RESUMEN

The aim of this work was to determine endometrial mRNA expression and uterine protein localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 during the estrous cycle and peri-implantation period in sows. Uterine tissues were collected from pregnant sows on days 12, 14, 16, and 18 after artificial insemination and from non-pregnant animals on days 2 and 12 of the estrous cycle (day 0 = day of estrus). Using immunohistochemistry, a positive signal for VEGF and its receptor VEGFR2 was found in uterine luminal epithelial cells, endometrial glands, stroma, blood vessels, and myometrium. A VEGFR1 signal was only found in endometrial and myometrial blood vessels and stroma. By day 18 of gestation, the mRNA expression levels of VEGF, VEGFR1, and VEGFR2 were higher than those observed on days 2 and 12 of the estrous cycle and on days 12, 14, and 16 of gestation. Then, a primary culture of sow endometrial epithelial cells was established to define the potential of the selective inhibition of VEGFR2 after treatment with inhibitor SU5416 and determine its effects on the expression pattern of the VEGF system. The endometrial epithelial cells treated with SU5416 showed a dose-dependent decrease in VEGFR1 and VEGFR2 mRNA expression. The present study provides additional evidence on the importance of the VEGF system during peri-implantation, as well as on the specific inhibitory activity of SU5416 in epithelial cells, which, as demonstrated, express the protein and mRNA of VEGF and its receptors VEGFR1 and VEGFR2.

Asunto(s)

Inhibidores de la Angiogénesis , Factor A de Crecimiento Endotelial Vascular , Animales , Porcinos , Femenino , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/metabolismo , Útero/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension-related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein-kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1-5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.

Asunto(s)

Cardiomegalia/metabolismo , Hipoxia/metabolismo , Cininas/metabolismo , Miocardio/metabolismo , Neovascularización Fisiológica , Apnea Obstructiva del Sueño/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Capilares/metabolismo , Capilares/fisiología , Cardiomegalia/complicaciones , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiología , Hipoxia/complicaciones , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Bradiquinina/genética , Receptores de Bradiquinina/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Apnea Obstructiva del Sueño/complicaciones , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

Prenatal exposure to glucocorticoids (GC) is a central topic of interest in medicine since GCs are essential for the maturation of fetal organs and intrauterine growth. Synthetic glucocorticoids, which are used in obstetric practice, exert beneficial effects on the fetus, but have also been reported to lead to intrauterine growth retardation (IUGR). In this study, a model of growth restriction in mice was established through maternal administration of dexamethasone during late gestation. We hypothesised that GC overexposure may adversely affect placental angiogenesis and fetal and placental growth. Female BALB/c mice were randomly assigned to control or dexamethasone treatment, either left to give birth or euthanised on days 15, 16, 17 and 18 of gestation followed by collection of maternal and fetal tissue. The IUGR rate increased to 100% in the dexamethasone group (8 mg/kg body weight on gestational days 14 and 15) and pups had clinical features of symmetrical IUGR at birth. Dexamethasone administration significantly decreased maternal body weight gain and serum corticosterone levels. Moreover, prenatal dexamethasone treatment not only induced fetal growth retardation but also decreased placental weight. In IUGR placentas, VEGFA protein levels and mRNA expression of VEGF receptors were reduced and NOS activity was lower. Maternal dexamethasone administration also reduced placental expression of the GC receptor, αGR. We demonstrated that maternal dexamethasone administration causes fetal and placental growth restriction. Furthermore, we propose that the growth retardation induced by prenatal GC overexposure may be caused, at least partially, by an altered placental angiogenic profile.

Asunto(s)

Dexametasona , Retardo del Crecimiento Fetal/metabolismo , Placenta/metabolismo , Placentación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Retardo del Crecimiento Fetal/inducido químicamente , Retardo del Crecimiento Fetal/fisiopatología , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Placenta/fisiopatología , Embarazo , Receptores de Glucocorticoides/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.

