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1.
Lab Chip ; 21(24): 4791-4804, 2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34309615

RESUMEN

We have developed and tested a novel microfluidic device for blood oxygenation, which exhibits a large surface area of gas exchange and can support long-term sustainable endothelialization of blood microcapillaries, enhancing its hemocompatibility for clinical applications. The architecture of the parallel stacking of the trilayers is based on a central injection for blood and a lateral injection/output for gas which allows significant reduction in shear stress, promoting sustainable endothelialization since cells can be maintained viable for up to 2 weeks after initial seeding in the blood microchannel network. The circular design of curved blood capillaries allows covering a maximal surface area at 4 inch wafer scale, producing high oxygen uptake and carbon dioxide release in each single unit. Since the conventional bonding process based on oxygen plasma cannot be used for surface areas larger than several cm2, a new "wet bonding" process based on soft microprinting has been developed and patented. Using this new protocol, each 4 inch trilayer unit can be sealed without a collapsed membrane even at reduced 15 µm thickness and can support a high blood flow rate. The height of the blood channels has been optimized to reduce pressure drop and enhance gas exchange at a high volumetric blood flow rate up to 15 ml min-1. The simplicity of connecting different units in the stacked architecture is demonstrated for 3- or 5-unit stacked devices that exhibit remarkable performance with low primary volume, high oxygen uptake and carbon dioxide release and high flow rate of up to 80 ml min-1.


Asunto(s)
Microfluídica , Oxigenadores , Dióxido de Carbono , Diseño de Equipo , Pulmón , Oxígeno
2.
Cell Commun Signal ; 19(1): 1, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397378

RESUMEN

BACKGROUND: Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. METHODS: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. RESULTS: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. CONCLUSIONS: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs' longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration. Video Abstract.


Asunto(s)
Células Progenitoras Endoteliales/efectos de los fármacos , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Progenitoras Endoteliales/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inmunomodulación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología
3.
Stem Cell Res Ther ; 11(1): 534, 2020 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-33303019

RESUMEN

Mesenchymal stem/stromal cells can modulate the effector immune cells especially T lymphocytes. Due to this important feature, they can regulate the development of a variety of disorders including inflammatory and autoimmune disorders, cancers, and transplantation outcomes. One of the most important MSC immunoregulatory functions is their capacity to convert conventional T cells into regulatory T cells. Several mechanisms, mostly related to MSCs but not T cells, have been shown essential for this aspect. The inflammatory microenvironment majorly caused by pro-inflammatory cytokines has been demonstrated to govern the direction of the immune response. In this respect, we have recently revealed that the TNFα-TNFR2 signaling controls several aspects of MSC immunomodulatory properties including their ability to suppress T cells and their conversion towards Foxp3-expressing Tregs. Here in this work, we have looked from another angle by investigating the impact of TNFR2 expression by T cells on their ability to be converted to suppressive Tregs by MSCs. We showed that unlike WT-T cells, their TNFR2 KO counterparts are remarkably less able to convert into Foxp3+ and Foxp3- Tregs. Furthermore, TNFR2 blockade diminished the anti-inflammatory cytokine secretion by iTregs and consequently resulted in less T cell immunosuppression. This work is the first evidence of the crucial association of TNFR2 expression by T cells with their iTreg conversion capacity by MSCs. It strengthens once more the potential of anti-TNFR2 administration for a strong and effective interference with the immunosuppression exerted by TNFR2-expressing cells.


Asunto(s)
Células Madre Mesenquimatosas , Linfocitos T Reguladores , Citocinas , Factores de Transcripción Forkhead/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética
4.
Cell Commun Signal ; 18(1): 94, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32546175

RESUMEN

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).


