RESUMO
PURPOSE: We have previously shown that the development of complications in the early pregnant decidua and myometrium in mice correlates with diabetes progression. In the current study, we investigated the influence of diabetes progression on the placental extracellular matrix (ECM) and on fetal development at the end of pregnancy. METHODS: Alloxan-induced type 1 diabetic female mice were bred either 30-50 days after diabetes induction (D) or 90-110D. Fetal and placental weights were registered at the 19th day of pregnancy together with analysis of gene expression, deposition and turnover of the placental ECM. RESULTS: The short-term diabetic group (30-50D) showed elevated embryonic losses and underweight fetuses (89%) with normal weight placentas. In contrast, the long-term group (90-110D) had increased malformations/fetal deaths and underweight fetuses (42%) and heavy placentas (50%). Normal-weight fetuses from the long-term group had placentas with either regular weight and fetal/placental weight ratio or increased weight and low fetal/placental weight ratio. Furthermore, the placentas of the short-term group showed alterations in the synthesis and deposition of collagen types I and V and in the activity of MMP2 whereas placentas of the 90-110D group presented alterations in collagen type III and V and MMP9. CONCLUSIONS: Diabetes progression promoted distinct outcomes in pregnancy. Modifications of both synthesis and turnover of ECM occurred even before changes of placental weight were detected. Adjustment of fetal/placental weight ratio or placental enlargement restored normal growth in part of the fetuses from the long-term group.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Matriz Extracelular/metabolismo , Desenvolvimento Fetal , Placenta/metabolismo , Animais , Feminino , Colágenos Fibrilares/metabolismo , Camundongos , GravidezRESUMO
Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1 h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.
Assuntos
Autorradiografia , Endométrio/citologia , Estradiol/análise , Estradiol/metabolismo , Receptores de Estrogênio/análise , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Animais , Sítios de Ligação , Endométrio/metabolismo , Estradiol/administração & dosagem , Feminino , Imuno-Histoquímica , Camundongos , Gravidez , Receptores de Estrogênio/química , Células Estromais/citologiaRESUMO
INTRODUCTION: We have previously shown that long-term type 1 diabetes affects the structural organization, contractile apparatus and extracellular matrix (ECM) of the myometrium during early pregnancy in mice. OBJECTIVE: This study aimed to identify which myometrial ECM components are affected by diabetes, including fibril-forming collagen types I, III and V, as well as proteoglycans, decorin, lumican, fibromodulin and biglycan. METHODS: Alloxan-induced type 1 diabetic female mice were divided into subgroups D1 and D2, formed by females that bred 90-100 and 100-110 days after diabetes induction, respectively. The deposition of ECM components in the myometrium was evaluated by immunohistochemistry/immunofluorescence. RESULTS: The subgroup D1 showed decreased deposition of collagen types I and III in the external muscle layer (EML) and decreased collagen types III and V in the internal muscle layer (IML). Collagen types I and III were decreased in both muscle layers of the subgroup D2. In addition, increased deposition of collagen types I and III and lumican as well as decreased collagen type V were observed in the connective tissue between muscle layers of D2. Lumican was decreased in the EML of the subgroups D1 and D2. Fibromodulin was repressed in the IML and EML of both D1 and D2. In contrast, decorin deposition diminished only in muscle layers of D2. No changes were noticed for biglycan. CONCLUSIONS: Subgroups D1 and D2 showed distinct stages of progression of diabetic complications in the myometrium, characterized by both common and specific sets of changes in the ECM composition.
