RESUMEN
Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.
Asunto(s)
Células Endoteliales/fisiología , Linfangiogénesis/fisiología , Células Madre Mesenquimatosas/fisiología , Neovascularización Fisiológica/fisiología , Células Madre/fisiología , Animales , Células Endoteliales/citología , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes , Inmunocompetencia , Inmunohistoquímica , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Células Madre/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41+ cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41+ cells in the BM and a significant increase in diploid CD41+ cells, whereas the number of polyploid CD41+ cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Progenitoras de Megacariocitos/metabolismo , Trombopoyesis , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Antimetabolitos/farmacología , Western Blotting , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Fluorouracilo/farmacología , Expresión Génica , Células HEK293 , Humanos , Células Progenitoras de Megacariocitos/efectos de los fármacos , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ésteres del Forbol/farmacología , Recuento de Plaquetas , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Ploidias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombopoyetina/farmacología , Factor C de Crecimiento Endotelial Vascular/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation. METHODS: Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation. RESULTS: Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4(+) cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR(+) and FLT4(+) cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes. CONCLUSIONS: Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.
Asunto(s)
Metilación de ADN/fisiología , Leucemia/metabolismo , Linfoma/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Western Blotting , Línea Celular Tumoral , Metilación de ADN/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Leucemia/genética , Linfoma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Pulmón/citología , Espectrometría de Masas/métodos , Proteoma/metabolismo , Células Madre/metabolismo , Animales , Medios de Cultivo Condicionados/química , Células Endoteliales/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica , Proteoma/química , Reproducibilidad de los Resultados , Solubilidad , Células Madre/citología , Fracciones Subcelulares/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt-1 (sFlt-1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti-VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt-1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt-1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt-1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30-fold, compared to control AdvGFP-treated animals. The threshold of kidney damage was in the range of 20-30 ng/ml sFlt-1 in plasma (8-15 ng/ml in urine). Co-administration of AdvsFlt-1 with AdvVEGF to neutralize circulating sFlt-1 resulted in more than a 70% reduction in free sFlt-1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt-1 on the kidneys. This demonstrates that below a critical threshold sFlt-1 fails to elicit damage to the fenestrated endothelium and that co-expression of VEGF is able to rescue effects mediated by sFlt-1 overexpression.
Asunto(s)
Preeclampsia/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/patología , Ratones , Preeclampsia/fisiopatología , Embarazo , Unión Proteica/efectos de los fármacos , Solubilidad/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. RESULTS: In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels.Mouse lung microvascular endothelial cells (MLMVECs) were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony). These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs) in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. CONCLUSION: The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.
Asunto(s)
Células Madre Adultas/metabolismo , Células Sanguíneas/metabolismo , Células Endoteliales/metabolismo , Pulmón/patología , Vasos Linfáticos/metabolismo , Células Madre Adultas/patología , Animales , Antígenos de Diferenciación/metabolismo , Células Sanguíneas/patología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Células Endoteliales/patología , Vasos Linfáticos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Nicho de Células Madre , Trasplante de Células MadreRESUMEN
The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Huesos/efectos de los fármacos , Portadores de Fármacos , Implantes Experimentales , Ácido Láctico , Mandíbula/efectos de los fármacos , Polímeros , Factor de Crecimiento Transformador beta/farmacología , Animales , Proteína Morfogenética Ósea 2 , Huesos/lesiones , Huesos/cirugía , Humanos , Masculino , Mandíbula/cirugía , Poliésteres , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Tomógrafos Computarizados por Rayos XRESUMEN
In the human, malformations of lymphatic vessels can be observed as lymphangiectasia, lymphangioma and lymphangiomatosis, with a prevalence of 1.2-2.8 per thousand. Their aetiology is unknown and a causal therapy does not exist. We investigated the origin of lymphatic endothelial cells (LECs) in avian and murine embryos, and compared the molecular profile of LECs from normal and malformed lymphatics of children. In avian embryos, Prox1+ lymphangioblasts are located in the confluence of the cranial and caudal cardinal veins, where the jugular lymph sac (JLS) forms. Cell lineage studies show that the JLS is of venous origin. In contrast, the lymphatics of the dermis are derived from mesenchymal lymphangioblasts located in the dermatomes, suggesting a dual origin of LECs in avian embryos. The same may hold true for murine embryos, where Lyve1+ LEC precursors are found in the cardinal veins, and in the mesenchyme. The mesenchymal cells express the pan-leukocyte marker CD45, indicating a cell type with lymphendothelial and leukocyte characteristics. In the human, such cells might give rise to Kaposi's sarcoma. Microarray analyses of LECs from lymphangiomas of children show a large number of regulated genes, such as VEGFR3. Our studies show that lymphvasculogenesis and lymphangiogenesis occur simultaneously in the embryo, and suggest a function for VEGFR3 in lymphangiomas.