Asunto(s)

Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/fisiopatología , Quistes Ováricos/genética , Quistes Ováricos/fisiopatología , Folículo Ovárico/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Estudios de Casos y Controles , Bovinos/fisiología , Enfermedades de los Bovinos/metabolismo , Femenino , Quiste Folicular/genética , Quiste Folicular/metabolismo , Quiste Folicular/fisiopatología , Expresión Génica , Quistes Ováricos/metabolismo , Ovario/metabolismo , Ovario/patología , Ovulación/genética , Ovulación/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: CIGB-247, a VSSP-adjuvanted VEGF-based vaccine, was evaluated in a phase I clinical trial in patients with advanced solid tumors (CENTAURO). Vaccination with the maximum dose of antigen showed an excellent safety profile, exhibited the highest immunogenicity and was the only one showing a reduction on platelet VEGF bioavailability. However, this antigen dose level did not achieve a complete seroconversion rate in vaccinated patients. These clinical results led us to the question whether a "reserve" of untapped immune response potential against VEGF could exist in cancer patients. To address this matter, CENTAURO-2 clinical trial was conducted where antigen and VSSP dose scale up were studied, and also incorporated the exploration of aluminum phosphate as adjuvant. These changes were made with the aim to increase immune response against VEGF. RESULTS: The present study reports the characterization of the humoral response elicited by CIGB-247 from the combining of different antigen doses and adjuvants. Cancer patients were immunologically monitored for approximately 1 year. Vaccination with different CIGB-247 formulations exhibited a very positive safety profile. Cancer patients developed IgM, IgG or IgA antibodies specific to VEGF. Elicited polyclonal antibodies had the ability to block the interaction between VEGF and its receptors, VEGFR1 and VEGFR2. The highest humoral response was detected in patients immunized with 800 µg of antigen + 200 µg of VSSP. Off-protocol long-term vaccination did not produce negative changes in humoral response. CONCLUSIONS: Vaccination with a human VEGF variant molecule as antigen in combination with VSSP or aluminum phosphate is immunogenic. The results of this study could contribute to the investigation of this vaccine therapy in an adequately powered efficacy trial. TRIAL REGISTRATION: Trial registration number: RPCEC00000155. Cuban Public Clinical Trial Registry. Date of registration: June 06, 2013. Available from: http://registroclinico.sld.cu/ .

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Cáncer/inmunología , Inmunidad Humoral/inmunología , Inmunoterapia Activa , Neoplasias/inmunología , Neoplasias/terapia , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Chlorocebus aethiops , Femenino , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/sangre , Conejos , Receptores de Factores de Crecimiento Endotelial Vascular/sangre , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.

Asunto(s)

Inhibidores de la Angiogénesis , Células Endoteliales/metabolismo , Biblioteca de Péptidos , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Células Endoteliales/citología , Humanos , Ratones , Dominios Proteicos , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacología

RESUMEN

PURPOSE: To compare ileal anastomoses in the immediate postoperative healing period after meloxicam use. METHODS: Forty two male Wistar rats were randomly divided into two groups of 21, COX and control group. To COX meloxicam in combination with morphine was given in 3 days period. Control group received only morphine during the same period. Each group was divided into three sub-groups of 7, which were euthanized at 5, 10, and 21 days postoperatively. Comparison was based in histological evaluation of collagen type I and III using sirius red, immunohistochemical through vascular endothelial growth factor and matrix metalloproteinase-9. RESULTS: Healing process in scheduled periods did not show significant differences (p>0.05) between the COX and control groups during any of the periods. CONCLUSION: The use of meloxicam in the postoperative period following ileal anastomosis did not affect healing.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Íleon/cirugía , Tiazinas/farmacología , Tiazoles/farmacología , Cicatrización de Heridas/efectos de los fármacos , Anastomosis Quirúrgica , Animales , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Íleon/irrigación sanguínea , Masculino , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Meloxicam , Modelos Animales , Neovascularización Fisiológica/efectos de los fármacos , Periodo Posoperatorio , Distribución Aleatoria , Ratas Wistar , Receptores de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Tiempo

RESUMEN

ABSTRACT PURPOSE : To compare ileal anastomoses in the immediate postoperative healing period after meloxicam use. METHODS: Forty two male Wistar rats were randomly divided into two groups of 21, COX and control group. To COX meloxicam in combination with morphine was given in 3 days period. Control group received only morphine during the same period. Each group was divided into three sub-groups of 7, which were euthanized at 5, 10, and 21 days postoperatively. Comparison was based in histological evaluation of collagen type I and III using sirius red, immunohistochemical through vascular endothelial growth factor and matrix metalloproteinase-9. RESULTS: Healing process in scheduled periods did not show significant differences (p>0.05) between the COX and control groups during any of the periods. CONCLUSION: The use of meloxicam in the postoperative period following ileal anastomosis did not affect healing.

