An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.     

Localization of platelet-derived endothelial cell growth factor in human placenta and purification of an alternatively processed form.

Platelet-derived endothelial cell growth factor (PD-ECGF) was purified to homogeneity from human term placenta, an organ characterized by extensive angiogenesis. N-terminal amino acid sequencing revealed that placental PD-ECGF was proteolytically processed at Thr-6, in contrast to PD-ECGF purified from human platelets, which is processed at Ala-11. The purified factor stimulated porcine aortic endothelial cells as well as two choriocarcinoma cell lines. Immunohistochemical staining revealed that PD-ECGF was present in the connective tissue cells of the placenta. The possibility that PD-ECGF is involved in the development of the placenta is discussed.     

Angiogenesis and vascular growth factor receptor expression in malignant melanoma.

The growth and metastases of many solid tumors are dependent on the recruitment of new blood vessels. Tumor angiogenesis is most likely initiated by paracrine release of growth factors that bind to their corresponding endothelial cell surface receptors. To determine whether angiogenesis and growth factor receptor expression are consistent findings in malignant melanoma, primary human melanomas were examined for mRNA expression of receptors for fibroblast growth factors (FGFR-1, FGFR-2), vascular endothelial growth factor (VEGFR-1, VEGFR-2), and the receptors Tiel and Tie2. Charts were reviewed and archival formalin-fixed, paraffin-embedded primary tumors were obtained from patients with thin (<1 mm; n = 10), intermediate (1 to 4 mm; n = 10), or thick malignant melanoma (>4 mm; n = 8). Also examined was whether melanoma cell lines could induce endothelial growth factor receptor synthesis by metabolic labeling. It was found that tumor vascularity did not correlate with clinical stage, melanoma thickness, or clinical outcome. It was also found that melanoma cell lines were not capable of directly regulating endothelial cell synthesis of growth factor receptors. However, expression of Tiel and VEGFR-2 mRNA by the tumor vasculature in select stage IA-IIB patients, and FGFR-1 mRNA expression by the tumor cells in the same clinical stages was found. The expression of these growth factor receptors did not correlate with clinical outcome. These data suggest that angiogenesis is not a prominent characteristic of primary malignant melanoma lesions and that the endothelial cell expression of Tiel and VEGFR-2 in vivo is probably not directly induced by the tumor.     

Characterization of a new endothelial cell growth factor (f-ECGF) partially purified from the supernatant of human fibroblast cells

A new endothelial cell growth factor (f-ECGF) was partially purified from the cultured medium of human fibroblast cells of embryonic lungs. The partially purified f-ECGF induced neovascularization in rabbit cornea. It showed a selective growth stimulatory activity on the endothelial cells in vitro, whereas acidic- and basic-fibroblast growth factors (a- and b-FGFs) showed a broad spectrum of growth stimulation among tissues or cells. f-ECGF did not compete with the binding of a-FGF to the cell surface receptor in HEP-G2 hepatoblastoma cell lines. These results indicated that f-ECGF is a new endothelial cell growth factor distinct from a- and b-FGFs which are known to be potent endothelial cell growth factors.     

Immortalization of human endothelial cells by murine sarcoma viruses, without morphologic transformation

Amphotropic murine leukemia virus pseudotypes of murine sarcoma viruses containing the ras or mos oncogenes were constructed to permit efficient introduction of the sarcoma virus genome into early-passage human umbilical vein endothelial cells. The resulting cell lines were morphologically and phenotypically unchanged, retaining properties characteristic of differentiated endothelial cells. For example, the cells in a Kirsten sarcoma virus-modified line were found to biosynthesize and secrete von Willebrand factor in both a constitutive and regulated manner, and they contained ultrastructurally identifiable Weibel-Palade bodies, an endothelial cell-specific organelle. In contrast to the parent cultures, sarcoma virus-modified cells were able to proliferate indefinitely in culture. Examination of both Kirsten sarcoma and Moloney leukemia virus-modified lines indicated that the immortalized cells retained a diploid female karyotype after over 18 months in culture. In addition, the sarcoma virus-modified cells were able to grow independently of added endothelial cell growth factor. This growth factor autonomy does not appear to be due to autocrine production of a biologically cross-reactive growth factor. These immortal, virus-modified endothelial cells express large amounts of sarcoma virus-specific mRNA but no detectable helper virus or transforming virus activity. This technique for immortalization of primary human cells without alteration of the differentiated characteristics of the cell type is readily applied to a variety of human cell types. Moreover, the ability to separate the immortalizing and transforming activities of viral oncogenes should provide further understanding as to mechanisms of oncogene action.     

Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells

Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor highly expressed in rodent alveolar epithelium after injury and repair. To investigate VEGF synthesis in human lung epithelial cells, we examined VEGF expression by cultured cells under basal conditions and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and 1HAEo-) and in primary human bronchial epithelial cells with RT-PCR, Western blot, and immunocytochemistry. Among the cytokines tested, only transforming growth factor-beta1 increased the levels of excreted VEGF(165) as measured by ELISA. Under hypoxia (0% O(2) for 24 h), the VEGF(165) level increased fivefold, and this effect was O(2) concentration dependent. VEGF concentrations in the medium of all the cell types studied reached values similar to those found in bronchoalveolar lavage fluids from normal patients. Endothelial cells (human umbilical vein endothelial cells) exposed to conditioned medium from primary bronchial epithelial cell cultures showed an increased growth rate, which was inhibited in the presence of a specific neutralizing antibody to VEGF. These results suggest that lung epithelial cells participate in the endothelial repair and angiogenesis that follow lung injury through the synthesis of VEGF.     

Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end-stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti-pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM-1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E-selectin and SLA class II was only induced after stimulation with human TNF-alpha and pig IFN-gamma, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF-alpha or pig IFN-gamma. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte-endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti-pig immune responses.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

R-(-)-β-O-methylsynephrine, a natural product, inhibits VEGF-induced angiogenesis in vitro and in vivo

R-(-)-β-O-methylsynephrine (OMe-Syn) is an active compound isolated from a plant of the Rutaceae family. We conducted cell proliferation assays on various cell lines and found that OMe-Syn more strongly inhibited the growth of human umbilical vein endothelial cells (HUVECs) than that of other normal and cancer cell lines tested. In angiogenesis assays, it inhibited vascular endothelial growth factor (VEGF)-induced invasion and tube formation of HUVECs with no toxicity. The anti-angiogenic activity of OMe-Syn was also validated in vivo using the chorioallantonic membrane (CAM) assay in growing chick embryos. Expression of the growth factors VEGF, hepatocyte growth factor, and basic fibroblast growth factor was suppressed by OMe-Syn in a dose-dependent manner. Taken together, our results indicate that this compound could be a novel basis for a small molecule targeting angiogenesis.     

Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.     

Transformation of human corneal endothelial cells by micro- injection of oncogenes

Human corneal endothelial cells (HCEC) were transfected with some cloned oncogenes. The direct microinjection of either early region (E1) genes of monkey (SA7) and human (Ad5) adenoviruses or Ha-ras oncogen in conjunction with the Ad5 Ela-gene into embryonic HCEC nuclei was shown to result in immortalization of these cells. 3 independent immortalized HCEC lines were established in their growth and morphological properties were studied. These properties were very similar to those of primary HCEC, but unlike primary HCEC the immortalized cells didn't need the endothelial cell growth factor.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.

Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.     

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.     

Maintenance of G1 Checkpoint Controls in Telomerase-Immortalized Endothelial Cells

Here we report the characterization of a series of telomerase-immortalized human umbilical vein endothelial cell lines (i-HUVEC). These cells maintain endothelial characteristics such as marker expression, dependence on basic fibroblast growth factor for proliferation, and the ability to form tube structures on Matrigel. In addition, these cells do not show signs of tumorigenic transformation because their growth is contact-inhibited, serum-dependent, and anchorage-dependent. In addition, i-HUVEC do not grow or survive when implanted subcutaneously in immunocompromised mice. Notably, the i-HUVEC lines maintain normal p53-dependent checkpoint control, inducing expression of p21Cip1/Waf1 in response to DNA damage. These cells subsequently decrease phosphorylation of pRb and arrest in G1. Furthermore, the i-HUVEC lines maintain normal p53-independent checkpoint control, inducing expression of p27Kip1 in response to lovastatin treatment, with a subsequent decrease in pRb phosphorylation. Lovastatin-treated i-HUVEC lines undergo a G1 arrest that can be reversed with comparable kinetics to that of low passage HUVEC. Together these data demonstrate that telomerase-immortalized endothelial cells can retain normal phenotypes and cell cycle regulation. This result could have significant implications in the study of angiogenic processes such as tumor growth, wound healing, and the vascularization of engineered tissue.