17, 18, 19, and 21 bp that bind cellular transcription factors. Although the HCMV enhancer acts as a powerfil stimulator of transcription in most cell types examined, human T cells do not support strong activity. The present study demonstrates that the tax gene product of human T-cell leukemia virus type I trans activates the major enhancer of HCMV more than 60-fold in the T-cell line Jurkat. When a series of chloramphenicol acetyltransferase expression plasmids containing synthetic oligonucleotides with the 17-, 18-, 19-, or 21-bp motif upstream of a minimal immediate-early 1 and 2 gene promoter was tested, two of the four repeat motifs could be identified as Tax-responsive elements. Both the 18and the 19-bp motifs were able to act as strong Tax-responsive elements even when they were present as single copies. Thus, in addition to interacting with human immunodeficiency virus, HCMV is able to interact with a second retrovirus of clinical importance.

Integrins have become a target for novel therapeutic strategies against malignant gliomas. Cilengitide, a synthetic Arg-Gly-Asp (RGD)-motif peptide, interferes with ligand binding to avb3 and avb5 integrins and is currently investigated in clinical trials. Integrins may also be involved in the activation of transforming growth factor (TGF)-b, a mediator of invasiveness and immune escape of glioma cells. Using flow cytometry, we demonstrate that the target integrins of cilengitide are expressed not only in glioblastoma blood vessels, but also by tumor cells. After exposure of glioma cells to cilengitide, we noticed reduced phosphorylation of Smad2 in most glioma cell lines, including stem-like glioma cells. Phophorylation of Smad2, but not cilengitide-induced detachment, is rescued by addition of recombinant TGF-b. Administration of cilengitide to glioma cells results in reduced TGF-b-mediated reporter gene activity. Furthermore, exposure to cilengitide leads to decreased TGF-b 1 and TGF-b 2 mRNA and protein expression. These effects are mimicked by blocking av, b3 or b5 antibodies or by silencing of integrins av, b3, b5 or b8 using RNA interference. Treatment of mice bearing experimental LN-308 glioma xenografts with cilengitide results in reduced pSmad2 levels. Taken together, cilengitide may exert anti-invasive and immune stimulatory activity in human glioblastoma patients by its anti-TGF-b properties.

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