Elsevier

Experimental Cell Research

Volume 315, Issue 7, 15 April 2009, Pages 1234-1246
Experimental Cell Research

Research Article
Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation

https://doi.org/10.1016/j.yexcr.2009.01.021Get rights and content

Abstract

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Introduction

Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is a process in which stimulated endothelial cells remodel extracellular matrix (ECM), migrate through the ECM, proliferate, differentiate and eventually form endothelial tubules capable of blood transport [1]. Proteases of at least three classes (serine, cysteine, and metallo-) [including matrix metalloproteinases (MMPs), members of the urokinase plasmin(ogen) system and cysteine cathepsins] play crucial roles in angiogenesis (for review see [2]). Proteases participate in the angiogenic process by generating both pro- and anti-angiogenic factors from ECM proteins [3], [4], [5] and by processing growth factors and receptors, including integrins [6], [7]. For example, cleavage of type IV collagen by endothelial cell MMPs results in the exposure of cryptic αvβ3-integrin binding sites, thus promoting angiogenesis [7]. Urokinase plasminogen activator (uPA) converts plasminogen to plasmin which can be further processed to generate angiostatin, an anti-angiogenic compound [8]. Cathepsin B, a lysosomal cysteine protease, regulates the “angiogenic switch” [9] by initiating proteolytic cascades involved in ECM degradation that promote angiogenesis [10], or conversely, by cleaving collagen XVIII and generating endostatin, an anti-angiogenic factor [11]. Overall, the balance of protease activity and the interaction of proteases with the ECM contribute to regulation of the angiogenic process.

ECM remodeling and degradation by tumor cells are facilitated by the translocation of proteases to cell surfaces and their secretion into the extracellular milieu [12], [13], [14], [15]. Many proteases implicated in ECM remodeling and degradation are also associated with caveolae [12], a lipid-rich region of the plasma membrane involved in endocytosis, cholesterol transport and cell signaling events (for reviews, see [16], [17]). These proteases include uPA and its receptor uPAR, cathepsin B and its cell surface binding protein S100A10/p11 (p11) [the light chain of the annexin II heterotetramer (AIIt)], MMP-2 and MMP-14 [12]. In colorectal carcinoma cells, downregulation of caveolin-1 (cav-1), the main structural protein of caveolae, decreases distribution of cathepsin B and uPA to caveolae and ECM degradation by these cells [18]. The association of proteases with caveolae of endothelial cells is intriguing since caveolae are involved in angiogenesis (for review, see [19]). During endothelial cell migration, cav-1 localizes to the rear of migrating cells [20], [21], [22]. Moreover, downregulation of cav-1 impedes endothelial cell polarization and directional movement [20]. Indeed, knockdown of cav-1 suppresses tube formation by endothelial cells and also reduces vessel formation in the chicken chorioallantoic membrane assay [23]. Thus, we hypothesize that compartmentalization of proteases to caveolae in endothelial cells modulates cell migration, proteolysis of ECM proteins and tube formation.

In the present study, we identified active cathepsin B as well as its cell surface-binding partner, S100A10/p11 (p11), in caveolae of human umbilical vein endothelial cells (HUVEC): an association that is augmented during in vitro endothelial tube cell formation. Using a live-cell proteolysis assay in combination with in vitro tube formation assays and cysteine cathepsin activity-based probes, we observed extracellular degradation of ECM proteins (i.e., type IV collagen) by migrating HUVEC and intracellular colocalization of ECM degradation products with cav-1 in vesicles containing active cysteine cathepsins. These data suggest an association of active cathepsin B with caveolae and degradation and remodeling of the ECM during endothelial cell migration and tube formation.

Section snippets

Materials

M199 medium, heparin, N-Octyl β-d-glucopyranoside, 2-[N-morpholino]ethanesulfonic acid (MES), methyl-β-cyclodextrin (MβCD), and all other chemicals unless otherwise stated were from Sigma (St. Louis, MO); fetal bovine serum (FBS), Lipofectin reagent, dye-quenched fluorescent (DQ)-gelatin and DQ-collagen IV were from Invitrogen (Carlsbad, CA); bovine endothelial cell growth factor (bECGF) was from Roche Applied Science (Indianapolis, IN); polyclonal anti-caveolin (610059), monoclonal

Gelatin and collagen IV degradation products localize to vesicles containing cav-1 in HUVEC

Remodeling of the ECM is a process critical to the formation of tubular structures by endothelial cells. To analyze this process, we employed a well-established live-cell proteolysis assay [35] and compared HUVEC grown on coverslips coated with gelatin containing DQ-gelatin with HUVEC grown on coverslips coated with rBM containing DQ-collagen IV. Following 16 h of incubation, live cells were imaged by confocal microscopy for degradation products (green fluorescence) of the two DQ-protein

Discussion

Differentiation of endothelial cells during angiogenesis is a process that involves proteolytic enzymes, which degrade basement membrane and thereby facilitate cell migration, invasion and capillary tube formation. The accessibility of proteases to the ECM is enhanced by their localization on the cell membrane. Here, we explored one possible mechanism for cell surface association, i.e., the distribution of cathepsin B and other proteases associated with ECM degradation to caveolae on the cell

Acknowledgments

This work was supported by a National Institutes of Health (NIH) National Technology Center for Networks and Pathways Grant (U54-RR020843) and a DOD Breast Cancer Center of Excellence (DAMD17-02-1-0693). The Microscopy and Imaging Resources Laboratory is supported by the NIH National Technology Center Grant and NIH Center Grants P30-ES06639 and P30-CA22453.

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