RESUMEN
OBJECTIVES: To evaluate the effect on endocrine and metabolic parameters of a new combined oral contraceptive (COC) containing estetrol (E4) and drospirenone (DRSP). STUDY DESIGN: Randomized, open-label, controlled, 3-arm, parallel study. Healthy subjects received either E4 15 mg/DRSP 3 mg (E4/DRSP) (n = 38), or ethinylestradiol (EE) 30 µg/levonorgestrel (LNG) 150 µg (n = 29), or EE 20 µg/DRSP 3 mg (n = 31) for 6 treatment cycles. Median percentage change from baseline to cycle 3 and to cycle 6 were evaluated for endocrine parameters, liver proteins, lipid profile, and carbohydrate metabolism. RESULTS: At cycle 6, E4/DRSP treatment had less effect on gonadotropins (follicle stimulating hormone [FSH] +30.5%, luteinizing hormone [LH] -7.5%) compared to EE/LNG (FSH -84.0%, LH -92.0%) and EE/DRSP (FSH -64.0%, LH -90.0%). With E4/DRSP increases in total cortisol (+26.0%) and cortisol binding globulin ([CBG] (+40.0%) were less compared to EE/LNG (cortisol +109.0%, CBG +152.0%) and EE/DRSP (cortisol +107.0%, CBG +140.0%). Liver proteins, except CRP, increased, but the effect was less pronounced with E4/DRSP for angiotensinogen (+75.0%) compared to EE/LNG (+170.0%) and EE/DRSP (+206.5%) and for sex hormone binding globulin ([SHBG] +55.0%), compared to EE/LNG (+74.0%) and EE/DRSP (+251.0%). E4/DRSP had minimal impact on lipid parameters; the largest effect was observed for triglycerides (+24.0%), which was less compared to EE/LNG (+28.0%) and EE/DRSP (+65.5%). E4/DRSP had no effect on carbohydrate metabolism. CONCLUSIONS: E4/DRSP treatment has limited effects on endocrine and metabolic parameters. The effects on gonadotropins, cortisol, CBG, angiotensinogen, SHBG and triglycerides were less pronounced compared to EE-containing products. IMPLICATIONS STATEMENT: Combining E4 15 mg with DRSP 3 mg resulted in a COC with a different metabolic profile in comparison to EE-containing products. The clinical relevance of these findings needs to be further assessed, using clinical endpoints to establish the safety profile of this new COC.
Asunto(s)
Estetrol , Androstenos , Anticonceptivos Orales Combinados , Estrógenos , Etinilestradiol , Femenino , Humanos , Levonorgestrel , Globulina de Unión a Hormona SexualRESUMEN
OBJECTIVE: To assess the effect on hemostasis parameters of a new combined oral contraceptive (COC). STUDY DESIGN: In this randomized, single centre, open-label, exploratory study, healthy women received either 15â¯mg estetrol/3 mg drospirenone (E4/DRSP) (nâ¯=â¯39), 30â¯mcg ethinylestradiol/150â¯mcg levonorgestrel (EE/LNG) (nâ¯=â¯30), or 20â¯mcg ethinylestradiol/3 mg drospirenone (EE/DRSP) (nâ¯=â¯32) for six 28-day cycles. Blood was collected at baseline, cycle 3, and cycle 6. Median change from baseline was evaluated for procoagulant, anticoagulant, and fibrinolytic parameters, and for sex hormone-binding globulin (SHBG). RESULTS: Median change of endogenous thrombin potential (ETP) based activated protein C sensitivity resistance (APCr) at cycle 6 was +30% for E4/DRSP, +165% for EE/LNG (p-value <0.05 vs E4/DRSP), and +219% for EE/DRSP (p-value <0.05 vs E4/DRSP). Changes to prothrombin fragment 1â¯+â¯2 and SHBG for E4/DRSP, EE/LNG, and EE/DRSP were +23%, +71%, and +64% (p-value <0.05 vs E4/DRSP); and +55%, +74% and +251% (p-value <0.05 vs E4/DRSP), respectively. At cycle 6, changes to other hemostasis parameters for E4/DRSP were similar or smaller than for EE/LNG or EE/DRSP. CONCLUSIONS: In this study, changes in hemostasis parameters after treatment with 6 cycles of E4/DRSP were smaller or similar to those observed for EE/LNG. Similar, but more pronounced changes were also observed versus EE/DRSP, which supports the hypothesis that the effect of COCs on hemostasis parameters is mainly mediated by the estrogenic component. Further studies are needed to provide more insight into the venous thromboembolic risk of E4/DRSP. IMPLICATIONS STATEMENT: This study reports that the effects on hemostasis parameters of a COC containing 15â¯mg E4/3 mg DRSP are less or similar to those for EE/LNG or EE/DRSP. It also demonstrates that the choice of estrogen modulates the effects of COCs on hemostasis parameters.
