Embryonic Endothelial Cells Research Articles

The Role of Neuregulin/erbB Signaling in Endothelial Progenitor Cell Biology

Neuregulin (NRG) is a ligand for the erbB3 and erbB4 members of the EGF family receptors, and is known to activate signaling pathways that mediate proliferation, survival, migration and differentiation in various cell types. Stimuli such as exercise and ischemia‐reperfusion, which induce NRG shedding also induce endothelial progenitor cell (EPC) mobilization and recruitment. We hypothesized that NRG signaling may a play role in EPC mobilization and homing.Using western blot and immunostaining, we found that a murine embryonic endothelial progenitor cell line expresses NRG ligand and the ErbB‐2, 3 and 4 receptors. Treatment with recombinant NRG led to ErbB2/B3 phosphorylation as well as downstream Akt and endothelial nitric oxide synthase phosphorylation. NRG activated signaling was blocked by the ErbB receptor inhibitors AG1478 and Lapatanib. To examine the role of NRG/ErbB signaling in EPC biology we performed a matrigel angiogenesis assay. In contrast to mature rat cardiac microvascular endothelial cells (CMEC), EPC's in 3 dimensional monoculture do not form microvessel networks. However when EPC's are co‐cultured with CMEC's in matrigel, EPC's incorporate into the developing microvessel network within 24 hrs. This effect was completely inhibited by the erbB receptor inhibitor AG1478.These results support a role for Neuregulin/ErbB signaling in EPC recruitment. A clearer understanding of what role NRG signaling may play in EPC biology has important implications for cardiovascular biology, as well as the pathophysiology of cardiac dysfunction seen in the setting of erbB2 targeted cancer therapeutics.

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

BackgroundWe demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.Methods and ResultsAfter the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity.ConclusionsOur findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.

Primary cilia sensitize endothelial cells for fluid shear stress

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress.

Upregulation of matrix and adhesion molecules induced by controlled topography

Electrostatic spinning is receiving increasing attention in the field of tissue engineering, due to its ability to produce 3-dimensional, multidirectional, microfibrous scaffolds. These structures are capable of supporting a wide range of cell growth; however, there is little knowledge relating material substrates with specific cellular interactions and responses. The aim of this research was to investigate if electrostatically spun scaffolds, with controlled topographical features, would affect the adhesion mechanisms of contacting cells. A range of electrostatically spun Tecoflex SG-80A polyurethane scaffolds was characterized in terms of inter-fibre separation, fibre diameter, surface roughness, void fraction and fibre orientation. Human embryonic lung fibroblasts and human vein endothelial cells were cultured on these scaffolds for 7, 14, 28 days, and analysed for their expression of extracellular matrix and adhesion molecules using image analysis and laser scanning confocal microscopy. There were significant differences in adhesion mechanisms between scaffolds, cell types and culture periods. Fibroblast-scaffolds were stimulated and oriented to a greater degree, and at earlier cultures, by the controlled topographical features than the endothelial cells. These conclusions confirm that cellular behaviour can be influenced by the induced scaffold topography at both molecular and cellular levels, with implications for optimum application specific tissue engineering constructs.

Possible role of NFĸB in the embryonic vascular remodeling and the endothelial-mesenchymal transition process

The NF-κB family of transcription factors, particularly the activated p50/p65 heterodimer, is expressed in vascular cells during intimal thickening formation when hemodynamic conditions are altered. Here, we report that p50, p65, IκB-α and IKK-α display different spatial and temporal patterns of expression and distribution during both chicken embryo aortic wall remodeling and intimal thickening development. Additionally, we show that both p50 and p65 were located in the nucleus of some mesenchymal cells expressing α-smooth muscle actin which are present in the spontaneous intimal thickening observed at embryonic days 12-14 of development. We also demonstrated that both NF-kB subunits are present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin and stimulated with complete medium. This study demonstrates for the first time the presence of activated NF-kB during the remodeling of the embryonic aortic wall and the formation of intimal thickening, providing evidence that suggest a possible role for this transcription factor in the EndoMT process.

Tissue Engineering by Self-Assembly of Cells Printed into Topologically Defined Structures

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.

Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation

Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.

Requirements for cellular co-trafficking of factor VIII and von Willebrand factor to Weibel-Palade bodies.

von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding (K(d) = 0.065 +/- 0.008 nm) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel-Palade bodies (WPBs). Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.

