RESUMEN
Cryopreservation has been used over many decades for the maintenance of viable biological specimens. Its expansion into the area of fertility preservation has been a natural outcome of the increased risks to human fertility from diseases, such as cancer and its treatment protocols, including radiation and chemo-therapy, and the general lifestyle trend to later marriages. The use of assisted reproductive techniques (ART) in preserving fertility have benefitted significantly from new scientific approaches, such as cryostorage, in which live cells and tissues are stored at low temperatures and revived when necessary. This review focuses on "cryopreservation science monitoring in reproductive biomedicine" to evaluate knowledge, trends, driving forces, impetus, and emerging technologies in order to draw a future roadmap for this field. Our analysis of the field of cryobiology emphasizes the significance of strategic planning of cryobiology research to support more its extensive use in therapeutics in the future. The Royan Institute (Tehran, Iran) recognises this need and has developed a strategic plan to engage in multidisciplinary research on the application of cryobiology, including cryobioengineering, in disease mitigation. We hoped that this study can help improve the quality and quantity of public discourse and expert awareness of the role for cryopreservation in fertility preservation within ART. DOI: 10.54680/fr23410110112.
Asunto(s)
Preservación de la Fertilidad , Humanos , Preservación de la Fertilidad/métodos , Criopreservación/métodos , Criobiología , Irán , Técnicas Reproductivas AsistidasRESUMEN
BACKGROUND: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. OBJECTIVE: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. METHODS: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. RESULTS: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. CONCLUSION: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells.
RESUMEN
Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial estrogen receptor beta (ERß) as an antagonist in MCF7-BK cells, increasing reactive oxygen species (ROS) concentrations from the mitochondria that were required for cytotoxicity. In part, this derived from TAM downregulating manganese superoxide dismutase (MnSOD) activity by causing the nitrosylation of tyrosine 34, thereby increasing ROS. ROS-activated protein kinase C delta and c-jun N-terminal kinases, resulting in the mitochondrial translocation of Bax and cytochrome C release. Interestingly, TAM failed to cause high ROS levels or induce cell death in MCF7-BK-TR cells due to stimulation of MnSOD activity through agonistic effects at mitochondrial ERß. In several mouse xenograft models, lentiviral shRNA-induced knockdown of MnSOD caused tumors that grew in the presence of TAM to undergo substantial apoptosis. Tumor MnSOD and mitochondrial ERß are therefore targets for therapeutic intervention to reverse TAM resistance and enhance a cell death response.
Asunto(s)
Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/fisiología , Receptor beta de Estrógeno/metabolismo , Mitocondrias/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Esophageal cancer (EC) is one of the most frequent and serious cancers worldwide, but its geographic distribution is disparate. Northern Iran is known as one of the hot spots for EC, but there is inadequate evidence available regarding its characteristics in northwestern region of Iran. Therefore, this study aimed to describe some demographic and histopathologic features of EC in this region of Iran. MATERIALS AND METHODS: 166 hospital referral patients from a hospital in the northwestern region of Iran who underwent endoscopic biopsy for the chief complaint of dysphagia or odynophagia, and were admitted with the pathologic diagnosis of esophageal cancer during 3 years were enrolled in this study. RESULTS: The mean age of the patient was 61.8 ± 12.0 years old. Male/female ratio was 0.84. With respect to the site of tumor, tumor was located in cervical esophagus in 7 cases (4.2%), upper thoracic in 5 patients (3%), middle thoracic in 64 patients (38.6%), lowers thoracic in 68 cases (41%), and cardia in 22 cases (13.2%). There was a significant difference among the site of tumor in different age groups (P = 0.021) and different sex groups (P = 0.001). In men, EC usually involves the lower parts, whereas in women it usually involves the upper parts of esophagus. Squamous cell carcinoma was the most common type of EC in all age groups, but the prevalence rate of adenocarcinoma seems to increase with age (P = 0.045). CONCLUSIONS: Demographic and histopathologic pattern of esophageal cancer in northwestern region of Iran is different from its histopathologic pattern in western countries in accordance with other reports from Golestan province in north-eastern region of Iran.
