Laurens A. van Meeteren

Autotaxin, a Secreted Lysophospholipase D, Is Essential for Blood Vessel Formation during Development

ABSTRACT Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the Gα13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Gα13.

Vascular endothelial growth factor B controls endothelial fatty acid uptake

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb-/- mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Structural Basis of Substrate Discrimination and Integrin Binding by Autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B–like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC50 ∼ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization.

Vascular endothelial growth factor (VEGF)-A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165-containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.

Autotaxin/Lysopholipase D and lysophosphatidic acid regulate murine hemostasis and thrombosis

The lipid mediator lysophosphatidic acid (LPA) is a potent regulator of vascular cell function in vitro, but its physiologic role in the cardiovasculature is largely unexplored. To address the role of LPA in regulating platelet function and thrombosis, we investigated the effects of LPA on isolated murine platelets. Although LPA activates platelets from the majority of human donors, we found that treatment of isolated murine platelets with physiologic concentrations of LPA attenuated agonist-induced aggregation. Transgenic overexpression of autotaxin/lysophospholipase D (Enpp2), the enzyme necessary for production of the bulk of biologically active LPA in plasma, elevated circulating LPA levels and induced a bleeding diathesis and attenuation of thrombosis in mice. Intravascular administration of exogenous LPA recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. Enpp2+/- mice, which have ∼50% normal plasma LPA levels, were more prone to thrombosis. Plasma autotaxin associated with platelets during aggregation and concentrated in arterial thrombus, and activated but not resting platelets bound recombinant autotaxin/lysoPLD in an integrin-dependent manner. These results identify a novel pathway in which LPA production by autotaxin/lysoPLD regulates murine hemostasis and thrombosis and suggest that binding of autotaxin/lysoPLD to activated platelets may provide a mechanism to localize LPA production.

Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA). ATX is secreted by adipose tissue and its expression is enhanced in obese/insulin-resistant individuals. Here, we analyzed the specific contribution of adipose-ATX to fat expansion associated with nutritional obesity and its consequences on plasma LPA levels. We established ATXF/F/aP2-Cre (FATX-KO) transgenic mice carrying a null ATX allele specifically in adipose tissue. FATX-KO mice and their control littermates were fed either a normal or a high-fat diet (HFD) (45% fat) for 13 weeks. FATX-KO mice showed a strong decrease (up to 90%) in ATX expression in white and brown adipose tissue, but not in other ATX-expressing organs. This was associated with a 38% reduction in plasma LPA levels. When fed an HFD, FATX-KO mice showed a higher fat mass and a higher adipocyte size than control mice although food intake was unchanged. This was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)γ2 and of PPAR-sensitive genes (aP2, adiponectin, leptin, glut-1) in subcutaneous white adipose tissue, as well as in an increased tolerance to glucose. These results show that adipose-ATX is a negative regulator of fat mass expansion in response to an HFD and contributes to plasma LPA levels.

Anti-human Activin Receptor-like Kinase 1 (ALK1) Antibody Attenuates Bone Morphogenetic Protein 9 (BMP9)-induced ALK1 Signaling and Interferes with Endothelial Cell Sprouting

Background: ALK1 is a TGF-β receptor essential for angiogenesis. An anti-ALK1 antibody is being tested in clinical trials as an inhibitor for tumor angiogenesis. Results: Anti-hALK1 antibody selectively recognizes ALK1 and interferes with ALK1 ligand binding, signaling, and endothelial sprouting. Conclusion: Anti-hALK1 antibody is a function-blocking antibody that prevents ALK1 signaling. Significance: This study shows mechanistically how anti-hALK1 antibody exerts its effect on angiogenesis. Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.

Discovery and Optimization of Boronic Acid Based Inhibitors of Autotaxin

Autotaxin (ATX) is an extracellular enzyme that hydrolyzes lysophosphatidylcholine (LPC) to produce the lipid mediator lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in diverse physiological and pathological processes, including vascular development, inflammation, fibrotic disease, and tumor progression. Therefore, targeting ATX with small molecule inhibitors is an attractive therapeutic strategy. We recently reported that 2,4-thiazolidinediones inhibit ATX activity in the micromolar range. Interestingly, inhibitory potency was dramatically increased by introduction of a boronic acid moiety, designed to target the active site threonine in ATX. Here we report on the discovery and further optimization of boronic acid based ATX inhibitors. The most potent of these compounds inhibits ATX-mediated LPC hydrolysis in the nanomolar range (IC(50) = 6 nM). The finding that ATX can be targeted by boronic acids may aid the development of ATX inhibitors for therapeutic use.

Anticancer activity of FTY720: Phosphorylated FTY720 inhibits autotaxin, a metastasis-enhancing and angiogenic lysophospholipase D

FTY720 is an immunomodulator that is phosphorylated in vivo and inhibits lymphocyte mobilization by targeting sphingosine 1-phospate receptors. At doses higher than required for immunomodulation, FTY720 inhibits tumor progression through an unknown mechanism. Here we show that FTY720-phosphate is a competitive inhibitor (Ki approximately 0.2microM) of autotaxin (ATX or NPP2), a nucleotide phosphodiesterase/pyrophosphatase (NPP) that enhances metastasis and angiogenesis and acts as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA). FTY720-phosphate did no affect the activity of NPP1, the closest relative of ATX. After oral administration in mice, FTY720 (3mg/kg) significantly reduced plasma LPA levels. These results suggest that FTY720 may exert its anticancer effects, at least in part, by targeting the ATX-LPA axis.