Evaluating Drug Efficacy and Toxicology in Three Dimensions: Using Synthetic Extracellular Matrices in Drug Discovery

The acceptance of the new paradigm of 3-D cell culture is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. I describe the development of a covalently cross-linked mimic of the extracellular matrix (sECM), now commercially available, for 3-D culture of cells in vitro and for translational use in vivo. These bio-inspired, biomimetic materials can be used "as is" in drug discovery, toxicology, cell banking, and, ultimately, medicine. For cell therapy and the development of clinical combination products, the sECM biomaterials must be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end users, physicians and patients, must dictate the key design criteria. In chemical terms, the sECM consists of chemically-modified hyaluronan (HA), other glycosaminoglycans (GAGs), and ECM polypeptides containing thiol residues that are cross-linked using biocompatible polyvalent electrophiles. For example, co-cross-linking the semisynthetic thiol-modified HA-like GAG with thiol-modified gelatin produces Extracel as a hydrogel. This hydrogel may be formed in situ in the presence of cells or tissues to provide an injectable cell-delivery vehicle. Alternately, an Extracel hyrogel can be lyophilized to create a macroporous scaffold, which can then be employed for 3-D cell culture. In this Account, we describe four applications of sECMs that are relevant to the evaluation of drug efficacy and drug toxicity. First, the uses of sECMs to promote both in vitro and in vivo growth of healthy cellularized 3-D tissues are summarized. Primary or cell-line-derived cells, including fibroblasts, chondrocytes, hepatocytes, adult and embryonic stem cells, and endothelial and epithelial cells have been used. Second, primary hepatocytes retain their biochemical phenotypes and achieve greater longevity in 3-D culture in Extracel. This constitutes a new 3-D method for rapid evaluation of hepatotoxicity in vitro. Third, cancer cell lines are readily grown in 3-D culture in Extracel, offering a method for rapid evaluation of new anticancer agents in a more physiological ex vivo tumor model. This system has been used to evaluate signal transduction modifiers obtained from our research on lipid signaling. Fourth, a new "tumor engineering" xenograft model uses orthotopic injection of Extracel-containing tumor cells in nude mice. This approach allows production of patient-specific mice using primary human tumor samples and offers a superior metastatic cancer model. Future applications of the injectable cell delivery and 3-D cell culture methods include chemoattractant and angiogenesis assays, high-content automated screening of chemical libraries, pharmacogenomic and toxicogenomic studies with cultured organoids, and personalized treatment models. In summary, the sECM technology offers a versatile "translational bridge" from in vitro to in vivo to facilitate drug discovery in both academic and pharmaceutical laboratories.

PRDM6 is enriched in vascular precursors during development and inhibits endothelial cell proliferation, survival, and differentiation

The mechanisms that regulate the differentiation program of multipotential stem cells remain poorly understood. In order to define the cues that delineate endothelial commitment from precursors, we screened for candidate regulatory genes in differentiating mouse embryoid bodies. We found that the PR/SET domain protein, PRDM6, is enriched in flk1(+) hematovascular precursor cells using a microarray-based approach. As determined by 5′ RACE, full-length PRDM6 protein contains a PR domain and four Krüppel-like zinc fingers. In situ hybridization in mouse embryos demonstrates staining of the primitive streak, allantois, heart, outflow tract, paraaortic splanchnopleura (P-Sp)/aorto–gonadal–mesonephric (AGM) region and yolk sac, all sites known to be enriched in vascular precursor cells. PRDM6 is also detected in embryonic and adult-derived endothelial cell lines. PRDM6 is co-localized with histone H4 and methylates H4–K20 (but not H3) in vitro and in vivo, which is consistent with the known participation of PR domains in histone methyltransferase activity. Overexpression of PRDM6 in mouse embryonic endothelial cells induces apoptosis by activating caspase-3 and inducing G1 arrest. PRDM6 inhibits cell proliferation as determined by BrdU incorporation in endothelial cells, but not in rat aortic smooth muscle cells. Overexpression of PRDM6 also results in reduced tube formation in cultured endothelial cells grown in Matrigel. Taken together, our data indicate that PRDM6 is expressed by vascular precursors, has differential effects in endothelial cells and smooth muscle cells, and may play a role in vascular precursor differentiation and survival by modulating local chromatin-remodeling activity within hematovascular subpopulations during development.