Assuntos
Colágeno/metabolismo , Complicações do Diabetes/patologia , Miométrio/patologia , Complicações na Gravidez/etiologia , Proteoglicanas/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Camundongos , Miométrio/metabolismo , Gravidez , Complicações na Gravidez/patologiaRESUMO
High diesel exhaust particle levels are associated with increased health effects; however, knowledge on the impact of its chemical contaminant 1,2-naphthoquinone (1,2-NQ) is limited. We investigated whether postnatal and adult exposures to 1,2-NQ influence allergic reaction and the roles of innate and adaptive immunity. Male neonate (6 days) and adult (56 days) C57Bl/6 mice were exposed to 1,2-NQ (100 nM; 15 min) for 3 days, and on day 59, they were sensitized and later challenged with ovalbumin (OVA). Airway hyper-responsiveness (AHR) and production of cytokines, immunoglobulin E (IgE) and leukotriene B4 (LTB4) were measured in the airways. Postnatal exposure to 1,2-NQ activated dendritic cells in splenocytes by increasing expressing cell surface molecules (e.g., CD11c). Co-exposure to OVA effectively polarized T helper (Th) type 2 (Th2) by secreting Th2-mediated cytokines. Re-stimulation with unspecific stimuli (PMA and ionomycin) generated a mixed Th1 (CD4(+)/IFN-γ(+)) and Th17 (CD4(+)/IL-17(+)) phenotype in comparison with the vehicle-matched group. Postnatal exposure to 1,2-NQ did not induce eosinophilia in the airways at adulthood, although it evoked neutrophilia and exacerbated OVA-induced eosinophilia, Th2 cytokines, IgE and LTB4 production without affecting AHR and mast cell degranulation. At adulthood, 1,2-NQ exposure evoked neutrophilia and increased Th1/Th2 cytokine levels, but failed to affect OVA-induced eosinophilia. In conclusion, postnatal exposure to 1,2-NQ increases the susceptibility to antigen-induced asthma. The mechanism appears to be dependent on increased expression of co-stimulatory molecules, which leads to cell presentation amplification, Th2 polarization and enhanced LTB4, humoral response and Th1/Th2 cytokines. These findings may be useful for future investigations on treatments focused on pulmonary illnesses observed in children living in heavy polluted areas.
Assuntos
Envelhecimento/imunologia , Poluentes Atmosféricos/toxicidade , Exposição por Inalação/efeitos adversos , Naftoquinonas/toxicidade , Pneumonia/induzido quimicamente , Hipersensibilidade Respiratória/induzido quimicamente , Emissões de Veículos/toxicidade , Imunidade Adaptativa/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Citocinas/imunologia , Suscetibilidade a Doenças/induzido quimicamente , Imunidade Inata/efeitos dos fármacos , Imunoglobulina E/imunologia , Exposição por Inalação/análise , Leucotrieno B4/imunologia , Masculino , Ovalbumina/imunologia , Pneumonia/imunologia , Hipersensibilidade Respiratória/imunologia , Emissões de Veículos/análiseRESUMO
We have previously shown the differential expression of versican in the mouse uterus under ovarian hormone influence. We also demonstrated there is not a direct correlation between mRNA levels and protein expression, suggesting posttranscriptional events, such as alteration in mRNA stability. This posttranscriptional effect may result in the elongation and stabilization of transcripts poly(A) tail. Thus, the aim of this study was to analyze whether estradiol (E2) regulates versican mRNA stability and expression in a dose-related and time-dependent manner. For this purpose female mice were ovariectomized and treated with a single injection of 0.1 or 10 µg E2. To block transcription a group of females received a single injection of alpha-amanitin before hormone administration. Uterine tissues were collected 30 min, 1, 3, 6, 12 and 24 h after treatments and processed for quantitative real time PCR (qPCR), RACE-PAT Assay and immunohistochemistry. qPCR showed that versican mRNA levels are higher than control from 3 to 24 h after E2 administration, whereas after transcription inhibition versican mRNA unexpectedly increases within 3 h, which can be explained when transcriptional blockers alter the degradation rate of the transcript, resulting in the superinduction of this mRNA. Accordingly, analysis of versican transcript poly(A) tail evidenced a longer product 3 h after treatment, but not after 12 h. Versican immunoreaction becomes conspicuous in the superficial stroma only 3 h after E2 injection, whereas the whole stroma is immunoreactive from 6 h onward. These results demonstrate that E2 modulates versican at the transcriptional and posttranscriptional levels in a time-dependent manner.
Assuntos
Estradiol/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/metabolismo , Versicanas/genética , Alfa-Amanitina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Poli A , Poliadenilação/efeitos dos fármacos , Fatores de Tempo , Versicanas/metabolismoRESUMO
BACKGROUND: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. METHODS: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. RESULTS: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. CONCLUSIONS: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.
