Immunohistochemical detection of factor VIII/von Willebrand factor in hyperplastic endothelial cells in glioblastoma multiforme and mixed glioma-sarcoma

The sarcomatous components of most glioma-sarcomas are thought to arise from the neoplastic transformation of hyperplastic endothelial and adventitial vascular cells in a preexisting glioblastoma multiforme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a marker for glial cells, was examined in 10 glioblastomas and seven mixed glioma-sarcomas using the peroxidase-antiperoxidase immunohistochemical technique. Hyperplasia of small blood vessels was observed in all 10 glioblastomas; in five, the vascular proliferation had resulted in the formation of prominent glomeruloid structures. FVIII/vWF was detected in the endothelial cells in these vascular structures, but not in the adventitial cells. In the mixed glioma-sarcomas. FVIII/vWF was detected only in endothelial cells; there was no staining for FVIII/vWF in the neoplastic mesenchymal cells. The gliomatous components of the mixed tumors stained intensely for GFAP. These observations indicate that both endothelial and nonendothelial cell types contribute to the small vessel hyperplasia in glioblastomas, and that the sarcomatous components of mixed glioma-sarcomas are derived from either non-endothelial cells or endothelial cells that have undergone antigenic loss following transformation.     

Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.     

Progressive enlargement of a mass lesion in late cerebral radionecrosis

The progressive enlargement of a mass lesion in late cerebral radionecrosis (LCR) was studied using a surgical specimen from a woman’s irradiated brain. Using immunohistochemical (IHC) staining on paraffin-embedded sections, expression of tumor necrosis factor-alpha (TNF??), interleukin (IL)6, Flk-1, and vascular endothelial growth factor (VEGF) was semi-quantitatively evaluated in the necrotic area and areas distant and adjacent to the necrotic area. High immunoreactivity for TNF??, IL6, and VEGF in regions distant to the necrotic core were strongly associated with inflammatory cell density, hyalinized vessels, and vascularization. Ghost cells stained with glial fibrillary acidic protein (GFAP), Ki-M1P, KP-1, ubiquitin-C-terminal hydrolase 1, and fibrinoid necrosis were observed in the necrotic area and adjacent regions. These results suggest that necrotic factors, inflammatory reaction, and angiogenetic factors act temporally and spatially through radiation-induced vascular endothelial cell damage to contribute to the progressive enlargement of an enhancing mass lesion in LCR.     

Mechanisms of angiogenesis in the brain

Brain angiogenesis is a tightly controlled process that is regulated by neuroectodermal derived growth factors that bind to tyrosine kinase receptors expressed on endothelial cells. In the rat brain, angiogenesis is complete around postnatal day 20, but endothelial cells can proliferate in the adult brain under pathological conditions such as hypoxia/ischemia and brain tumor growth. Current evidence suggests that physiological angiogenesis in the brain is regulated by similar mechanisms as pathological angiogenesis induced by tumors or by hypoxia/ischemia. The hypoxia-inducible endothelial cell mitogen and vascular permeability factor, vascular endothelial growth factor (VEGF) appears to play a pivotal role in most of these processes. VEGF is expressed when angiogenesis is high, as in embryonic neuroectoderm, in glioblastomas and around infarcts, but is expressed at low levels when angiogenesis is absent, as in adult neuroectoderm. Since growth factors such as VEGF and angiopoietins and their receptors appear to be necessary for angiogenesis, targeting of growth factor/receptor pathways for angiogenesis-dependent diseases such as glioblastoma might be useful for therapy. Several compounds, including anti-VEGF antibodies and VEGFR-2 inhibitors are currently in clinical trial. On the other hand, induction of angiogenesis by growth factors (pro-angiogenesis) might prove to be a rational therapy for patients with stroke.     

CD133+ glioblastoma stem-like cells induce vascular mimicry in vivo

Glioblastoma is one of the most angiogenic malignancy, the neoplastic vessels of which are likely to arise by angiogenesis and vasculogenesis. An alternative mechanism of tumor vasculature is described, termed vasculogenic mimicry, by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high cellular plasticity. Evidence suggests that cancer stem cells acquire a multi-potent plastic phenotype and show vasculogenic potential. In this study, we report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133(+)-GSC) and CD133- (CD133(-)-GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with CD133(-)-GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133(+)-GSC-derived tumor xenografts. Subsets of CD133(+)-GSC but not CD133(-)-GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133(-)-GSC and exclusively dispersed within the tumor tissues. Although the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.     