Calcitriol (1,25-Dihydroxycholecalciferol) Selectively Inhibits Proliferation of Freshly Isolated Tumor-Derived Endothelial Cells and Induces Apoptosis

Calcitriol (1,25-dihydroxycholecalciferol) has antiproliferative and/or proapoptotic effects on many cell types and the glucocorticoid dexamethasone enhances these effects. We have shown that calcitriol modulates several key signaling proteins involved in differentiation, proliferation and apoptosis in tumor-derived murine endothelial cells (TDEC) and that these effects were not seen with endothelial cells isolated similarly from normal tissues. In the present study, TDEC and mouse embryonic yolk sac endothelial cells (MYSEC) were treated with calcitriol and followed over time for an effect. MYSEC were utilized as ‘normal’ control endothelial cells because they were more primitive, being isolated from a highly neovascular tissue, and had a similar morphology without the stimulus of the tumor microenvironment. The vitamin D receptor (VDR) is present in TDEC and MYSEC, and was upregulated in calcitriol-treated TDEC and MYSEC; dexamethasone further increased VDR expression following 48 h of treatment. The modulatory effects on signaling proteins were maximal by treatment for 48 h; phospho-Erk, phospho-Akt, p21 and bcl-2 were decreased in treated TDEC with the induction of p27 but there were no effects on MYSEC. After 48 h increased apoptosis was seen in treated TDEC by annexin V labeling with caspase-3 cleavage and decreased levels of poly(ADP-ribose) polymerase, but no effects were seen in MYSEC. Cell cycle analysis showed increased G₀/G₁ arrest and an increase in the apoptotic sub-G₁ peak in treated TDEC but similar effects were not seen in MYSEC following 48-hour treatment. Proliferation assays were utilized and TDEC demonstrated decreased proliferation compared to normal endothelial cells at 48 h. To determine whether or not the VDR signaling was impaired in MYSEC, we performed the 24-hydroxylase (CYP24) promoter-luciferase reporter assay. CYP24 is a key enzyme involved in the breakdown of vitamin D. VDR signaling was intact in both cell types and calcitriol induced CYP24 mRNA expression in MYSEC but not in TDEC. Taken together, despite similar levels of VDR expression and intact signaling in both cell types, calcitriol selectively inhibits proliferation and induces apoptosis in TDEC with no effect on MYSEC. Thus calcitriol exerts differential effects on TDEC compared to normal cells.

EGFL7 in vascular development and repair

Egfl7 is an EGF-domain gene expressed in developing blood vessels in mouse embryos, and during endothelial regeneration in adults. Our aim is to determine the role of Egfl7 in the formation and maintenance of blood vessels. Using in situ hybridisation and immunostaining we have found that Egfl7 is present in the inner cell mass at embryonic day 7.5, in the visceral yolk sac at sites of angioblast differentiation, and in the vascular endothelium and endocardium of the embryo proper. Furthermore, Egfl7 expression is down-regulated in adult organs with notable exceptions: lung, sites of vascular injury, and pregnant uterus. Our results indicate that EGFL7 is a secreted cell surface protein, and cell migration assays show that the protein is a chemoattractant for embryonic endothelial cells and fibroblasts. Preliminary work implies that EGFL7 interacts with endothelial Notch4, and may bind to extracellular matrix proteins. Together, these results strongly suggest that Egfl7 has an important role during development and regeneration of the vasculature, and that EGFL7-positive cells could potentially be target cells for future gene delivery studies into whole animals. We will report on continuing work using siRNA-mediated knock-down, GFP knock-in, and transgenic mouse lines. Funding provided by NIH grants; R21 HL072270, R01 HL082098 to H.S., in part by T32 HL07695 to M.J.F. and S.C., and AHA fellowship 0525046Y to A.D.

Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 in the mouse embryo

AbstractVascular endothelial (VE) cadherin, PECAM-1 (platelet endothelial cell adhesion molecule-1, CD31), Tie2, CD34, and endoglin are established markers for adult and embryonic endothelial cells (ECs). Here, we report that the expression of these EC markers is initiated in the extraembryonic region at the late-streak stage (nominal stage E6.75). Immunohistochemical analysis shows that EC marker–positive cells arise in a subset of Flk1 (VEGF-R2) mesodermal cells. In contrast, GATA1, a marker for primitive erythropoietic progenitors, is expressed in a more restricted subset of Flk1-positive cells. Using flow cytometry, we observed that the GATA1-positive cell population existed as a subset of the EC marker–positive cell. Consistent with this notion, we showed with the primitive hematopoietic colony assay that primitive erythropoietic progenitors are enriched in PECAM-1– and Tie2-positive cells. These results suggest that primitive hematopoietic cells arise from EC marker–positive cells. Thus, VE-cadherin, PECAM-1, CD34, endoglin, and Tie2 are expressed not only in adult and embryonic ECs but in extraembryonic Flk1-positive cells during gastrulation. The latter cell population includes progenitors that give rise to primitive hematopoietic cells, suggesting that primitive and definitive hematopoietic cells in the mouse embryo arise from EC marker–positive cells.

Molecular Pathways Regulating Pro-migratory Effects of Hedgehog Signaling

The Hedgehog proteins play a crucial role in metazoan embryo development. Constitutive activation of the pathway is associated with multiple types of cancer. Recent experimental data suggest involvement of Hedgehog signaling in vascular remodeling, germ cell migration, and axon guidance. The molecular mechanisms underlying these effects remain elusive. Here we show that yolk sac-derived endothelial cells and embryonic fibroblasts can directly respond to the Hedgehog signal by increased migration in an in vitro scratch (wound) assay. We also identify Hedgehog transcriptional target genes in these cells, many of which participate in cell migration, axon guidance, and angiogenesis processes. Inhibition of one such molecular pathway, neuropilin-flavomonooxygenase, blocks Hedgehog-induced cell migration. These findings suggest that Hedgehog signaling directly affects embryonic endothelial and fibroblast cell migration via molecules and pathways known to regulate cell migration in response to a variety of environmental cues.

Stromal Cell-Derived Factor-1/CXCR4 Signaling Modifies the Capillary-Like Organization of Human Embryonic Stem Cell-Derived Endothelium In Vitro

The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here we used human ESCs (hESCs) as an in vitro model to explore early human vasculogenesis. We demonstrated that stromal cell-derived factor-1 (SDF-1) and CXCR4 were expressed concurrently with hESC-derived embryonic endothelial differentiation. Human ESC-derived embryonic endothelial cells underwent dose-dependent chemotaxis to SDF-1, which enhanced vascular network formation in Matrigel. Blocking of CXCR4 signaling abolished capillary-like structures induced by SDF-1. Inhibition of the SDF-1/CXCR4 signaling pathway by AMD3100, a CXCR4 antagonist, disrupted the endothelial sprouting outgrowth from human embryoid bodies, suggesting that the SDF-1/CXCR4 axis plays a critical role in regulating initial vessel formation, and may function as a morphogen during human embryonic vascular development.

Vascular Endothelial Growth Factor Signaling to Endothelial Nitric Oxide Synthase

See related article, pages 715–722 There is substantial evidence supporting the idea that the process of angiogenesis requires the synthesis of endothelium-derived nitric oxide (NO). NO, characterized as a major endothelial-derived relaxing factor, exerts paracrine and autocrine roles in maintaining cardiovascular homeostasis, vascular tone, and microvascular permeability. During the early steps of angiogenesis in which new blood vessels sprout from existing vascular beds, there is a persistence of vasodilation and increase in vascular permeability, suggesting that these hemodynamic changes in the existing vasculature are indispensable during an angiogenic process. A number of angiogenic factors can triggers the release of NO, synthesized by the endothelial isoform of NO synthase (eNOS). One such factor is the vascular endothelial growth factor (VEGF), which as its name implies, is a critical mitogenic, chemoattractant, and survival factor for endothelial cells1 in addition to being characterized as a potent vascular permeability factor.2 Early studies have demonstrated that VEGF can readily stimulate NO production in cultured cells and isolated blood vessels and that NO is essential in mediating the VEGF-induced endothelial cell proliferation and organization into tubes in 3D cultures.3,4 Subsequent in vivo studies have also demonstrated that eNOS knockout mice had significantly attenuated VEGF and ischemia induced angiogenesis and vascular permeability.5,6 These studies place eNOS derived NO as a key mediator of VEGF induced angiogenesis in postnatal mice. Since the discovery of VEGF, or VEGF-A, and subsequent members of the VEGF family (VEGF-B, -C, -D, -E, and placenta growth factor, PlGF), intense research has been performed to elucidate their modes of action. These growth factor ligands bind to 3 receptor tyrosine kinases (RTKs), namely VEGF receptor-1, -2, and -3 as well as to coreceptors such as heparan sulfate proteoglycans or neuropilins. Like many other RTKs, these receptors are able to form …

Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene (MAP2) resulted in an early gastrulation defect, which is bypassed in double MetAP-2/p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.