Assuntos
Biglicano/biossíntese , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Decorina/biossíntese , Estradiol/farmacologia , Proteínas da Matriz Extracelular/biossíntese , Sulfato de Queratano/biossíntese , Acetato de Medroxiprogesterona/farmacologia , Proteoglicanas/biossíntese , Útero/metabolismo , Animais , Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Lumicana , Camundongos , Útero/efeitos dos fármacosRESUMO
It is known that the development of diabetic complications in human pregnancy is directly related to the severity and the duration of this pathology. In this study, we developed a model of long-term type 1 diabetes to investigate its effects on the cytoarchitecture, extracellular matrix and cell proliferation during the first adaptation phase of the myometrium for pregnancy. A single dose of alloxan was used to induce diabetes in mice prior to pregnancy. To identify the temporal effects of diabetes the mice were divided into two groups: Group D1 (females that became pregnant 90-100 days after alloxan); Group D2 (females that became pregnant 100-110 days after alloxan). Uterine samples were collected after 168 h of pregnancy and processed for light and electron microscopy. In both groups the histomorphometric evaluation showed that diabetes promoted narrowing of the myometrial muscle layers which was correlated with decreased cell proliferation demonstrated by PCNA immunodetection. In D1, diabetes increased the distance between muscle layers and promoted oedema. Contrarily, in D2 the distance between muscle layers decreased and, instead of oedema, there was a markedly deposition of collagen in the myometrium. Ultrastructural analysis showed that diabetes affects the organization of the smooth muscle cells and their myofilaments. Consistently, the immunoreaction for smooth muscle α-actin revealed clear disorganization of the contractile apparatus in both diabetic groups. In conclusion, the present model demonstrated that long-term diabetes promotes significant alterations in the myometrium in a time-sensitive manner. Together, these alterations indicate that diabetes impairs the first phenotypic adaptation phase of the pregnant myometrium.
Assuntos
Diabetes Mellitus Experimental/patologia , Miométrio/patologia , Complicações na Gravidez/patologia , Adaptação Fisiológica/fisiologia , Animais , Divisão Celular/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Modelos Animais de Doenças , Feminino , Idade Gestacional , Humanos , Camundongos , Microscopia Eletrônica , Músculo Liso/patologia , Músculo Liso/fisiologia , Músculo Liso/ultraestrutura , Miométrio/fisiologia , Miométrio/ultraestrutura , Gravidez , Complicações na Gravidez/fisiopatologia , Fatores de Tempo , Contração Uterina/fisiologiaRESUMO
Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to investigate whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; TNF-alpha; IL-1 beta; and IL-6 gene expression; and I kappaB kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate-induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and TNF-alpha and IL-6 messenger RNA (mRNA) ( approximately 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, TNF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage infiltration and normalized IKK-alpha/beta phosphorylation; TNF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects.
Assuntos
Anti-Inflamatórios não Esteroides , Ácidos Heptanoicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Resistência à Insulina/fisiologia , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Pirróis/uso terapêutico , Glutamato de Sódio , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Atorvastatina , Glicemia/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Transportador de Glucose Tipo 4/biossíntese , Transportador de Glucose Tipo 4/metabolismo , Quinase I-kappa B/biossíntese , Imuno-Histoquímica , Lipídeos/sangue , Masculino , Camundongos , Obesidade/sangue , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. METHODS: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. RESULTS: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. CONCLUSION: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.
Assuntos
Ciclo Estral/fisiologia , Útero/fisiologia , Versicanas/genética , Versicanas/metabolismo , Animais , Diestro/fisiologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Estro/fisiologia , Feminino , Técnicas Imunoenzimáticas , Medroxiprogesterona/metabolismo , Medroxiprogesterona/farmacologia , Camundongos , Proestro/fisiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/efeitos dos fármacosRESUMO
In the pregnant mouse uterus, small leucine-rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 2008. (c) 2008 Wiley-Liss, Inc.