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1.
Sci Rep ; 8(1): 505, 2018 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-29323190

RESUMEN

Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) are ligands for Tie2, an endothelial-specific receptor tyrosine kinase that is an essential regulator of angiogenesis. Here we report the identification, via expression cloning, of thrombomodulin (TM) as another receptor for Ang1 and Ang2. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin-mediated activation of protein C, an anticoagulant protein, as well as thrombin-activatable fibrinolysis inhibitor (TAFI). Ang1 and Ang2 inhibited the thrombin/TM-mediated generation of activated protein C and TAFI in cultured endothelial cells, and inhibited the binding of thrombin to TM in vitro. Ang2 appears to bind TM with higher affinity than Ang1 and is a more potent inhibitor of TM function. Consistent with a potential role for angiopoietins in coagulation, administration of thrombin to mice rapidly increased plasma Ang1 levels, presumably reflecting release from activated platelets (previously shown to contain high levels of Ang1). In addition, Ang1 levels were significantly elevated in plasma prepared from wound blood, suggesting that Ang1 is released from activated platelets at sites of vessel injury. Our results imply a previously undescribed role for angiopoietins in the regulation of hemostasis.


Asunto(s)
Angiopoyetina 1/metabolismo , Angiopoyetina 2/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo , Angiopoyetina 1/sangre , Angiopoyetina 1/genética , Angiopoyetina 2/genética , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células COS , Carboxipeptidasa B2/metabolismo , Chlorocebus aethiops , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Factor Plaquetario 4/metabolismo , Unión Proteica , Proteína C/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor TIE-2/antagonistas & inhibidores , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Trombina/química , Trombina/farmacología , Trombomodulina/genética
2.
Mol Cancer Ther ; 13(5): 1345-55, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24634416

RESUMEN

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.


Asunto(s)
Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Endocrinology ; 153(4): 1972-83, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22334711

RESUMEN

Using specific inhibitors established that angiogenesis in the ovarian follicle and corpus luteum is driven by vascular endothelial growth factor. Recently, it has been demonstrated that the Notch ligand, delta-like ligand 4 (Dll4) negatively regulates vascular endothelial growth factor-mediated vessel sprouting and branching. To investigate the role of Dll4 in regulation of the ovarian vasculature, we administered a neutralizing antibody to Dll4 to marmosets at the periovulatory period. The vasculature was examined on luteal d 3 or d 10: angiogenesis was determined by incorporation of bromodeoxyuridine, staining for CD31 and cell death by staining for activated caspase-3. Ovulatory progesterone rises were monitored to determine effects of treatment on luteal function and time to recover normal cycles in a separate group of animals. Additionally, animals were treated in the follicular or midluteal phase to determine effects of Dll4 inhibition on follicular development and luteal function. Controls were treated with human IgG (Fc). Corpora lutea from marmosets treated during the periovulatory period exhibited increased angiogenesis and increased vascular density on luteal d 3, but plasma progesterone was significantly suppressed. By luteal d 10, corpora lutea in treated ovaries were significantly reduced in size, with involution of luteal cells, increased cell death, and suppressed plasma progesterone concentrations. In contrast, initiation of anti-Dll4 treatment during the midluteal phase produced only a slight suppression of progesterone for the remainder of the cycle. Moreover, Dll4 inhibition had no appreciable effect on follicular development. These results show that Dll4 has a specific and critical role in the development of the normal luteal vasculature.


Asunto(s)
Callithrix/fisiología , Cuerpo Lúteo/irrigación sanguínea , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Luteólisis/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Neovascularización Fisiológica/fisiología , Ovario/fisiología , Animales , Anticuerpos Neutralizantes/farmacología , Apoptosis/fisiología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/patología , Femenino , Inmunoglobulina G/farmacología , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/fisiología , Modelos Animales , Neovascularización Fisiológica/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Progesterona/sangre , Receptores Notch/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología
4.
Kidney Int ; 74(3): 300-9, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18480750

