The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching

Delta-like 4 (Dll4) is a transmembrane ligand for Notch receptors that is expressed in arterial blood vessels and sprouting endothelial cells. Here we show that Dll4 regulates vessel branching during development by inhibiting endothelial tip cell formation. Heterozygous deletion of dll4 or pharmacological inhibition of Notch signaling using gamma-secretase inhibitor revealed a striking vascular phenotype, with greatly increased numbers of filopodia-extending endothelial tip cells and increased expression of tip cell marker genes compared with controls. Filopodia extension in dll4(+/-) retinal vessels required the vascular growth factor VEGF and was inhibited when VEGF signaling was blocked. Although VEGF expression was not significantly altered in dll4(+/-) retinas, dll4(+/-) vessels showed increased expression of VEGF receptor 2 and decreased expression of VEGF receptor 1 compared with wild-type, suggesting they could be more responsive to VEGF stimulation. In addition, expression of dll4 in wild-type tip cells was itself decreased when VEGF signaling was blocked, indicating that dll4 may act downstream of VEGF as a "brake" on VEGF-mediated angiogenic sprouting. Taken together, these data reveal Dll4 as a negative regulator of vascular sprouting and vessel branching that is required for normal vascular network formation during development.     

Dll4 activation of Notch signaling reduces tumor vascularity and inhibits tumor growth

Gene targeting experiments have shown that Delta-like 4 (Dll4) is a vascular-specific Notch ligand critical to normal vascular development. Recent studies have demonstrated that inhibition of Dll4/Notch signaling in tumor-bearing mice resulted in excessive, yet nonproductive tumor neovascularization and unexpectedly reduced tumor growth. Because nonfunctional blood vessels have the potential to normalize, we explored the alternative approach of stimulating Notch signaling in the tumor vasculature to inhibit tumor growth. Here we show that retrovirus-induced over-expression of Dll4 in tumor cells activates Notch signaling in cocultured endothelial cells and limits vascular endothelial growth factor (VEGF)-induced endothelial cell growth. Tumors produced in mice by injection of human and murine tumor cells transduced with Dll4 were significantly smaller, less vascularized and more hypoxic than controls, and displayed evidence of Notch activation. In addition, tumor blood perfusion was reduced as documented by vascular imaging. These results demonstrate that Notch activation in the tumor microenvironment reduces tumor neovascularization and blood perfusion, and suggest that Dll4-induced Notch activation may represent an effective therapeutic approach for the treatment of solid tumors.     

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.     

The Dll4/Notch pathway controls postangiogenic blood vessel remodeling and regression by modulating vasoconstriction and blood flow

Blood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and Dll4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.     

Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.     

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting

Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.     

Overexpression of delta-like 4 induces arterialization and attenuates vessel formation in developing mouse embryos

The importance of Notch signaling pathway in the regulation of vascular development and angiogenesis is suggested by the expression of Notch receptors and ligands in vascular endothelial cells (ECs) and the observed vascular phenotypes in mutants of Notch receptors or ligands, especially Dll4. DLL4 is specifically expressed in arterial ECs during development, and haplo-insufficiency is embryonically lethal in mice. To address the role of Dll4 in vascular development, we produced mDll4 conditionally overexpressed transgenic mice that were crossed with constitutive recombinase cre lines. Double transgenic embryos displayed grossly enlarged dorsal aortae (DA) and died before embryonic day 10.5 (E10.5), showing a variable degree of premature arteriovenous fusion. Veins displayed ectopic expression of arterial markers. Other defects included reduced vascular sprouting, EC proliferation, and migration. mDll4 overexpression also inhibited VEGF signaling and increased fibronectin accumulation around the vessels. In vitro and in vivo studies of DLL4-FL (Dll4-full-length) in ECs recapitulate many of the mDll4 transgenics findings, including decreased tube formation, reduced vascular branching, fewer vessels, increased pericyte recruitment, and increased fibronectin expression. These results establish the role of Dll4 in arterial identity determination, and regulation of angiogenesis subject to dose and location.     

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4^+/? mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4^+/? CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.     

Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro- and anti-angiogenic therapies.     

A novel Notch ligand, Dll4, induces T-cell leukemia/lymphoma when overexpressed in mice by retroviral-mediated gene transfer.