Asunto(s)
Resistencia a la Proteína C Activada/inducido químicamente , Androstenos/farmacología , Anticonceptivos Orales Combinados/farmacología , Estetrol/farmacología , Estrógenos/farmacología , Etinilestradiol/farmacología , Hemostasis/efectos de los fármacos , Adolescente , Adulto , Androstenos/administración & dosificación , Androstenos/efectos adversos , Anticonceptivos Orales Combinados/administración & dosificación , Anticonceptivos Orales Combinados/efectos adversos , Estetrol/administración & dosificación , Estetrol/efectos adversos , Estrógenos/administración & dosificación , Estrógenos/efectos adversos , Etinilestradiol/administración & dosificación , Etinilestradiol/efectos adversos , Femenino , Humanos , Ciclo Menstrual/efectos de los fármacos , Persona de Mediana Edad , Globulina de Unión a Hormona Sexual/efectos de los fármacos , Adulto JovenRESUMEN
OBJECTIVES: This study evaluated acceptability, user satisfaction, body weight control and general well-being of estetrol (E4) combined with either drospirenone (DRSP) or levonorgestrel (LNG). METHODS: In this open-label, multi-centre, dose-finding, 6-cycle study, 396 healthy women of reproductive age were randomised into five treatment groups in a 24/4-day regimen: 15 mg or 20 mg E4 combined with either 3 mg DRSP or 150 µg LNG, and as reference estradiol valerate (E2V) combined with dienogest (DNG). Data on acceptability, user well-being, satisfaction and body weight were collected. RESULTS: The number of completers was the highest in the 15 mg E4/DRSP group (91.1%), and the lowest for 20 mg E4/LNG (70.1%). The largest proportion of treatment satisfaction was reported for 15 mg E4/DRSP (73.1%), and the lowest for 15 mg E4/LNG (50.6%). The number of women willing to continue with the assigned study treatment was the highest in the 15 mg E4/DRSP group (82.1%) and the lowest for 20 mg E4/LNG (58.3%). Well-being with E4/DRSP combinations was statistically significantly better than with E4/LNG combinations: OR (95% CI) 2.00 (1.13; 3.53) and 1.93 (1.06; 3.56) for 15 and 20 mg E4, respectively, and comparable to E2V/DNG. Proportion of women with a 2 kg or more weight loss after 3 and 6 cycles was the highest in the 15 mg E4/DRSP group (30.7 and 36.7%, respectively). CONCLUSIONS: The present study shows that 15 mg estetrol combined with 3 mg DRSP is associated with a high-user acceptability and satisfaction, and with a favourable body weight control.
Asunto(s)
Androstenos/uso terapéutico , Anticonceptivos Orales Combinados/uso terapéutico , Anticonceptivos Hormonales Orales/uso terapéutico , Estetrol/uso terapéutico , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Satisfacción del Paciente , Adolescente , Adulto , Androstenos/administración & dosificación , Peso Corporal , Anticonceptivos Orales Combinados/administración & dosificación , Anticonceptivos Hormonales Orales/administración & dosificación , Estetrol/administración & dosificación , Femenino , Finlandia , Humanos , Análisis de Intención de Tratar , Antagonistas de Receptores de Mineralocorticoides/administración & dosificación , Satisfacción del Paciente/estadística & datos numéricos , Encuestas y Cuestionarios , Hemorragia Uterina/fisiopatología , Adulto JovenRESUMEN
Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.
