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Aline David-Silva,1 João Victor Esteves,1 Mychel Raony PT Morais,2 Helayne Soares Freitas,1 Telma Maria Zorn,2 Maria Lucia Correa-Giannella,3 Ubiratan Fabres Machado1 1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; 2Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; 3Laboratório de Carboidratos e Radioimunoensaio, LIM-18, Hospital das Clinicas HCFMUSP, Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, BrazilCorrespondence: Ubiratan Fabres MachadoDepartment of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 1524, São Paulo, SP 05508-900, BrazilTel +55 11 30917494Email ubiratan@icb.usp.brPurpose: NAFLD is a hepatic component of type 2 diabetes mellitus (T2D), in which impaired hepatic glucose production plays an important role. Inhibitors of sodium glucose transporter 2 (SGLT2) reduce glycemia and exert beneficial effects on diabetic complications. Recently, dual SGLT1/2 inhibition has been proposed to be more effective in reducing glycemia. We hypothesized that improving hepatic glucose metabolism induced by SGLT1/2 inhibition could be accompanied by beneficial effects on NAFLD progression.Methods: Glycemic homeostasis, hepatic glucose production and NAFLD features were investigated in obese T2D mice, treated with SGLT1/2 inhibitor phlorizin for 1 week.Results: T2D increased glycemia; insulinemia; hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and glucose transporter 2 (Slc2a2 gene); hepatocyte nuclear factors 1A/4A/3B-binding activity in Slc2a2; endogenous glucose production; liver weight, plasma transaminase concentration as well as hepatic inflammation markers, and induced histological signals of non-alcoholic steatohepatitis (NASH, according to NASH-CRN Pathology Committee System). Phlorizin treatment restored all these parameters (mean NASH score reduced from 5.25 to 2.75 P< 0.001); however, plasma transaminase concentration was partially reverted and some hepatic inflammation markers remained unaltered.Conclusion: NAFLD accompanies altered hepatic glucose metabolism in T2D mice and that greatly ameliorated through short-term treatment with the dual SGLT1/2 inhibitor. This suggests that altered hepatic glucose metabolism participates in T2D-related NAFLD and highlights the pharmacological inhibition of SGLTs as a useful approach not only for controlling glycemia but also for mitigating development and/or progression of NAFLD.Keywords: GLUT2, PEPCK, G6Pase, phlorizin, NASH

Aims/hypothesis: The discovery of osteocalcin, a protein synthetized by osteoblasts, as a hormone that has positive effects on insulin resistance, contributed to support the concept of bone as an endocrine organ. However, very little is known about the molecular pathways involved in osteocalcin improved-insulin resistance. The present study aimed to investigate the mechanisms of action of osteocalcin on insulin resistance and inflammation in obese mice and 3T3-L1 adipocytes., Methods and Results: Lean control, saline-treated obese and uncarboxylated osteocalcin (uOC)-treated obese mice were subjected to insulin tolerance test in vivo. Blood was collect for biochemical/metabolic profile analysis; and, skeletal muscle, white adipose tissue (WAT) and bone were collected for protein (Western blotting) and mRNA (RT-qPCR) analysis. uOC effects on insulin resistance and inflammation were also investigated in 3T3-L1 adipocytes challenged with tumor necrosis factor. Osteocalcin treatment improved in vivo insulin resistance in obese mice. In WAT, osteocalcin had positive effects such as (1) WAT weight reduction; (2) upregulation of glucose transporter (GLUT) 4 protein and its mRNA (Slc2a4); (3) improved insulin-induced AKT phosphorylation; (4) downregulation of several genes involved in inflammation and inflammassome transcriptional machinery, and (5) reduction of the density of macrophage in crown-like structures (histomorphometrical analysis). Notably, in 3T3-L1 adipocytes, osteocalcin restored Slc2a4/GLUT4 content and reduced the expression of inflammatory genes after TNF-a challenge; moreover, osteocalcin treatment increased AKT phosphorylation induced by insulin. Finally, it was observed that in bone, osteocalcin improves insulin resistance by increasing insulin-induced AKT phosphorylation and reducing the expression of genes involved in bone insulin resistance, resulting in increased secretion of uncarboxylated osteocalcin in circulation., Conclusion: We provided some mechanisms of action for osteocalcin in the amelioration of insulin resistance in obesity: in WAT, osteocalcin improves insulin resistance by decreasing inflammation, and increasing insulin signaling and the expression of Slc2a4/GLUT4; and, in bone, osteocalcin increases the secretion of uncarboxylated osteocalcin by improving insulin resistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas-Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The embryo-implantation promotes deep changes in the uterus resulting in the formation of a new structure at the maternal-fetal interface, the decidua. Decidualization can also be induced in pseudopregnant rodents resulting in a structure called deciduoma that is morphologically and functionally similar to the decidua. Previous studies from our and other laboratories demonstrate that in rodents, decidualization of the endometrium requires remarkable remodeling of the endometrial extracellular matrix (ECM) that is mainly coordinated by estradiol and progesterone. The influence of the embryo in this process, however, has not yet been investigated. To enlarge the knowledge on this subject, the present study investigates the behavior of a set of ECM molecules, in the absence of paracrine cues originated from the embryo. For that deciduoma was induced in pseudopregnant Swiss mice, and the distribution of collagen types I, III, IV, V and the proteoglycans decorin and biglycan was investigated by immunolabeling from the fifth to the eighth day of pseudopregnancy. It was observed the deposition of collagen types III and IV as well as decorin and biglycan was similar to that previously described by our group in the decidua. However, in the absence of the embryo, some differences occur in the distribution of collagen types I and V, suggesting that beside the major role of ovarian hormones on the endometrial ECM remodeling, molecular signals originated from the conceptus may influence this process.

