Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis.     

Expression and localization of placenta growth factor and PlGF receptors in human meningiomas

It has previously been suggested that in human brain tumours, endothelial cell proliferation during angiogenesis is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and its receptors (VEGF receptor 1 and VEGF receptor 2). The mechanism of growth factor up-regulation is based on hypoxic activation of mRNA expression and mRNA stabilization and genetic events, leading to an increase of growth factor gene expression. The role of the other newly discovered VEGF family members with a high specificity for endothelial cells in the pathogenesis of glial neoplasms is unknown. To investigate which other members of the VEGF family are overexpressed in human brain tumours, the mRNA levels of placenta growth factor (PlGF), VEGF-A, and VEGF-B genes were determined by northern blot analysis in surgically obtained human meningiomas. In the 16 meningiomas examined, the mRNA for PlGF was highly expressed in four tumours and VEGF-A mRNA was highly abundant in three tumour samples. There was no close correlation between PlGF mRNA levels and VEGF-A expression levels. VEGF-B mRNA was abundantly expressed in all tumour samples at uniform levels. In a PlGF-positive tumour sample, immunoreactive VEGFR-1 and VEGFR-2 were detected in endothelial cells of the blood vessels. PlGF protein was detectable in most but not all capillaries of the tumour. PlGF is thus highly up-regulated in a subset of human meningiomas and may therefore have functions, in some tumour vessels, connected to endothelial cell maturation and tube formation. These findings suggest that PlGF, in addition to VEGF-A, may be another positive factor in tumour angiogenesis in human meningiomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome

Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin alpha1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia.     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies

Extensive angiogenesis and invasion of the maternal decidua by trophoblasts are essential for the development and function of the placenta. Vascular endothelial growth factors (VEGF), placenta growth factor (PlGF) and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt4 have important roles in vasculogenesis and angiogenesis. We have studied the localization of these proteins by immunohistochemistry and Western blotting in the placenta and of PlGF in maternal serum, and their association with diabetes, pre-eclampsia, fetal growth restriction (FGR) and fetal alcohol syndrome (FAS). VEGFR-1 and VEGFR-3 were detected mainly in the syncytiotrophoblastic layer whereas VEGFR-2 was detected in the vascular endothelial cells of the placenta. VEGFR-1, but not the other receptors, showed increased expression in placental syncytiotrophoblasts from 50% of patients with severe pre-eclampsia and FGR when compared with normal placentas. PlGF was undetectable in 38 of 44 samples of amniotic fluid of mothers with normal and complicated pregnancies. However, maternal serum PlGF concentrations were significantly lower in pre-eclamptic patients and in those with FGR when compared to diabetic women or healthy controls. These results suggest that low maternal serum PlGF and increased placental expression of its receptor VEGFR-1 are associated with pre-eclampsia and FGR.     

Increased Tie2 Expression, Enhanced Response to Angiopoietin-1, and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745-752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.     

Fibrohistiocytic differentiation in capillary hemangioblastoma.

Seven cases of capillary hemangioblastoma from the cerebellum and spinal cord were studied by immunohistochemical methods to determine the origin of the stromal cells. A subpopulation of factor XIIIa-positive tumor cells was a constant feature in hemangioblastomas. These stellate or spindle-shaped cells transformed into typical vacuolated stromal cells. Factor VIII-related antigen was limited to the vascular endothelium. Glial fibrillary acidic protein was present only in entrapped astrocytes. Staining for alpha-1-antitrypsin (alpha 1AT) and alpha-1-antichymotrypsin (alpha 1 ACT) was occasionally observed in stromal cells. It was concluded that the factor XIIIa-positive stromal cells in capillary hemangioblastoma indicate fibrohistiocytic differentiation, which is part of the differentiation spectrum of hemangiopericytomas.     

Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors.