Asunto(s)
Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Adenocarcinoma/epidemiología , Adenocarcinoma/terapia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/terapia , Estudios Transversales , Neoplasias Esofágicas/epidemiología , Neoplasias Esofágicas/terapia , Femenino , Humanos , Irán/epidemiología , Masculino , Persona de Mediana Edad , Tasa de Supervivencia , Resultado del TratamientoAsunto(s)
Antineoplásicos/efectos adversos , Erupciones por Medicamentos/diagnóstico , Dermatosis de la Mano/diagnóstico , Hidroxiurea/efectos adversos , Enfermedades del Pene/diagnóstico , Úlcera Cutánea/diagnóstico , Anciano , Antineoplásicos/administración & dosificación , Enfermedad Crónica , Diagnóstico Diferencial , Erupciones por Medicamentos/etiología , Erupciones por Medicamentos/patología , Dermatosis de la Mano/inducido químicamente , Dermatosis de la Mano/patología , Humanos , Hidroxiurea/administración & dosificación , Leucemia Mieloide/tratamiento farmacológico , Masculino , Enfermedades del Pene/inducido químicamente , Enfermedades del Pene/patología , Úlcera Cutánea/inducido químicamente , Úlcera Cutánea/patologíaRESUMEN
Basal cell carcinoma (BCC) is the most common cutaneous skin malignancy. BCC generally has a clinical course characterized by slow growth, minimal soft tissue invasiveness, and a high cure rate. Occasionally, however, BCC behaves aggressively with deep invasion, recurrence, and potential regional and distant metastasis. Several factors, including tumor size, duration, histology, and perineural spread, have been postulated as markers of the aggressive BCC phenotype. It is undetermined whether intrinsic biological factors within certain subsets of BCC predispose these tumors toward an inherently aggressive behavior, or whether any BCC with inadequate early management may assume this phenotype. Review of the pertinent literature on this topic suggests that both intrinsic biological factors and extrinsic management factors play a role in the development and progression of aggressive BCC.
Asunto(s)
Carcinoma Basocelular/patología , Carcinoma Basocelular/terapia , Carcinoma Basocelular/irrigación sanguínea , Carcinoma Basocelular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Recurrencia , Factores de RiesgoRESUMEN
Vascular endothelial cell growth factor (VEGF) is essential for angiogenesis. Atrial natriuretic peptide (ANP) inhibits the production of VEGF, but whether this important vascular peptide also inter- rupts VEGF signaling to angiogenesis is unknown. In cultured bovine aortic endothelial cells, VEGF significantly stimulated extracellular signal-regulated protein kinase activity and phosphorylation, which was inhibited 60% by coincubation with ANP or a natriuretic peptide clearance receptor specific ligand (NPRC), C-type NAP-(4-23) [C-ANP-(4-23)]. VEGF also stimulated c-Jun N-terminal kinase (JNK) and p38 activities/phosphorylation that were prevented by the two natriuretic peptides (NP). A specific NP guanylate cyclase (GC) receptor antagonist, HS-142-1, blocked the actions of ANP [but not those of C-ANP-(4-23)], supporting the involvement of both GC and NPRC receptors. VEGF and expression of constituitively active JNK each stimulated the synthesis of cyclin D1 and increased the activity of the cyclin-dependent kinase-4, which was inhibited 55% by ANP. VEGF induced endothelial cell proliferation and migration, which was significantly blocked by NP or by expressing a dominant negative JNK-1. VEGF stimulated human microvascular endothelial cells to form capillary tubes, which was significantly inhibited by expressing dominant negative JNK-1 and by NP. Therefore, VEGF induction of critical steps in angiogenesis is enhanced through JNK activation. The actions are significantly prevented by NP, which act through both the NPRC and GC receptors to block growth factor signaling. Thus, NP are candidate antiangiogenesis factors that inhibit both the synthesis and function of VEGF.
Asunto(s)
Factor Natriurético Atrial/farmacología , Factores de Crecimiento Endotelial/fisiología , Linfocinas/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Capilares/citología , Capilares/fisiología , Bovinos , Movimiento Celular/fisiología , Ciclina D1/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Péptidos/farmacología , Receptores del Factor Natriurético Atrial/metabolismo , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.
Asunto(s)
Citocalasina B/farmacología , Diacetil/análogos & derivados , Quinasa de Cadena Ligera de Miosina/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/enzimología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células CACO-2 , Citocalasina D/farmacología , Diacetil/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Glucosa/metabolismo , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Miosinas/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfoproteínas/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Estrogen is important for the primary prevention of vascular disease in young women, but the mechanisms of protection at the vascular cell are still largely unknown. Although traditionally thought of as a nuclear transcription factor, the estrogen receptor has also been identified in the cell plasma membrane to signal but serve largely undefined roles. Here we show that estradiol (E2) rapidly activates p38beta mitogen-activated protein kinase in endothelial cells (EC), which activates the mitogen-activated protein kinase-activated protein kinase-2 and the phosphorylation of heat shock protein 27. The sex steroid preserves the EC stress fiber formation and actin and membrane integrity in the setting of metabolic insult. E2 also prevents hypoxia-induced apoptosis and induces both the migration of EC and the formation of primitive capillary tubes. These effects are reversed by the inhibition of p38beta, by the expression of a dominant-negative mitogen-activated protein kinase-activated protein kinase-2 protein, or by the expression of a phosphorylation site mutant heat shock protein 27. E2 signaling from the membrane helps preserve the EC structure and function, defining potentially important vascular-protective effects of this sex steroid.