Asunto(s)
Cerulenina/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Lactonas/farmacología , Vasos Linfáticos/efectos de los fármacos , Melanoma Experimental/prevención & control , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Humanos , Linfangiogénesis/efectos de los fármacos , Metástasis Linfática , Vasos Linfáticos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Orlistat , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVES: This study aims to assess vaginal bleeding patterns and cycle control of oral contraceptives containing estetrol (E4) combined with either drospirenone (DRSP) or levonorgestrel (LNG). STUDY DESIGN: An open-label, multicentre, randomised, dose-finding study lasting six cycles in healthy women aged 18-35 years was used. Four treatments (15 mg or 20 mg E4, combined with either 3 mg DRSP or 150 mcg LNG) were administered in a 24/4-day regimen. A marketed dosing regimen of estradiol valerate with dienogest (E2V/DNG) served as reference since it contains (like E4) a natural oestrogen. RESULTS: A total of 396 women were randomised, of whom 389 received study medication, and 316 completed the study. By cycle 6, the frequencies of unscheduled bleeding and/or spotting and absence of withdrawal bleeding were the lowest in the 15 mg E4/DRSP group (33.8% and 3.5%, respectively). In the E2V/DNG reference group, these frequencies were 47.8% and 27.1%, respectively. By cycle 6, the frequency of women with absence of withdrawal bleeding was <20% for all E4 treatment groups: 3.5-3.8% combined with DRSP and 14.0-18.5% combined with LNG. By cycle 6, unscheduled intracyclic bleeding was reported by <20% of women in the 20 mg E4/LNG group (18.9%) and in the 15 mg E4/DRSP group (16.9%). CONCLUSION: This study showed that, of the four treatment modalities investigated, the 15 mg E4/DRSP combination has the most favourable bleeding pattern and cycle control. IMPLICATIONS: Due to its favourable bleeding pattern and cycle control, the 15 mg E4/DRSP combination is the preferred combination for further phase III clinical development.
Asunto(s)
Anticonceptivos Femeninos/administración & dosificación , Anticonceptivos Orales Combinados/administración & dosificación , Androstenos/administración & dosificación , Androstenos/efectos adversos , Anticonceptivos Femeninos/química , Anticonceptivos Orales Combinados/química , Relación Dosis-Respuesta a Droga , Estetrol/administración & dosificación , Estetrol/efectos adversos , Estradiol/administración & dosificación , Estradiol/efectos adversos , Estradiol/análogos & derivados , Femenino , Humanos , Levonorgestrel/administración & dosificación , Levonorgestrel/efectos adversos , Metrorragia/tratamiento farmacológico , Metrorragia/etiología , Nandrolona/administración & dosificación , Nandrolona/efectos adversos , Nandrolona/análogos & derivadosRESUMEN
OBJECTIVES: The aim of the study was to evaluate the efficacy of different dosages of estetrol (E4) combined with one of two progestins in suppressing the pituitary-ovarian axis and ovulation in healthy premenopausal women. METHODS: This was an open, parallel, phase II, dose-finding, pilot study performed in healthy women aged 18 to 35 years with a documented ovulatory cycle before treatment. For three consecutive cycles in a 24/4-day regimen, participants received 5 mg or 10 mg E4/3 mg drospirenone (DRSP); 5 mg, 10 mg or 20 mg E4/150 µg levonorgestrel; or 20 µg ethinylestradiol (EE)/3 mg DRSP as comparator. Pituitary-ovarian axis activity and the occurrence of ovulation were evaluated by monitoring follicular size, serum levels of follicle-stimulating hormone, luteinising hormone, estradiol and progesterone during treatment cycles 1 and 3. Endometrial thickness was evaluated throughout the trial, and the return of ovulation was evaluated after the last intake of medication. RESULTS: A total of 109 women were included in the trial. No ovulation occurred in any treatment group. Ovarian activity inhibition seemed proportional to the E4 dosage: the highest suppression was observed in the 20 mg E4 group and was very similar to that observed with EE/DRSP. Endometrial thickness was suppressed to the same extent in all groups. Post-treatment ovulation occurred in all participants between 17 and 21 days after the last active treatment. The study combinations were well tolerated and safe. CONCLUSIONS: Combined with a progestin, E4 adequately suppresses ovarian activity, particularly when given at a dosage above 10 mg/day.
