Endothelial Cell-conditioned Medium Research Articles

A Novel Purification Method for CNS Projection Neurons Leads to the Identification of Brain Vascular Cells as a Source of Trophic Support for Corticospinal Motor Neurons

One of the difficulties in studying cellular interactions in the CNS is the lack of effective methods to purify specific neuronal populations of interest. We report the development of a novel purification scheme, cholera toxin beta (CTB) immunopanning, in which a particular CNS neuron population is selectively labeled via retrograde axonal transport of the cell-surface epitope CTB, and then purified via immobilization with anti-CTB antibody. We have demonstrated the usefulness and versatility of this method by purifying both retinal ganglion cells and corticospinal motor neurons (CSMNs). Genomic expression analyses of purified CSMNs revealed that they express significant levels of many receptors for growth factors produced by brain endothelial cells; three of these factors, CXCL12, pleiotrophin, and IGF2 significantly enhanced purified CSMN survival, similar to previously characterized CSMN trophic factors BDNF and IGF1. In addition, endothelial cell conditioned medium significantly promoted CSMN neurite outgrowth. These findings demonstrate a useful method for the purification of several different types of CNS projection neurons, which in principle should work in many mammalian species, and provide evidence that endothelial-derived factors may represent an overlooked source of trophic support for neurons in the brain.

Conditioned medium of endothelial cells promotes the endothelial and hematopoietic differentiation of embryonic stem cells

Conditioned medium of endothelial cells promotes the endothelial and hematopoietic differentiation of embryonic stem cells

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Macrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM). Human bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days. MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP-EPCs express CD133 but not CD14. Also, unlike CACs, HPP-EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind-limb model of ischemia in mice (BALB/C-nu) in vivo.

Vascular-adrenal Niche - Endothelial Cell-mediated Sensitization of Human Adrenocortical Cells to Angiotensin II

Alterations in both vasculature and renin-angiotensin-aldosterone system are a consistent finding in the metabolic syndrome. Adrenal tissue is highly vascularized and encounters blood flow, exceeding by far the volume expected for its size. Endothelial cells in the adrenal vasculature are therefore a major cellular component of adrenocortical tissue. The aim of the study was to analyze the cellular interaction between endothelial and steroid producing cells, focusing on endothelial cell-factor-mediated activation of aldosterone synthesis. The interaction between human endothelial (HUVECs) cell-conditioned medium and human adrenocortical (NCI-H295R) cells IN VITRO induced a significant surge in aldosterone secretion. The endothelial cell-conditioned medium together with angiotensin II and forskolin also potentiated aldosterone release by 1.5-fold and 2.6-fold, respectively, while preincubation of NCI-H295R cells for 24 h with endothelial cell-conditioned medium enhanced and sensitized the response of NCI-H295R to subsequent angiotensin II and forskolin stimuli by 2.5-fold and 2.2-fold, respectively. The increase in aldosterone release after preincubation with endothelial cell-conditioned medium was sensitive to cycloheximide and KN-93. Cellular conditioning with endothelial-cell factors exerts a hitherto unknown paracrine regulation of aldosterone production in human adrenocortical cells. This interaction may contribute to altered basal aldosterone release and have a role in patients with hypertension.

Protective effect of losartan on endothelial cells exposed to high glucose levels in vitro

To investigate the effect of losartan (an angiotensin II type I receptor antagonist) on endothelial cells exposed to high glucose in vitro and related mechanism. Vascular endothelial cells of human umbilical vein were cultured in media with high glucose levels. The activities of SOD and CAT, the level of MDA were measured by spectrophotometry in the conditioned media of endothelial cells, the VEGF mRNA expression was performed using semi-quantitative reverse transcription PCR (RT-PCR) in the cell lysates, and the protein expression of VEGF was examined by enzyme-linked immunosorbent assay (ELISA) in the supernatants of cultured cells. When endothelial cells were cultured in high glucose, the activities of SOD and CAT were significantly decreased, but the level of MDA was markedly increased. However, the high glucose-induced effects were inhibited by losartan. The application of high glucose upregulated the mRNA and protein expression of VEGF in endothelial cells, which was also attenuated by losartan. High glucose disrupts the oxidative equilibrium and increases the expression of VEGF in endothelial cells, which can be inhibited by losartan.

