Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases. Introduction Various types of mammalian cells, such as cancer cells, macrophages, endothelial cells, platelets, and epithelial cells release extracellular vesicles (EVs) into their surroundings from the plasma and endosomal membrane compartments [1]C[4]. These mammalian EVs, also known as exosomes and microvesicles, are spherical bilayered proteolipids with an average diameter of 40C250 nm and are enriched with various bioactive constituents, including proteins, lipids, and genetic material [1]C[9]. Growing evidence has revealed that EVs play pleiotropic functions in intercellular communication: EVs stimulate recipient cells by the activation of a receptor and the transfer of membrane proteins, signaling molecules, mRNAs, and miRNAs [4]C[9]. EVs have often been referred to as cellular dust, although cells shed EVs either constitutively or in a regulated manner [1]C[9]. Moreover, the proteins, mRNAs, or miRNAs in EVs differ in composition depending on the states of donor cells [1], [4]. Recently, our group revealed that proteins of human colorectal cancer cell-derived EVs are interconnected via physical interactions and cluster into functional modules involved in EV biogenesis and function [4], [10]. Furthermore, the secretion of EVs is a universal cellular process occurring from Amyloid b-Peptide (1-40) (human) simple organisms (Archea or Gram-negative and Gram-positive bacteria) to complex multicellular organisms, suggesting that this EV-mediated communication is evolutionarily conserved [9], [11]C[13]. Taken together, these findings suggest that EVs play diverse roles in intercellular communication [6], [10]. However, the pathophysiological roles of EVs are not completely understood. Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a complex and multistep process involving adhesion, migration, invasion, proliferation, and differentiation of endothelial cells [14], [15]. This neovascularization occurs under various normal and pathological conditions [14]. For example, angiogenesis is essential for tumor growth and metastasis by providing oxygen and nutrients to the growing tumor [15]. In the tumor microenvironment, a heterogeneous population of cells, including cancer cells, endothelial cells, fibroblasts, and immune cells modulates an environment favorable to tumor growth and invasion [16]C[18]. These cancer and stromal cells secrete vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), tumor necrosis factor- (TNF-), and IL-6 into the surrounding area and these factors contribute to tumor-associated angiogenesis [16]C[19]. In addition to these proangiogenic soluble factors, the cells comprising the tumor tissue secrete EVs into the extracellular milieu and these shed EVs play multiple roles in tumor growth and metastasis by promoting Amyloid b-Peptide (1-40) (human) angiogenesis, tumor invasion, and immune escape [4]C[8], [20]C[23]. After the initial report on the angiogenic activities of EVs derived SH3RF1 from HT1080 human fibrosarcoma and DU-145 human prostate carcinoma cells [5], several studies confirmed that EVs derived from cancer cells, fibroblasts, and cancer stem cells promote and angiogenesis [4], [8], [24]C[28]. These angiogenic activities of EVs are mediated by vesicular lipid(s), proteins, including receptors and tetraspanin proteins, mRNAs, and miRNAs. However, the detailed mechanism of how EVs elicit angiogenic activity has not been extensively studied. Early growth response-1 (Egr-1), an immediate early gene and a zinc finger transcription factor, plays a crucial role in angiogenesis [29]C[32]. In addition to serum exposure, Egr-1 can be rapidly and transiently induced by cytokine, growth factor, and environmental stress, including hypoxia, fluid shear stress, and vascular injury [33], [34]. Egr-1 regulates the expression of proangiogenic genes, such as VEGF, FGF2, and IL-6 in endothelial cells or TNF- in macrophages [31], [34]C[36]. Within the tumor tissue, endothelial cells, cancer cells, fibroblasts, and tumor-infiltrating macrophages can express Egr-1. Furthermore, microvessel Amyloid b-Peptide (1-40) (human) densities in tumor tissues obtained from Egr-1-deficient mice are lower than those obtained from wild-type mice [37] and vessel-like structure formation in tumor tissue was suppressed by DNAzymes that target Egr-1 mRNA [31], suggesting that Egr-1 plays essential roles in tumor growth and angiogenesis. In this regard, several studies have reported that Egr-1 expression in cancer cells, endothelial cells, and macrophages is related to tumor progression [32], [36]C[38]. Collectively, these findings suggest that Egr-1 plays important roles in tumor-associated angiogenesis and tumor progression. In this report, we provide evidence that Egr-1 activation in endothelial cells should be a key mechanism involved in the angiogenic activity of Amyloid b-Peptide (1-40) (human) cancer-derived EVs. We found that Egr-1 activation by colorectal cancer cell-derived EVs promoted endothelial cell migration via the ERK1/2 and JNK signaling pathways and lipid.