Asunto(s)
Androstenos/administración & dosificación , Anticonceptivos Orales Combinados/administración & dosificación , Estetrol/administración & dosificación , Levonorgestrel/administración & dosificación , Antagonistas de Receptores de Mineralocorticoides/administración & dosificación , Inhibición de la Ovulación , Adolescente , Adulto , Androstenos/efectos adversos , Anticonceptivos Orales Combinados/efectos adversos , Relación Dosis-Respuesta a Droga , Endometrio/diagnóstico por imagen , Estetrol/efectos adversos , Estradiol/sangre , Etinilestradiol/administración & dosificación , Etinilestradiol/efectos adversos , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Levonorgestrel/efectos adversos , Hormona Luteinizante/sangre , Antagonistas de Receptores de Mineralocorticoides/efectos adversos , Folículo Ovárico/diagnóstico por imagen , Ovulación/efectos de los fármacos , Proyectos Piloto , Hipófisis/efectos de los fármacos , Hipófisis/fisiología , Progesterona/sangre , Ultrasonografía , Adulto JovenRESUMEN
OBJECTIVES: Estetrol (E4) is a natural estrogen produced by the human fetal liver. In combination with drospirenone (DRSP) or levonorgestrel (LNG), E4 blocks ovulation and has less effect on haemostatic biomarkers in comparison with ethinylestradiol (EE) combined with DRSP. This study evaluates the impact of several doses of E4/DRSP and E4/LNG on safety parameters such as liver function, lipid metabolism, bone markers and growth endocrine parameters. METHODS: This was a dose-finding, single-centre, controlled study performed in healthy women aged 18 to 35 years with a documented pretreatment ovulatory cycle. Participants received 5 mg or 10 mg E4/3 mg DRSP; 5 mg, 10 mg or 20 mg E4/150 µg LNG; or 20 µg EE/3 mg DRSP as a comparator for three consecutive cycles in a 24/4-day regimen. Changes from baseline to end of treatment in liver parameters, lipid metabolism, bone markers and growth endocrinology were evaluated. RESULTS: A total of 109 women were included in the study. Carrier proteins were minimally affected in the E4/DRSP and E4/LNG groups, in comparison with the EE/DRSP group, where a significant increase in sex hormone-binding globulin was observed. Similarly, minor effects on lipoproteins were observed in the E4 groups, and the effects on triglycerides elicited by the E4 groups were significantly lower than those in the EE/DRSP group. No imbalances in bone markers were observed in any groups. No alterations in insulin-like growth factor were observed in the E4 groups. CONCLUSIONS: E4-containing combinations have a limited effect on liver function, lipid metabolism, and bone and growth endocrine parameters.
Asunto(s)
Androstenos/farmacología , Anticonceptivos Orales Combinados/farmacología , Estetrol/farmacología , Etinilestradiol/farmacología , Levonorgestrel/farmacología , Hígado/efectos de los fármacos , Adolescente , Adulto , Fosfatasa Alcalina/sangre , Aspartato Aminotransferasas/sangre , HDL-Colesterol/sangre , HDL-Colesterol/efectos de los fármacos , LDL-Colesterol/sangre , LDL-Colesterol/efectos de los fármacos , Colágeno Tipo I/sangre , Colágeno Tipo I/efectos de los fármacos , Anticonceptivos Orales Combinados/farmacocinética , Anticonceptivos Sintéticos Orales/farmacología , Estetrol/farmacocinética , Estrógenos/farmacología , Femenino , Hormona del Crecimiento/sangre , Hormona del Crecimiento/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/fisiología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Osteocalcina/sangre , Osteocalcina/efectos de los fármacos , Péptidos/sangre , Péptidos/efectos de los fármacos , Globulina de Unión a Hormona Sexual/efectos de los fármacos , Globulina de Unión a Hormona Sexual/metabolismo , Triglicéridos/sangre , Adulto Joven , gamma-Glutamiltransferasa/sangreRESUMEN
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix, which mainly develops at the squamocolumnar (SC) junction. The progression of cervical HPV infections into (pre)neoplastic lesions suggests that viral antigens are not adequately recognized by innate immunity or presented to the adaptive immune system. Members of the defensin family have recently been found to inhibit viral and bacterial pathogens, to stimulate the migration of immune cells and to play a role in anticancer responses. In the present study, we focused on the poorly characterized human α-defensin 5 (HD-5) and its possible role in these processes. We showed that HD-5 was able to prevent HPV virion entry into cervical keratinocytes and to influence adaptive immunity. Indeed, this peptide specifically induced the chemoattraction and proliferation of both activated T lymphocytes and immature dendritic cells in a CCR2/CCR6-dependent manner and stimulated the infiltration of these professional antigen-presenting cells in a (pre)neoplastic epithelium transplanted in vivo in immunodeficient mice. No chemotactic effect was observed with plasmacytoid dendritic cells, macrophages or natural killer cells. Proliferative and angiogenic effects of HD-5 were also assessed in vitro and in vivo. However there was a striking regional disparity in expression of HD-5, being prominent in ectocervical, vaginal and vulvar neoplasia, while absent, or nearly so, in the cervical SC junction. Taken together, these results suggest one possible explanation for why the SC junction is uniquely vulnerable to both high-risk HPV infection (via reduced HD-5 expression and viral entry) and progression of neoplasia (via altered cell-mediated immune responses and altered microenvironment).