Asunto(s)
Células Progenitoras Endoteliales , Terapia de Inmunosupresión , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/inmunología , Adolescente , Adulto , Anciano , Animales , Células Cultivadas , Técnicas de Cocultivo , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/inmunología , Femenino , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Transducción de Señal , Adulto Joven
5.
PLoS One ; 14(5): e0216602, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31075112

RESUMEN

Endothelial dysfunction (ED) is part of the first steps in the development of cardiovascular diseases (CVD). Growth Differentiation Factor 15 (GDF15) is a cytokine belonging to the Transforming Growth Factor ß superfamily and its expression is increased both during ED and in CVD. Because high blood levels of GDF15 have been reported during ED, we hypothesized that GDF15 could be produced by endothelial cells in response to a vascular stress, possibly to attenuate endothelial function loss. Since senescence is mainly involved in both vascular stress and endothelial function loss, we used Endothelial Colony Forming Cells generated from adult blood (AB-ECFCs) as a model of endothelial cells to investigate GDF15 expression during cellular senescence. Then, we analyzed the potential role of GDF15 in AB-ECFC functions and senescence. When AB-ECFCs become senescent, they secrete increased levels of GDF15. We investigated GDF15 paracrine effects on non-senescent AB-ECFCs and showed that GDF15 enhanced proliferation, migration, NO production and activated several signaling pathways including AKT, ERK1/2 and SMAD2 without triggering any oxidative stress. Taken together, our results suggest that GDF15 production by senescent AB-ECFCs could act in a paracrine manner on non-senescent AB-ECFCs, and that this interaction could be beneficial to its model cells. Therefore, GDF15 could play a beneficial role in a dysfunctional vascular system as previously reported in patients with CVD, by limiting ED related to vascular stress occurring in these diseases.


Asunto(s)
Células Sanguíneas/citología , Células Endoteliales/citología , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Adulto , Anciano , Células Sanguíneas/metabolismo , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Senescencia Celular , Células Endoteliales/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Estrés Oxidativo , Transducción de Señal , Regulación hacia Arriba , Adulto Joven
6.
J Thorac Cardiovasc Surg ; 157(2): 655-666.e7, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30669226

RESUMEN

BACKGROUND: Right ventricular (RV) failure is the main prognostic factor in pulmonary hypertension, and ventricular capillary density (CD) has been reported to be a marker of RV maladaptive remodeling and failure. Our aim was to determine whether right intracoronary endothelial progenitor cell (EPC) infusion can improve RV function and CD in a piglet model of chronic thromboembolic pulmonary hypertension (CTEPH). METHODS: We compared 3 groups: sham (n = 5), CTEPH (n = 6), and CTEPH with EPC infusion (CTEPH+EPC; n = 5). After EPC isolation from CTEPH+EPC piglet peripheral blood samples at 3 weeks, the CTEPH and sham groups underwent right intracoronary infusion of saline, and the CTEPH+EPC group received EPCs at 6 weeks. RV function, pulmonary hemodynamics, and myocardial morphometry were investigated in the animals at 10 weeks. RESULTS: After EPC administration, the RV fractional area change increased from 32.75% (interquartile range [IQR], 29.5%-36.5%) to 39% (IQR, 37.25%-46.50%; P = .030). The CTEPH+EPC piglets had reduced cardiomyocyte surface areas (from 298.3 µm2 [IQR, 277.4-335.3 µm2] to 234.6 µm2 (IQR, 211.1-264.7 µm2; P = .017), and increased CD31 expression (from 3.12 [IQR, 1.27-5.09] to 7.14 [IQR, 5.56-8.41; P = .017). EPCs were found in the RV free wall at 4 and 24 hours after injection but not 4 weeks later. CONCLUSIONS: Intracoronary infusion of EPC improved RV function and CD in a piglet model of CTEPH. This novel cell-based therapy might represent a promising RV-targeted treatment in patients with pulmonary hypertension.


Asunto(s)
Células Progenitoras Endoteliales/trasplante , Neovascularización Fisiológica , Hipertensión Arterial Pulmonar/cirugía , Embolia Pulmonar/complicaciones , Trasplante de Células Madre , Disfunción Ventricular Derecha/prevención & control , Función Ventricular Derecha , Remodelación Ventricular , Animales , Animales Recién Nacidos , Presión Arterial , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión Arterial Pulmonar/etiología , Hipertensión Arterial Pulmonar/fisiopatología , Embolia Pulmonar/fisiopatología , Recuperación de la Función , Sus scrofa , Factores de Tiempo , Trasplante Autólogo , Disfunción Ventricular Derecha/etiología , Disfunción Ventricular Derecha/fisiopatología
7.
JCI Insight ; 2(21)2017 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-29093266

RESUMEN

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.