Ezrin is a cytoskeleton-associated protein that appears to link actin filaments to the plasma membrane. Immunocytochemical studies suggest that ezrin is expressed in epithelial cells but not in mesenchymal cells. In addition, ezrin is expressed by certain epithelial tumors, such as renal cell adenocarcinomas. Ezrin serves as a tyrosine kinase substrate, and is phosphorylated in epidermal growth factor-stimulated cells. Ezrin may thus mediate regulatory signals in different cell functions. We studied the distribution of ezrin in 104 cases of primary tumors of the central nervous system (CNS) by immunocytochemistry. Special interest was focused on capillary hemangioblastoma, owing to its resemblance to renal cell adenocarcinoma, and on malignant gliomas, owing to their frequent epidermal growth factor receptor amplification. The stromal cells of hemangioblastomas were found to be strongly positive for ezrin. No expression was detected in gliomas and, except for hemangioblastomas, ezrin expression was restricted to those few CNS tumors that show epithelial differentiation, ie, choroid plexus papillomas, craniopharyngiomas, ependymomas, and cysts. The diffuse cytoplasmic expression of ezrin in the stromal cells of capillary hemangioblastoma may indicate that stromal cells overexpress ezrin or express ezrin with deficient binding properties.     

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.     

Expression of vascular endothelial growth factor,placental growth factor,and their receptors Flt-1 and KDR in human placenta under pathologic conditions

The vascular endothelial growth factor (VEGF) family and its receptors have multifunctional activities besides angiogenesis, and some of these molecules are induced by hypoxia/ischemia. They are known to be expressed in human placenta, but little is known about their involvement in pathologic conditions. We have investigated the expression patterns of VEGF, placental growth factor (PlGF), and their receptors fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing region (KDR) in placentas with histopathological changes. Forty-two placentas from normal and complicated pregnancies delivered in the second and third trimesters were fixed with paraformaldehyde and embedded in paraffin. In situ hybridization and immunohistochemistry were performed on serial sections. In the villi with characteristic hypoxic/ischemic changes (HIC), including increased syncytial knots, infarction, or hypercapillarization, intense immunostaining for VEGF was detected in the media of blood vessels, and increased staining for KDR was demonstrated in the endothelial cells. Strong PlGF immunoreactivity was localized to the degenerative trophoblasts around the infarctions. Marked Flt-1 mRNA expression in the syncytiotrophoblast layers of HIC villi was identified, but some samples did not show ligand expression in these regions. Positive immunostaining for VEGF, PlGF, and Flt-1 was observed in infiltrated neutrophils and macrophages in the placentas with chorioamnionitis (CAM). These findings suggested that in the hypoxic/ischemic regions, VEGF and KDR expression is increased within the villous vessels by paracrine regulation, whereas the expression of PlGF and Flt-1 is enhanced in villous trophoblasts by autocrine regulation. The Flt-1 gene may also be up-regulated directly by hypoxia/ischemia independently of ligand mediation. Furthermore, the results indicated that VEGF and PlGF stimulate inflammatory cell migration by autocrine regulation via the Flt-1 receptor in the CAM placenta. Thus, various functions of VEGF family members participate in the development of pathologic changes in the placenta.     

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.     

Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries

BACKGROUND: Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. The existence of single or multiple VEGF isoforms and receptors suggests that these proteins may have overlapping but distinct functions, which may be reflected in their cell expression and distribution. METHODS: The localisation of VEGFs A-C and their receptors (VEGFRs 1-3, respectively) in 30 fresh human atherosclerotic arteries, 15 normal uterine arteries, and 15 saphenous veins using immunohistochemistry and western blotting. RESULTS: Saphenous veins showed no staining for VEGF-B or VEGFR-2. Smooth muscle cells (SMCs) showed the strongest staining for VEGF-A, VEGF-B, VEGFR-1, and VEGFR-2 in all specimens. Conversely, VEGFR-3 and VEGF-C were predominantly localised to the endothelial vasa vasorum in normal arteries, whereas medial SMCs showed the strongest staining in atherosclerotic arteries. Western blotting showed variations in VEGF protein localisation, with lower amounts of VEGF-B and VEGF-C in saphenous veins, compared with arterial tissue. Amounts of VEGF-C were lower than those of VEGF-A and VEGF-B in all specimens. CONCLUSION: This study provides direct evidence of the presence of VEGF proteins and receptors in human physiology and pathology, with variations in both the amounts of VEGF proteins expressed and their cellular distribution in normal arteries compared with atherosclerotic arteries. The presence of VEGFs A-C and their receptors in normal arterial tissue implies that VEGF functions may extend beyond endothelial cell proliferation. Reduced VEGFR-2 staining in atherosclerotic arteries may have implications for the atherosclerosis process and the development of vascular disease and its complications.     