Notch receptors mediate cell-fate decisions through interaction with specific ligands during development. The biological role of a novel Notch ligand, Dll4, in mice was explored by reconstituting lethally irradiated mice with bone marrow (BM) cells transduced with Dll4 retroviral vector. White blood cell and lymphocyte counts in Dll4-overexpressing mice were reduced at the early stage of reconstitution but increased significantly at approximately 10 weeks after BM transplantation. BM, spleen, lymph nodes, and peripheral blood of Dll4-overexpressing mice contained predominantly CD4(+)CD8(+) T cells and virtually lacked B cells. The Dll4-overexpressing mice eventually developed a lethal phenotype that was characterized by the progression of a T-cell lymphoproliferative disease (restricted to BM and lymphoid tissues) to transplantable monoclonal T-cell leukemia/lymphoma scattered to multiple organs. Results suggest that the interaction of Dll4 with Notch1 may provide key signals for T-cell development.     

Expression of Delta-like protein 4 in the human endometrium

Activation of Delta-Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues, and cancerous lesions. Delta and Notch genes are known to be expressed in endothelial cells, and little is known of their expression beyond the vascular system. The purpose of this study was to investigate whether Delta gene would be expressed in cells of the uterine endometrium. In this study, we found that the human endometrial cells expressed one of the Delta ligands, Delta-like 4 protein (Dll4). Dll4 was expressed in human endometrium in a spatiotemporal fashion. Immunohistochemistry studies showed the cytoplasm as well as membrane staining with apical localization both in the luminal and glandular epithelium and moderate diffuse staining in the cytoplasm of the stromal cells. Western blot analysis showed that the size of the endometrial Dll4 was identical to that in the human umbilical endothelial cells. The expression of Dll4 mRNA in human endometrial cells was quantitatively determined by real-time PCR. Dll4 mRNA expressed in the glandular epithelium showed large variations, and it was significantly elevated in the mid and late proliferative and early secretory endometrium. Endometrial stromal cells contained less Dll4 mRNA and had no clear correlation with the menstrual cycle. The effect of hormones was studied in the primary culture of isolated glandular epithelial and stromal cells. In glandular cells, estradiol had little effect, and medroxyprogesterone acetate significantly reduced the mRNAs compared with that of control. Relaxin induced the Dll4 mRNA. In stromal cells, both estradiol and medroxyprogesterone acetate reduced the Dll4 mRNA. To our knowledge, this is the first report of the expression of Dll4 in the endometrium. We propose that endometrial Dll4 may enhance the development of the endometrial microvascular system and facilitate the implantation of blastocyst in a fertile cycle.     

Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration

Neuropilin 2 (NRP2) is a receptor for the vascular endothelial growth factor (VEGF) and the semaphorin (SEMA) families, 2 unrelated ligand families involved in angiogenesis and neuronal guidance. NRP2 specifically binds VEGF-A and VEGF-C, although the biological relevance of these interactions in human endothelial cells is poorly understood. In this study, we show that both VEGF-A and VEGF-C induce the interaction of NRP2 with VEGFR-2. This interaction correlated with an enhancement of the VEGFR-2 phosphorylation threshold. Overexpression of NRP2 in primary human endothelial cells promoted cell survival induced by VEGF-A and VEGF-C. In contrast, SEMA3F, another ligand for NRP2, was able to inhibit human endothelial cell survival and migration induced by VEGF-A and VEGF-C. Moreover, a siRNA targeting specifically NRP2 was a potent inhibitor of human endothelial cell migration induced by VEGF-A and VEGF-C. Thus, our data indicate that NRP2 acts as a coreceptor that enhances human endothelial cell biological responses induced by VEGF-A and VEGF-C.     

Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells

The Notch ligand, Dll4, is essential for angiogenesis during embryonic vascular development and is involved in tumour angiogenesis. Several recent publications demonstrated that blockade of Dll4 signalling inhibits tumour growth, suggesting that it may constitute a good candidate for anti-cancer therapy. In order to understand the role of Dll4 at the cellular level, we performed an analysis of Dll4-regulated genes in HUVECs. The genes identified included several angiogenic signalling pathways, such as VEGF, FGF and HGF. In particular we identified downregulation (VEGFR2, placenta growth factor PlGF) of VEGF pathway components resulting in the overall effect of limiting the response of HUVEC to VEGF. However extensive upregulation of VEGFR1 was observed allowing continued response to its ligand PlGF but the soluble form of the VEGFR1, sVEGFR1 was also upregulated. PlGF enhanced tubulogenesis of HUVEC suggesting that downregulation of PlGF and upregulation of VEGFR1 including sVEGFR1 are important mechanisms by which Dll4 attenuates PlGF and VEGF signalling. Dll4-stimulated HUVECs had impaired ERK activation in response to VEGF and HGF indicating that Dll4 signalling negatively regulates these pathways. Dll4 expression reduced vessel sprout length in a 3D tubulogenesis assay confirming that Dll4 signalling inhibits angiogenesis. Altogether, our data suggest that Dll4 expression acts as a switch from the proliferative phase of angiogenesis to the maturation and stabilisation phase by blocking endothelial cell proliferation and allowing induction of a more mature, differentiated phenotype. The regulation of sVEGFR1 provides a novel mechanism for Dll4 signalling to regulate cells at distance, not just in adjacent cells.     