Asunto(s)
Desarrollo Embrionario , Vasos Linfáticos/anomalías , Vasos Linfáticos/embriología , Animales , Línea Celular , Embrión de Pollo , Niño , Embrión no Mamífero/citología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Hibridación in Situ , Ratones , Codorniz , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Linfangioma/metabolismo , Neoplasias Cutáneas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adolescente , Biopsia con Aguja , Estudios de Casos y Controles , Niño , Células Endoteliales/citología , Endotelio Linfático/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Linfangioma/patología , Masculino , Pronóstico , Proteína Reelina , Valores de Referencia , Muestreo , Sensibilidad y Especificidad , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Lymphangiogenesis plays an important role in several normal and pathological conditions, such as wound healing, pathogen infection, inflammation or the metastasis formation of endothelial malignancies. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4). In this study, we measured intratumoral levels of VEGF-C and VEGFR-3 through enzyme-linked immunosorbent assay (ELISA) in 193 primary breast cancer tissues and examined their prognostic values. A significant correlation was found between the VEGF-C and VEGFR-3 protein levels. High VEGF-C levels were associated with low-grade tumors and a smaller size. Univariate analysis showed that high VEGF-C was significantly associated with a favourable prognosis for disease-free survival (DFS) and overall survival (OS). No significant prognostic value of VEGFR-3 was detected. Multivariate analysis confirmed the independent prognostic value of VEGF-C. The intratumoral VEGF-C level is a significant prognostic indicator of primary breast cancer. An investigation of the mechanisms of VEGF-C protein processing in human cancer tissue should be carried out in the future.
Asunto(s)
Neoplasias de la Mama/patología , Factor C de Crecimiento Endotelial Vascular/análisis , Receptor 3 de Factores de Crecimiento Endotelial Vascular/análisis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Análisis de Regresión , Análisis de SupervivenciaRESUMEN
BACKGROUND: During the female reproductive cycle, follicular development and corpus luteum formation crucially depend on the fast generation of new blood vessels. The importance of granulosa cells and follicular fluid in controlling this angiogenesis is still not completely understood. Vascular endothelial growth factor (VEGF) produced by granulosa cells and secreted into the follicular fluid plays an essential role in this process. On the other hand, soluble VEGF receptor-1 (sFlt-1) produced by endothelial cells acts as a negative modulator for the bioavailability of VEGF. However, the regulation of sFlt-1 production remains to be determined. METHODS: We analyzed the influence of human follicular fluid obtained from FSH-stimulated women as well as of human granulosa cell conditioned medium on sFlt-1 production in and release from human umbilical vein endothelial cells (HUVEC) in vitro. Soluble Flt-1 gene expression was determined by RT-PCR analysis, amount of sFlt-1-protein was quantified by Sandwich-ELISA. RESULTS: Human follicular fluid as well as granulosa cell-conditioned medium significantly inhibit the production of sFlt-1 by endothelial cells on a posttranscriptional level. Treatment of cultured granulosa cells with either hCG or FSH had not impact on the production of sFlt-1 inhibiting factors. We further present data suggesting that this as yet unknown sFlt-1 regulating factor secreted by granulosa cells is not heat-sensitive, not steroidal, and it is of low molecular mass (< 1000 Da). CONCLUSION: We provide strong support that follicular fluid and granulosa cells control VEGF availability by down regulation of the soluble antagonist sFlt-1 leading to an increase of free, bioactive VEGF for maximal induction of vessel growth in the ovary.
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Células Endoteliales/fisiología , Líquido Folicular/fisiología , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Regulación hacia Abajo , Células Endoteliales/metabolismo , Femenino , Humanos , Neovascularización Fisiológica/fisiologíaRESUMEN
Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.
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Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/metabolismo , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/metabolismo , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Escherichia coli/genética , Humanos , Proteínas Recombinantes , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay. Here we describe a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human, rat and murine VEGF-C. The different antibodies developed against human and rat VEGF-C could be combined to detect processed and partially processed VEGF-C in a specific way. The ELISA was able to detect human and rat VEGF-C with a minimum detection limit of 100 pg/ml. The assay did not show any cross-reactivity with the related protein VEGF-D. Furthermore, complex formation with its soluble receptors VEGFR-2 and VEGFR-3 did not restricted the sensitivity of the assay. Using this assay, VEGF-C was measured in supernatants and lysates of different cell types and in tumour tissue samples of murine, rat and human origin. Cell lines secrete VEGF-C in very low amounts (<1 ng/ml) whereas VEGF-C transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C was detected in some carcinomas in the low protein range. This ELISA will be a useful tool for investigations concerning the physiological function of VEGF-C in lymphangiogenesis under normal and pathophysiological conditions.
