Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

Leucine-rich repeats and immunoglobulin-like domains-1 (LRIG1) is a transmembrane protein with an ectodomain containing 15 leucine-rich repeats (LRRs) homologous to mammalian decorin and the Drosophila kekkon1 gene. In this study, we demonstrate that a soluble ectodomain of LRIG1, containing only the LRRs, inhibits ligandindependent epidermal growth factor receptor (EGFR) activation and causes growth inhibition of A431, HeLa and MDA-468 carcinoma cells. In contrast, cells that do not express detectable levels of EGFR fail to respond to soluble LRIG1. However, when a functional EGFR gene is introduced in these cells, they become growth-inhibited by soluble LRIG1 protein. Furthermore, we demonstrate the existence of high-affinity (K d ¼ 10 nM) binding sites on the A431 cells that can be competitively displaced (up to 75%) by molar excess of EGF. Even more powerful effects are obtained with a chimeric proteoglycan harboring the N-terminus of decorin, substituted with a single glycosaminoglycan chain, fused to the LRIG1 ectodomain. Both proteins also inhibit ligand-dependent activation of the EGFR and extracellular signal-regulated protein kinase 1/2 signaling in a rapid and dose-dependent manner. These results suggest a novel mechanism of action evoked by a soluble ectodomain of LRIG1 protein that could modulate EGFR signaling and its growthpromoting activity. Attenuation of EGFR activity without physical downregulation of the receptor could represent a novel therapeutic approach toward malignancies in which EGFR plays a primary role in tumor growth and survival.

Endorepellin, a C-terminal fragment of the vascular basement membrane proteoglycan perlecan, inhibits angiogenesis via the ␣2␤1-integrin receptor. Because this integrin is also implicated in plateletcollagen responses and because endorepellin or its fragments are generated in response to injury and inflammation, we hypothesized that endorepellin could also affect platelet biology. We discovered that endorepellin supported ␣2␤1-dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an ␣2␤1-integrin function-blocking antibody. Collectively, these results suggest that endorepellin/␣2␤1-integrin interaction and effects are specific and dependent on cell type, differ from those emanated by exposure to collagen, and may be due to cellular differences in ␣2␤1-integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic basement membrane fragments in platelet biology. IntroductionEndorepellin, the C-terminal domain of perlecan, 1,2 exerts antiangiogenic activity 3-5 by interacting with ␣2␤1 integrin and triggering a signaling cascade that leads to disruption of actin cytoskeleton in endothelial cells and ultimately to angiostasis. 6,7 The ␣2␤1 integrin also exists in platelets, fibroblasts, and epithelial cells and regulates cell adhesion and signaling. [8][9][10][11][12] We hypothesized that endorepellin could affect platelet function via this integrin receptor. This hypothesis is based on the fact that endorepellin or fragments thereof are present in the blood and various body fluids and could interact with platelets at sites of injury, inflammation, and cancer growth. For example, a biologically active fragment of endorepellin (LG3) is present in the urine of patients with end-stage renal disease 13 and in the amniotic fluid of pregnant women with premature rupture of fetal membranes. 14,15 Perlecan fragments of similar size were found in urinary 16 and blood 17 proteomes, and LG3 is released by apoptotic endothelial cells. 18 Here we show that endorepellin supports ␣2␤1-integrinmediated platelet adhesion, but does not activate or aggregate platelets. Via an src kinase-dependent mechanism, endorepellin enhances all collagen-evoked platelet responses studied, without directly binding to collagen. 3 Our results suggest that endorepellin/␣2␤1 interactions are cell specific and differ from collagen-␣2␤1 binding. Generation of endorepellin at sites of injury might enhance initial platelet adhesion and in combination with newly exposed collagen matrix could hasten in vivo platelet responses. Materials and methodsInformed consent was provided according to the Declaration of Helsinki and Institutional Review Board approval was obtained from Thomas Jefferson University. Endorepellin, platelets, and materialsReagents are listed in the Supplemental Materials (available on Blood website; see ...