RESUMO
The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and ß-catenin in cHL cells. Furthermore, LEF-1- and ß-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Microambiente Tumoral , Via de Sinalização Wnt , Adesão Celular/genética , Linhagem Celular , Movimento Celular/genética , Quimiocina CCL19/metabolismo , Quimiotaxia/genética , Quimiotaxia/imunologia , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Neovascularização Patológica , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The embryonic origin of lymphatic endothelial cells (LECs) has been a matter of controversy since more than a century. However, recent studies in mice have supported the concept that embryonic lymphangiogenesis is a complex process consisting of growth of lymphatics from specific venous segments as well as the integration of lymphangioblasts into the lymphatic networks. Similarly, the mechanisms of adult lymphangiogenesis are poorly understood and have rarely been studied. We have recently shown that endothelial progenitor cells isolated from the lung of adult mice have the capacity to form both blood vessels and lymphatics when grafted with Matrigel plugs into the skin of syngeneic mice. Here, we followed up on these experiments and studied the behavior of host leukocytes during lymphangiogenesis in the Matrigel plugs. We observed a striking co-localization of CD45(+) leukocytes with the developing lymphatics. Numerous CD45(+) cells expressed the LEC marker podoplanin and were obviously integrated into the lining of lymphatic capillaries. This indicates that, similar to inflammation-induced lymphangiogenesis in man, circulating CD45(+) cells of adult mice are capable of initiating lymphangiogenesis and of adopting a lymphvasculogenic cellular differentiation program. The data are discussed in the context of embryonic and inflammation-induced lymphangiogenesis.
Assuntos
Antígenos Comuns de Leucócito/imunologia , Leucócitos/imunologia , Vasos Linfáticos/imunologia , Animais , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Leucócitos/citologia , Vasos Linfáticos/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Inflammation is a local or systemic tissue reaction caused by external or internal stimuli with the objective to remove the noxa, inhibit its further dissemination and eventually repair damaged tissue. Blood vessels and perivascular connective tissue are important regulators of the inflammatory process. After a short initial ischemic phase, inflamed tissue is characterized by hyperaemia and increased permeability of capillaries. Therefore, blood vessels have been in the focus of inflammation research for quite some time, whereas lymphatic vessels have been neglected. Their reactivity is not immediately obvious, and, their identification within the tissue has hardly been possible until lymphatic endothelial cell (LEC)-specific molecules have been identified a few years ago. This has opened up the possibility to study lymphatics in normal and diseased tissues, and to isolate LECs for transcriptome and proteome analyses. Initial studies now provide evidence that lymphatics are not just a passive route for circulating lymphocytes, but seem to be directly involved in both the induction and the resolution of inflammation. This review provides a summary on the basics of inflammation, the structure of lymphatics and their molecular markers, human inflammation-associated diseases and their relation to lymphatics, animal models to study the interaction of lymphatics and inflammation, and finally inflammation-associated molecules expressed in LECs. The integration of lymphatics into inflammation research opens up an exciting new field with great clinical potential.
Assuntos
Endotélio Vascular/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Leucócitos/imunologia , Vasos Linfáticos/imunologia , Animais , Permeabilidade Capilar/imunologia , Endotélio Linfático/imunologia , Endotélio Linfático/metabolismo , Endotélio Vascular/metabolismo , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Leucócitos/metabolismo , Vasos Linfáticos/metabolismoRESUMO
OBJECTIVES: The present study was conducted to test the hypothesis that preshaped polylactic acid (PLA) implants loaded with recombinant human bone morphogenic protein 2 (rhBMP-2) can induce bone formation in a rat ectopic model. MATERIALS AND METHODS: Two groups of porous cylindrical poly-DL-lactic acid implants of 8-mm diameter were produced by gas foaming with CO(2), incorporating 48 and 96 microg rhBMP-2, respectively, into each implant. Blank PLA implants were used as controls. The release of BMPs and the induction of alkaline phosphatase were assessed in vitro. Osteoinduction in vivo was tested by insertion of 15 implants from each group into the gluteal muscles of Wistar rats. Five implants from each group were retrieved after 6, 13 and 26 weeks and assessed using flat panel volume detector computed tomography and light microscopy. RESULTS: Both groups of implants showed increased release of rhBMP-2 during the first 24-48 h, with a slightly higher amount being released from the implants with 48 microg. Release during subsequent intervals was <100 ng/72 h in the low-concentration group and >100 ng in the group with 96 microg rhBMP-2. Implants with 95 microg rhBMP-2 exhibited bone formation in vivo on the outside of the implants across the observation period of 26 weeks with invasion of bone into the pores, whereas implants with 48 microg rhBMP-2 failed to induce the formation of bone tissue. No bone formation was found in the control implants. CONCLUSIONS: The results suggest that release rates of rhBMP-2 for ectopic bone induction have to be >100 ng/72 h to maintain the osteoinductive activity of the tested porous PLA implants. This slow release system may have impact on alveolar bone augmentation procedures when used as individually preformed osteoinductive implants.
