A designed angiopoietin-2 variant, pentameric COMP-Ang2, strongly activates Tie2 receptor and stimulates angiogenesis

AbstractDespite that angiopoietin-2 (Ang2) produces more versatile and dynamic functions than angiopoietin-1 (Ang1) in angiogenesis and inflammation, the molecular mechanism that underlies this difference is still unknown. To define the role of oligomerization of Ang2 in activation of its receptor, Tie2, we designed and generated different oligomeric forms of Ang2 by replacement of the amino-terminal domains of Ang2 with dimeric, tetrameric, and pentameric short coiled-coil domains derived from GCN4, matrillin-1, and COMP. COMP-Ang2 strongly binds and activates Tie2, whereas GCN4-Ang2 and MAT-Ang2 weakly to moderately bind and activate Tie2. Although native Ang2 strongly binds to Tie2, it does not activate Tie2. Accordingly, COMP-Ang2 strongly promotes endothelial cell survival, migration, and tube formation in a Tie2-dependent manner, and the potency of COMP-Ang2 is almost identical to that of COMP-Ang1. Furthermore, the potency of COMP-Ang2-induced enhanced angiogenesis in the wound healing region is almost identical to the potency of COMP-Ang1-induced enhanced angiogenesis. Overall, there is no obvious difference between COMP-Ang2 and COMP-Ang1 in in vitro and in vivo angiogenesis. Our results provide compelling evidence that proper oligomerization of Ang2 is a critical determinant of its binding and activation of Tie2

Toll-Like Receptor 4 Decoy, TOY, Attenuates Gram-Negative Bacterial Sepsis

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using ‘the Hybrid leucine-rich repeats (LRR) technique’. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-κB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins

Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth

FZC18 reduces cell sensitivity to soluble Wnt3a.

<p>HEK293T cell batches stably expressing FZC18 (1; 4; 5) or empty vector (V) were incubated with either 50% control or Wnt3a conditioned medium (CM) for 16 hr before lysis. CRT (β-catenin-T-Cell factor Regulated Transcription) assays using Super8•Topflash or the negative control Super8•Fopflash reporters are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity (mean±SD).</p

Partially purified FZC18 inhibits Wnt3a-induced Wnt/β-catenin signaling.

<p>CRT assay using the β-catenin reporters Super8•Topflash and Super8•Fopflash, as indicated. (A) HEK293-EBNA cells incubated for 16 hr with either 50% control CM or 50% Wnt3a CM. Wnt3a induces an 80-fold increase in CRT. (B and C) Partially purified, Fc tagged human FZC18_CRD (hFZC18_CRD-Fc) dose-dependently inhibits Wnt3a-induced CRT in HEK293-EBNA cells, as shown with Super8•Topflash <i>(B)</i> and Super8•Fopflash <i>(C)</i> CRT reporters. Cells were incubated for 16 hr with 50% Wnt3a CM that had been pre-incubated overnight on a rotary wheel at +4°C with the indicated concentrations of hFc tag alone (recombinant human Fc from IgG, negative control) or hFZC18_CRD-Fc. Results are shown as mean±SD of hFZC18_CRD-Fc/hFc tag ratios. R² indicates 2^nd degree polynomial regression coefficient. Curve fitting is shown by a red line. Super8•Fopflash <i>(C)</i> negative control CRT reporters (C) are shown for the highest concentrations of hFZC18_CRD-Fc/hFc. (D) Immunoblots show hFc and hFZC18_CRD-Fc from each sample using anti-Fc tag antibody.</p

Frizzled 1 and 8 receptors partially rescue the inhibition of Wnt3a-induced CRT by FZC18.

<p>CRT reporter gene assays using the β-catenin-TCF responsive reporter Super8•Topflash in HEK293T cells stably expressing FZC18 (A, B and C) and vector (A, B). Twenty-four hours after transfection with the CRT reporter and increasing amounts of either FZD1 receptor (A), FZD8 receptor (B), FZD8 receptor, FZD8_CRD or FZC8_CRD-GPI (C) cDNAs, cells were incubated either with 50% control CM (L) or with 50% Wnt3a CM for 16 hr. Results are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity. Error bars represent standard deviations.</p