Angiopoietin-1 (Ang1) has potential therapeutic applications in inducing angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. However, production of Ang1 is hindered by aggregation and insolubility resulting from disulfidelinked higher-order structures. Here, by replacing the N-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), we have generated a soluble, stable, and potent Ang1 variant, COMP-Ang1. This variant is more potent than native Ang1 in phosphorylating the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor and Akt in primary cultured endothelial cells, enhancing angiogenesis in vitro and increasing adult angiogenesis in vivo. Thus, COMP-Ang1 is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo.

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structurefunction relationships of Ang1 with Tie2.Angiopoietin-1 (Ang1) 1 was discovered as a secreted protein ligand of tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2Ј (Tie2) (1). Tie2 is a member of the receptor tyrosine kinase family and is expressed predominantly on vascular endothelial cells and early hematopoietic cells (2-4). Ang1-and Tie2-deficient mice have similar phenotypes characterized by embryonic lethality with severe defects in vascular remodeling, insufficient vessel stabilization, and perturbed vascular maturation, indicating that Ang1 and Tie2 play critical roles in vascular development (5, 6). Accordingly, transgenic overexpression or gene transfer of Ang1 not only enhances vessel formation, but also protects the adult vasculature against vascular leakage (7-10). In addition, Ang1 can counteract vascular endothelial growth factor-induced side effects (9, 11) while having an additive effect on vessel formation (8, 10). Thus, Ang1 is a very promising growth factor for therapeutic angiogenesis (12, 13). Moreover, in a series of experiments, we found that the Ang1/Tie2 system in normal adult blood vessels was important in maintaining the integrity of nonproliferating endothelial cells by strongly inducing endothelial cell survival against insult-mediated damage (14 -16). Furthermore, recent series o...

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