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.

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.

Rapid actions of T3 on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T3 on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T3. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T3 dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T3 chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T3 is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

Introduction: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies., Methods: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM., Results: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90-110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy., Conclusions: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Signal transducers and activators of transcription 3 (Stat3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of Stat3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (Socs3) expression. Stat3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, Stat3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for Stat3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, Socs3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of Stat3, its serine activation and Socs3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice.

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.

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis., (Copyright © 2011 John Wiley & Sons, Ltd.)

Our goal was to study the effect of Gp₄G on skin tissues and unravel its intracellular action mechanisms. The effects of Gp₄G formulation, a liposomic solution of Artemia salina extract, on several epidermal, depmal, and hair follicle structures were quantified. A 50% increase in hair length and a 30% increase in the number of papilla cells were explained by the changes in the telogen/anagen hair follicle phases. Increasing skin blood vessels and fibroblast activation modified collagen arrangement in dermal tissues. Imunohistochemical staining revealed expressive increases of versican (VER) deposition in the treated animals (68%). Hela and fibroblast cells were used as in vitro models. Gp₄G enters both cell lines, with a hyperbolic saturation profile inducing an increase in the viabilities of Hela and fibroblast cells. Intracellular ATP and other nucleotides were quantified in Hela cells showing a 38% increase in intracellular ATP concentration and increases in the intracellular concentration of tri- , di- , and monophosphate nucleosides, changing the usual quasi-equilibrium state of nucleotide concentrations. We propose that this change in nucleotide equilibrium affects several biochemical pathways and explains the cell and tissue activations observed experimentally.

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β(2)-adrenoceptor (β2-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR and α(2C)-AR (α(2A) /α(2C)-ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α(2A) /α(2C)-ARKO versus wild-type (WT) mice, micro-computed tomographic (µCT) analysis showed increased, better connected, and more plate-shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate-resistant acid phosphatase (TRACP) and receptor activator of NF-κB (RANK), which are osteoclast-related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine-regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β(2)-AR mRNA expression also was similar in KO and WT littermates, whereas α(2A)-, α(2B)- and α(2C)-AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected α(2A)-, α(2B)-, α(2C)- and β(2)-ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5-day-old mouse fetuses and 35-day-old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α(2)-AR agonist clonidine and to the nonspecific α-AR antagonist phentolamine. These findings suggest that β(2)-AR is not the single adrenoceptor involved in bone turnover regulation and show that α(2)-AR signaling also may mediate the SNS actions in the skeleton., (Copyright © 2011 American Society for Bone and Mineral Research.)

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.

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., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Background: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption., Methodology/principal Findings: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues., Conclusions/significance: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

Glutamine is the most important donor of NH(3) in kidney playing an important role in acid-base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1beta and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.