RESUMEN

The loss of interstitial capillaries is a feature of several experimental models of renal disease and this contributes to secondary kidney injury. Angiopoietin-1 is a secreted growth factor which binds to Tie-2 present on endothelia to enhance cell survival thereby stabilizing capillary architecture in-vitro. Previous studies showed that angiopoietin-1 prevented renal capillary and interstitial lesions following experimental ureteric obstruction. We tested here the effect of angiopoietin-1 treatment on capillary loss and associated tubulointerstitial damage known to follow recovery from folic acid-induced tubular necrosis and acute renal injury. We found that delivery of angiopoietin-1 by adenoviral vectors stabilized peritubular capillaries in folic acid nephropathy but this was accompanied by profibrotic and inflammatory effects. These results suggest that the use of endothelial growth factor therapy for kidney disease may have varying outcomes that depend on the disease model tested.


Asunto(s)
Angiopoyetina 1/efectos adversos , Fibrosis/inducido químicamente , Inflamación/inducido químicamente , Necrosis Tubular Aguda/tratamiento farmacológico , Adenoviridae/genética , Angiopoyetina 1/administración & dosificación , Angiopoyetina 1/uso terapéutico , Animales , Modelos Animales de Enfermedad , Ácido Fólico/efectos adversos , Vectores Genéticos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Necrosis Tubular Aguda/inducido químicamente , Necrosis Tubular Aguda/patología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Circulación Renal/efectos de los fármacos
5.
Proc Natl Acad Sci U S A ; 104(47): 18363-70, 2007 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-18000042

RESUMEN

VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Factores de Crecimiento Endotelial Vascular/biosíntesis , Envejecimiento/fisiología , Inhibidores de la Angiogénesis/inmunología , Animales , Anticuerpos/inmunología , Biomarcadores , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones SCID , Unión Proteica , Factores de Crecimiento Endotelial Vascular/sangre , Factores de Crecimiento Endotelial Vascular/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/patología
6.
J Vasc Res ; 44(4): 283-91, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17406120

RESUMEN

BACKGROUND: Infusion of exogenous vascular endothelial growth factor (VEGF) into adult brain at doses above 60 ng/day induces dramatic angiogenesis accompanied by vascular leak and inflammation. Blood vessels formed by this treatment are dilated and tortuous, exhibiting a pathological morphology. Pathological VEGF-induced angiogenesis is preceded by vascular leak and inflammation, which have been proposed to mediate subsequent angiogenesis. METHODS: To test this hypothesis, we infused VEGF into the brains of adult rats to induce pathological angiogenesis. Some of these rats were treated with dexamethasone, a potent anti-inflammatory glucocorticoid, to inhibit inflammation and edema. RESULTS: We demonstrate that inhibition of inflammation by treatment with dexamethasone significantly attenuated VEGF-induced pathological angiogenesis. To present converging evidence that inflammation may be important in this angiogenic process, we also demonstrate that mice genetically deficient in the inflammatory mediator intercellular adhesion molecule-1 have attenuated VEGF-induced angiogenesis. These same mice showed normal amounts of physiological angiogenesis in response to enriched environments, however, suggesting that a generalized reduction in vascular plasticity could not account for their poor angiogenic response to VEGF. CONCLUSIONS: Taken together, the data from these experiments suggest that the inflammation which occurs before or during VEGF-induced pathological brain angiogenesis plays a contributory role in the pathological angiogenic process.


Asunto(s)
Circulación Cerebrovascular/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Neovascularización Patológica/tratamiento farmacológico , Factores de Edad , Animales , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Noqueados , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/inmunología , Ratas , Ratas Sprague-Dawley , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/farmacología
7.
Proc Natl Acad Sci U S A ; 103(42): 15491-6, 2006 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-17030814

RESUMEN

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.


Asunto(s)
Angiopoyetina 2/metabolismo , Comunicación Autocrina , Células Endoteliales/fisiología , Estrés Oxidativo , Androstadienos/metabolismo , Angiopoyetina 2/genética , Animales , Apoptosis/fisiología , Células Cultivadas , Células Endoteliales/citología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Activación Transcripcional , Wortmanina
8.
Reproduction ; 132(4): 589-600, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17008470

RESUMEN

The intense angiogenesis characteristic of early corpus luteum development is dependent upon vascular endothelial growth factor (VEGF) as inhibitors of VEGF administered at the peri-ovulatory period suppress endothelial cell proliferation and progesterone secretion. We now report that administration of VEGF Trap, a soluble decoy receptor-based inhibitor, at the mid- or the late luteal phase in the marmoset results in a rapid decline in plasma progesterone. Since vascularisation of the corpus luteum is largely complete by the mid-luteal phase, it suggested that this functional luteolysis involved mechanisms other than inhibition of angiogenesis. A second experiment investigated the role of VEGF in maintaining the integrity of the luteal vasculature and hormone-producing cells. VEGF Trap was administered to marmosets in the mid-luteal phase and ovaries were obtained 1, 2, 4 or 8 days later for localisation of activated caspase-3 staining in the corpus luteum and compared with those obtained 2, 4 and 8 days after administration of control protein. The number of cells with activated caspase-3 staining was significantly increased after administration of VEGF Trap. Dual staining of activated caspase-3 with the endothelial cell marker CD31 showed that at 1 day post-treatment, more than 90% caspase-3-stained cells were vascular endothelium, prior to detection of an increasing incidence in death of hormone-producing cells on days 2 and 4. Staining with CD31 showed that the endothelial cell area was decreased after treatment. By 8 days after treatment, corpora lutea had regressed to varying degrees, while all control corpora lutea remained healthy. These results show that VEGF inhibition in the mid- or the late luteal phase induces functional luteolysis in the marmoset that is associated with premature and selective death of endothelial cells.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Cuerpo Lúteo/irrigación sanguínea , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/patología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Animales , Callithrix , Muerte Celular , Cuerpo Lúteo/citología , Cuerpo Lúteo/efectos de los fármacos , Células Endoteliales/patología , Femenino , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica/métodos , Fase Luteínica , Luteólisis , Modelos Animales , Progesterona/sangre , Receptores de Factores de Crecimiento , Factor A de Crecimiento Endotelial Vascular/metabolismo
9.
Microcirculation ; 13(6): 499-509, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16864416

RESUMEN

OBJECTIVE: To determine which elements of the angiogenic process are controlled by VEGF under physiological conditions. METHODS: VEGF Trap was administered at 10 mg x kg(-1) by subcutaneous injection twice weekly to mice, which were subject to one of two established angiogenic stimuli: (1) increasing blood flow by administration of prazosin (50 mg L(-1)); (2) muscle overload by extirpation of a synergist. Angiogenesis was determined by calculating capillary to fiber ratio (C:F), and proliferating cell nuclear antigen (PCNA) staining localized to capillaries or the interstitium used to measure cell proliferation. Characteristic ultrastructural changes were quantified using electron microscopy. RESULTS: Administration of VEGF Trap abolished the increases in C:F seen in both models, and prevented growth of luminal filopodia and large vacuole formation. Overload-induced proliferation associated with capillaries was reduced, along with endothelial cell number abluminal sprouts and basement membrane breakage. However, interstitial proliferation remained high, along with the increased capillary association of pericytes and fibroblasts. CONCLUSIONS: VEGF is required for both models of angiogenesis, although some morphological changes lie upstream, or are independent of, VEGF involvement. The abolition of angiogenesis in a model unaffected by NO inhibition shows that NO is not required for all VEGF-dependent angiogenesis in vivo.


Asunto(s)
Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , Pericitos/metabolismo , Prazosina/farmacología , Receptores de Factores de Crecimiento , Estrés Mecánico
11.
Nat Med ; 12(7): 793-800, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16799557

RESUMEN

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.


Asunto(s)
Eritropoyetina/fisiología , Hígado/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Hematócrito , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Modelos Animales , Policitemia/fisiopatología , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Vasos Retinianos/fisiología
12.
J Clin Endocrinol Metab ; 90(2): 1114-22, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15562010

