Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages

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.     

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.     

Intracellular localization of endothelial cell annexins is differentially regulated by oxidative stress

Annexins are calcium-dependent phospholipid binding proteins that are implicated in the regulation of both intracellular and extracellular thrombostatic mechanisms in the vascular endothelium. Tight control of annexin gene expression and targeting of annexin proteins is therefore of importance in maintaining the health of the endothelium. Because annexins are abundant in vascular endothelial cells and could be either dysregulated by or contribute to anomalies in Ca2+ signaling, we investigated annexin gene expression and subcellular localization in human umbilical vein endothelial cells (HUVEC) in a model of chronic oxidative stress. HUVEC were cultured under mild hyperoxic conditions in a custom-built chamber to induce oxidative stress over a period of 12 days. Although annexin expression levels did not change significantly in response to hyperoxic stress, immunofluorescence analysis revealed striking effects on the subcellular localization of certain annexins, including the redistribution of annexins 5 and 6 from the cytosol to the nucleus. In addition, oxidative stress modulated the responses of certain annexins to stimulation with a range of pharmacological and physiological Ca2+-mobilizing agonists, in a manner that suggested that annexin localization is regulated via the complex integration of both Ca2+ and intracellular signaling pathways. These results show that differential regulation of annexin localization by oxidative stress may have a causative role in the cellular pathophysiology of vascular endothelial cell disease.     

Sequence and Chromosomal Localization of Mouse Annexin XI

Mouse annexin XI (anx11)²was cloned from a macrophage cDNA library and characterized by genetic linkage mapping, DNA sequencing, and structural comparison with other annexins. TheAnx11gene localized to mouse chromosome 14 in close linkage with theRarb, Plau,andWnt5agenes near the centromere and 1.8 cM distal from theAnx7gene. The open reading frame was flanked by long, untranslated regions and encoded a 503-amino-acid protein with 93.1% identity to its human orthologue. Its 189-aa amino terminus corresponded to the widely expressed variant 1 of two possible, alternatively spliced forms. A previously described peptide fromAplysia brasilianawas identified as a closely related invertebrate homologue. Since annexin XI is known to be localized in the nucleus at certain stages of development, the identification of a region in tetrad repeats 3 and 4 resembling the “chromo box” domain may be relevant to a nuclear regulatory function of annexin XI. Knowledge of the mouse cDNA sequence and genetic map location will assist in the analysis of genomic organization and expression and provide a useful animal model to investigate gene function and hereditary phenotype for annexin XI.     

Systems Analysis of a Mouse Xenograft Model Reveals Annexin A1 as a Regulator of Gene Expression in Tumor Stroma

Annexin A1 is a multi functional molecule which is involved in inflammation, innate and adaptive immune systems, tumor progression and metastasis. We have previously showed the impaired tumor growth, metastasis, angiogenesis and wound healing in annexin A1 knockout mice. While tumor is a piece of heterogeneous mass including not only malignant tumor cells but also the stroma, the importance of the tumor stroma for tumor progression and metastasis is becoming increasingly clear. The tumor stroma is comprised by various components including extracellular matrix and non-malignant cells in the tumor, such as endothelial cells, fibroblasts, immune cells, inflammatory cells. Based on our previous finding of pro-angiogenic functions for annexin A1 in vascular endothelial cell sprouting, wound healing, tumor growth and metastasis, and the previously known properties for annexin A1 in immune cells and inflammation, this study hypothesized that annexin A1 is a key functional player in tumor development, linking the various components in tumor stroma by its actions in endothelial cells and immune cells. Using systems analysis programs commercially available, this paper further compared the gene expression between tumors from annexin A1 wild type mice and annexin A1 knockout mice and found a list of genes that significantly changed in the tumor stroma that lacked annexin A1. This revealed annexin A1 to be an effective regulator in tumor stroma and suggested a mechanism that annexin A1 affects tumor development and metastasis through interaction with the various components in the microenvironment surrounding the tumor cells.     

Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution.

We have identified and characterized a 12th subfamily of vertebrate annexins by systematic analysis of the primary structure, chromosomal mapping, and molecular evolution of unique cDNA and protein sequences from human and mouse. Distinctive features included rare expression, a codon deletion in conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats 1, 3, and 4. The paralogy of novel annexin A10 (following revised nomenclature) was confirmed by FISH-mapping human ANXA10 to chromosome 4q33 and genetic linkage mapping mouse Anxa10 to midchromosome 8. Phylogenetic analysis established that the 5' and 3' halves of the annexin A6 octad are more closely related to annexins A5 and A10, respectively, than they are to each other. Molecular date estimates, paralogy linkage maps between human chromosomes 4 and 5, and annexin structural considerations led to the proposal that annexins A5 and A10 may have been the direct progenitors of annexin A6 octad formation via chromosomal duplication during the genome expansion in early chordates.     

Drapc1 expression during mouse embryonic development

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1(null) mice, the peritonitis response was exaggerated ( approximately 40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased at both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1(null) mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection.     

3-morpholinosydnonimine participates in the attenuation of neointima formation via inhibition of annexin A2-mediated vascular smooth muscle cell migration

3-Morpholinosydnonimine (SIN-1) affects vascular smooth muscle cell migration and proliferation, processes essential for atherosclerosis. However, the mechanism by which SIN-1 exerts these effects has not been elucidated. We used 2-DE followed by MALDI-TOF/TOF MS to identify responses in protein expression to SIN-1 in rat aortic smooth muscle. Platelet-derived growth factor-BB increased cell migration and proliferation in rat aortic smooth muscle cells, and subsequent SIN-1 treatment inhibited it. Administration of SIN-1 in vivo attenuated neointima formation in balloon-injured rat carotid arteries. Proteomic analysis showed that glutathione peroxidase and 40S ribosomal protein S12 were differentially expressed in aortic strips exposed to SIN-1. Expression of annexin A2 was decreased by SIN-1. Platelet-derived growth factor-BB-induced cell migration was increased and inhibited in rat aortic smooth muscle cells with overexpression and knockdown of annexin A2 gene, respectively. The expression of annexin A2 was increased in vascular neointima compared with the intact control, which was inhibited by SIN-1 treatment. These results demonstrate that SIN-1 may attenuate vascular neointima formation by inhibiting annexin A2-mediated migration. Therefore, annexin A2 may be a potential target for therapeutic strategies for atherosclerosis.     

Knockdown of legumain inhibits cleavage of annexin A2 in the mouse kidney

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro. In this study, to determine whether annexin A2 is cleaved by legumain in vivo, siRNA-lipoplex targeting mouse legumain was injected into mouse tail veins. Mouse kidneys were then isolated and the effect of knockdown of legumain expression on annexin A2 cleavage was examined. The results showed that both legumain mRNA and protein expression levels were decreased in the siRNA-treated mouse kidneys and that legumain activity toward a synthetic substrate, Z-Ala-Ala-Asn-MCA, was decreased by about 40% in the kidney but not in the liver or spleen. Furthermore, cleavage of annexin A2 at the N-terminal region was decreased in the mouse kidney that had been treated with the legumain siRNA-lipoplex. These results suggest that legumain siRNA was delivered to the kidney by using LipoTrust and that the reduced legumain expression inhibited legumain-induced degradation of annexin A2 in vivo.     

巢蛋白mRNA在小鼠中枢神经系统发育过程中的表达

巢蛋白属于中等纤维基因家族,在增殖较快的神经前体细胞中表达。该基因被克隆后,作为神经前体的标记基因得到广泛应用。本文中,我们根据小鼠巢蛋白ｃＤＮＡ序列,设计了一对引物,在确定了反轩录ＰＣＲ反应的最佳反应条件后,详细地考察了小鼠巢蛋白ｍＲＮＡ在中枢神经系统发育过程中的表达规律。