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Ensayo de Inmunoadsorción Enzimática/métodos , Factor C de Crecimiento Endotelial Vascular/análisis , Animales , Anticuerpos/análisis , Northern Blotting , Reacciones Cruzadas , Humanos , Neoplasias/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
The vascular endothelial growth factor (VEGF) family is involved in angiogenesis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strategies against cancer. However, the physiological functions of VEGFs in quiescent tissues are unclear and may interfere with such systemic therapies. In pathological conditions, increased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accompany VEGF activity. In this study we investigated normal human and monkey tissues for expression patterns of these receptors. Immunohistochemical staining methods at the light and electron microscopic level were applied to normal human and monkey tissue samples, using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbs PAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similar distribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, -2, and -3 was observed in microvessels adjacent to epithelia in the eye, gastrointestinal mucosa, liver, kidney, and hair follicles, which is in line with the reported preferential expression of VEGF-A in some of these epithelia. VEGFR-1, -2, and -3 expression was also observed in blood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2 and -3, was present in microvessels in brain and retina. Electron microscopy showed that VEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normal vasculature of tonsils. These findings indicate that VEGFRs have specific distribution patterns in normal tissues, suggesting physiological functions of VEGFs that may be disturbed by systemic anti-VEGF therapy. One of these functions may be involvement of VEGF in paracrine relations between epithelia and adjacent capillaries.
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Endotelio Vascular/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Epitelio , Humanos , Inmunohistoquímica , Tejido Linfoide/irrigación sanguínea , Macaca fascicularis , Microcirculación , Especificidad de Órganos , Receptores de Factores de Crecimiento Endotelial Vascular , Especificidad de la Especie , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
PURPOSE: The vascular endothelial growth factor (VEGF) family is involved in vascular leakage and angiogenesis in diabetic retinopathy (DR) in the eye, but may also have physiological functions. Based on the hypothesis that differential VEGF receptor (VEGFR) expression in the retina is an important determinant of effects of VEGF, this study was conducted to investigate VEGFR expression in the diabetic retina and in an experimental monkey model of VEGF-A-induced retinopathy. METHODS: In retinas of 27 eyes of diabetic donors, 18 eyes of nondiabetic control donors, and 4 monkey eyes injected with PBS or VEGF-A, expression patterns of VEGFR-1, -2, and -3 in relation to leaky microvessels, as identified by the marker pathologische anatomie Leiden-endothelium (PAL-E) were studied by immunohistochemistry. RESULTS. In control human retinas and retinas of PBS-injected monkey eyes, all three VEGFRs were expressed in nonvascular areas, but only VEGFR-1 was constitutively expressed in retinal microvessels. In diabetic eyes, increased microvascular VEGFR-2 expression was found in association with PAL-E expression, whereas microvascular VEGFR-3 was present in a subset of PAL-E-positive cases. In VEGF-A-injected monkey eyes, VEGFR-1, -2, and -3 and PAL-E were expressed in retinal microvessels. CONCLUSIONS: The VEGFR-1, -2, and -3 expression patterns in control retinas suggest physiological functions of VEGFs that do not involve the vasculature. Initial vascular VEGF signaling may act primarily through VEGFR-1. In diabetic eyes, expression of retinal VEGFR-2 and -3 is increased, mainly in leaky microvessels, and VEGF-A induces vascular expression of the VEGF-A receptor VEGFR-2 and the VEGF-C/D receptor VEGFR-3. These findings indicate a dual role of VEGFs in the physiology and pathophysiology of the retina and suggest that microvascular VEGFR-2 and -3 signaling by VEGFs occurs late in the pathogenesis of DR, possibly initiated by high levels of VEGF-A in established nonproliferative DR.
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Retinopatía Diabética/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Vasos Retinianos/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales , Permeabilidad Capilar , Retinopatía Diabética/inducido químicamente , Retinopatía Diabética/patología , Factores de Crecimiento Endotelial , Femenino , Humanos , Técnicas para Inmunoenzimas , Macaca fascicularis , Masculino , Persona de Mediana Edad , Receptores de Factores de Crecimiento Endotelial Vascular , Vasos Retinianos/patología , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.
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Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/aislamiento & purificación , Escherichia coli/genética , Cuerpos de Inclusión/metabolismo , Factor de Crecimiento Transformador beta , Reactores Biológicos , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Medios de Cultivo , Dimerización , Escherichia coli/crecimiento & desarrollo , Ingeniería Genética , Conformación Proteica , Pliegue de Proteína , Renaturación de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of ß(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.
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Actinas/metabolismo , Permeabilidad Capilar/inmunología , Cortactina/deficiencia , Cortactina/inmunología , Neutrófilos/inmunología , Transducción de Señal/inmunología , Animales , Western Blotting , Adhesión Celular/fisiología , Cortactina/genética , Cortactina/metabolismo , Células Endoteliales/metabolismo , GTP Fosfohidrolasas/metabolismo , Genotipo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Fluorescente , Neutrófilos/metabolismo , Oligonucleótidos/genética , ARN Interferente Pequeño/genética , Venas Umbilicales/citología , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rhoRESUMEN
Adipose tissue growth depends on angiogenesis. We tested the hypothesis that adipose tissue produces factors relevant to angiogenesis. We obtained fat biopsies in 2 different patient cohorts, cultured adipose-derived stem cells and studied mature adipocytes. We performed microarray, RT-PCR, and Western blotting; studied a rat obesity/metabolic syndrome model; and conducted viral gene transfer experiments in leptin-deficient mice. The microarray identified the splice variant of the vascular endothelial growth factor receptor, the soluble fms-like tyrosine kinase 1 (sFlt-1), as an antiangiogenesis candidate. We verified the expression findings and found that sFlt-1 was secreted by isolated mature human adipocytes. Tumor necrosis factor-α decreased sFlt-1 expression in mature adipocytes, whereas hypoxia had no effect. Separating cells from adipose tissue showed that the highest sFlt-1 expression was present in adipose-tissue nonfat cells rather than in the adipocytes themselves. We also found that sFlt-1 expression and sFlt-1 release by adipose-tissue explants were inversely correlated with body mass index of the corresponding patients but was directly correlated with adiponectin expression. In the obesity/metabolic syndrome rat model, we observed that circulating sFlt-1 levels and sFlt-1 expression in adipose tissue were also inversely correlated with body weight. To model our putative antiangiogenic factor further, we next overexpressed sFlt-1 by viral transfer in a mouse genetic model of leptin deficiency and observed that the transfected mice gained less weight than controls. We suggest that sFlt-1 could act as a paracrine factor inhibiting adipose tissue growth. Local sFlt-1 may regulate angiogenic potential and thereby influence adipose tissue mass.
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Adipocitos/metabolismo , Adipoquinas/metabolismo , Regulación de la Expresión Génica , Neovascularización Patológica/genética , Obesidad/genética , ARN/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Adipocitos/patología , Animales , Western Blotting , Índice de Masa Corporal , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Obesos , Neovascularización Patológica/etiología , Neovascularización Patológica/metabolismo , Obesidad/enzimología , Obesidad/patología , Ratas , Ratas Transgénicas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
PURPOSE: Tumor progression correlates with the induction of a dense supply of blood vessels and the formation of peritumoral lymphatics. Hemangiogenesis and lymphangiogenesis are potently regulated by members of the vascular endothelial growth factor (VEGF) family. Previous studies have indicated the upregulation of VEGF-A and -C in progressed neuroblastoma, however, quantification was performed using semiquantitative methods, or patients who had received radiotherapy or chemotherapy were studied. EXPERIMENTAL DESIGN: We have analyzed primary neuroblastoma from 49 patients using real-time reverse transcription-PCR and quantified VEGF-A, -C, and -D and VEGF receptors (VEGFR)-1, 2, 3, as well as the soluble form of VEGFR2 (sVEGFR-2), which has recently been characterized as an endogenous inhibitor of lymphangiogenesis. None of the patients had received radiotherapy or chemotherapy before tumor resection. RESULTS: We did not observe upregulation of VEGF-A, -C, and -D in metastatic neuroblastoma, but found significant downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic stages III, IV, and IVs. In stage IV neuroblastoma, there were tendencies for the upregulation of VEGF-A and -D and the downregulation of the hemangiogenesis/lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2 in MYCN-amplified tumors. Similarly, MYCN transfection of the neuroblastoma cell line SH-EP induced the upregulation of VEGF-A and -D and the switching-off of sVEGFR-2. CONCLUSION: We provide evidence for the downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic neuroblastoma stages, which may promote lymphogenic metastases. Downregulation of hemangiogenesis and lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2, and upregulation of angiogenic activators VEGF-A and VEGF-D in MYCN-amplified stage IV neuroblastoma supports the crucial effect of this oncogene on neuroblastoma progression.