Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking α_vβ₅ integrin

Abstract

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-α_v integrin, but not by anti-CD36 antibody treatment. Introduction of α_vβ₅ integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with α_vβ₅ integrin, but not to α_vβ₃ integrin in brain tissue. Fluorescent BAI1-TSR colocalized with α_vβ₅ integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking α_vβ₅ integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.

Introduction

Angiogenesis depends on growth factors and their receptors, but is also influenced by receptors for extracellular matrix (ECM) proteins. In general, cell adhesion to the ECM is mediated by integrins, heterodimeric transmembrane proteins composed of over 15α and 8β subunits. Some integrin combinations recognize a single ECM ligand, while others bind several different ECM proteins [1]. During angiogenesis, it is likely that integrins expressed on the surface of activated endothelial cells (ECs) regulate critical adhesive interactions with a variety of ECM proteins. These interactions regulate distinct biological events such as cell migration, proliferation, and differentiation. In addition, angiogenesis in specific tissues, such as in the brain, may depend on specific EC interactions with ECM proteins that vary considerably in their adhesive properties [2], [3], [4].

Two α_v integrins, α_vβ₃ and α_vβ₅, have been demonstrated to be necessary for cytokine- or tumor-induced angiogenesis [2], [5]. These integrins mediate two distinct pathways of angiogenesis. Particularly, α_vβ₃ is necessary for the formation, survival, and maturation of newly formed blood vessels [2], [6], [7]. These survival pathways can be blocked either by function-blocking antibodies or by peptide analogues that block the active RGD binding site on integrins α_vβ₃ and α_vβ₅, thus inducing apoptosis of the angiogenic ECs [5], [6]. Endothelial apoptosis results in inhibition of new blood vessel formation, inhibition of tumor growth, and tumor regression [8], [9]. Thrombospondins (TSPs) are ECM proteins that inhibit EC proliferation, migration, and angiogenesis. Among the five different thrombospondin genes, only TSP1 and TSP2 contain three thrombospondin type 1 repeats (TSRs), and these two genes inhibit angiogenesis in vitro and in vivo [10], [11], [12], [13].

BAI1 is a novel brain-specific gene that was identified during a screen for genomic DNA fragments containing functional p53 binding sites [14]. Expression of BAI1 is directly induced by wild-type p53 [14]. The predicted BAI1 protein includes a seven-span transmembrane region (STR) flanked by large extracellular and cytoplasmic domains. Two additional human genes homologous to BAI1 have been identified and designated as BAI2 and BAI3. Analysis of the three predicted BAI proteins showed that the TSR and STR regions were well conserved. However, the extracellular and cytoplasmic domains are relatively different among the three proteins [15], [16]. All three BAI genes were specifically expressed in brain, and it is likely that the three are closely related.

The third cytoplasmic loop of an STR-containing G-protein coupled receptor is important to its interaction with G proteins [17]. However, this third loop is very short in BAI1 compared with BAI2, and it appears likely that BAI1 does not perform the function normally mediated by the third cytoplasmic loop. BAI1 possesses two presumably functional elements in its extracellular region, one RGD motif and 5 TSRs [3]. The former is a potential recognition sequence for binding integrins, and the TSRs of BAI1 can inhibit experimental angiogenesis induced by fibroblast growth factor [14], [18]. It is assumed that the functions of the TSR region of BAI1 are similar to those of TSP1 or TSP2. We recently showed that the brain-specific developmental expression pattern of BAI1 and BAI2 was correlated with decreasing neovascularization in the adult brain [16]. BAI1 and BAI2 were involved in the late and early stages, respectively, of neovascularization in the cerebral cortex after ischemia [16]. This finding suggests that BAI1 and BAI2 act as angiostatic factors in ischemia-induced brain angiogenesis, perhaps in concert to angiogenic vascular endothelial growth factor (VEGF). However, BAI1's mechanisms of anti-angiogenic action in the brain are still unknown.

In this study, we investigated BAI1's effects on endothelial cell proliferation to derive an understanding of the mechanisms underlying this protein's anti-angiogenic action. We found that the antiproliferative activity of BAI1 is localized to the TSR region, which binds and functions via α_vβ₅ integrin. Our results indicate that neurons may inhibit nearby angiogenesis through the paracrine antiproliferative effect of BAI1 on EC.