Assuntos
Implantes Absorvíveis , Proteínas Morfogenéticas Ósseas/farmacologia , Portadores de Fármacos , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Proteína Morfogenética Óssea 2 , Nádegas , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Humanos , Ácido Láctico , Masculino , Ossificação Heterotópica , Poliésteres , Polímeros , Ratos , Ratos WistarRESUMO
The aim of the present report was to test a system for controlled release of recombinant human bone morphogenic protein (rhBMP2) incorporated into polylactic acid (PLA) implants. Incorporation of rhBMP2 into the polymer was accomplished by mixing rhBMP2 solution with granular powder of amorphous poly-DL-lactic acid, subsequent lyophilization, and high pressure CO(2) treatment at 100 bar. Porous cylindrical implants of 8 mm diameter and 3 mm thickness were fabricated with 100, 200, 400, and 800 microg BMP2/g polymer and submitted to in vitro testing. Polymer degradation was assessed during immersion of PLA implants into PBS for 176 days by measuring the inherent viscosity at days 0, 99, and 131. BMP2 release was evaluated by immersion of both the lyophilized powder and the implants into cell culture medium for up to 27 days. BMP2 release was assessed using a custom made ELISA. The biological activity of the released growth factor was determined by measuring the induction of alkaline phosphatase (AP) in C2C12 cells. There was a significant retardation in the release of BMP2 from the implants compared to the granular powder. Detectable amounts of BMP2 were found for all concentrations of BMP2 until the end of the observation period. Significant induction of AP was detected by BMP released from the implants after 3, 6, and 9 days. The present in-vitro study has shown that incorporation of rhBMP2 into PLA implants with subsequent slow release of biologically active growth factor is possible.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Preparações de Ação Retardada/metabolismo , Ácido Láctico/metabolismo , Polímeros/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Implantes Absorvíveis , Fosfatase Alcalina/biossíntese , Animais , Proteína Morfogenética Óssea 2 , Linhagem Celular , Indução Enzimática , Gases , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , PoliésteresRESUMO
Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.
Assuntos
Capilares/metabolismo , Endotélio Vascular/citologia , Retina/citologia , Angiopoietina-1/biossíntese , Animais , Capilares/citologia , Sobrevivência Celular , Densitometria , Retinopatia Diabética/patologia , Genes Reporter , Hipóxia , Immunoblotting , Óperon Lac , Ligantes , Camundongos , Neovascularização Patológica , Pericitos/citologia , Pericitos/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor TIE-2/metabolismo , Retina/embriologia , Vasos Retinianos/patologia , Fatores de Tempo , Regulação para CimaRESUMO
Vascular endothelial growth factor (VEGF) receptors consist of three cell-membrane type receptors (VEGFR-1, VEGFR-2 and VEGFR-3), and soluble form of VEGFR-1 (sVEGFR-1), an intrinsic negative counterpart of the VEGF. In this study, we measured intratumoral protein levels of free and total VEGF, VEGFR-2 and sVEGFR-1 from 202 primary breast cancer tissues and examined their prognostic values. A significant inverse correlation was found between free or total VEGF and oestrogen receptor (ER) status (P=0.042 and 0.032, respectively). A univariate analysis showed that low sVEGFR-1 and high total VEGF were significantly associated with poor prognosis in disease-free survival (DFS) and overall survival (OS). The ratio of sVEGFR-1 to total VEGF was a strong prognostic indicator (DFS: P=0.008; OS: P=0.0002). A multivariate analysis confirmed the independent prognostic values of total VEGF and the ratio of sVEGFR-1 to total VEGF. In subgroup analysis, total VEGF was a significant prognostic indicator for ER-positive tumours but not for ER-negative tumours, whereas sVEGFR-1 was significant for ER-negative tumours but not for ER-positive tumours. In conclusion, the intratumoral sVEGFR-1 level, VEGF level and the ratio of sVEGFR-1 to total VEGF are potent prognostic indicators of primary breast cancer, and might be relevant to ER status.
