VEGF-R2 and neuropilin-1 are involved in VEGF-A-induced differentiation of human bone marrow progenitor cells

Tumor growth and metastasis require the generation of new blood vessels, a process known as neo-angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor-A (VEGF-A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF-receptor tyrosine kinases and about the implication of the VEGF-R co-receptor, neuropilin-1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM-AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF-A on BM-AC133+ cells relies on an early action of VEGF-A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF-R2/neuropilin-1-dependent signaling pathway. This signaling promotes the differentiation of BM-AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF-R2 and/or neuropilin-1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors

There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

Vascular endothelial growth factor (VEGF-A) is a crucial stimulator of vascular cell migration and proliferation. Using bone marrow-derived human adult mesenchymal stem cells (MSCs) that did not express VEGF receptors, we provide evidence that VEGF-A can stimulate platelet-derived growth factor receptors (PDGFRs), thereby regulating MSC migration and proliferation. VEGF-A binds to both PDGFRalpha and PDGFRbeta and induces tyrosine phosphorylation that, when inhibited, results in attenuation of VEGF-A-induced MSC migration and proliferation. This mechanism was also shown to mediate human dermal fibroblast (HDF) migration. VEGF-A/PDGFR signaling has the potential to regulate vascular cell recruitment and proliferation during tissue regeneration and disease.     

Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus.     

Gastrulation and angiogenesis,not endothelial specification,is sensitive to partial deficiency in vascular endothelial growth factor-a in mice

Mouse embryogenesis is dose sensitive to vascular endothelial growth factor-A (VEGF-A), and mouse embryos partially deficient in VEGF-A die in utero because of severe vascular defects. In this study, we investigate the possible causes that underlie this phenomenon. Although the development of vascular defects in VEGF-A-deficient embryos seems to suggest that endothelial differentiation depends on the presence of a sufficient level of VEGF-A, we were surprised to find that endothelial differentiation per se is insensitive to a significant loss of VEGF-A activity. Instead, the development of the multipotent mesenchymal cells, from which endothelial progenitors arise in the yolk sac, is most highly dependent on VEGF-A. As a result of VEGF-A deficiency, dramatically fewer multipotent mesenchymal cells are generated in the prospective yolk sac. However, among the small number of mesenchymal cells that do enter the prospective yolk sac, endothelial differentiation occurs at a normal frequency. In the embryo proper, vasculogenesis is initiated actively in spite of a significant VEGF-A deficiency, but the subsequent steps of vascular development are defective. We conclude that a full-level VEGF-A activity is not critical for endothelial specification but is important for two distinct processes before and after endothelial specification: the development of the yolk sac mesenchyme and angiogenic sprouting of blood vessels.     

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A)

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure prior to chick embryo incubation (GD 0) induces dilated cardiomyopathy, and reduces myocardial hypoxia, vascular endothelial growth factor A (VEGF-A) expression, and coronary vascularization. We investigated whether reduced coronary vascularization 1) occurs in the absence of changes in cardiac morphology and 2) is associated with altered secretion of VEGF-A and/or an antivasculogenic factor. METHODS: Chicken eggs were treated with control (corn oil) or TCDD (0.075-0.3 pmol of TCDD/gm) on GD 5. In vivo cardiac morphology and artery number were determined on GD 10, while in vitro vascular outgrowth and VEGF-A secretion were determined from cardiac explants on GD 6. Effects of recombinant VEGF-A (rcVEGF-A), soluble flt-1 (sFlt-1) receptor plus rcVEGF-A, and control conditioned media were assessed in TCDD explants, while effects of TCDD-conditioned media was assessed in control explants. RESULTS: TCDD reduced coronary artery number in vivo by 53 +/- 8% and induced a dose-related reduction in tube outgrowth in vitro, but had no effect on cardiac morphology. All TCDD doses reduced explant VEGF-A secretion equally (43 +/- 3%), compared to control. sFlt-1 blocked outgrowth in control cultures and blocked rcVEGF-A-mediated rescue of outgrowth in TCDD explants. Control conditioned media partially rescued outgrowth from TCDD explants, while conditioned media from TCDD explants had no effect on controls. CONCLUSIONS: TCDD inhibition of coronary vascularization can occur in the absence of changes in cardiac morphology and is associated with reduced VEGF-A secretion but not an antivasculogenic factor. Since control media only partly rescues TCDD's inhibitory effect, we suggest that TCDD-exposed endothelial cells are less responsive to vasculogenic stimuli.     

Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos

Semaphorins are a large family of secreted and cell surface molecules that guide neural growth cones to their targets during development. Some semaphorins are expressed in cells and tissues beyond the nervous system suggesting the possibility that they function in the development of non-neural tissues as well. In the trunk of zebrafish embryos endothelial precursors (angioblasts) are located ventral and lateral to the somites. The angioblasts migrate medially and dorsally along the medial surface of the somites to form the dorsal aorta just ventral to the notochord. Here we show that in zebrafish Sema3a1 is involved in angioblast migration in vivo. Expression of sema3a1 in somites and neuropilin 1, which encodes for a component of the Sema3a receptor, in angioblasts suggested that Sema3a1 regulates the pathway of the dorsally migrating angioblasts. Antisense knockdown of Sema3a1 inhibited the formation of the dorsal aorta. Induced ubiquitous expression of sema3a1 in hsp70:(gfp)sema3a1(myc) transgenic embryos inhibited migration of angioblasts ventral and lateral to the somites and retarded development of the dorsal aorta, resulting in severely reduced blood circulation. Furthermore, analysis of cells that express angioblast markers following induced expression of sema3a1 or in a mutant that changes the expression of sema3a1 in the somites confirmed these results. These data implicate Sema3a1, a guidance factor for neural growth cones, in the development of the vascular system.     

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2

The Kruppel-like factor KLF2 was recently identified as a novel regulator of endothelial pro-inflammatory and pro-thrombotic function. Here it is shown that overexpression of KLF2 potently inhibits vascular permeability factor/vascular endothelial growth factor (VEGF-A)-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis. In vitro, KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammatory factors in endothelial cells. This effect is due to a potent inhibition of VEGFR2/KDR expression and promoter activity. These observations identify KLF2 as a regulator of VEGFR2/KDR and provide a foundation for novel approaches to regulate angiogenesis.     

The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation

Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required.     

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.     

Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-mediated mechanism

Growing evidences support that androgen displays beneficial effects on cardiovascular functions although the mechanism of androgen actions remains to be elucidated. Modulation of endothelial cell growth and function is a potential mechanism of androgen actions. We demonstrated in the present study that androgens [dihydrotestosterone (DHT) and testosterone], but not 17β-estradiol, produced a time- and dose-dependent induction of cell proliferation in primary human aortic endothelial cells (HAECs) as evident by increases in viable cell number and DNA biosynthesis. Real-time qRT-PCR analysis showed that DHT induced androgen receptor (AR), cyclin A, cyclin D1, and vascular endothelial growth factor (VEGF) gene expression in a dose- and time-dependent manner. The addition of casodex, a specific AR antagonist, or transfection of a specific AR siRNA blocked DHT-induced cell proliferation and target gene expression, indicating that the DHT effects are mediated via AR. Moreover, coadministration of SU5416 to block VEGF receptors, or transfection of a specific VEGF-A siRNA to knockdown VEGF expression, produced a dose-dependent blockade of DHT induction of cell proliferation and cyclin A gene expression. Interestingly, roscovitine, a selective cyclin-dependent kinase inhibitor, also blocked the DHT stimulation of cell proliferation with a selective inhibition of DHT-induced VEGF-A expression. These results indicate that androgens acting on AR stimulate cell proliferation through upregulation of VEGF-A, cyclin A, and cyclin D1 in HAECs, which may be beneficial to cardiovascular functions since endothelial cell proliferation could assist the repair of endothelial injury/damage in cardiovascular system.