VEGF, its receptors and the tie receptors in recurrent miscarriage

The aetiology of recurrent miscarriage (at least three consecutive miscarriages) usually remains unsolved. The vascular endothelial growth factor (VEGF) family of proteins, together with their receptors and the Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) receptors, are crucial for embryonic development. Therefore, we used immunohistochemistry to analyse the expression of VEGF, the VEGF receptors (VEGFR)-1, -2, and -3, and the Tie-1 and Tie-2 receptors in placental and decidual tissue of women with a history of recurrent miscarriage and missed abortion (MA; n = 12) or blighted ovum (BO; n = 6), and from normal early terminated pregnancies (n = 12). Compared with controls, the MA and BO groups showed: (i) diminished placental trophoblastic VEGF immunoreactivity; (ii) weaker VEGFR-1 and -2 immunoreactivity in decidual vascular endothelium; (iii) reduced placental trophoblastic Tie-1 receptor immunoreactivity; and (iv) reduced decidual vascular endothelial Tie-1 and -2 receptor immunoreactivity. The absence of VEGFR-3 immunoreactivity in decidual vascular endothelium was also noted in all study groups. Interestingly, placental villi from the BO group presented blood vessel-like structures negative for von Willebrand factor, but positive for VEGF, VEGFR-1, -2, -3, Tie-1 and Tie-2 receptor. We conclude that the expression of these antigens may be altered in recurrent miscarriages.     

Fetal and maternal angiogenic/anti-angiogenic factors in normal and preeclamptic pregnancy

Few studies evaluated angiogenic/anti-angiogenic factors and endothelial (dys)function in both maternal and umbilical cord blood (UCB) in preeclampsia (PE). We aimed to clarify the role of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 (VEGFR-1) and tissue plasminogen activator (tPA), by evaluating them in maternal and UCB in 42 normal and 46 preeclamptic (PEc) cases.

In PE, maternal and UCB PlGF were significantly lower; maternal VEGF, sVEGFR-1 and tPA were significantly higher. In UCB, sVEGFR-1 and tPA were significantly higher in PEc cases, while VEGF and PlGF were significantly lower. A significant correlation between maternal and UCB sVEGFR-1, and between sVEGFR-1 and tPA both in maternal and UCB, was observed in PEc cases.

In maternal and UCB circulation in PE, a close interaction seems to exist between endothelial dysfunction and angiogenesis disturbance, and sVEGFR-1 seems to play a central role in those disturbances.     

Expression and functional significance of vascular endothelial growth factor receptors in human tumor cells

Vascular endothelial growth factor (VEGF) is one of the key factors in tumor neoangiogenesis, acting through its receptors KDR (VEGFR-2) and fit-1 (VEGFR-1) expressed on endothelial cells. Our data demonstrate that VEGFR-1 and to a lesser extent VEGFR-2 are expressed in a number of human tumor tissues and derived cells in culture. VEGFR-1 protein is expressed in 26 of 42 glioma tissues, 22 of which show a coexpression of VEGFR-1 with VEGFR-2; 1 glioma tissue expresses exclusively VEGFR-2. In the derived glioma cell cultures, we found VEGFR-1 mRNA expression in 6 of 11 cultures, with one coexpressing VEGFR-1 and VEGFR-2. Of four established glioma cell lines, two expressed VEGFR-1. In addition VEGFR-1 protein expression was demonstrated in 30 of 37 tumor tissues of squamous cell carcinomas of the head and neck, with VEGFR-2 coexpression in 15 tissues and an expression of VEGFR-2 alone in 1 tissue. Derived tumor cell cultures showed mRNA expression of VEGFR-1 alone in seven of seven cases. Established melanoma cell lines expressed VEGFR-1 mRNA in four of five lines, with VEGFR-2 coexpression in two lines. Concerning the functional significance of VEGF receptor expression, VEGF treatment of VEGFR-1-expressing tumor cells induced the inhibition of cell proliferation by 25 to 55% and the inhibition of tumor cell migration by 29 to 55%. Thus our data indicate that the coexpression of VEGF and VEGFR-1 in tumor cells could have an inhibitory effect on tumor cell proliferation and migration, a mechanism possibly induced as a response to a deficiency in nutrient and oxygen supply.