Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Generation of transgenic mice overexpressing EfnB2 in endothelial cells

Genetic studies have shown that ephrin-B2 and its cognate EphB4 receptor are necessary for normal embryonic angiogenesis. Moreover, there is overwhelming evidence that ephrin-B2 is involved in tumor vascularization, yet its role in adult angiogenesis has been difficult to track genetically. Here, we report the generation of transgenic mice that over-express EfnB2 specifically in endothelial cells (ECs). We show that exogenous expression of EfnB2 under the control of the Tie2 promoter/enhancer regions in ECs does not affect viability or growth of the transgenic animals. We further show that targeted expression of EfnB2 in ECs is not sufficient to rescue severe cardiovascular defects at mid-gestation stages but rescues early embryonic lethality associated with loss-of-function mutation in EfnB2. This mouse model will be useful to study the role of ephrin-B2 in physiological and pathological angiogenesis.     

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.     

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.     

Membrane type 1-matrix metalloproteinase is regulated by chemokines monocyte-chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in endothelial cells during angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.     

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate that beta1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting beta1 integrins may effectively impair neovascularization.     

Angiogenic interaction between retinal endothelial cells and pericytes from normal and diabetic rabbits, and phenotypic changes of diabetic cells.

Retinal endothelial cells (ECs) and pericytes (PCs) were cloned and cultured from normal and diabetic rabbits to clarify the mechanism of diabetic proliferative retinopathy from the viewpoint of the interaction between ECs and PCs, and phenotypic changes of diabetic cells. PC-conditioned medium (PC-CM) from normal rabbits stimulated in vitro angiogenesis of diabetic ECs more than that of normal ECs. in vitro angiogenesis was also more stimulated in diabetic ECs than in normal ECs by basic fibroblast growth factor (bFGF) or transforming growth factor-beta 1, indicating that diabetic ECs are different from normal ECs in terms of angiogenic potential. One mechanism of this property of diabetic ECs was the acceleration of cell proliferation but not of cell migration, because diabetic ECs grew more rapidly but did not migrate more than normal ECs in response to PC-CM or bFGF. Moreover, PC-CM from diabetic PCs stimulated angiogenesis of normal ECs more than that from normal PCs, indicating that diabetic PCs secreted more angiogenic factor(s) than normal PCs. The angiogenic, mitogenic and migratory activities of PC-CM both from normal and diabetic PCs were similarly inhibited by an anti-bFGF antibody. Western blot analysis revealed this factor to be a bFGF-like molecule. These data indicate that the interaction between ECs and PCs and the phenotypic changes of diabetic ECs and PCs both contribute to the proliferative retinopathy in diabetes.     

Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation

It is now well documented that the growth and meta-stasis of malignant tumors beyond a few mm3 dependlargely upon the formation of networks known as angio-genesis [1–3]. Several studies have shown that the tumormass can be restricted to within a certain …     

FGF-16 is required for embryonic heart development

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.     

In silico analysis of angiogenesis associated gene expression identifies angiogenic stage related profiles

In vitro models have been extensively used to map gene expression in ECs but few studies have used cells from in vivo sources directly. Here, we compare different gene expression surveys on both cultured and fresh tissue derived ECs, and it emerges that gene expression profiles can be paralleled with the angiogenic stage of the cells. ECs stimulated with different growth factors in monolayer cultures exhibit gene expression profiles indicative of an active proliferative state, whereas gene expression in tube forming cells in vitro involves genes implicated in cell adhesion processes. Genes overexpressed in tumor ECs are biased towards extracellular matrix remodeling, a late event in angiogenesis. The elucidation of gene expression profiles under these different conditions will contribute to a better understanding of the molecular mechanisms during angiogenesis in both pathological and physiological circumstances and will have implications for the development of angiogenesis interfering treatment strategies.