Glioblastoma niches: from the concept to the phenotypical reality

Recently, the concept of niches as sites of tumor progression, invasion, and angiogenesis in glioblastoma (GB) has been extensively debated. Niches, considered the sites in which glioblastoma stem cells (GSCs) reside, have been classified as perivascular, perinecrotic, and invasive. However, from a neuropathological point of view, it is not easy to establish when a tumor structure can be considered a niche. The relevant literature has been reviewed in the light of our recent experience on the subject. As for perinecrotic niches, the occurrence of GSCs around necrosis is interpreted as triggered by hypoxia through HIF-1α. Our alternative hypothesis is that, together with progenitors, they are the cell constituents of hyper-proliferative areas of GB, where perinecrotic niches have developed, and they would, therefore, represent the remnants of GSCs/progenitors spared by the developing necrosis. Perivascular structures originate from both transport vessels and exchange vessels, i.e., venules, arterioles, or the undefinable neo-formed small vessels, but only those in which a direct contact between GSCs/progenitors and endothelial cells occurs can be called niches. Both pericytes and microglia/macrophages play a role in niche function: Macrophages of blood origin invade GB only after the appearance of “mother vessels” with consequent blood-brain barrier disruption. Not all vessel/tumor cell structures can be considered niches, that is, crucial sites of tumor progression, invasion, and angiogenesis.     

Expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor-β (PDGFR-β) in human gliomas

The growth of solid tumors is highly dependent on vascular proliferation. Vascular endothelial growth factor (VEGF), the main mediator of angiogenesis, and platelet-derived growth factor receptor-β (PDGFR-β), receptor for the potent mitogen PDGF, are two indicators of the angiogenic potential of human gliomas. We studied a series of 57 surgical biopsies of astrocytic neoplasms by immunohistochemistry to elucidate the relationship between tumor proliferation, quantified as Ki67-LI, and the expression of these two proteins. Ki67-LI increases throughout histological malignancy, although staining in endothelial cells has rarely been recorded. Elevated amounts of VEGF-positive tumor cells (VEGF-LI) were found in anaplastic astrocytomas and glioblastomas, mainly around areas of necrosis, cysts, or edema. Endothelium of blood vessels was consistently stained. PDGFR-β positivity was found in glomeruloid formations and in tumor cells, excluding pilocytic astrocytomas. Multinucleated giant cells and perivascular tumor cells were positive in glioblastomas. In addition, peritumoral microglia-like cells were also stained in some cases. Statistical correlation was only found between PDGFR-β and Ki67 LIs. In conclusion, VEGF as permeability factor is involved in the development of secondary neoplastic changes, whereas PDGFR-β is directly correlated to proliferation indexes. Strong expression of VEGF and PDGFR-β found in endothelium and tumor cells would seem to support a combined role in tumoral neoangiogenesis     

CXCR4活化促进人恶性胶质瘤细胞分泌血管生成因子

目的观测CXCR4活化对人恶性胶质瘤细胞系U87中血管内皮生长因子（VEGF）、白细胞介素-8（IL-8）基因表达及蛋白分泌的影响。方法采用间接免疫荧光标记观测CXCR4在U87细胞中的表达和定位；用间质细胞衍生因子-1α（SDF-1α）激活CXCR4，通过酶联免疫吸附试验（ELISA）检测U87细胞培养上清中VEGF、IL-8蛋白的含量，用逆转录聚合酶链反应（RT-PCR）检测二者mRNA的表达。结果CXCR4表达于U87细胞的胞膜和胞质，经活化后可增加VEGF、IL-8mRNA的表达和蛋白分泌量。结论人恶性胶质瘤细胞中CXCR4活化后能够促进血管生成因子VEGF和IL-8的产生。     

Type 1 collagen as a potential niche component for CD133‐positive glioblastoma cells

Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133‐positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum‐containing medium (SCM) or serum‐free medium with some growth factors (SFM) and in non‐coated (Non‐coat) or type 1 collagen‐coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non‐coat/SCM. Semi‐quantitative measurement of CD133‐positive cells by immunocytochemistry showed a statistically significant increase of CD133‐positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133‐positive GSCs and maintain GSCs in adherent culture.     