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., ((c) 2010. Published by Elsevier Inc.)

In the pregnant mouse endometrium, collagen fibrillogenesis is characterized by the presence of very thick collagen fibrils which are topographically located exclusively within the decidualized stroma. This dynamic biological process is in part regulated by the small leucine-rich proteoglycans decorin and biglycan. In the present study we utilized wild-type (Dcn(+/+)) and decorin-deficient (Dcn(-/-)) time-pregnant mice to investigate the evolution of non-decidualized and decidualized collagen matrix in the uterine wall of these animals. Ultrastructural and morphometric analyses revealed that the organization of collagen fibrils in the pregnant endometrium of both non-decidualized and decidualized stroma showed a great variability of shape and size, regardless of the genotype. However, the decidualized endometrium from Dcn(-/-) mice contained fibrils with larger diameter and more irregular contours as compared to the wild-type littermates. In the Dcn(-/-) animals, the proportion of thin (10-50 nm) fibrils was also higher as compared to Dcn(+/+) animals. On day 7 of pregnancy, biglycan was similarly localized in the decidualized endometrium in both genotypes. Lumican immunostaining was intense both in decidualized and non-decidualized stroma from Dcn(-/-) animals. The present results support previous findings suggesting that decorin participates in uterine collagen fibrillogenesis. In addition, we suggest that the absence of decorin disturbs the process of lateral assembly of thin fibrils, resulting in very thick collagen fibrils with irregular profiles. Our data further suggest that decorin, biglycan and lumican might play an interactive role in collagen fibrillogenesis in the mouse endometrium, a process modulated according to the stage of pregnancy.

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.

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.

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly ( approximately 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly ( approximately 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring.

Problem Cells responsible for the synthesis of follistatin and activin A in the pregnant mouse endometrium have not been characterized. Method of study Immunocytochemistry was used to determine the distribution of follistatin and activin A in the pregnant mouse uterus. Results Follistatin was detected in the endometrium prior to decidualization and embryo implantation. Follistatin was not seen in fully decidualized cells, being restricted to non-decidualized fibroblasts and cells in the process of decidualization. In contrast, activin A was detected exclusively in mature antimesometrial decidual cells during involution. After day eleven of pregnancy, both substances were identified in the extracellular matrix of the spongiotrophoblast. Conclusion As previously described for decidual prolactin-related protein and the proteoglycan perlecan, follistatin and activin A were detected in the extracellular matrix of the spongiotrophoblast, suggesting that this region acts as reservoir for these growth factors in the mouse placenta.

The morphogenesis of tissues and organs requires dynamic changes in cells and in extracellular matrix components. It is known that various extracellular matrix molecules are of fundamental importance for gonad differentiation and growth. In the adult testis, the extracellular matrix represents an important component of the interstitium, participating in the transport of biologically active substances needed for the communication between different cellular components, as well as for the regulation of spermatogenesis and hormone production. The present study was designed in order to identify the proteoglycans biglycan, decorin and perlecan, as well as the glycosaminoglycan hyaluronan, during testis development in mouse embryos. Our data profile the chronology of testis differentiation, as well as the distribution of these extracellular matrix components during testis development in mice. We show that these extracellular matrix molecules are present early in the development of the gonads, suggesting that they play a role in gonad development. In addition, we found no decorin in the testicular cords. Furthermore, of the proteoglycans analysed, only biglycan was seen surrounding immature Sertoli cells and Leydig cell precursors in the testicular cords. This indicates that specific sets of extracellular matrix molecules are required in the various compartments of the developing gonad.

Objective: We investigated factors that may be involved in the reduced leukocyte migration observed in intrauterine undernourished rats., Methods: Male Wistar rat offspring (8-9 wk of age) of dams fed during pregnancy with 50% less food than control dams were used to measure L-selectin expression (by flow cytometry), bone marrow cell count, blood cell count, laminin and type IV collagen in the basal membrane of venules of the spermatic fascia (by immunohistochemistry), total protein level and serum albumin, and the production of leukotriene B4 after stimulation with tumor necrosis factor-alpha and corticosterone plasma levels (by enzyme-linked immunosorbent assay)., Results: Hypocellularity in bone marrow and peripheral blood and reduced L-selectin expression were found in the undernourished rat offspring (UR) compared with nourished offspring (NR; P < 0.05). Type IV collagen in the basal membrane of the venules of the spermatic fascia was less in UR than in NR (P < 0.05). The total protein levels and serum albumin did not differ between the two groups. Leukotriene B4 production after stimulation with tumor necrosis factor-alpha was lower in UR (P < 0.05). These differences could not be attributed to circulating glucocorticoids levels, which were not different in the NR and UR groups., Conclusion: Our data suggest that all observed differences contribute to reduced leukocyte migration in undernourishment.