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/fisiología , Estradiol/farmacología , Proteínas de Choque Térmico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Aorta , Apoptosis/efectos de los fármacos , Bovinos , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Antagonistas de Estrógenos/farmacología , Femenino , Proteínas de Choque Térmico HSP27 , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Microcirculación , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fosfatos/metabolismo , Fosforilación , Piridinas/farmacología , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Chemotherapy or irradiation treatment induces breast cancer cell apoptosis, but this can be limited by estradiol (E2) through unknown mechanisms. To investigate this, we subjected estrogen receptor-expressing human breast cancer cells (MCF-7 and ZR-75-1) to paclitaxel (taxol) or to UV irradiation. Marked increases in cell apoptosis were induced, but these were significantly reversed by incubation with E2. Taxol or UV stimulated c-Jun N-terminal kinase (JNK) activity, which was inhibited by E2. Expression of a dominant-negative Jnk-1 protein strongly prevented taxol- or UV-induced apoptosis, whereas E2 inhibition of apoptosis was reversed by expression of constituitively active Jnk-1. As targets for participation in apoptosis, Bcl-2 and Bcl-xl were phosphorylated in response to JNK activation by taxol or UV; this was prevented by E2. Taxol or UV activated caspase activity in a JNK-dependent fashion and caused the cleavage of procaspase-9 to caspase-9, each inhibited by E2. Independently, the steroid also activated extracellular signal-regulated protein kinase activity, which contributed to the antiapoptotic effects. We report novel and rapid mechanisms by which E2 prevents chemotherapy or radiation-induced apoptosis of breast cancer, probably mediated through the plasma membrane estrogen receptor.
Asunto(s)
Apoptosis/fisiología , Receptores de Estrógenos/fisiología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama , Caspasa 9 , Caspasas/metabolismo , Membrana Celular/fisiología , Precursores Enzimáticos/metabolismo , Estradiol/farmacología , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de la radiación , Paclitaxel/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/efectos de la radiación , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Rayos Ultravioleta , Proteína bcl-XRESUMEN
Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.
Asunto(s)
Factor Natriurético Atrial/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas RGS/fisiología , Transporte Biológico , Endotelina-3/farmacología , GTP Fosfohidrolasas/efectos de los fármacos , FosforilaciónRESUMEN
The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERalpha or ERbeta into Chinese hamster ovary (CHO) cells. Transfection of ERalpha resulted in a single transcript by Northern blot, specific binding of labeled 17beta-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (K(d)S) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERbeta3 also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERalpha or CHO-ERbeta activated Galphaq and G(alpha)s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-beta-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-beta-E2 in ERbeta-expressing CHO, but was inhibited in CHO-ERalpha cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-beta-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERbeta. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERbeta and ERalpha.
Asunto(s)
Membrana Celular/fisiología , Regulación de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos , Receptores de Estrógenos/genética , Adenilil Ciclasas/análisis , Animales , Unión Competitiva , Northern Blotting , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Membrana Celular/genética , Cricetinae , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión al GTP/análisis , Inositol 1,4,5-Trifosfato/análisis , Inositol 1,4,5-Trifosfato/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 1 Activada por Mitógenos , Radioinmunoensayo , Receptores de Estrógenos/fisiología , Conteo por Cintilación , Transducción de Señal , Timidina/metabolismo , TransfecciónRESUMEN
Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS.
