Domains I and IV of Annexin A2 Affect the Formation and Integrity of In Vitro Capillary-Like Networks

Abstract

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

The native structure of annexin A2 peptides in hydrophilic environment determines their anti-angiogenic effects

“…Activation of plasminogen did not appear to be important, while the disruption of the VE-cadherin-mediated contacts between ECs appeared to be an essential part of this effect [29]. In the present study, D I -P2 was the only peptide that strongly inhibited the formation of a network of HUVECs grown on SMCs (Figs.…”

“…Previously, we showed that domains I and IV of AnxA2 in low concentration (15 mM) strongly inhibit the formation of a capillary-like network in the co-culture system [29]. Activation of plasminogen did not appear to be important, while the disruption of the VE-cadherin-mediated contacts between ECs appeared to be an essential part of this effect [29].…”

“…The expression and purification of the AnxA2 peptides as fusion peptides coupled to His-MBP were performed as described for AnxA2-D I [29] and AnxA2-D IV [33]. The primers used for the creation of the cDNAs encoding the peptides are listed in Table 1.…”

“…We have previously shown that domains I and IV of AnxA2 inhibit the formation of a vascular-like network in a co-culture system of HUVECs and smooth muscle cells (SMCs). In addition, these domains are able to disrupt a pre-formed network by interfering with VE-cadherin-mediated adhesion between the ECs [29].…”

“…We have been able to purify a soluble and partially folded form of AnxA2 domain IV by mutating hydrophobic amino acids involved in interfacial contacts with the other domains [33]. This domain as well as the soluble and fully folded domain I of AnxA2 showed potent anti-angiogenic effects when tested in the co-culture system [29].…”