Asunto(s)
Macrófagos/metabolismo , Oncostatina M/metabolismo , Osificación Heterotópica/inmunología , Osificación Heterotópica/metabolismo , Animales , Antígenos CD34 , Lesiones Encefálicas , Diferenciación Celular , Proliferación Celular , Células Endoteliales , Femenino , Hematopoyesis , Células Madre Hematopoyéticas , Xenoinjertos , Humanos , Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad beta del Receptor de Oncostatina M , Osificación Heterotópica/patología , Osteogénesis , Médula Espinal , Transcriptoma
8.
Stem Cell Res ; 21: 148-159, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28499264

RESUMEN

Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.


Asunto(s)
Diferenciación Celular , Células Progenitoras Endoteliales/citología , Adolescente , Adulto , Factores de Edad , Anciano , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Clonales , Colágeno/farmacología , Ensayo de Unidades Formadoras de Colonias , Combinación de Medicamentos , Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/metabolismo , Sangre Fetal/citología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Laminina/farmacología , Masculino , Persona de Mediana Edad , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteoglicanos/farmacología , Donantes de Tejidos , Adulto Joven
9.
Theriogenology ; 84(8): 1397-404, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26298408

RESUMEN

Plasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10-14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity.


Asunto(s)
Hormona Antimülleriana/sangre , Desarrollo Embrionario , Ensayo de Inmunoadsorción Enzimática/veterinaria , Animales , Bovinos , Femenino , Pruebas de Función Ovárica/métodos , Pruebas de Función Ovárica/veterinaria , Sensibilidad y Especificidad
10.
Hum Reprod ; 28(3): 762-9, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23321213

RESUMEN

STUDY QUESTION: Are anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR-II) mRNAs similarly regulated by gonadotrophins in lutein granulosa cells (GCs) from control, normo-ovulatory and oligo/anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: AMH mRNA expression was induced by LH only in lutein GC of oligo/anovulatory PCOS women; down-regulation of AMHR-II, induced by LH in control and normo-ovulatory PCOS women, was absent in oligo/anovulatory women. WHAT IS KNOWN ALREADY: It was suggested that AMH could be responsible for the blockade of follicles at the small antral stage in PCOS women. In keeping with this hypothesis, both AMH and AMHR-II are overexpressed in lutein GCs from oligo/anovulatory PCOS women. STUDY DESIGN, SIZE, DURATION: Women undergoing IVF were included in this prospective study, either in the control group (30 women) or in the PCOS group (21 normo-ovulatory and 19 oligo/anovulatory patients) between January 2010 and July 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human lutein GCs were isolated from follicular fluid during IVF protocols. Twenty-four hours after seeding, lutein GCs from each woman were serum starved and cultured for 48 h ± FSH, LH or cAMP. Then AMH and AMHR-II mRNAs were quantified by quantitative RT-PCR and AMH protein concentration was measured in the culture medium by ELISA. Experimental results were analyzed, within each group of women, by the non-parametric Wilcoxon test for paired comparisons between cells cultured in control medium and FSH, LH or cAMP treated cells. Clinical comparisons between the three groups of women were performed on log values using the ANOVA test with Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: FSH up-regulated both AMH expression and secretion by lutein GCs from the three groups of women (P < 0.05). LH had no effect on AMH mRNAs levels in lutein GCs from controls and normo-ovulatory PCOS women, but increased AMH expression in oligo/anovulatory PCOS women (P < 0.05). Interestingly, LH and cAMP treatments reduced AMHR-II expression by lutein GCs from controls and normo-ovulatory PCOS women (P < 0.05), but had no effect on AMHR-II mRNA levels in oligo/anovulatory PCOS women. LIMITATIONS, REASONS FOR CAUTION: The lutein GCs are not the best model to study AMH and AMHR-II regulation by gonadotrophins. Indeed, AMH and AMHR-II are down-regulated in luteinized cells. Furthermore, these cells have been exposed to non-physiological levels of gonadotrophins and hCG. However, AMH and AMHR-II mRNAs are quantifiable by real-time RT-PCR, and the cells are still responsive to FSH and LH. The age of patients is significantly different between control and oligo/anovulatory PCOS women: this may be a bias in the interpretation of results but older women in the control group had a good ovarian reserve. WIDER IMPLICATIONS OF THE FINDINGS: The overexpression of AMH and AMHR-II in oligo/anovulatory PCOS women could be due to increased LH levels and/or inhibition of its repressive action. The fact that this dysregulation is observed in oligo/anovulatory, but not in normo-ovulatory, PCOS women emphasizes the role of LH in the follicular arrest of PCOS women and suggests that this involves the AMH/AMHR-II system. STUDY FUNDING/COMPETING INTEREST(S): The Assistance-Publique Hôpitaux de Paris provided a Contrat d'Interface and the Agence de Biomédecine provided a grant to Nathalie di Clemente. Schering-Plough provided an FARO grant to Alice Pierre. The authors have nothing to disclose.