Asunto(s)

Animales , Masculino , Tiazinas/farmacología , Tiazoles/farmacología , Cicatrización de Heridas/efectos de los fármacos , Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Íleon/cirugía , Periodo Posoperatorio , Factores de Tiempo , Anastomosis Quirúrgica , Distribución Aleatoria , Ratas Wistar , Neovascularización Fisiológica/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Modelos Animales , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Íleon/irrigación sanguínea

RESUMEN

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Asunto(s)

Hernias Diafragmáticas Congénitas/metabolismo , Pulmón/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Hernias Diafragmáticas Congénitas/inducido químicamente , Hernias Diafragmáticas Congénitas/embriología , Pulmón/embriología , Éteres Fenílicos , Embarazo , Ratas Sprague-Dawley

RESUMEN

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Asunto(s)

Animales , Femenino , Embarazo , Hernias Diafragmáticas Congénitas/metabolismo , Pulmón/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Modelos Animales de Enfermedad , Hernias Diafragmáticas Congénitas/inducido químicamente , Hernias Diafragmáticas Congénitas/embriología , Pulmón/embriología , Éteres Fenílicos , Ratas Sprague-Dawley

RESUMEN

We have previously reported the isolation of a novel single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF), from a phage-displayed human antibody repertoire. This scFv, denominated 2H1, was shown to block the binding of VEGF to its receptor but exhibited a moderate binding affinity. Here, we describe the affinity maturation of the 2H1 scFv. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of the parental scFv as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was designed and constructed by combining recurrent replacements found among selected variants in a single molecule, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant obtained here, L3H6, showed an overall affinity improvement of 18-fold over the parental scFv and exhibited an enhanced potency to block the binding of VEGF to its receptor. Using phage display and extensive mutagenesis of VEGF, we determined the fine specificity of L3H6. This functional mapping revealed a novel neutralizing epitope on human VEGF defined by the residues Y25, T71, E72, N100, K101, E103 and R105. The conformational epitope recognized by L3H6 was recapitulated by grafting human VEGF residues into the mouse molecule, providing further confirmation of the nature of the identified epitope.

Asunto(s)

Mapeo Epitopo/métodos , Epítopos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Anticuerpos de Cadena Única/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Epítopos/genética , Epítopos/inmunología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Unión Proteica , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología

RESUMEN

The weight of 6- and 7-d-old chicken embryos and their chorioallantoic membrane (CAM), vascular density, protein immunoexpression, and mRNA of hypoxia inducible factor 2 (HIF-2α), vascular endothelial growth factor (VEGF-A), and its type receptor 2 (FLK-1) in the CAM were compared in subjects incubated at 2 different altitudes: 355 and 1,378 m above sea level (masl) in Colombia. A difference was not found when comparing the weight of CAM and embryos incubated at 355 masl with those incubated at 1,378 masl on d 6 and 7 of incubation (P > 0.05). Higher vascular density in CAM (P < 0.05), percentage of cells immunoexpressing HIF-2α and FLK-1 in the CAM (P < 0.05), and relative expression of HIF-2α and FLK-1 mRNA in the CAM (P < 0.05 and P < 0.01, respectively) were encountered in embryos incubated at 1,378 masl compared with those incubated at 355 masl, but only on d 6. Percentages of cells immunoexpressing VEGF-A and relative expression of VEGF-A mRNA in the CAM values were not different when considering altitude and age (P > 0.05). Relative hypoxia (1,378 masl) appears to affect HIF-2α and FLK-1 expression in CAM of 6-d-old chicken embryos, and this change in expression results in increased vascular development in CAM on this day. At hatching, chickens between treatments differed in their BW (P < 0.0001). The lack of differences in findings on d 7 could be due to morphological, physiological, and molecular events occurring at this time.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Embrión de Pollo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipoxia/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Membrana Corioalantoides/irrigación sanguínea , Oxígeno/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

