Abstract-Angiopoietin-like 4 (ANGPTL4) is a secreted protein that belongs to the angiopoietin family and is involved in angiogenesis and metabolism regulation. We previously reported the induction of angptl4 by hypoxia in endothelial cells and in human ischemic tissues from peripheral artery disease. We here observed in a mouse model of hindlimb ischemia that the mRNA upregulation in the vessels correlates with the accumulation of the full-length protein in ischemic tissues. We then investigated its functions in endothelial cells. In response to hypoxia, endogenous ANGPTL4 accumulates in the subendothelial extracellular matrix (ECM). Although the secreted protein undergoes proteolysis leading to truncated fragments present in the medium, only full-length ANGPTL4 interacts with the ECM. Competition and direct binding assays indicate that the strong interaction of ANGPTL4 with the ECM is heparin/heparan sulfate proteoglycan dependent. The balance between matrix-associated and soluble forms of ANGPTL4 points to the role of the ECM in the regulation of its bioavailability. The angiogenic function of the ECM-bound full-length protein was investigated using either the form associated with the conditioned ECM from ANGPTL4-transfected HEK293 cells or the purified immobilized protein. We show that matrix-associated and immobilized ANGPTL4 limit the formation of actin stress fibers and focal contacts in the adhering endothelial cells and inhibit their adhesion. Immobilized ANGPTL4 also decreases motility of endothelial cells and inhibits the sprouting and tube formation. Altogether, these findings show that hypoxic endothelial cells accumulate ANGPTL4 in the ECM, which in turn negatively regulates their angiogenic capacities through an autocrine pathway. Key Words: extracellular matrix Ⅲ endothelial cells Ⅲ angiogenesis Ⅲ hypoxia C ardiovascular disorders such as coronary and peripheral artery diseases lead to a deficient blood supply to tissues and a decrease in oxygen partial pressure, ie, hypoxia. Because of their location at the interface of circulating blood and peripheral tissues, endothelial cells are exposed to hypoxia. A critical adaptation to hypoxia is angiogenesis, which consists in the formation of new blood vessels extending from the preexisting vasculature. 1 This phenomenon occurs through the activation of the endothelial cells by a multistep process including changes of cell/extracellular matrix (ECM) interactions. In ischemic cardiovascular pathologies, reactive angiogenesis is a beneficial event. Therefore, unraveling the interplay of multiple molecular signals and events that occur in hypoxia and lead to functional new blood vessels is a challenging issue.We previously identified angiopoietin-like 4 gene (angptl4) as a hypoxia-induced target in vitro in the human microvascular endothelial cell line (HMEC-1) and in vivo in the vessels of ischemic tissues from peripheral artery disease. 2 Human ANGPTL4 is a secreted glycoprotein that belongs to the angiopoietin family. 3 It is composed of 406 amino acids...

Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.