Asunto(s)
Anovulación/etiología , Células de la Granulosa/metabolismo , Fase Luteínica/metabolismo , Hormona Luteinizante/metabolismo , Síndrome del Ovario Poliquístico/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Regulación hacia Arriba , Adulto , Hormona Antimülleriana/biosíntesis , Hormona Antimülleriana/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Regulación hacia Abajo , Femenino , Hormona Folículo Estimulante/metabolismo , Líquido Folicular , Células de la Granulosa/patología , Humanos , Síndrome del Ovario Poliquístico/fisiopatología , Estudios Prospectivos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Índice de Severidad de la Enfermedad
11.
J Clin Endocrinol Metab ; 97(9): E1649-57, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22689696

RESUMEN

BACKGROUND: Anti-müllerian hormone (AMH) is a member of the TGF-ß family, which limits follicle maturation. Recently serum AMH has been recognized as a useful diagnostic and prognostic tool in human reproductive endocrinology. OBJECTIVE: The aim of this study was to investigate the regulation of human ovarian AMH by estradiol and FSH. METHODS: AMH mRNA were quantified by real time RT-PCR in human granulosa cells (GC). AMH transcription was studied in KK1 GC cotransfected with estrogen receptors (ER)-ß or ERα, and normal human AMH promoter-luciferase construct (hAMH-luc) or mutated AMH promoter reporter constructs. Binding sites for estradiol (estrogen response element half-site) and steroidogenic factor 1 were disrupted by targeted mutagenesis. The level of ER in GC was determined by quantitative RT-PCR and Western blotting. RESULTS: In KK1 cells, estradiol up-regulated and inhibited hAMH-luc in the presence of ERα and ERß respectively. Disruption of estrogen response element half-site and/or steroidogenic factor 1 binding sites did not modify ERß-mediated effect of estradiol on hAMH-luc, whereas it affected that conveyed by ERα. The FSH enhancement of hAMH-luc was abolished by estradiol in cells overexpressing ERß. When both ER were transfected, estradiol inhibited hAMH-luc or had no effect. Estradiol repressed AMH mRNAs in human GC, which express a little more ERα than ERß mRNA. CONCLUSIONS: Our results show that AMH expression can be differentially regulated by estradiol depending on the ER and suggest that its decrease in GC of growing follicles, which mainly express ERß, and during controlled ovarian hyperstimulation is due to the effect of estradiol.


Asunto(s)
Hormona Antimülleriana/biosíntesis , Estradiol/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor beta de Estrógeno/efectos de los fármacos , Adulto , Animales , Sitios de Unión/efectos de los fármacos , Western Blotting , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Genes Reporteros/genética , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Ovario/efectos de los fármacos , Ovario/metabolismo , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor Esteroidogénico 1/metabolismo , Adulto Joven
12.
Am J Physiol Endocrinol Metab ; 301(3): E539-47, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21693691

RESUMEN

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.


