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.

The polybasic insertion in autotaxin α confers specific binding to heparin and cell surface heparan sulfate proteoglycans.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXα isoform, which differs from the canonical form (ATXß) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXα insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXα remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXα binds specifically to heparin with high affinity (K(d) ~10(-8) M), whereas ATXß does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXα. We further show that ATXα, but not ATXß, binds abundantly to SKOV3 carcinoma cells. ATXα binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXα insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.

Autotaxin in embryonic development.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (-/-) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα(12/13)-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX-LPA signaling in the regulation of left-right asymmetry. Here we discuss our present understanding of the role of ATX-LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Transforming growth factor ß family members in regulation of vascular function: in the light of vascular conditional knockouts.

Blood vessels are composed of endothelial cells, mural cells (smooth muscle cells and pericytes) and their shared basement membrane. During embryonic development a multitude of signaling components orchestrate the formation of new vessels. The process is highly dependent on correct dosage, spacing and timing of these signaling molecules. As vessels mature some cascades remain active, albeit at very low levels, and may be reactivated upon demand. Members of the Transforming growth factor ß (TGF-ß) protein family are strongly engaged in developmental angiogenesis but are also regulators of vascular integrity in the adult. In humans various genetic alterations within this protein family cause vascular disorders, involving disintegration of vascular integrity. Here we summarize and discuss recent data gathered from conditional and endothelial cell specific genetic loss-of-function of members of the TGF-ß family in the mouse.

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß 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-ß 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.

GATA2 and Lmo2 control angiogenesis and lymphangiogenesis via direct transcriptional regulation of neuropilin-2.

GATA-binding protein 2 (GATA2) and LIM domain only 2 (Lmo2) form common transcription complexes during hematopoietic differentiation. Here we show that these two transcription factors also play a key role in endothelial cells (EC) and lymphatic EC (LEC) function. Primary EC and tumor-associated blood vessels expressed GATA2 and Lmo2. VEGF-induced sprouting angiogenesis in both differentiating embryonic stem cells (embryoid bodies) and primary EC increased GATA2 and Lmo2 levels. Conversely, silencing of GATA2 and Lmo2 expression in primary EC inhibited VEGF-induced angiogenic activity, including EC migration and sprouting in vitro, two key steps of angiogenesis in vivo. This inhibition of EC function was associated with downregulated expression of neuropilin-2 (NRP2), a co-receptor of VEGFRs for VEGF, at the protein, mRNA and promoter levels. NRP2 overexpression partially rescued the impaired angiogenic sprouting in the GATA2/Lmo2 knockdown EC, confirming that GATA2 and Lmo2 mediated EC function, at least in part, by directly regulating NRP2 gene expression. Furthermore, it was found that primary LEC expressed GATA2 and Lmo2 as well. Silencing of GATA2 and Lmo2 expression in LEC inhibited VEGF-induced LEC sprouting, also in a NRP2-dependent manner. In conclusion, our results demonstrate that GATA2 and Lmo2 cooperatively regulate VEGF-induced angiogenesis and lymphangiogenesis via NRP2.

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 (IC(50) approximately 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.

Regulation of endothelial cell plasticity by TGF-ß.

Recent evidence has demonstrated that endothelial cells can have a remarkable plasticity. By a process called Endothelial-to-Mesenchymal Transition (EndMT) endothelial cells convert to a more mesenchymal cell type that can give rise to cells such as fibroblasts, but also bone cells. EndMT is essential during embryonic development and tissue regeneration. Interestingly, it also plays a role in pathological conditions like fibrosis of organs such as the heart and kidney. In addition, EndMT contributes to the generation of cancer associated fibroblasts that are known to influence the tumor-microenvironment favorable for the tumor cells. EndMT is a form of the more widely known and studied Epithelial-to-Mesenchymal Transition (EMT). Like EMT, EndMT can be induced by transforming growth factor (TGF)-ß. Indeed many studies have pointed to the important role of TGF-ß receptor/Smad signaling and downstream targets, such as Snail transcriptional repressor in EndMT. By selective targeting of TGF-ß receptor signaling pathological EndMT may be inhibited for the therapeutic benefit of patients with cancer and fibrosis.

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 ATX(F/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.

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.

Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein.

Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an approximately 100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 A resolution and allowed the collection of complete diffraction data to about 2.6 A resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.

Suppression of the p53-dependent replicative senescence response by lysophosphatidic acid signaling.

Lysophosphatidic acid (LPA) is a lipid mediator of a large number of biological processes, including wound healing, brain development, vascular remodeling, and tumor progression. Its role in tumor progression is probably linked to its ability to induce cell proliferation, migration, and survival. In particular, the ascites of ovarian cancers is rich in LPA and has been implicated in growth and invasion of ovarian tumor cells. LPA binds to specific G protein-coupled receptors and thereby activates multiple signal transduction pathways, including those initiated by the small GTPases Ras, Rho, and Rac. We report here a genetic screen with retroviral cDNA expression libraries to identify genes that allow bypass of the p53-dependent replicative senescence response in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen. Using this approach, we identified the LPA receptor type 2 (LPA(2)) and the Rho-specific guanine nucleotide exchange factor Dbs as potent inducers of senescence bypass. Enhanced expression of LPA(2) or Dbs also results in senescence bypass in primary mouse embryo fibroblasts in the presence of wild-type p53, in a Rho GTPase-dependent manner. Our results reveal a novel and unexpected link between LPA signaling and the p53 tumor-suppressive pathway.

Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development.

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 Galpha13 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 Galpha13.

Regulation and biological activities of the autotaxin-LPA axis.

Autotaxin (ATX), or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), is an exo-enzyme originally identified as a tumor cell autocrine motility factor. ATX is unique among the NPPs in that it primarily functions as a lysophospholipase D, converting lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA). LPA acts on specific G protein-coupled receptors to elicit a wide range of cellular responses, ranging from cell proliferation and migration to neurite remodeling and cytokine production. While LPA signaling has been studied extensively over the last decade, we are only now beginning to explore the properties and biological importance of ATX as the major LPA-producing phospholipase. In this review, we highlight recent advances in our understanding of the ATX-LPA axis, giving first an update on LPA action and then focusing on ATX, in particular its regulation, its link to cancer and its vital role in vascular development.

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.

Fluorogenic phospholipid substrate to detect lysophospholipase D/autotaxin activity.

[reaction: see text] Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target.

Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin.

We previously reported that fatty alcohol phosphates (FAP) represent a minimal pharmacophore required to interact with lysophosphatidic acid (LPA) receptors. To improve the activity of the first-generation saturated FAP series, a structure-activity relationship (SAR) study was carried out that includes modifications to the headgroup and alkyl side chain of the FAP pharmacophore. A series of unsaturated (C(10)-C(18)) FAP, headgroup-modified hydrolytically stable saturated (C(10)-C(18)) alkyl phosphonates, and saturated and unsaturated (C(10)-C(18)) thiophosphate analogues were synthesized and evaluated for activity in RH7777 cells transfected with individual LPA(1)(-3) receptors, in PC-3 cells and in human platelets that endogenously express all three isoforms. In this series we identified several LPA(1)- and LPA(3)-selective antagonists with IC(50) values in the nanomolar range. Oleoyl-thiophosphate (15g) was shown to be a pan-agonist, whereas tetradecyl-phosphonate (16c) was identified as a pan-antagonist. These compounds were also tested for the ability to activate the transcription factor PPARgamma, an intracellular receptor for LPA, in CV1 cells transfected with the PPRE-Acox-Rluc reporter gene. All the FAP tested, along with the previously reported LPA GPCR antagonists dioctanoyl glycerol pyrophosphate (2), Ki16425 (6), and the agonist OMPT (3), were activators of PPARgamma. The pan-agonist oleoyl-thiophosphate (15g) and pan-antagonist tetradecyl-phosphonate (16c) mimicked LPA in inhibiting autotaxin, a secreted lysophospholipase D that produces LPA in biological fluids.

NRP1 presented in trans to the endothelium arrests VEGFR2 endocytosis, preventing angiogenic signaling and tumor initiation.

Neuropilin 1 (NRP1) modulates angiogenesis by binding vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. We examined the consequences when VEGFR2 and NRP1 were expressed on the same cell (cis) or on different cells (trans). In cis, VEGF induced rapid VEGFR2/NRP1 complex formation and internalization. In trans, complex formation was delayed and phosphorylation of phospholipase Cγ (PLCγ) and extracellular regulated kinase 2 (ERK2) was prolonged, whereas ERK1 phosphorylation was reduced. Trans complex formation suppressed initiation and vascularization of NRP1-expressing mouse fibrosarcoma and melanoma. Suppression in trans required high-affinity, steady-state binding of VEGF to NRP1, which was dependent on the NRP1 C-terminal domain. Compatible with a trans effect of NRP1, quiescent vasculature in the developing retina showed continuous high NRP1 expression, whereas angiogenic sprouting occurred where NRP1 levels fluctuated between adjacent endothelial cells. Therefore, through communication in trans, NRP1 can modulate VEGFR2 signaling and suppress angiogenesis.

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis.

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-ß (TGF-ß) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.

TGF-ß receptor signaling pathways in angiogenesis; emerging targets for anti-angiogenesis therapy.

Angiogenesis, the formation of new blood vessels is essential for diverse physiological processes such as development, but also for pathological conditions like tumor growth. Most studied in this context are tyrosine kinase signaling pathways such as those involving vascular endothelial growth factor (VEGF). There is however accumulating evidence that more pathways are as essential for angiogenesis. Knockout studies of factors in transforming growth factor ß (TGF-ß) signaling have for example showed that also this pathway is indispensable for angiogenesis. This review highlights our understanding of TGF-ß signaling in vascular development and angiogenesis. In particular, we focus on recent insights into the role of the TGF-ß type I receptor ALK1 and co-receptor endoglin in tumor angiogenesis, which provide opportunities for the development of new anti-angiogenesis therapies for treatment of cancer patients.