Autocrine/paracrine sphingosine‐1‐phosphate fuels proliferative and stemness qualities of glioblastoma stem cells

Accumulating reports suggest that human glioblastoma contains glioma stem‐like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine‐1‐phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro‐stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro‐stemness environment and favoring GSC proliferation, survival and stem properties. GLIA 2014;62:1968–1981     

Proliferative potential of vascular components in human glioblastoma multiforme

Summary Sixteen patients with glioblastoma multiforme received a 1-h intravenous infusion of 5-bromodeoxyuridine (BrdU), 150–200 mg/m² at the start of surgery, to label S-phase cells in tumor tissue. Labeled cells of vascular components and of tumor parenchyma were detected in excised tumor specimens by indirect immunoperoxidase staining using anti-BrdU monoclonal antibodies followed by periodic acid-Schiff staining. The BrdU labeling index (LI, defined as the percentage of labeled cells in relation to the total number of cells scored) was calculated separately for vascular components and tumor parenchyma in each specimen. The BrdU LI of vascular components of glioblastoma multiforme was remarkably higher than that of normal brain (1.1%–8.7% vs.<0.05%). The mean BrdU LIs of vascular components and tumor cells in eight primary glioblastomas were 4.5±0.8% (mean±SE) and 9.9±1.1%, respectively, while the corresponding BrdU LIs in eight recurrent tumors were 2.7±0.5% and 9.3±0.7%. The differences in the BrdU LIs of primary and recurrent tumors were not statistically significant, but the BrdU LI of vascular components was consistently much lower than that of tumor cells. BrdU labeling of vascular components was inconsistent and occurred mostly in glomerular-shaped vessels, but only about 20% of them contained labeled cells. These results suggest that unusual vascular proliferation, such as the formation of glomerular-shaped vessels and endothelial or adventitial proliferation, in glioblastoma multiforme may have been programmed to slow down or even to cease at a certain stage, and is not likely to be the result of neoplastic transformation.Supported in part by grant PDT-159 from the American Cancer Society and by grant CA 13525 from the National Cancer Institute     

Human von Willebrand factor (factor VIII-related antigen) in glial neoplastic cells of brain gliomas

In order to determine whether the glial neoplastic cells within the brain gliomas show human von Willebrand factor (factor VIII-related antigen [FVIII-rA]) expression, and the possible distribution of FVIII-rA positive glial cells within the neoplasm, morphological examination and computerised morphometric analysis were conducted in 35 brain neoplasms, divided into 2 groups: glioblastoma multiforme (15 cases) and astrocytoma II (20 cases). Only in glioblastomas was the expression of FVIII-rA within the glial neoplastic cells found to be significantly higher than in background. The groups of FVIII-rA positive glial cells were usually found near the vascular glomeruloid structures. On the basis of the examination, the following conclusions were drawn: at least a part of the glial neoplastic cells within the glioblastoma multiforme may present expression of FVIII-rA; lack of FVIII-rA expression in the glial cells of astrocytomas II suggests that the activity of FVIII-rA within these cells is connected with the malignancy of gliomas; the role of FVIII-rA positive glial cells remains unknown, but the tendency to location near the vascular glomeruloid structures of glioblastomas might suggest their potential functional connection with endothelial cells.     

Presence of D110 antigen expressing immunocompetent cells in glioblastoma associates with prolonged survival

Monoclonal antibody D110 has recently been described as a novel marker of hypoxic tissue damage, reacting with a so far unknown antigen with preferential expression in the central nervous system. The aim of the study was to investigate D110 immunoreactivity in glioblastoma, its association with the expression of hypoxia-related proteins and its impact on patient outcome. A total of 114 consecutive adult patients who underwent first operation of primary glioblastoma were included in this study. We evaluated D110 immunoreactivity qualitatively and semi-quantitatively and correlated it with expression of hypoxia inducible factor 1 alpha (HIF-1alpha), expression of vascular endothelial growth factor (VEGF), and with patient survival using univariate and multivariate statistical analysis. We observed D110 immunolabelling in 85.1% of the cases. D110 immunoreactivity was detectable in infiltrating HLA-DR and CD68 expressing cells, most likely microglial cells or haematogenous cells of monocyte/macrophage lineage. In the peripheral lymphoreticular system, immunohistochemistry disclosed selective D110 labelling of Langerhans cells and of dendritic cells of the thymic medulla. Univariate statistical analysis revealed significantly longer survival of patients whose glioblastomas contained D110 immunoreactive infiltrating cells. There was no association between presence of D110 immunoreactive cells and expression of HIF-1alpha and VEGF. We conclude that D110 immunoreactivity in glioblastoma does not seem to be related to tissue hypoxia. D110 identifies immunocompetent cells, which positively influence survival of glioblastoma patients.     

