Kidney organoids recapitulate human basement membrane assembly in health and disease.

Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.

Influence of high glucose in the expression of miRNAs and IGF1R signaling pathway in human myometrial explants.

PURPOSE: Several roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium. METHODS: Human myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR. RESULTS: Myometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged. CONCLUSIONS: The human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

Monitoring the Progress and Healing Status of Burn Wounds Using Infrared Spectroscopy.

Burns are one of the leading causes of morbidity worldwide and the most costly traumatic injuries. A better understanding of the molecular mechanisms in wound healing is required to accelerate tissue recovery and reduce the health economic impact. However, the standard techniques used to evaluate the biological events associated to wound repair are laborious, time-consuming, and/or require multiple assays/staining. Therefore, this study aims to evaluate the feasibility of Fourier transform infrared (FT-IR) spectroscopy to monitor the progress and healing status of burn wounds. Burn injuries were induced on Wistar rats by water vapor exposure and biopsied for further histopathological and spectroscopic evaluation at four time-points (3, 7, 14, and 21 days). Spectral data were preprocessed and compared by principal component analysis. Pairwise comparison of post-burn groups to each other revealed that metabolic activity induced by thermal injury decreases as the healing progresses. Higher amounts of carbohydrates, proteins, lipids, and nucleic acids were evidenced on days 3 and 7 compared to healthy skin and reduced amounts of these molecular structural units on days 14 and 21 post-burn. FT-IR spectroscopy was used to determine the healing status of a wound based on the biochemical information retained by spectral signatures in each phase of healing. Our findings demonstrate that FT-IR spectroscopy can monitor the biological events triggered by burn trauma as well as to detect the wound status including full recovery based on the spectral changes associated to the biochemical events in each phase.

Lipoatrophy-Associated Insulin Resistance and Hepatic Steatosis are Attenuated by Intake of Diet Rich in Omega 3 Fatty Acids.

SCOPE: Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. METHODS AND RESULTS: Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. CONCLUSION: Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Lipoatrophy-associated insulin resistance and hepatic steatosis are attenuated by intake of diet rich in omega 3 fatty acids

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-gama deletion exclusively in adipocytes (A-PPARgama KO) and littermate controls (A-PPARgama WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Lipoatrophy-associated insulin resistance and hepatic steatosis are attenuated by intake of diet rich in omega 3 fatty acids

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-gama deletion exclusively in adipocytes (A-PPARgama KO) and littermate controls (A-PPARgama WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Tumour-derived transforming growth factor-ß signalling contributes to fibrosis in patients with cancer cachexia.

BACKGROUND: Cachexia is a paraneoplastic syndrome related with poor prognosis. The tumour micro-environment contributes to systemic inflammation and increased oxidative stress as well as to fibrosis. The aim of the present study was to characterise the inflammatory circulating factors and tumour micro-environment profile, as potentially contributing to tumour fibrosis in cachectic cancer patients. METHODS: 74 patients (weight stable cancer n = 31; cachectic cancer n = 43) diagnosed with colorectal cancer were recruited, and tumour biopsies were collected during surgery. Multiplex assay was performed to study inflammatory cytokines and growth factors. Immunohistochemistry analysis was carried out to study extracellular matrix components. RESULTS: Higher protein expression of inflammatory cytokines and growth factors such as epidermal growth factor, granulocyte-macrophage colony-stimulating factor, interferon-α, and interleukin (IL)-8 was observed in the tumour and serum of cachectic cancer patients in comparison with weight-stable counterparts. Also, IL-8 was positively correlated with weight loss in cachectic patients (P = 0.04; r = 0.627). Immunohistochemistry staining showed intense collagen deposition (P = 0.0006) and increased presence of α-smooth muscle actin (P < 0.0001) in tumours of cachectic cancer patients, characterizing fibrosis. In addition, higher transforming growth factor (TGF)-ß1, TGF-ß2, and TGF-ß3 expression (P = 0.003, P = 0.05, and P = 0.047, respectively) was found in the tumour of cachectic patients, parallel to p38 mitogen-activated protein kinase alteration. Hypoxia-inducible factor-1α mRNA content was significantly increased in the tumour of cachectic patients, when compared with weight-stable group (P = 0.005). CONCLUSIONS: Our results demonstrate TGF-ß pathway activation in the tumour in cachexia, through the (non-canonical) mitogen-activated protein kinase pathway. The results show that during cachexia, intratumoural inflammatory response contributes to the onset of fibrosis. Tumour remodelling, probably by TGF-ß-induced transdifferentiation of fibroblasts to myofibroblasts, induces unbalanced inflammatory cytokine profile, angiogenesis, and elevation of extracellular matrix components (EMC). We speculate that these changes may affect tumour aggressiveness and present consequences in peripheral organs.

Proteomic and functional analysis identifies galectin-1 as a novel regulatory component of the cytotoxic granule machinery.

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.

Distinct effects of short- and long-term type 1 diabetes to the placental extracellular matrix and fetal development in mice.

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.

Mapping of estradiol binding sites through receptor micro-autoradiography in the endometrial stroma of early pregnant mice.

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.

Hormetic modulation of hepatic insulin sensitivity by advanced glycation end products.

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.

The absence of the embryo in the pseudopregnant uterus alters the deposition of some ECM molecules during decidualization in mice.

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.

Long-term type 1 diabetes alters the deposition of collagens and proteoglycans in the early pregnant myometrium of mice.

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.

Early postnatal, but not late, exposure to chemical ambient pollutant 1,2-naphthoquinone increases susceptibility to pulmonary allergic inflammation at adulthood.

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.

Estradiol induces transcriptional and posttranscriptional modifications in versican expression in the mouse uterus.

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.

Diguanoside tetraphosphate (Gp4G) is an epithelial cell and hair growth regulator.

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.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone.

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.

Double disruption of α2A- and α2C-adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype.

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via ß(2)-adrenoceptor (ß2-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 ß(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 ß(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 ß(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.

Effects of long-term diabetes on the structure and cell proliferation of the myometrium in the early pregnancy of mice.

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.

Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis.

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.