Problem: Identification of the cell types responsible for the synthesis of decidual prolactin-related protein (dPRP) in the pregnant mouse endometrium., Method of Study: Histochemistry and immunocytochemistry were used to determine peri-implantation dPRP and perlecan distribution in the mouse uterus., Results: We identified dPRP in pre-decidual and mature decidual cells from days 5 to 12 of pregnancy. On day 8, dPRP immunoreactivity was detected within cytoplasmic granules of a specific population of granulated decidual cells (GDCs). In mesometrial decidual cells, weak immunoreactivity was seen from days 7 to 14. Between days 11 and 14, dPRP was found in cytoplasm and in the extracellular matrix surrounding islands of spongiotrophoblast. Perlecan, a heparan sulfate proteoglycan, was co-localized with dPRP., Conclusion: GDCs are a putative source of dPRP in pregnant mice. Co-localization of perlecan with dPRP suggests that the former acts as a dPRP reservoir and facilitates its paracrine effect in developing placental tissues.

There are evidences that low-intensity red laser radiation is capable to accelerate wound healing. Nowadays, this therapy has been gradually introduced in clinical practice although mechanisms underlying laser effects are poorly understood. To better understand the photobiological effects of laser radiation, this study investigated by electron microscopy, immunohistochemistry and autoradiography the morphological and functional features of irradiated and none irradiated injured mice skin. Full-thickness skin lesions were created on the back of mice and irradiated on days 1, 5, 8, 12, and 15 post-wounding with a He-Ne laser (lambda=632.8nm), dose 1J/cm(2), exposure time 3min. Non-irradiated lesions were used as a control. The mice were inoculated with (3)H-proline and sacrificed one hour after on the 8th, 15th and 22nd days to histological and radioautographical analysis. The irradiated-lesions showed a faster reepithelization compared with control lesions. The irradiated dermis contained a higher number of activated fibroblasts compared to control group and, most of them showed several cytoplasmic collagen-containing phagosomes. In irradiated-lesions, smooth muscle alpha-actin positive cells predominated, which correspond to a higher number of myofibroblasts observed in the electron microscope. Moreover, laser radiation reduced the local inflammation and appears to influence the organization of collagen fibrils in the repairing areas. Quantitative autoradiography showed that the incorporation of (3)H-proline was significantly higher in irradiated-dermis on the 15th day post-wounding (p<0.05). These results suggest that laser radiation may accelerate cutaneous wound healing in a murine model.

Blastocyst reception and implantation require various adaptations of the uterine microenvironment. We have previously shown that in mice, remodeling of the extracellular matrix begins early in pregnancy, characterized by synthesis, degradation and alteration of collagen fibrillogenesis, accompanied by pregnancy stage-dependent expression of glycosaminoglycans and proteoglycans. Fibrillin-1 is a matrix glycoprotein that participates in the formation of elastic fibers and promotes cell adhesion through its integrin-binding domain. In the present study, we used light microscope immunohistochemistry to analyze the distribution of fibrillin-1 in the endometrial stroma of mice during estrous and diestrous, as well as in the pre- and postimplantation periods. Fibrillin-1 was found among endometrial fibroblasts and decidual cells, respectively, in the pre- and postimplantation periods. However, fibrillin-1 organization and distribution in the various regions of the endometrial stroma were found to be pregnancy stage dependent. Fibrillin-1 was also abundant in the basement membrane regions of blood vessels, as well as in the luminal and glandular epithelia. Fibrillin-1 at the maternal-fetal interface and in Reichert's membrane of embryos at up to 6 days of development might facilitate embryo expansion and fixation. Changes in the fibrillin-1 expression during the peri-implantation period suggest that fibrillin-1 plays a role in preparing the endometrium for embryo implantation., (Copyright 2007 S. Karger AG, Basel.)