Potential role for insulin-like growth factor II and vitronectin in the endothelial–mesenchymal transition process

Endothelial-to-mesenchymal transition (EndoMT) is a process through which certain subsets of endothelial cells lose endothelial characteristics and transform into mesenchymal or smooth muscle–like cells. Emerging evidence suggests that this process plays an important role during vascular development and in many vascular pathologies. As in epithelial–mesenchymal transition, EndoMT seems to progress through a series of important steps whose interdependence and order are not clear, and that some of them are regulated by soluble growth factors. Insulin-like growth factor II (IGFII), apart from being considered important in cancer, angiogenesis, and atherosclerotic lesions, is also considered as essential to embryonic development. Here, we report that addition of IGFII promoted the EndoMT process in the presence of very low amounts of chicken serum to arrested primary embryonic aortic chicken endothelial cells attached to fibronectin (FN), gelatin, or native type I collagen. This was demonstrated by cell spreading, loss of cell–cell contacts, detachment, migration, and transformation. These cellular events also occurred when IGFII was added to medium containing vitronectin (VN). Additionally, we demonstrated that these proteins were present in the spontaneous intimal thickenings that are observed at day 11–13 of chicken embryo development. We also show that alterations in the distribution of VE-cadherin and β-catenin occur after IGFII and serum or VN stimulation, and propose that the via VN IGFII effects may be facilitated by interaction of the mannose-6-phosphate/IGFII receptor (M6P/IGFIIR) with the urokinase-type plasminogen activator receptor (uPAR) and its ligand (uPA). Collectively, these findings provide the first evidence for a potential role of the IGFII-VN complex during the EndoMT process. From our observations and previous studies, we postulate a working hypothesis supporting a fundamental role for these molecules during EndoMT.

Vascular endothelial growth factor stimulates embryonic urinary bladder development in organ culture

To determine whether vascular endothelial growth factor A (VEGF) and its receptors are expressed during bladder development in mice when capillaries are forming, and whether exogenous VEGF might enhance the growth of endothelia and other types of bladder cells, using an embryonic organ-culture model. Whole bladders from wild-type mice, at embryonic day (E) 14, were grown in serum-free organ culture in an air/5% CO2 atmosphere; some cultures were supplemented with VEGF and/or with VEGF receptor 1/Fc chimera (VEGFR1/Fc), which blocks VEGF bioactivity. Organs were harvested after 6 days and the expression of VEGF and related molecules assessed using immunohistochemistry. VEGF, VEGFR1 and VEGFR2 positive cells were immunodetected in E14 and E18 bladders. Exogenous VEGF increased whole-organ growth, as assessed by explant areas, total cell numbers, DNA and protein content; proliferation was enhanced, and apoptosis decreased, in urothelium and surrounding tissues. VEGF also increased the proportions of cells expressing endothelial (CD31) and smooth muscle (alpha smooth muscle actin) markers. VEGFR1/Fc blocked the growth-enhancing effects of exogenous VEGF. In organ culture, exogenous VEGF not only stimulated embryonic bladder endothelial cells but also strikingly enhanced the growth of the whole organ. Whether the effects of VEGF on diverse bladder cell populations are direct or indirect requires further investigation. The finding that VEGF protein is present in embryonic bladders in vivo raises the possibility that it has similar actions during normal development. The results also illuminate the pathobiology of certain bladder diseases in which VEGF levels have been shown to be increased.

Differentiation of Lymphatic Endothelial Cells From Embryonic Stem Cells on OP9 Stromal Cells

The discovery of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3) has started to provide an understanding of the molecular mechanisms of lymphangiogenesis. The homeobox gene prox1 has been proven to specify lymphatic endothelial cells (ECs) from blood ECs. We investigated the process of lymphatic EC (LEC) differentiation using embryonic stem (ES) cells. VEGFR-2+ cells derived from ES cells differentiated into LECs at day 3 on OP9 stromal cells defined by the expression of prox1, VEGFR-3, and another lymphatic marker podoplanin. VEGFR-2+ cells gave rise to LYVE-1+ embryonic ECs, which were negative for prox1 on day 1 but turned to prox1+ LECs by day 3. VEGFR-3-Fc or Tie2-Fc, sequestering VEGF-C or angiopoietin1 (Ang1), suppressed colony formation of LECs on OP9 cells. However, addition of VEGF-C and Ang1 in combination with VEGF to the culture of VEGFR-2+ cells on collagen-coated dishes failed to induce LECs. LEC-inducing activity of OP9 cells was fully reproduced on paraformaldehyde-fixed OP9 cells with the conditioned medium. We succeeded in differentiating LECs from ES cells and revealed the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation.