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

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

Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development.

Basement membranes are specialized extracellular matrices consisting of tissue-specific organizations of multiple matrix molecules and serve as structural barriers as well as substrates for cellular interactions. The network of collagen IV is thought to define the scaffold integrating other components such as, laminins, nidogens or perlecan, into highly organized supramolecular architectures. To analyze the functional roles of the major collagen IV isoform α1(IV 2α2(IV) for basement membrane assembly and embryonic development, we generated a null allele of the Col4a1/2 locus in mice, thereby ablating both α-chains. Unexpectedly, embryos developed up to E9.5 at the expected Mendelian ratio and showed a variable degree of growth retardation. Basement membrane proteins were deposited and assembled at expected sites in mutant embryos, indicating that this isoform is dispensable for matrix deposition and assembly during early development. However, lethality occurred between E10.5-E11.5, because of structural deficiencies in the basement membranes and finally by failure of the integrity of Reichert's membrane. These data demonstrate for the first time that collagen IV is fundamental for the maintenance of integrity and function of basement membranes under conditions of increasing mechanical demands, but dispensable for deposition and initial assembly of components. Taken together with other basement membrane protein knockouts, these data suggest that laminin is sufficient for basement membrane-like matrices during early development, but at later stages the specific composition of components including collagen IV defines integrity, stability and functionality

Sequential expression of annexin A5 in the vasculature and skeletal elements during mouse development

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. (C) 2001 Elsevier Science Ireland Ltd. All rights reserve

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

Skin wound repair is not altered in the absence of endogenous AnxA1 or AnxA5, but pharmacological concentrations of AnxA4 and AnxA5 inhibit wound hemostasis

Skin injury induces the cell surface exposure of phosphati- dylserine (PS) on damaged and dying cells to activate coagu- lation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the rel- evance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with dis- tinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 μM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice- forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affin- ity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and re- pair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds