Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth

Receptor tyrosine kinases of the Eph family are up-regulated in different types of cancer. EphB4 and its ligand ephrin-B2 have been linked to breast cancer, but little is known about how this receptor-ligand complex may contribute to oncogenesis. The Eph receptors transmit forward signals via their kinase domain and reverse signals via their transmembrane ephrin-B ligands. Therefore, we used EphB4 that were lacking the kinase domain and tagged with EGFP (EphB4 Delta C-EGFP) to differentiate between EphB4 and ephrin-B2 signaling. Interestingly, we found that expression of EphB4 Delta C-EGFP in breast cancer cells increases tumor growth in a mouse xenograft model. Given the undetectable EphB4 activation in the tumor cells, dominant negative effects of EphB4 Delta C-EGFP are unlikely to explain the increased tumor growth. Examination of the tumors revealed that ephrin-B2 is primarily expressed in the vasculature and that the EphB4 Delta C-EGFP tumors have a higher blood content than control tumors, concomitant with increased size of blood vessels. In support of an effect on the vasculature, the extracellular domain of EphB4 attracts endothelial cells in vitro and stimulates endothelial cell invasion, survival, and proliferation, all crucial factors for angiogenesis. These results support a model in which EphB4 promotes tumor growth by stimulating angiogenesis through ephrin-B2.     

Stromal cells expressing ephrin-B2 promote the growth and sprouting of ephrin-B2(+) endothelial cells

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arterial endothelial cells (ECs) and surrounding cells and interacts with multiple EphB class receptors. Conversely, EphB4, a specific receptor for ephrin-B2, is expressed on venous ECs, and both ephrin-B2 and EphB4 play essential roles in vascular development. The bidirectional signals between EphB4 and ephrin-B2 are thought to be specific for the interaction between arteries and veins and to regulate cell mixing and the making of particular boundaries. However, the molecular mechanism during vasculogenesis and angiogenesis remains unclear. Manipulative functional studies were performed on these proteins in an endothelial cell system. Using in vitro stromal cells (OP9 cells) and a paraaortic splanchnopleura (P-Sp) coculture system, these studies found that the stromal cells expressing ephrin-B2 promoted vascular network formation and ephrin-B2(+) EC proliferation and that they also induced the recruitment and proliferation of alpha-smooth muscle actin (alpha-SMA)-positive cells. Stromal cells expressing EphB4 inhibited vascular network formation, ephrin-B2(+) EC proliferation, and alpha-SMA(+) cell recruitment and proliferation. Thus, these data suggest that ephrin-B2 and EphB4 mediate reciprocal interactions between arterial and venous ECs and surrounding cells to form each characteristic vessel. (Blood. 2001;98:1028-1037)     

Hedgehog signaling in murine vasculogenesis and angiogenesis

Vasculogenesis-the formation of blood vessels de novo from endothelial cells-and angiogenesis-the process of blood vessel remodeling-are regulated by a number of signal transduction pathways, some specific to the vascular system and others used more broadly during embryogenesis. Recent evidence in both zebrafish and mouse suggests a role for Hedgehog (Hh) signaling in both vasculogenesis and angiogenesis. Hh signaling can target endothelial cells directly or can stimulate blood vessel support cells to produce vascular growth factors. Current studies are aimed at determining how the Hh cascade interacts with the other signaling pathways to promote vessel differentiation.     

EphB2 and EphB4 receptors forward signaling promotes SDF-1-induced endothelial cell chemotaxis and branching remodeling

The complex molecular mechanisms that drive endothelial cell movement and the formation of new vessels are poorly understood and require further investigation. Eph receptor tyrosine kinases and their membrane-anchored ephrin ligands regulate cell movements mostly by cell-cell contact, whereas the G-protein-coupled receptor CXCR4 and its unique SDF-1 chemokine ligand regulate cell movement mostly through soluble gradients. By using biochemical and functional approaches, we investigated how ephrinB and SDF-1 orchestrate endothelial cell movement and morphogenesis into capillary-like structures. We describe how endogenous EphB2 and EphB4 signaling are required for the formation of extracellular matrix-dependent capillary-like structures in primary human endothelial cells. We further demonstrate that EphB2 and EphB4 activation enhance SDF-1-induced signaling and chemotaxis that are also required for extracellular matrix-dependent endothelial cell clustering. These results support a model in which SDF-1 gradients first promote endothelial cell clustering and then EphB2 and EphB4 critically contribute to subsequent cell movement and alignment into cord-like structures. This study reveals a requirement for endogenous Eph signaling in endothelial cell morphogenic processes, uncovers a novel link between EphB forward signaling and SDF-1-induced signaling, and demonstrates a mechanism for cooperative regulation of endothelial cell movement.     