Assuntos
Neoplasias da Mama/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Neovascularização Fisiológica , PrognósticoAssuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Feminino , Humanos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
To explore the possibility of vascular endothelial growth factor (VEGF) receptor scintigraphy of primary tumours and their metastases, we analysed the binding properties of (123)I-labelled VEGF(165) ((123)I-VEGF(165)) and (123)I-VEGF(121) to human umbilical vein endothelial cells (HUVECs), several human tumour cell lines (HMC-1, A431, KU812, U937, HEP-1, HEP-G2, HEP-3B and Raji), a variety of primary human tumours (n = 40) and some adjacent non-neoplastic tissues as well as normal human peripheral blood cells in vitro. Two classes of high-affinity (123)I-VEGF(165)-binding site were found on the cell surface of HUVECs. In contrast, one class of high-affinity binding sites for (123)I-VEGF(165) was found on HMC-1, A431, HEP-1, HEP-G2, HEP-3B and U937 cells as well as many primary tumours. For (123)I-VEGF(121), a single class of high-affinity binding site was found on certain cell lines (HUVEC, HEP-1 and HMC-1) and distinct primary tumours (primary melanomas, ductal breast cancers and ovarian carcinomas as well as meningiomas). Tumour cells expressed significantly higher numbers of VEGF receptors compared with normal peripheral blood cells and adjacent non-neoplastic tissues. Immunohistochemical staining revealed that the VEGF receptor Flk-1 is expressed to a much higher extent within malignant tissues compared with neighbouring non-neoplastic cells. We observed significantly greater specific binding of (123)I-VEGF(165) and (123)I-VEGF(121) to a variety of human tumour cells/tissues compared with the corresponding normal tissues or normal peripheral blood cells. In comparison with (123)I-VEGF(121), (123)I-VEGF(165) bound to a higher number of different tumour cell types with a higher capacity. Thus, (123)I-VEGF(165) may be a potentially useful tracer for in vivo imaging of solid tumours.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Neoplasias/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Humanos , Técnicas Imunoenzimáticas , Radioisótopos do Iodo , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Cintilografia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of growth and differentiation factors and have been characterized by their ability to induce new bone formation in ectopic (non-skeletal) sites. BMPs are secreted molecules and are key regulators in early embryogenesis and organogenesis. One of the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in vertebrates. This osteo-inductive capacity of BMPs has long been considered very promising for applications in bone repair, in the treatment of skeletal diseases, and in oral applications such as dentiogenesis and cementogenesis during regeneration of periodontal wounds. We discuss here biological roles of the BMPs in the organism and their signaling cascades leading to bone and cartilage formation in particular. It is also the aim of this review to evaluate the potential and the problems of BMPs in skeletal tissue engineering for the regeneration of bone damaged by disease or trauma and to serve as therapeutic agents for periodontal defects.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas Morfogenéticas Ósseas/fisiologia , Animais , Doenças Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/uso terapêutico , Remodelação Óssea/fisiologia , Consolidação da Fratura/fisiologia , Humanos , Camundongos , Doenças Periodontais/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNF alpha (VEGF-A/TNF alpha). This inhibition was not observed when tube formation was induced by VEGF-C/TNF alpha or bFGF/TNF alpha. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNF alpha-induced as well as the VEGF-C/TNF alpha-induced tube formation in vitro. P1GF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNF alpha, nor inhibited or stimulated by itself the VEGF-A/TNF alpha-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/crescimento & desenvolvimento , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Endotélio Vascular/fisiologia , Humanos , Técnicas In Vitro , Camundongos , Modelos Cardiovasculares , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.
Assuntos
Ascite/etiologia , Ascite/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Ascite/prevenção & controle , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/genética , Feminino , Humanos , Linfocinas/antagonistas & inibidores , Linfocinas/genética , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular , Recidiva , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
One of the key molecules promoting angiogenesis is the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which acts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (or Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble variant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the flt-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducing the level of free, active VEGF-A, and therefore, plays a pivotal role in the generation of vascular diseases like pre-eclampsia or intra-uterine growth retardation. Here we show that sVEGFR-1 is produced by cultured human microvascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5-10% remains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putative ligands, VEGF-A and placenta growth factor (PIGF), from macrovascular endothelial cells. The release of sVEGFR-1 was quantitatively measured in two different ELISA types. The release of sVEGFR-1 was strongly enhanced by phorbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PIGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cultured from placenta, a tissue that has a high flt-1 gene expression. Moreover, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exogenous VEGF165 and PIGF-2. Here, for the first time, to what extent recombinant ligands have to be added to compensate for the sink function of amniotic fluids was analyzed. In conclusion, human endothelial cells produce high levels of sVEGFR-1, which influences the availability of VEGF-A or related ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembrane receptors and interfere with their signal transduction.