Asunto(s)
Factores de Crecimiento Endotelial/fisiología , Linfocinas/fisiología , Síndrome de Hiperestimulación Ovárica/fisiopatología , Actinas/análisis , Adulto , Líquidos Corporales/química , Permeabilidad de la Membrana Celular , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Factores de Crecimiento Endotelial/análisis , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Humanos , Uniones Intercelulares/ultraestructura , Transporte Iónico/efectos de los fármacos , Linfocinas/análisis , Linfocinas/farmacología , Óxido Nítrico/farmacología , Folículo Ovárico/química , Sodio/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Ligand binding to vascular endothelial cell growth factor (VEGF) receptors activates the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK). Possible cross-communication of ERK and JNK effecting endothelial cell (EC) actions of VEGF is poorly understood. Incubation of EC with PD 98059, a specific mitogen-activated protein kinase kinase inhibitor, or transfection with Y185F, a dominant negative ERK2, strongly inhibited VEGF-activated JNK. JNK was also activated by ERK2 expression in the absence of VEGF, inhibited 82% by co-transfection with dominant negative SEK-1, indicating upstream activation of JNK by ERK. VEGF-stimulated JNK activity was also reversed by dominant negative SEK-1. Other EC growth factors exhibited similar cross-activation of JNK through ERK. VEGF stimulated the nuclear incorporation of thymidine, reversed 89% by PD 98059 and 72% by Y185F. Dominant negative SEK-1 or JNK-1 also significantly reduced VEGF-stimulated thymidine incorporation. Expression of wild type Jip-1, which prevents JNK nuclear translocation, inhibited VEGF-induced EC proliferation by 75%. VEGF stimulated both cyclin D1 synthesis and Cdk4 kinase activity, inhibited by PD 98059 and dominant negative JNK-1. Important events for VEGF-induced G1/S progression and cell proliferation are enhanced through a novel ERK to JNK cross-activation and subsequent JNK action.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Linfocinas/farmacología , Proteínas Quinasas Activadas por Mitógenos , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Endothelins (ET) and VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) are products mainly of endothelial cells, which are also regulated via autocrine and paracrine pathways by these peptides. As a follow-up to our focus on vascular factors in ulcer pathogenesis and healing, we review here our recent studies with ET-1 and VEGF/VPF in animal models and human subjects. Our new results demonstrated a rapid and time-dependent release of ET-1 into the systemic circulation after intragastric administration of ethanol or HCI in rats, and ethanol in humans. The ET-1 release preceded the development of hemorrhagic erosions in both species and might be used as a diagnostic tool to noninvasively quantify acute gastric mucosal lesions. The development of solitary duodenal ulcers in the rat was preceded only by an organ- (involving only the duodenum and not the stomach) and molecule-specific (induced only by cysteamine and not by the nonulcerogenic analog ethanolamine) rapid local release of ET-1. The severity of cysteamine-induced duodenal ulcers was dose-dependently decreased by pretreatment with ET-1 antibodies or antagonist bosentan. A single intragastric dose of VEGF/VPF resulted in gastroprotection against ethanol, while daily intragastric treatment with the peptide for three weeks stimulated angiogenesis in the base of cysteamine-induced duodenal ulcers and accelerated ulcer healing. Thus, modulation of vascular factors seems to be sufficient for both acute gastroprotection and chronic duodenal ulcer healing.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Endotelinas/metabolismo , Linfocinas/metabolismo , Úlcera Péptica/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Humanos , Úlcera Péptica/diagnóstico , Úlcera Péptica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The proliferation of cultured astrocytes is positively and negatively regulated, respectively, by the endogenous neuropeptides, endothelin-3 (ET-3) and atrial natriuretic peptide (ANP). Here, we determined the important steps for the modulation by ET and ANP of G1 to S phase cell cycle progression. ET-3 stimulated an increased number of fetal rat diencephalic astrocytes to progress through G1/S, and this was blocked significantly by ANP. ET augmented the gene expression and/or protein production of D-type, A and E cyclins, whereas ANP inhibited these events significantly. ET also stimulated the activation of the cyclin-dependent kinases Cdk2, Cdk4, and Cdk6, directed against the retinoblastoma protein pRb, and this was inhibited by as much as 80% by ANP. As an additional mechanism of cell cycle restraint, ANP stimulated the production of multiple cyclin-dependent kinase inhibitory (CKI) proteins, including p16, p27, and p57. This was critical because antisense oligonucleotides to each CKI reversed ANP-induced inhibition of ET-stimulated DNA synthesis by as much as 85%. CKI antisense oligonucleotides also reversed the ANP inhibition of Cdk phosphorylation of pRb. In turn, ET inhibited ANP-stimulated production of the CKIs, thereby promoting cell cycle progression. Specific and changing associations of the CKI with Cdk2 and Cdk4 were stimulated by ANP and inhibited by ET. Our findings identify several mechanisms by which endogenous modulators of astrocyte proliferation can control the G1-S progression and indicate that multiple CKIs are necessary to restrain cell cycle progression in these cells.