Regulation of vasculogenesis by platelet-mediated recruitment of bone marrow-derived cells

Bone marrow-derived cells contribute to physiological and pathological vascular remodeling throughout ontogenesis and adult life. During tissue regeneration and tumor growth, the release of cytokines and chemokines mediates the recruitment of hematopoietic and endothelial progenitor cells that contribute to the assembly of neovessels. Current evidence implies that platelets contribute structurally and instructively to vascular remodeling. Platelets adhere almost immediately to exposed or activated endothelium, and they are major storage and delivery vehicles for pro- and antiangiogenic growth factors including VEGF-A and thrombospondin (TSP), and cytokines and chemokines, such as stromal-derived factor 1 (SDF-1). By site-specific deployment of these factors, platelets orchestrate the local angiogenic stimulus within a tissue and direct the recruitment and differentiation of circulating bone marrow-derived cells. These insights have profound clinical implications; inhibition of platelet-deployed growth factors or their receptors may be an effective strategy to block tumor growth, whereas activation of these pathways may be used to accelerate revascularization and tissue regeneration.     

Regulation of angiogenesis and endothelial cell motility by matrix-bound fibroblasts

This study sought to examine the effect of matrix-bound fibroblasts on angiogenesis and endothelial cell motility. Promotion of angiogenesis by matrix-bound fibroblasts was assessed using rat aortic ring and endothelial tube formation assays. Enhancement of human endothelial cell motility by matrix-bound fibroblasts was assessed using cytodex-2 bead and colloidal gold phagokinetic motility assays. Antibody to hepatocyte growth factor/scatter factor (HGF/SF) but not vascular endothelial growth factor (VEGf) decreased fibroblast-enhanced motility of endothelial cells. The promotion of tube formation by matrix-bound fibroblasts was neutralised with antibodies to HGF/SF and VEGf, both known promoters of angiogenesis. HGF/SF presence was detected by ELISA; whilst the presence of VEGf was detected by Western blotting. These data show that matrix-embedded fibroblasts regulate the motility of vascular endothelial cells and enhance angiogenesis, an effect partly attributed to production of angiogenesis-promoting cytokines HGF/SF and/or VEGf. This revised version was published online in June 2006 with corrections to the Cover Date.     

Slit2/Robo1 signaling is involved in angiogenesis of glomerular endothelial cells exposed to a diabetic-like environment

Abnormal angiogenesis plays a pathological role in diabetic nephropathy (DN), contributing to glomerular hypertrophy and microalbuminuria. Slit2/Robo1 signaling participates in angiogenesis in some pathological contexts, but whether it is involved in glomerular abnormal angiogenesis of early DN is unclear. The present study evaluated the effects of Slit2/Robo1 signaling pathway on angiogenesis of human renal glomerular endothelial cells (HRGECs) exposed to a diabetic-like environment or recombinant Slit2-N. To remove the effect of Slit2 derived from mesangial cells, human renal mesangial cells (HRMCs) grown in high glucose (HG) medium (33 mM) were transfected with Slit2 siRNA and then the HG-HRMCs-CM with Slit2 depletion was collected after 48 h. HRGECs were cultured in the HG-HRMCs-CM or recombinant Slit2-N for 0, 6, 12, 24, or 48 h. The mRNA and protein expressions of Slit2/Robo1, PI3K/Akt and HIF-1α/VEGF signaling pathways were detected by quantitative real-time PCR, western blotting, and ELISA, respectively. The CCK-8 cell proliferation assay, flow cytometry and the scratch wound-healing assay were used to assess cell proliferation, cycles, and migration, respectively. Matrigel was used to perform a tubule formation assay. Our results showed that the HG-HRMCs-CM with Slit2 depletion enhanced the activation of Slit2/Robo1, PI3K/Akt, and HIF-1α/VEGF signaling in HRGECs in time-dependent manner (0–24 h post-treatment). In addition, the HG-HRMCs-CM with Slit2 depletion significantly promoted HRGECs proliferation, migration, and tube formation. Pretreatment of HRGECs with Robo1 siRNA suppressed the activation of PI3K/Akt and HIF-1α/VEGF signaling and inhibited angiogenesis, whereas PI3K inhibitor suppressed HIF-1α/VEGF signaling, without influencing Robo1 expression. In the HRGECs treated with Slit2-N, Slit2-N time-dependently enhanced the activation of Robo1/PI3K/Akt/VEGF pathway but not HIF-1α activity, and promoted HRGECs proliferation, migration, and tube formation. The effects induced by Slit2 were also abolished by Robo1 siRNA and PI3K inhibitor. Taken together, our findings indicate that in a diabetic-like environment, in addition to mesangial cells, autocrine activation of Slit2/Robo1 signaling of HRGECs may contribute to angiogenesis of HRGECs through PI3K/Akt/VEGF pathway; therefore, Slit2/Robo1 signaling may be a potent therapeutic target for the treatment of abnormal angiogenesis in early DN and may have broad implications for the treatment of other diseases dependent on pathologic angiogenesis.     

Regulation of adipogenesis and osteogenesis in mesenchymal stem cells by vascular endothelial growth factor A

Understanding the mechanisms by which bone marrow mesenchymal stem cells (BMSCs) differentiate into bone‐forming osteoblasts and marrow adipocytes is crucial to develop strategies for the treatment of several bone diseases. Age‐related bone loss resulting in osteopenia and osteoporosis has been associated with reduced numbers of osteoblasts and increased numbers of adipocytes, likely originating from differentiation defects in BMSCs. Although many factors involved in the complex regulation of osteoblast and adipocyte cell lineages have previously been identified, their functional interactions in the context of BMSC differentiation and maintenance of bone homeostasis during ageing are unknown. Recent discoveries have provided important new insights into the mechanisms by which the nuclear envelope protein lamin A and vascular endothelial growth factor A (VEGF) mutually control BMSC fate. Particularly interesting is the finding that VEGF in this context functions as an intracellular protein, unaffected by neutralizing antibodies, and not as a secreted growth factor. These insights may not only facilitate the identification of new targets for treating bone diseases but also lead to improved design of tissue engineering approaches aimed at stimulating bone regeneration and repair.     

Regulation of the endothelial function and angiogenesis by vascular endothelial growth factor-A (VEGF-A

VEGF-A (vascular endothelial growth factor-A) is an endothelial-specific growth factor that stimulates endothelial function and angiogenesis. VEGF-A plays an important role during development of the vascular system, wound healing, vascularization of tumors, and for angiogenesis in ischemic tissues including the heart. VEGF-A stimulates many actions of endothelial cells including proliferation, migration, and nitric oxide release via binding to and activation of the two primarily endothelial-specific receptor-tyrosine kinases KDR and Flt-1. KDR and Flt-1 stimulate multiple signal transduction pathways in endothelial cells. This review provides an overview of the role of VEGF-A in the regulation of endothelial function, angiogenesis, and arteriogenesis with regard to activation of signal transduction pathways and their functional consequences in the endothelium. Moreover, this article discusses recent developments exploring the therapeutic potential of VEGF-A for treatment of cardiovascular diseases.     

Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells

Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule that controls numerous cellular properties and activities. The oncogene v-p3k is a homolog of the gene coding for the catalytic subunit of PI 3-kinase, p110alpha. P3k induces transformation of cells in culture, formation of hemangiosarcomas in young chickens, and myogenic differentiation in myoblasts. Here, we describe a role of PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or of cellular PI 3-kinase equipped with a myristylation signal, Myr-P3k, can induce angiogenesis in the chorioallantoic membrane (CAM) of the chicken embryo. This process is characterized by extensive sprouting of new blood vessels and enlargement of preexisting vessels. Overexpression of the myristylated form of the PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Overexpression of the tumor suppressor PTEN or of dominant-negative constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of chicken embryos, suggesting that PI 3-kinase and Akt signaling is required for normal embryonal angiogenesis. The levels of mRNA for vascular endothelial growth factor (VEGF) are elevated in cells expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are also increased by insulin treatment through the PI 3-kinase-dependent pathway. VEGF mRNA levels are decreased in cells treated with the PI 3-kinase inhibitor LY294002 and restored by overexpression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS vector induces angiogenesis in chicken embryos. These results suggest that PI 3-kinase plays an important role in angiogenesis and regulates VEGF expression.     