Asunto(s)
Cuello del Útero/metabolismo , Infecciones por Papillomavirus/inmunología , Lesiones Precancerosas/inmunología , Neoplasias Uterinas/virología , alfa-Defensinas/biosíntesis , Animales , Western Blotting , Células Cultivadas , Cuello del Útero/inmunología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Uterinas/inmunología , Displasia del Cuello del Útero/inmunología , Displasia del Cuello del Útero/virologíaRESUMEN
INTRODUCTION AND HYPOTHESIS: The pathophysiology of pelvic organ prolapse (POP) is incompletely understood. The purpose of this study is to describe the current knowledge about histology of the vaginal wall and its possible involvement in the pathogenesis of pelvic organ prolapse. METHODS: Eligible studies were selected through a MEDLINE search covering January 1986 to December 2012. The research was limited to English-language publications. RESULTS: Investigations of changes in the vaginal tissue that occur in women with genital prolapse are currently still limited and produced contrary results. The heterogeneity of the patients and the control groups in terms of age, parity and hormonal status, of the localization of biopsies and the histological methods as well as the lack of validation of the quantification procedures do not allow clear and definitive conclusions to be drawn. CONCLUSIONS: This review shows that current knowledge of the histological changes observed in women with POP are inconclusive and relatively limited. More studies are needed in this specific field to better understand the mechanisms that lead to POP.
Asunto(s)
Prolapso de Órgano Pélvico/patología , Vagina/patología , Colágeno/metabolismo , Tejido Conectivo/metabolismo , Elastina/metabolismo , Femenino , Humanos , Vagina/irrigación sanguínea , Vagina/inervación , Vagina/metabolismoRESUMEN
PURPOSE: To evaluate the antilymphangiogenic potential of multi-target tyrosine kinase inhibitor sunitinib in corneal neovascularization (NV). METHODS: Inflammatory corneal NV was induced by thermal cauterization applied in the central cornea of mice, to which sunitinib malate was daily administered by gavage or not. At days 6, 11, or 17 post cauterization, lymphatic and blood vessels, as well as inflammatory cells were immunostained and quantified in whole-mounted corneas. RT-PCRs were performed to evidence VEGF-A, VEGF-C, VEGF-D, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor (VEGFR)-1 and -2 (sVEGFR-1, sVEGFR-2) expressions. Macrophages were isolated from mice peritoneal cavity following thioglycollate injection to produce conditioned medium. The effects of sunitinib were evaluated in vitro in the aortic and lymphatic ring assays in the presence or not of macrophage conditioned medium. RESULTS: Sunitinib treatment drastically reduced pathologic corneal lymphangiogenesis and angiogenesis. Reduced F4/80+ cell infiltration was evidenced in sunitinib-treated mice and was associated to decreased VEGF-A (by 50%, P < 0.01) and VEGF-C (by 35%, P < 0.01) expressions, while VEGF-D and sVEGFR-2 expressions were not affected. In vitro, sunitinib dose-dependently inhibited aortic ring outgrowth, but failed to affect lymphangiogenesis in the lymphatic ring assay. However, macrophage conditioned medium-enhanced angiogenesis and lymphangiogenesis were both strongly counteracted by sunitinib treatment. Mechanistically, sunitinib blocked VEGFR-2 phosphorylation induced by VEGF-A released by macrophages. CONCLUSIONS: Sunitinib exerts antihemangiogenic and antilymphangiogenic effects in vivo by reducing F4/80+ cell recruitment and interacting with their released factors.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neovascularización de la Córnea/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Indoles/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Pirroles/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Glicoproteínas/metabolismo , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/metabolismo , Macrófagos Peritoneales , Masculino , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Placentario , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Gestacionales/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sunitinib , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.
Asunto(s)
Linfangiogénesis/genética , Vasos Linfáticos/embriología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/fisiología , Animales , Animales Modificados Genéticamente , Células Cultivadas , Colágeno Tipo I/metabolismo , Colagenasas/genética , Colagenasas/metabolismo , Colagenasas/fisiología , Embrión no Mamífero , Líquido Extracelular/enzimología , Líquido Extracelular/metabolismo , Femenino , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/fisiología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pez CebraRESUMEN
The 16-kDa angiostatic N-terminal fragment of human prolactin (16K hPRL) has been reported to be a new potent anticancer compound. This protein has already proven its efficiency in several mouse tumor models in which it prevented tumor-induced angiogenesis and delayed tumor growth. In addition to angiogenesis, tumors also stimulate the formation of lymphatic vessels, which contribute to tumor cell dissemination and metastasis. However, the role of 16K hPRL in tumor-induced lymphangiogenesis has never been investigated. We establish in vitro that 16K hPRL induces apoptosis and inhibits proliferation, migration, and tube formation of human dermal lymphatic microvascular endothelial cells. In addition, in a B16F10 melanoma mouse model, we found a decreased number of lymphatic vessels in the primary tumor and in the sentinel lymph nodes after 16K hPRL treatment. This decrease is accompanied by a significant diminished expression of lymphangiogenic markers in primary tumors and sentinel lymph nodes as determined by quantitative RT-PCR. These results suggest, for the first time, that 16K hPRL is a lymphangiostatic as well as an angiostatic agent with antitumor properties.
Asunto(s)
Proteínas Angiostáticas/farmacología , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Linfangiogénesis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Prolactina/farmacología , Proteínas Angiostáticas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Endoteliales/patología , Femenino , Humanos , Ganglios Linfáticos/patología , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/patología , Ratones , Neovascularización Patológica/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Prolactina/uso terapéuticoRESUMEN
BACKGROUND: Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures. RESULTS: In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation. CONCLUSIONS: In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.
Asunto(s)
Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Animales , Movimiento Celular , Células Cultivadas , Córnea/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Linfático/metabolismo , Endotelio Linfático/patología , Femenino , Adyuvante de Freund , Hiperplasia , Técnicas In Vitro , Linfangioma/etiología , Linfangioma/patología , Vasos Linfáticos/patología , Vasos Linfáticos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de TransmisiónRESUMEN
Angiogenesis, extracellular matrix remodeling and cell migration are associated with cancer progression and involve at least, the plasminogen activating system and its main physiological inhibitor, the plasminogen activator inhibitor-1 (PAI-1). Considering the recognized importance of PAI-1 in the regulation of tumor angiogenesis and invasion in murine models of skin tumor transplantation, we explored the functional significance of PAI-1 during early stages of neoplastic progression in the transgenic mouse model of multistage epithelial carcinogenesis (K14-HPV16 mice). We have studied the effect of genetic deletion of PAI-1 on inflammation, angiogenesis, lymphangiogenesis and tumor progression. In this model, PAI-1 deficiency neither impaired keratinocyte hyperproliferation or tumor development nor affected the infiltration of inflammatory cells and development of angiogenic or lymphangiogenic vasculature. We are reporting evidence for concomitant lymphangiogenic and angiogenic switches independent to PAI-1 status. Taken together, these data indicate that PAI-1 is not rate limiting for neoplastic progression and vascularization during premalignant progression, or that there is a functional redundancy between PAI-1 and other tumor regulators, masking the effect of PAI-1 deficiency in this long-term model of multistage epithelial carcinogenesis.
Asunto(s)
Papillomavirus Humano 16/genética , Queratina-14/genética , Inhibidor 1 de Activador Plasminogénico/fisiología , Neoplasias Cutáneas/etiología , Animales , Linfangiogénesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidor 1 de Activador Plasminogénico/deficiencia , Lesiones Precancerosas/etiología , Neoplasias Cutáneas/irrigación sanguíneaRESUMEN
The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.