Assuntos
Líquidos Corporais/metabolismo , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células Cultivadas , Humanos , Ligantes , Neovascularização Patológica , Neovascularização Fisiológica , Proteínas Recombinantes , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Neoplasias Meníngeas/química , Meningioma/química , Proteínas de Neoplasias/análise , Proteínas da Gravidez/análise , RNA Mensageiro/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Criança , Fatores de Crescimento Endotelial/análise , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Linfocinas/análise , Masculino , Neoplasias Meníngeas/patologia , Meningioma/patologia , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Crescimento Placentário , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Proteínas da Gravidez/genética , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fator B de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The goal of this investigation was to measure changes in vascular permeability, pore cutoff size, and number of transvascular transport pathways as a function of time and in response to vascular endothelial growth factor (VEGF), placenta growth factor (PIGF-1 and PIGF-2), or basic fibroblast growth factor (bFGF). Two human and two murine tumors were implanted in the dorsal skin chamber or cranial window. Vascular permeability to BSA (approximately 7 nm in diameter) and extravasation of polyethylene glycol-stabilized long-circulating liposomes (100-400 nm) and latex microspheres (approximately 800 nm) were determined by intravital microscopy. Vascular permeability was found to be temporally heterogeneous. VEGF superfusion (100 ng/ml) significantly increased vascular permeability to albumin in normal s.c. vessels, whereas a 30-fold higher dose of VEGF (3000 ng/ml) was required to increase permeability in pial vessels, suggesting that different tissues exhibit different dose thresholds for VEGF activity. Furthermore, VEGF superfusion (1000 ng/ml) increased vascular permeability to albumin in a hypopermeable human glioma xenograft in cranial window, whereas VEGF superfusion (10-1000 ng/ml) failed to increase permeability in a variety of hyperpermeable tumors grown in dorsal skin chamber. Interestingly, low-dose VEGF treatment (10 ng/ml) doubled the maximum pore size (from 400 to 800 nm) and significantly increased the frequency of large (400 nm) pores in human colon carcinoma xenografts. PIGF-1, PIGF-2, or bFGF did not show any significant effect on permeability or pore size in tumors. These findings suggest that exogenous VEGF may be useful for augmenting the transvascular delivery of larger antineoplastic agents such as gene targeting vectors and encapsulated drug carriers (typical range, 100-300 nm) into tumors.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Linfocinas/farmacologia , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica , Proteínas da Gravidez/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos , Substâncias Macromoleculares , Camundongos , Microesferas , Fator de Crescimento Placentário , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. METHODS AND RESULTS: Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. CONCLUSIONS: The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.