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.

Decidualization in mice comprises a deep remodeling of extracellular matrix (ECM) components of the endometrium. In a previous biochemical study we showed that collagen types I and III are present in both pregnant and nonpregnant mouse endometrium, whereas collagen type V is expressed exclusively after the onset of decidualization. The distribution of collagen types in the pregnant mouse endometrium and possible changes of these molecular types in the different regions of the decidua is, however, not known. Using immunofluorescence and confocal microscopy we showed the presence of collagen types I, III, and V in the endometrial stroma of implantation and interimplantation sites from days 5 to 8 of pregnancy in the mouse. Collagen type III was chiefly expressed in the implantation sites and was the only collagen type to be present in the materno-fetal interface on the day of the embryo implantation. However, collagen type I was the predominant collagen in the interimplantation sites. Collagen type V was weakly expressed in the nondecidualized stroma during all periods but was expressed in larger amounts in the decidualized areas on day 7 of pregnancy, simultaneously with the accumulation of thick collagen fibrils in the same region. The highest immunofluorescence labeling for the three types of collagen was observed on day 7 when the antimesometrial decidual tissue achieved its greatest development. These data support previous studies that showed an intense ECM remodeling of the mouse endometrial stroma during the beginning of pregnancy. This outstanding remodeling may be important to stabilize placental anchorage.

We studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08+/-0.008 (n=34) to 7.18+/-0.018 (n=33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29+/-0.022 pH U/min (n=14) to 0.50+/-0.024 pH U/min (n=14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.

We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low-intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L(parallel) is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L(perpendicular) is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L(parallel) and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized.

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues., ((c) 2006 Wiley-Liss, Inc.)

We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.

The digitalics (digoxin and digitoxin) have an estrogenic action, confirmed by the presence of gynecomastia observed in men who are users of steroids, and it is also confirmed by the increase of vaginal trophism in postmenopausal female users, although little is known about the endometrium. These effects might result from the similarity of the chemical structures between the digitalics and estrogens. This study evaluated 27 female mice. Twenty-four mice were oophorectomized and in three of them the ovaries were manipulated, but they were not extirpated. Forty days after the surgery each group of three animals received a medication with a specific drug (digoxin, digitoxin or estradiol) for 2 weeks. Twenty-four hours after the last medication, all the animals were hysterectomized and the wombs were processed, cut and put on laminae. By histomorphometry, we analysed the area of the transverse sections of the endometrium, the area of the endometrial glands and the height of the uterine epithelium. The respective averages and the standard deviations were calculated. The results showed that digoxin neither presented an estrogenic action (p > 0.05) nor raised the power of the estradiol action (p > 0.05). Digitoxin promoted an estrogenic action on the glandular area (p < 0.05), and also raised the power of the estradiol action on the transverse section of the endometrium and on the glandular area (p < 0.05).

Unlabelled: We investigated the effects of GC-1, a TRbeta-selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF-I deficiency and the skeletal defects caused by hypothyroidism, GC-1 had no effect on serum IGF-I or on IGF-I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRbeta1., Introduction: We investigated the role of the thyroid hormone receptor beta1 (TRbeta1) on skeletal development of rats using the TRbeta-selective agonist GC-1., Materials and Methods: Twenty-one-day-old female rats (n = 6/group) were rendered hypothyroid (Hypo) and treated for 5 weeks with 0.3 ug/100 g BW/day of T3 (1xT3), 5xT3, or equimolar doses of GC-1 (1xGC-1 and 5xGC-1). Serum triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), and insulin-like growth factor (IGF)-I concentrations were determined by radioimmunoassay (RIA). BMD and longitudinal bone growth were determined by DXA. Trabecular bone histomorphometry and epiphyseal growth plate (EGP) morphometry were performed in the distal femur. Expressions of IGF-I protein and of collagen II and X mRNA were evaluated by immunohistochemistry and in situ hybridization, respectively. To determine hormonal effects on ossification, skeletal preparations of hypothyroid-, 5xGC-1-, and 5xT3-treated neonatal rats were compared., Results: Hypothyroidism impaired longitudinal body growth and BMD gain, delayed ossification, reduced the number of hypertrophic chondrocytes (HCs; 72% versus Euthyroid [Eut] rats; p < 0.001), and resulted in disorganized columns of EGP chondrocytes. Serum IGF-I was 67% reduced versus Eut rats (p < 0.001), and the expression of IGF-I protein and collagen II and X mRNA were undetectable in the EGP of Hypo rats. T3 completely or partially normalized all these parameters. In contrast, GC-1 did not influence serum concentrations or EGP expression of IGF-I, failed to reverse the disorganization of proliferating chondrocyte columns, and barely affected longitudinal growth. Nevertheless, GC-1 induced ossification, HC differentiation, and collagen II and X mRNA expression and increased EGP thickness to Eut values. GC-1-treated rats had higher BMD gain in the total tibia, total femur, and in the femoral diaphysis than Hypo animals (p < 0.05). These changes were associated with increased trabecular volume (48%, p < 0.01), mineralization apposition rate (2.3-fold, p < 0.05), mineralizing surface (4.3-fold, p < 0.01), and bone formation rate (10-fold, p < 0.01)., Conclusions: Treatment of hypothyroid rats with the TRbeta-specific agonist GC-1 partially reverts the skeletal development and maturation defects resultant of hypothyroidism. This finding suggests that TRbeta1 has an important role in bone development.