Asunto(s)
Inhibidor 1 de Activador Plasminogénico/fisiología , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Línea Celular Tumoral , Femenino , Heterocigoto , Humanos , Inflamación , Linfangioma/metabolismo , Masculino , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Metástasis de la NeoplasiaRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs. We observed that knockdown of PAI-1 in ECs enhances cell-associated plasmin activity and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144-Lys145, releasing a soluble proapoptotic FasL fragment from the surface of ECs. The data provide a mechanism explaining the proangiogenic activity of PAI-1.
Asunto(s)
Apoptosis , Células Endoteliales/metabolismo , Proteína Ligando Fas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Serpinas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/patología , Fibrinolisina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/metabolismo , Neuroblastoma/irrigación sanguínea , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Serpina E2 , Serpinas/deficiencia , Serpinas/genética , Factores de Tiempo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Receptor fas/metabolismoRESUMEN
An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1(-/-) mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1(-/-) BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis.
Asunto(s)
Células de la Médula Ósea/fisiología , Carcinoma/irrigación sanguínea , Neovascularización Coroidal/etiología , Neovascularización Patológica/etiología , Inhibidor 1 de Activador Plasminogénico/fisiología , Neoplasias Cutáneas/irrigación sanguínea , Animales , Trasplante de Médula Ósea , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Queratinocitos/patología , Queratinocitos/trasplante , Ratones , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidor 1 de Activador Plasminogénico/genéticaRESUMEN
In addition to their direct antimicrobial activity, defensins might also influence adaptive immunity by attracting immature dendritic cells (DC). As these cells have been shown to be deficient in uterine cervix carcinogenesis, we evaluated the ability of alpha-defensin (HNP-2, human neutrophil defensin 2) and beta-defensin (HbetaD2, human beta defensin 2) to stimulate their migration in human papillomavirus (HPV)-associated (pre)cancers. We first observed, using RT-PCR and immunohistology, that HbetaD2 is absent in HPV-transformed keratinocytes and that it is weakly expressed in cervical (pre)neoplastic lesions in comparison with normal keratinocytes. We next demonstrated that defensins exert a chemotactic activity for DC in a Boyden Chamber assay and stimulate their infiltration in an in vitro-formed (pre)neoplastic epithelium (organotypic culture of HPV-transformed keratinocytes). To evaluate the ability of defensins also to recruit DC in vivo, we developed a model of immunodeficient mice grafted with organotypic cultures of HPV+ keratinocytes, which form an epithelium similar to a high-grade neoplastic lesion, with tumoral invasion and neovascularization. Intravenously injected human DC were able to infiltrate grafts of HPV+ keratinocytes after administration of HNP-2 in the transplantation chamber. Taken together, these results suggest that defensins could reverse a frequent immune alteration observed in cancer development.
Asunto(s)
Células Dendríticas/trasplante , Infecciones por Papillomavirus/complicaciones , Lesiones Precancerosas/virología , Neoplasias del Cuello Uterino/virología , alfa-Defensinas/farmacología , beta-Defensinas/farmacología , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Línea Celular Transformada , Movimiento Celular , Cuello del Útero/citología , Cuello del Útero/inmunología , Cuello del Útero/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epitelio/metabolismo , Epitelio/virología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica/patología , Técnicas de Cultivo de Órganos , Papillomaviridae/fisiología , Infecciones por Papillomavirus/metabolismo , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , alfa-Defensinas/inmunología , beta-Defensinas/inmunología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Plg)/plasminogen activator (PA) system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1) in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background) deleted for PAI-1 gene (PAI-1-/-), we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.
Asunto(s)
Neovascularización Patológica/metabolismo , Inhibidor 1 de Activador Plasminogénico/fisiología , Neoplasias Cutáneas/patología , Animales , Progresión de la Enfermedad , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Inhibidor 1 de Activador Plasminogénico/genética , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/etiología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/trasplanteRESUMEN
The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti- or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP-9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP-2 and MMP-3 were stromal. Neither the single deficiency of MMP-2, MMP-3, or MMP-9, nor the combined absence of MMP-9 and MMP-3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP-2:MMP-9-deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP-2 and MMP-9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP-2 and MMP-9. Our data demonstrate for the first time in an experimental model that MMP-2 and MMP-9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.