Asunto(s)
Astrocitos/citología , Ciclinas/metabolismo , Fase G1 , Fase S , Animales , Células Cultivadas , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteína de Retinoblastoma/metabolismoRESUMEN
Important vascular proteins such as endothelin-1 (ET-1) promote the development of cardiovascular diseases. Oestrogen, and perhaps progesterone, prevent the development of vascular disease in women through incompletely understood cellular mechanisms. We hypothesized that oestradiol or progesterone might regulate the production of ET-1 as a potential novel mechanism. We found that serum and angiotensin II (AII) significantly stimulated ET-1 secretion from cultured bovine aortic endothelial cells, inhibited 50-75% by oestradiol or by progesterone. Serum and AII stimulated ET-1 mRNA levels, inhibited at least 70% by oestradiol and by progesterone. Serum stimulated ET-1 transcription mainly through the first 43 nucleotides of the ET-1 promoter, but oestradiol and progesterone did not inhibit this. In contrast, AII stimulated ET-1 transcription through nucleotides -143 to -98, specifically involving an activator protein-1 (AP-1) site at -102. Oestradiol and progesterone caused a 60-70% inhibition of AII-stimulated wild-type construct -. 143ET-1/CAT activity (CAT is chloramphenicol acyltransferase). AII-stimulation of ET-1 transcription was critically dependent on stimulation of mitogen-activated protein kinase (erk) activity, inhibited by oestradiol and progesterone. In summary, we found that sex steroids inhibit AII-induced erk signalling to the ET-1 transcriptional programme. This novel mechanism of negative transcriptional regulation by oestradiol and progesterone decreases the production of ET-1, potentially contributing to the vascular protective effects of these steroids.
Asunto(s)
Endotelina-1/biosíntesis , Endotelio Vascular/efectos de los fármacos , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Progesterona/farmacología , Transcripción Genética , Angiotensina II , Animales , Aorta , Bovinos , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Medios de Cultivo , Medio de Cultivo Libre de Suero , Endotelina-1/genética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Semivida , Humanos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Estrogen (E) has been identified in epidemiologic and prospective studies to protect against the development of cardiovascular disease in women. It is unclear whether progesterone (P) is similarly beneficial. The mechanisms by which E or P might act are incompletely defined. One possibility is that sex steroids inhibit the proliferation of vascular smooth muscle, an early/important event in vascular pathology. We examined the ability of E and P to inhibit the growth of human umbilical vein smooth muscle cells (hUVSMC) in culture, when stimulated by serum or the mitogen, endothelin-1 (ET-1). Serum and ET-1 stimulated hVSMC cell numbers by approximately 110% and 43% respectively, compared with control, after 3 days in culture. This stimulation was maximally reversed 75% by E and 64% by P. No synergistic or additive effects of the two steroids were found. ET-1 and serum stimulated mitogen-activated protein kinase (MAP-K) and MAP-kinase kinase activities, and these were critical for mitogenesis. Mitogen-stimulated MAP-kinase kinase and MAP-K activities were significantly inhibited by either E or P. The steroids also inhibited mitogen-stimulated c-fos and c-myc, downstream targets for MAP-K action. Critical signaling and molecular events through which mitogens stimulate VSMC proliferation can be significantly inhibited by E or P, providing a potential cellular mechanism for their vascular protective actions.
Asunto(s)
Estrógenos/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Progesterona/farmacología , Northern Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , ADN/metabolismo , Endotelina-1/farmacología , Activación Enzimática , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/genética , Genes myc/genética , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mitógenos/farmacología , Músculo Liso Vascular/química , Embarazo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-myc/análisis , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Albúmina Sérica Bovina/farmacología , Timidina/metabolismo , Tritio , Venas Umbilicales/química , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
The proliferation of vascular endothelial cells (EC) is an important event in angiogenesis. The synthesis of the EC growth factor, vascular endothelial cell growth factor (VEGF), is stimulated by a variety of activators; but the effects of important vasoactive peptides are not well understood, and there are no known natural inhibitors of VEGF production. We found that the vasoactive peptides endothelin (ET)-1 and ET-3 stimulated the synthesis of VEGF protein 3-4-fold in cultured human vascular smooth muscle cells, comparable in magnitude to hypoxia. ET-1 and ET-3 acted through the ETA and ETB receptors, respectively, and signaling through protein kinase C was important. Atrial natriuretic peptide (ANP), C-type natriuretic peptide, and C-ANP-(4-23), a ligand for the natriuretic peptide clearance receptor, equipotently inhibited production of VEGF by as much as 88% and inhibited ET- or hypoxia-stimulated VEGF transcription. EC proliferation and invasion of matrix were stimulated by VEGF secreted into the medium by ET-incubated vascular smooth muscle cells. This was inhibited by ANP. Our results identify the natriuretic peptides as the first peptide inhibitors of VEGF synthesis and indicate a novel mechanism by which vasoactive peptides could modulate angiogenesis.