Problem: During early pregnancy in mice, there is recruitment of specific immune cells, remodeling of the endometrium, cell differentiation and synthesis of new molecules., Method of Study: Immunohistochemistry was used to determine the distribution of perlecan and syndecan-4 in the uteri before and after embryo implantation., Results: During pre-implantation, perlecan was identified in basement membranes and extracellular spaces of the endometrial stroma. In contrast, expression of syndecan-4 was quite weak. In the peri-implantation period, perlecan remained in the basement membranes, and it was no longer observed in the stroma and it was identified in the embryonic cells. On day 4 of pregnancy, syndecan-4 increased in the fibroblasts of the subepithelial stroma. After implantation, syndecan-4 was pronounced in pre-decidual and mature decidual cells., Conclusions: The coordinate balance between the pre- and post-implantation periods suggests a role of these two molecules in the adaptive modification of the uterine microenvironment to receive and implant the embryo.

Leukocyte adhesion to endothelial cells plays a key role in inflammatory processes associated with end-organ injury. Endothelin-1 (ET-1), which stimulates inflammatory processes, contributes to cardiovascular damage in deoxycorticosterone (DOCA)-salt hypertension. We investigated whether ETA receptor blockade modulates in vivo leukocyte-endothelial cell interactions and expression of cell adhesion molecules (CAM) involved in these processes. DOCA-salt and control uninephrectomized rats were treated with the ETA antagonist BMS182874 (40 mg/kg per day) or vehicle. Analysis of CAMs expression by reverse transcription-polymerase chain reaction and immunohistochemistry showed increased cardiac platelet selectin (P-selectin), detected mainly in endothelial cells, and vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1 (ICAM-1), in DOCA-salt rats. Cardiac expression of endothelial selectin (E-selectin) was decreased, whereas immunoreactivity to ED-1 and myeloperoxidase (MPO) activity, markers of macrophage and leukocyte infiltration, respectively, were increased in DOCA-salt. Leukocyte-endothelial cell interaction, functionally assessed in venules of internal spermatic fascia by intravital microscopy, was significantly altered in DOCA-salt rats as evidenced by increased leukocyte adhesion and decreased rolling. BMS182874 treatment normalized leukocyte-endothelium interactions, decreased cardiac VCAM-1 expression in DOCA and control groups, and had no effects on ICAM-1 expression. BMS182874 also increased E-selectin and abolished P-selectin expression in DOCA-salt, but not in control rats. The ETA antagonist reduced cardiac ED-1 content and MPO activity and prevented cardiac damage in DOCA-salt rats. These data indicate that ET-1 participates, via activation of ETA receptors, in altered leukocyte-endothelial cell interactions in DOCA-salt rats, possibly by modulating expression of CAMs, and that the inflammatory status is associated with cardiac damage in mineralocorticoid hypertension.