RESUMEN

Follicular development is associated with intense angiogenesis and increased permeability of blood vessels under the control of locally produced angiogenic factors such as vascular endothelial growth factor (VEGF). The aim of the present study was to evaluate the effects of transient inhibition of VEGF on pituitary-ovarian function in the macaque. Animals were given a single, iv injection of a potent, receptor-based VEGF antagonist, the VEGF Trap. VEGF Trap was given at a dose of 4, 1, or 0.25 mg/kg in the midfollicular phase or at 1.0 mg/kg in the late follicular phase. Controls were treated with vehicle or a control protein, recombinant human Fc (1 mg/kg). Blood samples were collected once daily for 12 d after injection, and three times per week thereafter until normal ovulatory cycles had resumed. The VEGF Trap produced a rapid suppression of estradiol and inhibin B concentrations at all doses tested, followed by a marked and sustained increase in LH and FSH. Ovulation and formation of a functional corpus luteum, as evidenced by increased serum progesterone levels, failed to occur at the anticipated time. Normal ovarian activity resumed when plasma concentrations of unbound VEGF Trap fell below about 1 mg/liter. When treatment was initiated in the midfollicular phase, control macaques ovulated 7.2 +/- 0.4 d later, but ovulation was delayed in a dose-dependent manner by VEGF Trap, occurring 23 +/- 0.7, 30 +/- 1.4, and 43 +/- 0.8 d after injection of 0.25, 1, or 4 mg/kg, respectively. Thus, the VEGF Trap exerts a potent, dose-dependent, but reversible inhibitory effect on ovarian function.


Asunto(s)
Ovario/fisiología , Ovulación/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Estradiol/fisiología , Antagonistas de Estrógenos/farmacología , Femenino , Inhibinas/antagonistas & inhibidores , Inyecciones Intravenosas , Macaca , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Ovario/efectos de los fármacos , Receptores de Factores de Crecimiento , Proteínas Recombinantes de Fusión/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación
14.
Circulation ; 110(16): 2430-5, 2004 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-15477421

RESUMEN

BACKGROUND: The rate of reendothelialization is critical in neointima formation after arterial injury. Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, has been advocated for accelerating endothelial repair and preventing intimal hyperplasia after percutaneous coronary interventions. However, the precise mechanism of action of VEGF treatment and the physiologic role of endogenous VEGF after arterial injury are not well described. To better understand the role of VEGF in arterial repair, we overexpressed both VEGF and a soluble, chimeric VEGF receptor (VEGF-trap), which binds free VEGF with high affinity, in a mouse model of arterial injury. METHODS AND RESULTS: Four groups of C57BL/6 mice underwent denuding endothelial injury 1 day after systemic injection of recombinant adenovirus expressing (1) VEGF, (2) VEGF-trap, (3) VEGF plus VEGF-trap, or (4) control adenovirus. Circulating levels of adenovirus-encoded proteins were significantly elevated after gene transfer. VEGF overexpression accelerated reendothelialization and increased luminal endothelial cell proliferation 2 weeks after arterial injury (P<0.05), resulting in decreased neointima formation at 4 weeks compared with control (P<0.01). Cotreatment with VEGF-trap completely sequestered free VEGF and abrogated the beneficial effect of VEGF overexpression. Interestingly, sequestration of endogenous VEGF by VEGF-trap overexpression alone also led to delayed reendothelialization at 2 weeks (P<0.01) and increased neointima formation at 4 weeks (P<0.01). CONCLUSIONS: VEGF overexpression accelerated endothelial repair and inhibited neointima formation after arterial injury. Conversely, sequestration of exogenous and/or endogenous VEGF by VEGF-trap delayed reendothelialization and significantly increased neointima size. This demonstrates the therapeutic potential of VEGF but also emphasizes the important physiologic role of endogenous VEGF in vascular repair.


Asunto(s)
Endotelio Vascular/lesiones , Terapia Genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Cicatrización de Heridas/fisiología , Angioplastia/efectos adversos , Animales , División Celular , Células Endoteliales/patología , Endotelio Vascular/patología , Humanos , Hiperplasia , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Método Simple Ciego , Túnica Íntima/patología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética
15.
J Am Coll Cardiol ; 44(4): 897-903, 2004 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-15312878