Inhibition of Dll4-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion

Vascular development is dependent on various growth factors and certain modifiers critical for providing arterial or venous identity, interaction with the surrounding stroma and tissues, hierarchic network formation, and recruitment of pericytes. Notch receptors and ligands (Jagged and Delta-like) play a critical role in this process in addition to VEGF. Dll4 is one of the Notch ligands that regulates arterial specification and maturation events. In the current study, we have shown that loss of function by either targeted allele deletion or use of a soluble form of Dll4 extracellular domain leads to inhibition of Notch signaling, resulting in increased vascular proliferation but defective maturation. Newly forming vessels have thin caliber, a markedly reduced vessel lumen, markedly reduced pericyte recruitment, and deficient vascular perfusion. sDll4 similarly induced defective vascular response in tumor implants leading to reduced tumor growth. Interference with Dll4-Notch signaling may be particularly desirable in tumors that have highly induced Dll4-Notch pathway.     

BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.     

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function.We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells.While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration).In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.     

Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.     

miR-27b controls venous specification and tip cell fate

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.     

Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A

Vascular endothelial growth factor (VEGF) signaling is required for both differentiation and proliferation of vascular endothelium. Analysis of differentiated embryonic stem cells with one or both VEGF-A alleles deleted showed that both the differentiation and the expansion of endothelial cells are blocked during vasculogenesis. Blood island formation was reduced by half in hemizygous mutant VEGF cultures and by 10-fold in homozygous mutant VEGF cultures. Homozygous mutant cultures could be partially rescued by the addition of exogenous VEGF. RNA levels for the endothelial adhesion receptors ICAM-2 and PECAM were reduced in homozygous mutant cultures, but ICAM-2 RNA levels decreased substantially, whereas PECAM RNA levels remained at hemizygous levels. The quantitative data correlated with the antibody staining patterns because cells that were not organized into vessels expressed PECAM but not ICAM-2. These PECAM+ cell clumps accumulated in mutant cultures as vessel density decreased, suggesting that they were endothelial cell precursors blocked from maturation. A subset of PECAM+ cells in clumps expressed stage-specific embryonic antigen-1 (SSEA-1), and all were ICAM-2(-) and CD34(-), whereas vascular endothelial cells incorporated into vessels were PECAM(+), ICAM-2(+), CD34(+), and SSEA-1(-). Analysis of flk-1 expression indicated that a subset of vascular precursor cells coexpressed PECAM and flk-1. These data suggest that VEGF signaling acts in a dose-dependent manner to affect both a specific differentiation step and the subsequent expansion of endothelial cells. (Blood. 2000;95:1979-1987)     

Overexpression of CREG attenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway

Aims

Cellular repressor of E1A-stimulated genes (CREG) is a homeostasis-modulating gene abundantly expressed in adult artery endothelium. Previous studies have demonstrated a protective effect of CREG against atherosclerosis through prevention of vascular smooth muscle cell apoptosis. However, the role of CREG in endothelial cells (ECs) apoptosis and the underlying signaling mechanisms are unknown.

Method and results

We ascertained that CREG expression was decreased in atherogenesis-prone endothelium in apolipoprotein E-null (apoE^−/−) mice compared with their wild-type littermates using in situ immunofluorescent staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining and caspase-3 activity assays determined that treatment of apoE^−/− mice arteries with staurosporine (STS) significantly induced endothelial apoptosis associated with a reduction of CREG expression. Gain- and loss-of-function analyses revealed that silencing CREG expression significantly enhanced ECs apoptosis, whereas CREG overexpression abrogated apoptosis stimulated by STS or etoposide (VP-16). Blocking assays using the neutralizing antibody for vascular endothelial growth factor (VEGF) and the specific inhibitor of phosphoinositide 3-kinase (PI3K), such as LY294002 or wortmannin, demonstrated that the protective effect of CREG on ECs apoptosis was mainly mediated by activation of the VEGF/PI3K/AKT signaling pathway.

Conclusions

These data demonstrate that CREG plays a critical role in protecting the vascular endothelium from apoptosis, and the protective effort of CREG against ECs apoptosis is through the activation of the VEGF/PI3K/AKT signaling pathway.