Release of soluble urokinase receptor from vascular cells

Urokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1beta (IL-1beta), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1beta stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall.     

In Situ Expression of Angiopoietins in Astrocytomas Identifies Angiopoietin-2 as an Early Marker of Tumor Angiogenesis

Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for α-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.     

In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis.

Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for alpha-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.     

Effects of minocycline on apoptosis and angiogenesis-related protein expression in a rat model of intracerebral hemorrhage

In the present study, a rat model of non-traumatic intracerebral hemorrhage was established by type IV collagenase injection into the right globus pallidus. Bax and Bcl-2 expression in tissues surrounding hematomas was significantly increased within 14 days after injury, and it then gradually decreased. Vascular endothelial growth factor, Flk-1 and Flt-1 mRNA expression gradually increased over time. After intraperitoneal injection with minocycline, Bax expression was decreased 1 day after intracerebral hemorrhage. Flk-1 and Flt-1 mRNA expression was decreased after minocycline injection, but Bcl-2 expression was increased, and vascular endothelial growth factor mRNA expression was decreased between 4-14 days. These results indicated that protective effects of minocycline on nerve tissues were associated with increased Bcl-2 expression and decreased Bax expression in the early stage after intracerebral hemorrhage. In the late stage, minocycline downregulated vascular endothelial growth factor and its receptor expression to inhibit brain tissue self-repair.     

Vascular-specific growth factor angiopoietin 1 is involved in the organization of neuronal processes

Neuronal processes and vessels have similar branching and bifurcation patterning in the adult body and appear to use many of the same molecules during their development, including vascular endothelial growth factor, Notch, neuropilin, and ephrins/Ephs. We were interested in determining whether the endothelial growth factor angiopoietin (Ang) has a unique role in the nervous system in addition to its angiogenic role. By using a mouse molecular genetics approach, we overexpressed Ang1 in the mouse forebrain and observed increases in overall vascularization, consistent with prior reports describing the role of Ang1. Nonvascular events, involving alterations in the dendritic organization of layer II motor cortex neurons, dentate granule cells, and pyramidal cells of CA1, were seen, suggesting that Ang1 was able to influence the growth of these processes. The angiopoietin tyrosine kinase receptor Tie2 was not found on neurons or their processes, but beta1 integrin was and has previously been found to act as an Ang receptor. Our findings provide some of the first data evaluating the interactions between the developing nervous system and the vascular protein Ang1. Understanding interactions between the developing nervous and vascular systems will lead to novel insight into how the two systems interact throughout development, during senescence, and in disease.     

Angiogenic and neurotrophic effects of vascular endothelial growth factor (VEGF165): studies of grafted and cultured embryonic ventral mesencephalic cells

The present series of experiments investigated the effects of vascular endothelial growth factor (VEGF165) on adult rat striatal cerebrovasculature and embryonic dopamine (DA) neuron allografts in a rat model of Parkinson's disease (PD). We examined VEGF165's ability to (1) alter the vascular network of the adult rat striatum, (2) influence the vascular growth of solid embryonic day 14 (E14) ventral mesencephalic (VM) grafts when placed into a VEGF-pretreated host striatum, (3) alter the function and survival of E14 VM grafts when transplanted into an adult DA-deleted striatum, and (4) influence cell survival and neurite growth in cultures of E14 VM cells. We demonstrate here that a single bolus injection of VEGF165 into the adult rat striatum significantly increases the amount of vasculature in the vicinity of the injection site in a delayed and transient manner when compared to saline controls. Transplanting solid E14 VM grafts into the VEGF165-pretreated striatum resulted in a homogeneous distribution of small blood vessels throughout the graft, a pattern that closely resembles mature adult vasculature. In contrast, grafts in the control condition contained a patchy distribution of heavily dilated vessels. Behavioral measurements indicate that VEGF pretreatment of the intrastriatal graft site accelerates recovery of amphetamine-induced rotational asymmetry in unilateral 6-OHDA lesioned rats. Unexpectedly, however, VEGF pretreatments failed to increase survival of tyrosine hydroxylase-immunoreactive (THir) neurons in the grafts. In contrast to this finding in vivo, adding VEGF165 to glial-reduced E14 rat VM cultures produced a fourfold increase in THir cell survival and a doubling in the length of THir neurites. We conclude that with the proper method of delivery, VEGF165 may prove to be one of several strategies necessary to significantly improve the survival and function of fetal VM tissue grafts.