RESUMEN

OBJECTIVES: The aim of this research was to test the effects of vascular endothelial growth factor (VEGF)/angiopoietin-1 (Ang-1) on adult hypoperfused tissues. BACKGROUND: Angiopoietin-1 and VEGF act separately and synergistically in vascular development during embryogenesis. However, little is known regarding their relative roles in collateral development after chronic arterial obstruction and tissue ischemia in the adult. METHODS: Central and caudal ear arteries of 32 rabbits were ligated to induce ischemia. At two months, when flow was about 65% of pre-ligation values, we injected intradermally 10(9) plaque-forming unit adenovirus with the following transgenes: Ang-1, VEGF, or a combination of both. Ear perfusion was followed up for four weeks, and vessel leakage was assessed by Evens Blue test. RESULTS: Before injection, flow was 65% of baseline, and endogenous VEGF levels in ischemic tissue were increased. Adenovirus-encoding VEGF gene (Ad.VEGF) at one week caused a visible inflammatory response associated with a 24% flow increase (p = 0.018). Adenovirus-encoding Ang-1 gene (Ad.Ang-1) increased flow 22% (p = 0.004) with no visible inflammation; Ad.VEGF caused three times as much vessel leakage as Ad.Ang-1 (142.5 +/- 38 vs. 49.5 +/- 9.8 ng Evens Blue/mg tissue; p < 0.001). However, at four weeks, compared with baseline, VEGF decreased flow 18% (p = 0.004), whereas Ang-1 increased tissue perfusion 26% (p < 0.001). This effect was abolished when Ad.Ang-1 was injected with soluble VEGF receptor [Ad.Flt(1-3)-Fc], which blocks VEGF-dependent signaling. Exogenous Ang-1 did not increase perfusion in a normally perfused ear, in which endogenous VEGF is not expressed. CONCLUSIONS: Exogenous Ang-1 enhances perfusion in hypoperfused tissues only in the presence of increased levels of endogenous VEGF. Overexpression of VEGF, however, after causing an inflammatory response, does not improve collateral blood flow.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Angiopoyetina 1/farmacología , Endotelio Vascular/efectos de los fármacos , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Adenoviridae/genética , Animales , Oído Externo/irrigación sanguínea , Expresión Génica , Terapia Genética , Isquemia/fisiopatología , Masculino , Neovascularización Fisiológica/genética , Conejos , Distribución Aleatoria , Flujo Sanguíneo Regional
16.
Genes Dev ; 18(9): 1060-71, 2004 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-15132996

RESUMEN

Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. In this report, we demonstrate that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying for the first time a nuclear signaling pathway through which Ang-1 modulates gene expression. We use microarray analysis to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. We use RNA interference to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. Our data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability.


Asunto(s)
Angiopoyetina 1/farmacología , Proteínas de Unión al ADN/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Proteínas Serina-Treonina Quinasas , Factores de Transcripción/fisiología , Angiopoyetina 1/fisiología , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Bovinos , Células Cultivadas , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Endotelio Vascular/citología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/farmacología , Transducción de Señal , Factores de Transcripción/genética
17.
Proc Natl Acad Sci U S A ; 100(13): 7785-90, 2003 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-12805568

RESUMEN

Vascular endothelial growth factor (VEGF) is a critical promoter of blood vessel growth during embryonic development and tumorigenesis. To date, studies of VEGF antagonists have primarily focused on halting progression in models of minimal residual cancer. Consistent with this focus, recent clinical trials suggest that blockade of VEGF may impede cancer progression, presumably by preventing neoangiogenesis. However, VEGF is also a key mediator of endothelial-vascular mural cell interactions, a role that may contribute to the integrity of mature vessels in advanced tumors. Here, we report that high-affinity blockade of VEGF, using the recently described VEGF-Trap, abolishes mature, preexisting vasculature in established xenografts. Eradication of vasculature is followed by marked tumor regression, including regression of lung micrometastases. Thus, the contribution of relatively low levels of VEGF to vessel integrity may be critical to maintenance of even very small tumor masses. Potent blockade of VEGF may provide a new therapeutic option for patients with bulky, metastatic cancers.