Asunto(s)
Hormona Antimülleriana/metabolismo , AMP Cíclico/metabolismo , Factor de Transcripción SOX9/metabolismo , Células de Sertoli/metabolismo , Factor Esteroidogénico 1/metabolismo , Hormona Antimülleriana/genética , Línea Celular , AMP Cíclico/genética , Proteínas de Unión al ADN , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Expresión Génica , Humanos , Masculino , Regiones Promotoras Genéticas , Factores de Empalme de ARN , Factor de Transcripción SOX9/genética , Transducción de Señal/fisiología , Factor Esteroidogénico 1/genética , Factores de Transcripción , Regulación hacia Arriba
13.
Mol Endocrinol ; 25(4): 645-55, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21330407

RESUMEN

Anti-Müllerian hormone (AMH), also called Müllerian-inhibiting substance, a member of the TGF-ß family, is responsible for the regression of Müllerian ducts in the male fetus. In females, AMH is synthesized by granulosa cells of preantral and small antral follicles, and production wanes at later stages of follicle maturation. Using RT-PCR in luteal granulosa cells in primary culture and reporter gene techniques in the KK1 granulosa cell line, we show that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by granulosa cells. The specific effect of gonadotropins upon granulosa cells may be obscured in vivo by the effect of FSH upon follicular maturation and by the presence of other hormones and growth factors, acting individually or in concert.


Asunto(s)
Hormona Antimülleriana/genética , AMP Cíclico/metabolismo , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Factor de Transcripción GATA4/metabolismo , Genes Reporteros , Gonadotropinas/metabolismo , Humanos , Hormona Luteinizante/metabolismo , Ratones , Folículo Ovárico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor Esteroidogénico 1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
14.
J Immunol ; 185(1): 624-33, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20530261

RESUMEN

Mast cells exert protective effects in experimental antiglomerular basement membrane-induced glomerulonephritis (GN), yet the responsible mediators have not been identified. In this study, we investigated the role of mouse mast cell protease (mMCP)-4, the functional homolog of human chymase, using mMCP-4-deficient mice. Compared with wild type animals, mMCP-4-deficient mice exhibited lower proteinuria, blood creatinine, and blood urea nitrogen levels, indicating an aggravating role of mMCP-4. Kidney histology confirmed less severe renal damage in mMCP-4-deficient mice with reduced deposits, glomerular and interstitial cellularity, and fibrosis scores. High amounts of mMCP-4 were detected in renal capsules, but not in the whole kidney, from wild type mice. Its expression in renal capsules was markedly decreased after GN induction, suggesting that locally released enzyme by degranulated mast cells could contribute to the functional and physiopathological hallmarks of GN. Supporting a proinflammatory role, glomerular and interstitial macrophage and T cell infiltration, levels of proinflammatory TNF and MCP-1 mRNA, and the expression of the profibrotic peptide angiotensin II together with type I collagen were markedly downregulated in kidneys of mMCP-4-deficient mice. We conclude that mMCP-4 chymase, contrary to the global anti-inflammatory action of mast cells, aggravates GN by promoting kidney inflammation. These results highlight the complexity of mast cell-mediated inflammatory actions and suggest that chymase inhibition may represent a novel therapeutic target in GN.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/enzimología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Enfermedades del Complejo Inmune/enzimología , Enfermedades del Complejo Inmune/patología , Mediadores de Inflamación/fisiología , Serina Endopeptidasas/fisiología , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Células Cultivadas , Fibrosis , Enfermedades del Complejo Inmune/inmunología , Pruebas de Función Renal , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos
15.
Endocrinology ; 151(3): 1299-309, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20056831

