Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components. Apoptosis of endothelial cells (EC)4 is increasingly recognized as an important component of the "response to injury" process, as most clinical risk factors of atherosclerosis (such as hypertension (1, 2), hyperglycemia (3, 4), oxidized low density lipoproteins (LDLs) (5, 6) and oxidative stress (7)) induce EC apoptosis. Interventions aimed at preventing EC apoptosis in animal models of transplant vasculopathy, an immune-mediated form of atherosclerosis, prevent neointima formation, indicating that EC apoptosis is an important pro-atherosclerotic trigger (8 -13). During vascular remodeling, EC injury and apoptosis are followed by migration of ␣-smooth muscle actinpositive cells (smooth muscle cells (SMC) and myofibroblasts) that accumulate within the intima through a state of resistance to apoptosis largely dependent on Bcl-xl overexpression (14 -16).Recent evidence from our group and others suggests that apoptotic EC favor neointima formation through the release of paracrine mediators, which in turn, increase Bcl-xl expression and inhibit the apoptosis of vascular SMC and fibroblasts (17)(18)(19)(20). The production of biologically active mediators by apoptotic EC is at least partially dependent on pericellular proteolysis, leading to basement membrane and extracellular matrix (ECM) degradation with the release of cryptic anti-apoptotic factors (18 -20). A C-terminal fragment of endorepellin (perlecan domain V) released in association with EC apoptosis was found to heighten Bcl-xl expression in SMC and fibroblasts (18 -20). Perlecan is a basement membrane modular proteoglycan composed of five structural domains (21). The C-terminal domain, also called endorepellin, comprises three laminin-like globular (LG1-LG3) modules interspaced by four epiderm...

Gastrulation involves of a series of coordinated cell movements to organize the germ layers and establish the major body axes of the embryo. One gastrulation movement is epiboly, which involves the thinning and spreading of a multilayered cell sheet. Epiboly plays a prominent role in zebrafish gastrulation and studies of zebrafish epiboly have provided insights into basic cellular properties and mechanisms of morphogenesis that are widely used in animal development. Although considerable progress has been made in identifying molecules that are required for epiboly, we still understand very little about how these factors cooperate to drive the process. Here, we review work on the molecular and cellular basis of zebrafish epiboly in order to identify unifying themes and to highlight some of the current open questions.

Apoptotic endothelial cells are an important component of the “response to injury” process. Several atherosclerosis risk factors such as hyperglycemia and oxidized low-density lipoproteins, and immune injuries, such as antibodies and complement, induce endothelial cell apoptosis. While endothelial cell apoptosis is known to affect neighboring vascular wall cell biology, its consequences on macrophage reprogramming are ill defined. In this study, we report that apoptosis of human and mouse endothelial cells triggers the release of milk fat globule-epidermal growth factor 8 (MFG-E8) and reprograms macrophages into an anti-inflammatory cells. We demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from serum-starved apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if MFG-E8 is absent from the media. Macrophage treatment with recombinant MFG-E8 recapitulates the effect of conditioned media. Finally, we showed that MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Taken together, our study suggests a key role of MFG-E8 release from apoptotic endothelial cells in macrophage reprogramming and demonstrates the importance of the apoptotic microenvironment in anti-inflammatory macrophage responses.

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.

In Xenopus and zebrafish gastrulae, PAPC attenuates planar cell polarity signaling and controls formation of an adhesive, yet flexible, contact at the ectoderm–mesoderm boundary.