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.

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.

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.

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.

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice.

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.

Collagen fibril organization in the pregnant endometrium of decorin-deficient mice.

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.

Hormone-regulated expression and distribution of versican in mouse uterine tissues.

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.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton.

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.

Changes in fibrillin-1 in the endometrium during the early stages of pregnancy in mice.

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.

Distribution of collagen types I, III, and V in pregnant mouse endometrium.

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.

Granulated and non-granulated decidual prolactin-related protein-positive decidual cells in the pregnant mouse endometrium.

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.

Ultrastructural and autoradiographical analysis show a faster skin repair in He-Ne laser-treated wounds.

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.

Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells.

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.

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium.

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.

ETA receptor mediates altered leukocyte-endothelial cell interaction and adhesion molecules expression in DOCA-salt rats.

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.

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography.

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.