Primordial germ cells (PGCs) are the progenitor cells of the vertebrate germ line. These cells originate outside of the embryo and, through separation, migration, and colonization, arrive at the genital ridge, contributing to gonad development. Diverse extracellular matrix molecules are present along the PGC migratory pathway, permitting or inhibiting PGC displacement. Collagens and tenascin form the substratum for in vitro migration of neural crest cells and PGCs. However, little is known about the expression and distribution of these molecules during in situ PGC migration. Using immunohistochemistry, we identified tenascin-C and types I, III, and V collagen along the mouse PGC migration pathway. These molecules were spatiotemporally expressed in basement membranes of hindgut, coelomic epithelia, and mesonephric tubules and mesenchyme throughout the study. Our results complement previous data from our laboratory and contribute to building comprehension of the composition of the mouse PGC migratory pathway extracellular matrix, thereby enhancing understanding of the process.

Objective: This study was carried out to investigate the influence of low-intensity polarized visible laser radiation on the acceleration of skin wound healing., Background Data: Low-level laser therapy (LLLT) at adequate wavelength, intensity, and dose can accelerate tissue repair. However, there is still unclear information about light characteristics, such as coherence and polarization. Some studies indicate that linearly polarized light can survive through long propagation distance in biological tissue., Materials and Methods: Three burns about 6 mm in diameter were created on the back of rats with liquid N(2). Lesion "L(//)" was irradiated by He-Ne laser (lambda = 632.8 nm), D= 1.0 J/cm(2), with linear polarization parallel to the spinal column of the rat. Lesion "L(inverted v)" was irradiated using the same laser and dose, but the light polarization was aligned perpendicularly to the relative orientation. Lesion "C" was not irradiated in order to be considered as control. The animals were sacrificed at day 3-17 after lesion creation. Samples were collected and prepared for histological analysis., Results: Histological analysis showed that the healing of irradiated wounds was faster than that of non-irradiated wounds. Moreover, it was observed that skin wound repair is dependent on polarization orientation with respect to a referential axis as the animal's spinal column. Consequently, "L(//)" was completely healed after 17 days, whereas "L (perpendicular) " showed a moderate degree of healing after the same period., Conclusions: These results indicate that the relative direction of the laser polarization plays an important role in the wound healing process when highly coherent He-Ne laser is used.

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-microm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

Biglycan and decorin are small leucine-rich proteoglycans that play several biological and structural roles in different tissues and organs. Several reports have indicated that biglycan participates in odontoblast and ameloblast differentiation and in the calcification process. In the present study we show that the expression of biglycan changes from within the ameloblasts and odontoblasts to the extracellular space according to the stage of animal development. In predentin and in the pulp space, however, biglycan was continually expressed throughout the period of investigation. In contrast, decorin was absent in odontoblasts and in ameloblasts and was exclusively expressed in predentin throughout the period of observation. In young rats, however, decorin was expressed in the extracellular spaces of the pulp, where it was concentrated mainly in the peripheral pulp.

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with (3)H-estradiol-17beta in association with (3)H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-alpha. (3)H-estradiol nuclear binding is present but variable during days 1.5-7.5 of pregnancy. Sites of strong nuclear binding of (3)H-estradiol exhibit strong immunocytochemical staining with ER-alpha antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear (3)H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in (3)H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen (3)H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in (3)H-estradiol binding corresponds to an increased labeling index with (3)H-thymidine. A highly differentiated binding of (3)H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong (3)H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.

In mice, embryo implantation induces profound changes in the endometrium. These changes include redifferentiation of endometrial fibroblasts and extensive remodeling of extracellular matrix components. We have previously shown that, during this process, there is an impressive increase in the thickness of collagen fibrils present in decidualised areas that surround the implantation site, while collagen fibrils in non-decidualised areas and in the interimplantation site remain thin. In vitro and in vivo experiments have identified small leucine rich proteoglycans (SLRPs) as regulators of collagen fibrillogenesis. In a previous study, we demonstrated a difference between the pre-implantation and the post-implantation expression and distribution of four SLRPs types in uterine tissues. The present study, utilising immunocytochemical electron microscopy, shows that biglycan is associated with the presence of thick collagen fibrils in decidualised regions of the endometrium and that decorin is associated exclusively with thin collagen fibrils in non-decidualised endometrial areas. These results strongly indicate that biglycan plays a role in collagen fibrillogenesis and probably participates in the determination of collagen fibril thickness in the mouse decidua.

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [alpha1(V)](3), was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.