Asunto(s)
Antineoplásicos/farmacología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/antagonistas & inhibidores , Linfocinas/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Actinas/metabolismo , Animales , Apoptosis , Plaquetas/metabolismo , Progresión de la Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Lectinas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Microscopía Confocal , Músculo Liso/metabolismo , Necrosis , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neovascularización Patológica , Perfusión , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Receptores de Factores de Crecimiento Endotelial Vascular , Factores de Tiempo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
18.
J Cell Physiol ; 195(2): 241-8, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12652651

RESUMEN

Vascular endothelial growth factor (VEGF) plays a central role in the development of retinal neovascularization and diabetic macular edema. There is also evidence suggesting that VEGF is an important stimulator for choroidal neovascularization. In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF-TRAP(R1R2), in models for these disease processes. VEGF-TRAP(R1R2) is a fusion protein, which combines ligand binding elements taken from the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous injections or a single intravitreous injection of VEGF-TRAP(R1R2) strongly suppressed choroidal neovascularization in mice with laser-induced rupture of Bruch's membrane. Subcutaneous injection of VEGF-TRAP(R1R2) also significantly inhibited subretinal neovascularization in transgenic mice that express VEGF in photoreceptors. In two models of VEGF-induced breakdown of the blood-retinal barrier (BRB), one in which recombinant VEGF is injected into the vitreous cavity and one in which VEGF expression is induced in the retina in transgenic mice, VEGF-TRAP(R1R2) significantly reduced breakdown of the BRB. These data confirm that VEGF is a critical stimulus for the development of choroidal neovascularization and indicate that VEGF-TRAP(R1R2) may provide a new agent for consideration for treatment of patients with choroidal neovascularization and diabetic macular edema.


Asunto(s)
Enfermedades de la Coroides/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Linfocinas/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Animales , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/fisiología , Coroides/efectos de los fármacos , Coroides/metabolismo , Coroides/fisiopatología , Enfermedades de la Coroides/metabolismo , Enfermedades de la Coroides/fisiopatología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/fisiopatología , Receptores de Factores de Crecimiento/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Retina/efectos de los fármacos , Retina/metabolismo , Retina/fisiopatología , Arteria Retiniana/efectos de los fármacos , Arteria Retiniana/patología , Arteria Retiniana/fisiopatología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
19.
Blood ; 102(1): 161-8, 2003 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-12649136

RESUMEN

Gene therapy approaches involving vascular endothelial growth factor (VEGF) to promote therapeutic angiogenesis are under consideration for conditions ranging from ischemic heart disease to nonhealing skin ulcers. Here we make the surprising observation that the transgenic delivery of VEGF to the skin results in a profound inflammatory skin condition with many of the cellular and molecular features of psoriasis, including the characteristic vascular changes, epidermal alterations, and inflammatory infiltrates. Even longstanding psoriatic disease remains dependent on the transgenic VEGF in this model because it can be effectively reversed by the addition of VEGF Trap, a potent VEGF antagonist. Previous attempts to faithfully replicate the psoriatic phenotype through the transgenic delivery of epidermal keratinocyte growth factors or inflammatory mediators generated phenotypes with only partial resemblance to human psoriasis, leaving unanswered questions about the etiology of this disease. The ability of transgenic VEGF to induce a psoriasiform phenotype suggests a new etiology and treatment approach for this disease and further substantiates emerging concerns about possible proinflammatory adverse effects that might be associated with therapeutic attempts to deliver VEGF.


Asunto(s)
Factores de Crecimiento Endotelial/administración & dosificación , Factores de Crecimiento Endotelial/efectos adversos , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular/efectos adversos , Linfocinas/administración & dosificación , Linfocinas/efectos adversos , Psoriasis/inducido químicamente , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Modelos Animales de Enfermedad , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Inmunohistoquímica , Inflamación/inducido químicamente , Inflamación/etiología , Ratones , Ratones Transgénicos , Psoriasis/etiología , Receptores de Factores de Crecimiento , Proteínas Recombinantes de Fusión/farmacología , Piel/efectos de los fármacos , Piel/lesiones , Piel/patología , Transgenes , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
20.
Nat Struct Biol ; 10(1): 38-44, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12469114

RESUMEN

Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.


Asunto(s)
Angiopoyetinas/química , Angiopoyetinas/metabolismo , Receptor TIE-2/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Angiopoyetina 1/química , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 2/química , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Angiopoyetinas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Dimerización , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Modelos Moleculares , Fosforilación , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
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