血管内皮生长因子基因转染骨髓间充质干细胞移植促进缺血心肌血管生成的研究

目的研究血管内皮生长因子(vascularendothelial growth factor,VEGF)基因转染骨髓间充质干细胞(mesenchymalstem cells,MSCs)移植对缺血心肌的血管生成作用。方法于2004年5月至2005年8月取第四军医大学西京医院分离、培养Wistar大鼠的MSCs,用真核表达载体pcDNA3.1(-)/hVEGF165转染MSCs。45只近交系Wistar大鼠随机均分为转染组(MSCs/VEGF组)、对照组(MSCs组)、无血清培养基组(DMEM组),结扎前降支建立急性心肌梗死模型后在梗死区边缘区行5×106细胞移植,DMEM组行等量培养基注射。细胞移植前行CM-DiI标记。移植1个月后行心脏B超测量射血分数值,组织化学染色评价新生血管密度。结果培养的MSCs呈典型贴壁生长成纤维样外观,pcDNA3.1(-)/hVEGF165能有效转染大鼠MSCs,移植1个月后MSCs/VEGF组较其余各组左室射血分数(LVEF),再生血管密度明显增加,差异均有显著性(P<0.01)。结论VEGF基因转染MSCs移植能显著促进缺血心肌血管再生,进而改善心脏功能。     

Effects of statins on angiogenesis and vasculogenesis

Statins promote the proliferation, migration, and survival of endothelial cells and bone marrow-derived endothelial progenitor cells (angioblasts) by stimulating the serine/threonine protein kinase Akt (also known as protein kinase B) pathway. Like vascular endothelial growth factor (VEGF), the statins promote angiogenesis and vasculogenesis. Therefore, Akt activation may explain some of the beneficial effects of the statins, including postnatal neovascularization.     

Glycogen-Synthase Kinase3beta/beta-catenin axis promotes angiogenesis through activation of vascular endothelial growth factor signaling in endothelial cells

Glycogen-Synthase Kinase 3beta (GSK3beta) has been shown to function as a nodal point of converging signaling pathways in endothelial cells to regulate vessel growth, but the signaling mechanisms downstream from GSK3beta have not been identified. Here, we show that beta-catenin is an important downstream target for GSK3beta action in angiogenesis and dissect the signal transduction pathways involved in the angiogenic phenotype. Transduction of human umbilical vein endothelial cells (HUVECs) with a kinase-mutant form of the enzyme (KM-GSK3beta) increased cytosolic beta-catenin levels, whereas constitutively active GSK3beta (S9A-GSK3beta) reduced beta-catenin levels. Lymphoid enhancer factor/T-cell factor promoter activity was upregulated by KM-GSK3beta and diminished by S9A-GSK3beta, whereas manipulation of Akt signaling had no effect on this parameter. beta-Catenin transduction induced capillary formation in a Matrigel-plug assay in vivo and promoted endothelial cell differentiation into network structures on Matrigel-coated plates in vitro. beta-Catenin activated the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C in endothelial cells, and these effects were mediated at the levels of protein, mRNA, and promoter activity. Consistent with these data, beta-catenin increased the phosphorylation of the VEGF receptor 2 (VEGF-R2) and promoted its association with PI3-kinase, leading to a dose-dependent activation of the serine-threonine kinase Akt. Inhibition of PI3-kinase or Akt signaling led to a significant reduction in the pro-angiogenic activity of beta-catenin. Collectively, these data show that the growth factor-PI3-kinase-Akt axis functions downstream of GSK3beta/beta-catenin signaling in endothelial cells to promote angiogenesis.