Assuntos
Coração Fetal/química , Proteínas Fetais/análise , Proteínas Musculares/análise , Proteínas do Tecido Nervoso/análise , Proteínas Proto-Oncogênicas/análise , Receptores Proteína Tirosina Quinases/análise , Receptores de Superfície Celular/análise , Receptores de Fatores de Crescimento/análise , Capilares/química , Capilares/embriologia , Circulação Coronária , Endocárdio/química , Fatores de Crescimento Endotelial/fisiologia , Secções Congeladas , Humanos , Linfocinas/fisiologia , Miocárdio/química , Neovascularização Fisiológica/fisiologia , Neuropilina-1 , Pericárdio/química , Pericárdio/embriologia , Receptor de TIE-1 , Receptor TIE-2 , Receptores de TIE , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) is an important factor for endothelial cell proliferation and a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Its biological activity is mediated by two receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR). In contrast to VEGFR-2, a naturally occurring soluble form of the VEGFR-1 (sVEGFR-1) is produced by endothelial cells by differential splicing of the flt-1 gene, and it is a secreted gene product. In order to develop a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of sVEGFR-1, we established five anti-human receptor antibodies and characterized them in detail. These antibodies recognize different epitopes located within the seven Ig-like domains of the extracellular receptor protein but have no neutralizing activity in ligand binding assays. Together with a polyclonal antiserum, a specific human sVEGFR-1 ELISA was developed using the mAb #190.11. The ELISA can detect human sVEGFR-1 with a minimum detection limit of 1 ng/ml. The ELISA does not show any cross-reactivity with other related soluble receptors. Using this assay, human sVEGFR-1 was measured in the supernatant of different VEGFR-1 expressing cell types. No sVEGFR-1 protein was detectable after heparin Sepharose treatment or size-exclusion filtration (< 30 kDa). The ELISA assay for sVEGFR-1 was also used to measure the amount of the soluble receptor in amniotic fluid samples of patients undergoing amniocentesis during the course of normal pregnancies. The concentration of the samples was in the range of 5-35 ng/ml. This ELISA could be useful powerful tool for investigations concerning the physiological function of the soluble receptor under normal and pathophysiological conditions.Furthermore, it may facilitate studies of the mechanisms of receptor production.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Proto-Oncogênicas/análise , Receptores Proteína Tirosina Quinases/análise , Animais , Anticorpos Monoclonais/imunologia , Células Cultivadas , Reações Cruzadas , Meios de Cultura , Endotélio Vascular/citologia , Humanos , Ligantes , Camundongos , Coelhos , Solubilidade , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
The three human VEGF receptors 1-3 mediate biological signals important for new blood vessel formation and lymphangiogenesis. Soluble VEGF receptors contain all the information necessary for high affinity ligand binding and have been used as experimental tools and regulators in several angiogenic in vitro and in vivo models. Recombinant receptor molecules can be used for specific inhibition of VEGF mediated signal transduction and for blocking tumor angiogenesis by limiting the amount of VEGF secreted from tumor cells or stroma cells. A naturally occurring soluble VEGFR-1 has been discovered in the supernatant from endothelial cells and at present appears to be the key regulator for the availability of VEGF secreted from different cells and tissues. The exact physiological role has not yet been demonstrated.
RESUMO
Vascularization is a prerequisite for corpus luteum formation. Angiogenesis is thought to be regulated by vascular growth factors. Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) specifically induces endothelial cell proliferation as well as angiogenesis and increases capillary permeability. Recently, VEGF/VPF-mRNA expression was demonstrated in luteinized human granulosa cells (GC) in vitro. In addition, the production of VEGF/VPF by human granulosa can be demonstrated immunocytochemically. VEGF/VPF is thought to mediate its effects through specific cell surface receptors. So far, two VEGF/VPF-receptors (VEGF/VPF-R) have been identified (KDR, and flt-1). A third receptor (flt-4) is highly correlated to KDR and flt-1, but the true ligand for this receptor is still unknown. The appearance of all three receptors is more or less restricted to endothelial cells. To clarify whether VEGF/VPF acts in an auto- or paracrine fashion in human luteinized GC, mRNA was scrutinized for specific expression of the three receptors by Northern blot technique. No specific VEGF/VPF-R or flt-4 transcripts were detectable, indicating that VEGF/VPF is a genuine paracrine growth factor from human luteinized GC directed to endothelial cells.
Assuntos
Corpo Lúteo/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Células da Granulosa/metabolismo , Linfocinas/metabolismo , Comunicação Parácrina , Adulto , Northern Blotting , Feminino , Humanos , Técnicas In Vitro , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The FLT-1 and KDR genes encode transmembrane tyrosine kinases which function as high-affinity receptors for vascular endothelial growth factor (VEGF). We have used the baculovirus system to express the extracellular parts of the FLT-1 receptor and KDR receptor in soluble form (sFLT-1 and sKDR), for in vitro binding and competition assays. Here, we show that the binding of VEGF165 to sKDR but not sFLT-1 is dependent on heparin, regardless of whether VEGF165 or sKDR is immobilized. Further, only sFLT-1 acts as a receptor antagonist in solution and sKDR can neither compete with the binding of VEGF165 to human endothelial cells carrying both receptors nor block VEGF165 induced mitogenicity. Soluble KDR only partially inhibits cell migration even at high concentrations, in contrast to sFLT which can almost completely block (82%) VEGF-induced cell proliferation and migration. Taken together these results show that the two soluble VEGF receptor proteins, sFLT-1 and sKDR, despite binding the same ligand, behave very differently when immobilized with regard to their dependence on heparin for VEGF binding. In solution their respective ability to function as receptor antagonists is also strikingly different, possibly a reflection of their different dependency on heparin.