CIGB-247 is a novel cancer therapeutic vaccine that uses a mutated form of human VEGF as antigen. Being metastatic disease the most dramatic factor of tumor biology affecting patient survival and cure, preclinical evaluation of the impact of CIGB-247 vaccination on experimental metastasis mouse models is highly relevant, and constitutes the focus of this work. CIGB-247 was administered in a weekly schedule known to effectively reduce primary tumor growth. The vaccine was tested in experimental and spontaneous metastasis models of colon (CT26), lung (3LL-D122) and breast (F3II) carcinomas growing in C57Bl/6 or BALB/c mice. Primary tumor growth parameters, metastatic counts, and/or animal survival were recorded. Histology and specific humoral and cellular responses to the vaccine were evaluated. As compared to control groups, CIGB-247 vaccination significantly reduced the number and size of metastatic tumor foci in lungs after intravenous inoculation of CT26 and 3LL-D122 tumor cells. Spontaneous lung dissemination from 3LL-D122 and F3II breast tumor cells implanted in the footpad, or subcutaneously, was also reduced by immunization with CIGB-247. The vaccine elicited in both mouse strains antibodies specific for human and murine VEGF that effectively blocked the interaction of VEGF with VEGF receptor 2. Differing from other experimental reports that describe the use of VEGF for active tumor immunotherapy, CIGB-247 elicited a specific cellular response, measured both by a DTH increment and the induction of spleen cells cytotoxic to syngeneic tumor cells producing murine VEGF. In summary our results reinforce the potential of CIGB-247 vaccination to reduce both tumor growth and the number and size of tumor metastasis in lungs, the latter both after direct inoculations of cells in the blood stream, or as part of primary tumor progression in immunocompetent mice.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Inmunoterapia Activa , Neoplasias Pulmonares/terapia , Metástasis de la Neoplasia/prevención & control , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos Antineoplásicos/sangre , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Femenino , Humanos , Inmunidad Humoral , Neoplasias Pulmonares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes/inmunología

RESUMEN

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERß, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERß agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.

Asunto(s)

Astrocitoma/fisiopatología , Neoplasias Encefálicas/fisiopatología , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/fisiología , Ciclina D1/genética , Ciclina D1/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrógeno/genética , Humanos , Coactivador 1 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo

Asunto(s)

Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.

Asunto(s)

Hernia Diafragmática/metabolismo , Hernias Diafragmáticas Congénitas , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Esteroides/farmacología , Estenosis Traqueal/fisiopatología , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Hernia Diafragmática/inducido químicamente , Hernia Diafragmática/embriología , Inmunohistoquímica , Exposición Materna , Éteres Fenílicos , Embarazo , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Estenosis Traqueal/metabolismo

RESUMEN

OBJECTIVE: To evaluate whether maternal malnutrition during lactation programs ovarian folliculogenesis and the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and its receptors KDR, Flt-1, and FGFR. DESIGN: Experimental study. SETTING: University-based research laboratory. ANIMAL(S): Adult female rats from a urogenital research laboratory. INTERVENTION(S): Six rat dams randomly assigned to the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein; and a protein-energy-restricted group (PER), with free access to an isoenergy and protein-restricted diet containing 8% protein. After weaning, the female pups had free access to the standard laboratory diet until 90 days of age, when they were sacrificed at the proestrum stage. MAIN OUTCOME MEASURE(S): Quantification of ovarian follicles, vessels, and expression of growth factors and their receptors. RESULT(S): Maternal malnutrition during lactation caused a significant reduction in the number of primordial (C = 6.60 +/- 0.24, PER = 5.20 +/- 0.20), primary (C = 5.80 +/- 0.66, PER = 4.00 +/- 0.31), and Graafian follicles/section (C = 2.18 +/- 0.29, PER = 1.08 +/- 0.37), in KDR (C = 0.22 +/- 0.04, PER = 0.09 +/- 0.01), Flt-1 (C = 0.28 +/- 0.05, PER = 0.12 +/- 0.02), and FGFR mRNA expression (C = 0.34 +/- 0.05, PER = 0.13 +/- 0.05) and in the vessel density of follicles (C = 17.26 +/- 2.30, PER = 9.96 +/- 0.97). CONCLUSION(S): Maternal malnutrition during lactation programs the follicular development by a reduction of VEGF and FGF mRNA receptors expression, probably from a direct action on the follicular development or a reduction in vasculature resulting in a decreased delivery of folliculotrophic substances in PER animals.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/fisiología , Lactancia/fisiología , Desnutrición/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Neovascularización Fisiológica/fisiología , Folículo Ovárico/fisiología , Ovario/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Dieta con Restricción de Proteínas , Femenino , Ratas , Ratas Wistar