Effects of active components extracted from Qixue Bingzhi Recipe on proliferation of vascular smooth muscle cells and expressions of platelet-derived growth factor and its receptor genes

To observe the effects of paeoniflorin and total flavones extracted from Qixue Bingzhi Recipe on proliferation of vascular smooth muscle cells (VSMCs) cultured in endothelial cell conditioned medium (EC-CM) induced by oxidized low-density lipoprotein (ox-LDL) and the expressions of platelet-derived growth factor and its receptor genes. The VSMCs were cultured in normal culture medium, EC-CM, simvastatin-medicated EC-CM, paeoniflorin and total flavones (low, medium and high dose) -medicated EC-CM respectively. The growth activity of VSMCs was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. The expressions of platelet-derived growth factor (PDGF)-BB and platelet-derived growth factor receptor-alpha (PDGFR-alpha) mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR). The growth activity of VSMCs cultured in the EC-CM induced by ox-LDL was significantly higher than that in the normal culture medium (P<0.01), and the expressions of PDGF-BB and PDGFR-alpha mRNAs were obviously increased as compared with those in the normal culture medium. The growth activity of VSMCs cultured in each paeoniflorin and total flavones-medicated EC-CM was significantly lower than that in the EC-CM (P<0.01), and the expressions of PDGF-BB and PDGFR-alpha mRNAs were obviously decreased as compared with those in the EC-CM. The paeoniflorin and total flavones extracted from Qixue Bingzhi Recipe may be beneficial to the prevention and treatment of arteriosclerosis, and this efficacy may be correlated with down-regulating the expressions of PDGF-BB and PDGFR-alpha mRNAs which are related to the proliferation of VSMCs cultured in EC-CM induced by ox-LDL.

Trophoblast Migration Under Flow Is Regulated by Endothelial Cells1

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.

Injured brain endothelial cells release neurotoxic thrombin.

The multifunctional serine protease thrombin has been shown to be neurotoxic in vitro and in vivo and is demonstrable in the Alzheimer disease (AD) brain. We have documented that in AD the cerebral microvasculature is a source of inflammatory and neurotoxic proteins. The objective of this study was to determine if injured brain endothelial cells could be a source of neurotoxic thrombin. Brain endothelial cells were incubated with either sodium nitroprusside (SNP, 10 microM), inflammatory proteins (IL-1beta, IL-6, TNFalpha, LPS, IFNgamma) or the PKC inhibitor bisindolymaleimide (1 microM) for 24 h and conditioned media collected. Endothelial cell conditioned medium was incubated with purified apolipoprotein E4 (apoE4) for 24 h, and then analyzed for neurotoxic activity against primary cortical cultures and for apoE4 fragments by western blot. Endothelial cell conditioned medium collected after treatment with either SNP, inflammatory proteins, or the PKC inhibitor bisindolymaleimide, demonstrated a significant (p < 0.005) level of thrombin activity, the presence of apoE4 fragments, and was capable of evoking neuronal cell death. These data demonstrate that endothelial cell injury results in thrombin release and suggest that the brain microcirculation could be a source of neurotoxic factors in AD.

The effects and the mechanism of YSEC-CM on the growth of yolk sac hematopoietic stem/progenitor cells

To explore the distinct background of bone marrow and embryonic yolk sac hematopoiesis performance, serum free murine yolk sac endothelial cell and bone marrow endothelial cell conditioned medium were compared for their effects on the development profiles of yolk sac hematopoietic stem/progenitor cells. The mRNAs expression techniques were applied to understand the cytokine and receptor genes expression and the possible mechanisms. The results suggested that the differential gene expressions were existed between the hematopoietic cells of yolk sac and bone marrow and between the microenvironment of yolk sac and bone morrow.

Smooth muscle cell growth in monolayer and aortic organ culture is promoted by a nonheparin binding endothelial cell-derived soluble factor/s

Objective To characterize endothelial derived soluble factor(s) that regulate neointimal formation in porcine aortic organ cultures. Methods and results Endothelial cell (EC) conditioned medium, collected in preconfluent EC cultures at 4 days after plating, stimulates vascular smooth muscle cell (SMC) growth in cell culture and in the intima of porcine aortic organ cultures. EC conditioned medium was fractionated consecutively by salt precipitation, ion exchange chromatography and affinity chromatography on a heparin column. Heparin column nonbound fraction (HNBF) contains an endothelial cell-derived soluble factor/s (ECDSF) that promotes neointimal formation by increasing intimal SMC (iSMC) proliferation, as detected by BrdU labeling and inhibiting iSMC apoptosis, as shown by TUNEL. Trypsin digestion of HNBF resulted in loss of mitogenic activity. HNBF show a prominent 70-kDa band in SDS-NuPAGE. Conclusions Endothelial-derived soluble factor(s) has a molecular weight higher than other growth factors, does not have affinity to heparin, is a protein, at least in the active part of the molecule and increases iSMC number due to increased proliferation and suppression of apoptosis leading to neointimal formation.

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

BackgroundIn human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.ResultsWhen cultured alone, trophoblasts expressed low levels of β1 integrin as determined by quantitative immunofluorescence microscopy. When trophoblasts were cultured on top of endothelial cells for 24 h, the expression of trophoblast β1 integrin was significantly increased as determined by image analysis. β1 Integrin expression was not increased when trophoblasts were cultured with endothelial cell-conditioned medium, suggesting that upregulation requires direct contact between trophoblasts and endothelial cells. To identify endothelial cell surface molecules responsible for induction of trophoblast integrin expression, trophoblasts were cultured in dishes coated with recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), or αVβ3 integrin. Trophoblast β1 integrin expression (assessed by immunofluorescence microscopy and Western blotting) was increased when PECAM-1 or αVβ3 integrin, but not ICAM-1, was used as substrate.ConclusionsDirect contact between trophoblasts and endothelial cells increases the expression of trophoblast β1 integrin.

Postnatal vasculogenesis: Identification, growth and function of endothelial progenitor cells from peripheral blood mononuclear cells

To identify best growth conditions for the in vitro differentiation of human peripheral blood mononuclear cells (PBMNCs) into endothelial progenitor cells (EPCs), PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, and IGF-I; M199 with bovine retina-derived growth supplement (RDGS); human umbilical vein endothelial cell (HUVEC) conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in M199 with RDGS (cocultures system); PHA burst (10μL/106 cells) for 24 h and culture in M199 with RDGF. EPCs were identified by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. Matrigel was used to assess in vitro angiogenesis capability. Spindle-shaped and attached cells sprouted with growth factors, differentiating in EPCs within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDGS, numerous large cell clusters appeared within 1 week but the number of clusters decreased during culture. After stimulation with PHA, many cells clusters and some adherent EC-like cells were observed within 7 days; their number and size increased within 14 days. With HUVEC conditioned medium, spindleshaped cells were observed after 10 days. FACS con- firmed the endothelial phenotype. Cocultures induced the appearance of double-labeled EPCs expressing endothelial antigens starting from 7 days. EPCs were able to migrate in response to VEGF and to incorporate in capillary-like structures formed by HUVECs on Matrigel. PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions. The contact with mature endothelial cells (ECs) makes this process quicker.

Transendothelial migration confers a survival advantage to activated T lymphocytes: role of LFA-1/ICAM-1 interactions.

The clearance of activated T lymphocytes by apoptosis is an essential component in the resolution of the immune response; however, certain signals received within inflamed tissue may result in the persistence of activated T cells. Our previous work has shown that, when compared with resting cells, effector cells migrate more efficiently across endothelium, thus such cells may be selectively recruited to sites of inflammation. We hypothesized that transmigration of T cells across endothelium might influence cell survival. We have generated T cell lines by culturing in IL-2 following PHA activation. These T cell lines die rapidly by apoptosis when deprived of IL-2 (53.7 +/- 4.0% survival after 24 h). In contrast, cells that have migrated across human umbilical vein endothelial cells (HUVEC) survived significantly better than control cells (80.3 +/- 3.6%, n= 18, P<0.001). Endothelial cell conditioned medium was also able to reduce apoptosis, but this effect was small when compared with the protective effect of transmigration. Culture of T lymphocytes on fibronectin, or RGD peptides, or in suspension with a range of chemokines active on T cells, including RANTES and lymphotactin had no effect on survival. In contrast, blocking LFA-l/ICAM-l interactions reduced the protective effect of transmigration (42.3 +/- 6.7% reduction). Culture of activated T cells on immobilized ICAM-l alone also increased survival. These results indicate that signals received by activated T cells during extravasation can influence their subsequent survival within tissue, and implicates the involvement of LF A-l/ICAM-l interactions.

IL-6 is produced by splenocytes derived from CMV-infected mice in response to CMV antigens, and induces MCP-1 production by endothelial cells: a new mechanistic paradigm for infection-induced atherogenesis

Atherosclerosis is an inflammatory disease. One of the candidate inflammatory triggers is infection. To further characterize the interaction between infection, cytokine induction, and atherosclerosis, we tested the hypothesis that cytomegalovirus (CMV) infection induces the pro-inflammatory cytokine interleukin-6 (IL-6), which in turn induces “pro-atherosclerotic” changes in vascular endothelial cells (ECs). ELISA was used to determine the levels of monocyte chemoattractant protein-1 (MCP-1) in the supernatant of mouse and human ECs incubated with IL-6, and IL-6 levels in supernatants of splenocytes, derived from CMV-infected and uninfected mice, stimulated with mice CMV antigens. IL-6 induced, in a dose response fashion, MCP-1 expression in human ECs: 0, 2, 10, and 50 pg/ml IL-6 increased MCP-1 levels in EC conditioned medium from 1120±65 to 1148±105, 1395±40, and 2119±130 pg/ml, respectively ( P<0.001). IL-6 also induced MCP-1 expression in mouse ECs ( P<0.002). Importantly, IL-6 concentration in the supernatants of splenocytes stimulated with CMV antigens rose from undetectable levels in uninfected mice to 14.9±5 pg/ml in the infected mice ( P<0.04). These results suggest a previously unrecognized, but potentially important mechanism whereby CMV, and other pathogens, contribute to atherogenesis: T lymphocytes, clonally expanded in response to antigens presented by CMV infection, home to sites of vascular injury and locally release IL-6 when presented with either pathogen antigens that may be present in the plaque, or when they cross-react with host peptides homologous to the relevant pathogen antigens; IL-6 then triggers ECs to release MCP-1, which recruits more monocytes and T-cells into the vessel wall and thereby exacerbates local inflammation, and thus atherogenesis.

Expression of hematopoietic inhibitory factors in mouse fibroblasts, macrophages and endothelial cells.

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT-PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated. The results showed: (1) six cytokines, MIP-1alpha, MIP-2, TGF-beta, TNF-alpha, IFN-gamma and Tbeta(4), inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-alpha, INF-gamma and Tbeta(4), was seen in all three types of stromal cells, while TGF-beta mRNA was only seen in endothelial cells and macrophages, and MIP-1alpha mRNA in endothelial cells and fibroblasts. The inhibitors TGF-beta, MIP-1alpha, and Tbeta(4)have an inhibitory effect on the growth of CFU-GM, but TNF-alpha, INF-gamma and MIP-2 do not.

Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture

Removal of vascular smooth muscle cells (SMC) from their native environment alters the biochemical and mechanical signals responsible for maintaining normal cell function, causing a shift from a quiescent, contractile phenotype to a more proliferative, synthetic state. We examined the effect on SMC function of culture on two-dimensional (2D) substrates and in three-dimensional (3D) collagen Type I gels, including the effect of exogenous biochemical stimulation on gel compaction, cell proliferation, and expression of the contractile protein smooth muscle α-actin (SMA) in these systems. Embedding of SMC in 3D collagen matrices caused a marked decrease in both cell proliferation and expression of SMA. The presence of the extracellular matrix modulated cellular responses to platelet-derived growth factor BB, heparin, transforming growth factor-β1, and endothelial cell-conditioned medium. Cell proliferation and SMA expression were shown to be inversely related, while gel compaction and SMA expression were not correlated. Taken together, these results show that SMC phenotype and function can be modulated using biochemical stimulation in vitro, but that the effects produced are dependent on the nature of the extracellular matrix. These findings have implications for the study of vascular biology in vitro, as well as for the development of engineered vascular tissues.

Hematopoietic Commitment of ES Cells in Culture

Publisher Summary The ES/EB system has been instrumental in identifying a progenitor with hemangioblast properties. When early EB-derived cells are cultured in the presence of vascular endothelial growth factor (VEGF), interleukin-6, and endothelial cell-conditioned medium, they generate blast cell colonies with endothelial and primitive and definitive hematopoietic potential. The progenitor that gives rise to this colony, the blast colony-forming cell (BL-CFC) represents a transient population that is present within the EBs for approximately 24 hr, between days 2.5 and 3.5 of differentiation, preceding the onset of primitive erythropoiesis. The characteristics of the BL-CFC—namely its early development and its potential to generate primitive and/or definitive hematopoietic cells, as well as endothelial progeny—suggests that it represents the in vitro equivalent of the yolk sac hemangioblast. The protocol outlined in this chapter describes the growth of ES cells and their differentiation into two stages of development: the BL-CFC/ hemangioblast stage and the primitive/definitive hematopoietic stage.

A novel secreted splice variant of vascular endothelial cell growth inhibitor.

Vascular endothelial cell growth inhibitor (VEGI), a member of the tumor necrosis factor (TNF) family, is an endothelial cell-specific inhibitor of angiogenesis. Overexpression by cancer cells of a secretable VEGI fusion protein resulted in abrogation of xenograft tumor progression, but overexpression of full-length VEGI was completely without effect. This finding indicates that secretion is essential for VEGI action. Here we report the identification of two new VEGI isoforms consisting of 251 and 192 amino acid residues. Both isoforms show endothelial cell-specific expression and share a C-terminal 151-residue segment with the previously described VEGI, which comprises 174 residues. The isoforms are generated from a 17 kb human gene by alternative splicing. Their expression is regulated in parallel by inflammatory cytokines TNF-alpha and interferon-gamma. VEGI-251, the most abundant isoform, contains a putative secretion signal. VEGI protein is detected in conditioned media of endothelial cells and VEGI-251-transfected mammalian cells. Overexpression of VEGI-251 in endothelial cells causes dose-dependent cell death. VEGI-251-transfected cancer cells form xenograft tumors of reduced growth rate and microvessel density compared with tumors of empty vector transfectants. These findings support the view that endothelial cell-secreted VEGI may function as an autocrine inhibitor of angiogenesis and a naturally existing modulator of vascular homeostasis.