RESUMEN

In the mammalian ovary, kit ligand (KL), coded by a cAMP-stimulatable gene, is a protein that promotes initiation of follicle growth. The neuropeptide somatostatin (SST) is a small peptide that inhibits cAMP generation in many cell types. Consequently, SST receptor agonists might alter KL production and subsequent follicle growth. The present study was undertaken to look for the existence of a functional SST system in the mouse ovary, to test the effects of the SST receptor 2 (SSTR-2) antagonist BIM-23627 on in vitro folliculogenesis, and to compare them with those of KL, which was demonstrated to stimulate follicle growth in the neonatal rat ovary. Pairs of ovaries from 5-d-old mice were incubated in vitro during 15 d in the presence of either KL or BIM-23627. For every mouse, one ovary was cultured in culture medium (control), and the other ovary was cultured in the presence of either KL or BIM-23627. After 5, 10, and 15 d culture, the ovaries were histologically assessed for the content of primordial, primary, and secondary follicles. The SSTR-2 and -5, but not SST, were identified at the transcriptional and translational (mainly in granulosa cells) levels. Both KL and BIM-23627 triggered a reduction of the percentages of primordial follicles and an increase of the percentages of primary and secondary follicles when compared with control ovaries from the same animal. In conclusion, extraovarian SST, acting through its receptors 2 and 5 present on granulosa cells, may be involved in mouse folliculogenesis by reducing recruitment of resting follicles.


Asunto(s)
Folículo Ovárico/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Factor de Células Madre/metabolismo , Animales , Animales Recién Nacidos , Femenino , Ratones , Técnicas de Cultivo de Órganos , Folículo Ovárico/crecimiento & desarrollo , Péptidos , Receptores de Somatostatina/antagonistas & inhibidores
16.
J Immunol ; 181(7): 5167-73, 2008 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-18802121

RESUMEN

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p

Asunto(s)
Glicoproteínas/biosíntesis , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adipoquinas , Anciano , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/patología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Línea Celular , Proteína 1 Similar a Quitinasa-3 , Femenino , Glicoproteínas/sangre , Glicoproteínas/fisiología , Humanos , Lectinas , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/patología , Índice de Severidad de la Enfermedad
17.
J Allergy Clin Immunol ; 120(6): 1301-7, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17996929

RESUMEN

BACKGROUND: Airway remodeling in patients with severe steroid-refractory asthma might result from a reduced ability of steroid therapy to limit the transcription of remodeling factors by the bronchial epithelium. OBJECTIVE: We sought to compare the levels of transcripts encoding remodeling factors in bronchial epithelium of healthy volunteers and of asthmatic patients with either steroid-sensitive or steroid-refractory disease and to correlate these levels with hallmarks of airway remodeling. METHODS: By means of real-time quantitative PCR, we assessed the levels of 14 transcripts encoding remodeling factors, matrix metalloproteinases, and extracellular matrix proteins in laser-capture microdissected bronchial epithelium of healthy volunteers, patients with mild steroid-untreated asthma, and patients with steroid-sensitive and steroid-refractory asthma (n = 8-10 in each group). Histologic features of airway remodeling and endothelin-1 (EDN1) immunolocalization were determined by using frozen specimens. RESULTS: Patients with steroid-refractory asthma had greater levels of EDN1 transcripts (4.1-fold increase, P = .026) and protein (P = .0009) in their bronchial epithelium compared with patients with steroid-sensitive asthma. EDN1 mRNA levels and protein expression in asthmatic patients were negatively correlated with prebronchodilator and postbronchodilator FEV(1) value (r(2) >or= 0.193, P

Asunto(s)
Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Asma/metabolismo , Disnea/metabolismo , Endotelina-1/biosíntesis , Endotelina-1/genética , Regulación de la Expresión Génica/inmunología , Mucosa Respiratoria/metabolismo , Antiinflamatorios/uso terapéutico , Asma/inmunología , Asma/patología , Asma/fisiopatología , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/patología , Disnea/inmunología , Disnea/patología , Disnea/fisiopatología , Humanos , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Mucosa Respiratoria/fisiopatología , Esteroides/uso terapéutico
18.
Curr Biol ; 16(17): 1719-25, 2006 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-16950109

RESUMEN

Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.


Asunto(s)
Citocinesis/fisiología , Endocitosis/fisiología , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/fisiología , Animales , Línea Celular , Drosophila , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo
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