RESUMEN

The vascular endothelial growth factors are key mediators of angiogenesis and are also related to several physiological processes such as monocyte chemotaxis, dendritic cell development, hematopoietic stem cell survival, and many others. PlGF, VEGF, VEGFB, VEGFC and VEGFD were identified as members of the vascular endothelial growth factor family. They act by differential activation of three receptors: Flt-1, KDR and Flt-4. PlGF and VEGFB only activate Flt-1. VEGF activates both Flt-1 and KDR. VEGFC and VEGFD activate KDR and Flt-4. The available three dimensional structures of VEGF and PlGF, in complex with the domain-2 of Flt-1, show that both proteins bind in a very similar way to Flt-1 receptor. Here we construct the three dimensional model of the domain-2 of KDR receptor using the same domain of Flt-1 as template. We also construct the model complexes VEGF/KDR, VEGFB/Flt-1, VEGFB/KDR and PlGF/KDR. Molecular dynamics simulations with explicit solvent are carried out on eleven molecular systems: unbound VEGF, VEGF/Flt-1(D2), VEGF/KDR(D2), unbound PlGF, PlGF/Flt-1(D2), PlGF/KDR(D2), unbound VEGFB, VEGFB/Flt-1(D2), VEGFB/KDR(D2), unbound Flt-1(D2) and unbound KDR(D2). We analyze protein-protein interactions, shape complementarity, charge complementarity and hydrogen bonds. As a coarse estimation of the desolvation penalties, we assume a correlation to the number of hydrogen bonds with solvent molecules that are lost upon complex formation. The results herein are consistent with the experimental selectivity profile (VEGF being able to activate both Flt-1 and KDR receptors while VEGFB and PlGF being only able to activate Flt-1), and provide a collection of evidences sustaining the complementarity of polar interactions as the main responsible for protein recognition and selectivity.

Asunto(s)

Receptores de Factores de Crecimiento Endotelial Vascular/química , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/química , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/química , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Fetal and neonatal morbidity and mortality is high in severe pre-eclampsia compared with mild pre-eclampsia and normotensive pregnancies. Causes for these fetal disturbances had been associated with iatrogenic prematurity and reduction in placental blood flow. Actual evidences suggest that in severe (early-onset) pre-eclampsia a reduction in placental angiogenesis could be a mechanism responsible for the reduced placental blood flow, while in mild (late-onset) pre-eclampsia normal placental blood flow could result from either no alteration or increased placental angiogenesis, or reduced vessel resistance. Since adenosine is involved in endothelium proliferation and angiogenesis, and umbilical and maternal blood level of this nucleoside is elevated in pre-eclampsia compared with normal pregnancies, it is feasible that placental angiogenesis in mild and/or severe pre-eclampsia involves adenosine-dependent cell signaling mechanisms. There are not reports regarding adenosine role in placental angiogenesis neither in normal nor in pathological pregnancies. However, it is well established that adenosine stimulates adenosine receptors triggering expression of angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF stimulates VEGF receptors type 1 and 2, activating signaling cascades that involve increased synthesis of endothelial-derived nitric oxide (NO). On the other hand, the soluble VEGF receptor type 1 (sFlt-1), whose plasma concentration is increased in severe compared with mild pre-eclampsia, reduces angiogenesis, spotting sFlt-1 as a factor that could potentially be involved in this phenomenon. This review focuses on the available evidence regarding a potential differential mechanism of placental angiogenesis in mild compared with severe pre-eclampsia, and analyzes the potential role of adenosine/VEGF/VEGF receptors/NO signaling cascade in this phenomenon.

Asunto(s)

Neovascularización Fisiológica/fisiología , Placenta/irrigación sanguínea , Preeclampsia/fisiopatología , Transducción de Señal , Adenosina/metabolismo , Endotelio Vascular/metabolismo , Femenino , Humanos , Placenta/metabolismo , Preeclampsia/metabolismo , Embarazo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The aim of the present study was to evaluate the effect of vascular endothelial growth factor (VEGF) on the survival and growth of goat preantral follicles after in vitro culture and to verify the expression of VEGF receptor (VEGFR)-2 in goat ovaries. Ovarian fragments were cultured for 1 or 7 days in minimal essential medium (MEM) with different concentrations of VEGF (1, 10, 50, 100 or 200 ng mL(-1)). Non-cultured (fresh control) and cultured tissues were processed for histological and ultrastructural studies. The results showed that 200 ng mL(-1) VEGF resulted in a similar percentage of normal preantral follicles after 1 and 7 days of culture compared with control. Compared with basic culture medium alone, an increase in follicular and oocyte diameters was observed in the presence of 10 ng mL(-1) VEGF after 7 days culture. Ultrastructural analysis confirmed follicular integrity after 7 days culture in the presence of 200 ng mL(-1) VEGF. Immunohistochemical studies demonstrated the expression of VEGFR-2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. In conclusion, the present study has shown that VEGF maintains follicular ultrastructural integrity and promotes follicular growth. In addition, VEGFR-2 is expressed in oocytes of caprine ovarian follicles at all developmental stages and in granulosa cells of developing follicles.

Asunto(s)

Cabras/metabolismo , Folículo Ovárico/efectos de los fármacos , Ovario/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Fase Folicular/fisiología , Células de la Granulosa/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/ultraestructura , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo