Impaired Expression of Ectonucleotidases in Ectopic and Eutopic Endometrial Tissue Is in Favor of ATP Accumulation in the Tissue Microenvironment in Endometriosis.

Endometriosis is a prevalent disease defined by the presence of endometrial tissue outside the uterus. Adenosine triphosphate (ATP), as a proinflammatory molecule, promotes and helps maintain the inflammatory state of endometriosis. Moreover, ATP has a direct influence on the two main symptoms of endometriosis: infertility and pain. Purinergic signaling, the group of biological responses to extracellular nucleotides such as ATP and nucleosides such as adenosine, is involved in the biology of reproduction and is impaired in pathologies with an inflammatory component such as endometriosis. We have previously demonstrated that ectonucleotidases, the enzymes regulating extracellular ATP levels, are active in non-pathological endometria, with hormone-dependent changes in expression throughout the cycle. In the present study we have focused on the expression of ectonucleotidases by means of immunohistochemistry and in situ activity in eutopic and ectopic endometrial tissue of women with endometriosis, and we compared the results with endometria of women without the disease. We have demonstrated that the axis CD39-CD73 is altered in endometriosis, with loss of CD39 and CD73 expression in deep infiltrating endometriosis, the most severe, and most recurring, endometriosis subtype. Our results indicate that this altered expression of ectonucleotidases in endometriosis boosts ATP accumulation in the tissue microenvironment. An important finding is the identification of the nucleotide pyrophophatase/phosphodiesterase 3 (NPP3) as a new histopathological marker of the disease since we have demonstrated its expression in the stroma only in endometriosis, in both eutopic and ectopic tissue. Therefore, targeting the proteins directly involved in ATP breakdown could be an appropriate approach to consider in the treatment of endometriosis.

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE).

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression.

Overexpression of lysine-specific demethylase 1 in ovarian endometriomas and its inhibition reduces cellular proliferation, cell cycle progression, and invasiveness.

OBJECTIVE: To investigate whether lysine-specific demethylase 1 (LSD1) is aberrantly expressed in endometriomas and whether treatment with tranylcypromine, an LSD1 inhibitor, has any effect on cell viability, cell cycle, and invasiveness. DESIGN: Laboratory study using human tissues. SETTING: Academic hospital. PATIENT(S): Forty-two ectopic endometrial tissue samples, their homologue eutopic endometrial tissue samples, and 70 control endometrial tissue samples. INTERVENTION(S): Immunohistochemistry analysis of LSD1 of all human tissue samples, and Western blot analysis, quantitative real-time reverse-transcription polymerase chain reaction analysis, cell viability assay, cell cycle analysis, and invasion assay of eutopic and ectopic endometriotic stromal cells and normal endometrial stromal cells. MAIN OUTCOME MEASURE(S): Immunostaining levels of LSD1, gene and protein expression levels, cell viability, cell cycles, and invasiveness. RESULT(S): The expression of the LSD1 gene and protein in endometriosis was elevated. Treatment of endometriotic stromal cells with tranylcypromine statistically significantly reduced the cellular proliferation, cell cycle progression, and invasiveness. CONCLUSION(S): Because DNA and histones are intimately intertwined and work in concert in transcription regulation, conceivably histone demethylation activity of LSD1 could be wide ranging. The inhibition of LSD1 activity by tranylcypromine and the resultant inhibition of proliferation, cell cycle progression, and invasiveness suggest that LSD1 may be a candidate therapeutic target for endometriosis.

Ectopic lymphoid neogenesis and lymphoid chemokines in Sjogren's syndrome: at the interplay between chronic inflammation, autoimmunity and lymphomagenesis.

It has long been demonstrated that a subset of patients with Sjogren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the salivary glands (SG). These structures are characterised by periductal clusters of T and B lymphocytes, development of high endothelial venules and differentiation of follicular dendritic cells (FDC) networks. Evidence in patients with and animal models of SS demonstrated that the formation and maintenance of ELS in the SG is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. Several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing some of these factors during chronic inflammation in SS. In this review we focus on the cellular and molecular mechanisms regulating the formation of ELS in SS SG, with particular emphasis on the role of lymphoid chemokines. In addition, we summarise accumulating data in support of the notion that ELS in SS represent functional niches whereby autoreactive B cells undergo affinity maturation, clonal selection and differentiation into autoantibody producing cells, thus contributing to autoimmunity over and above secondary lymphoid organs. Furthermore, we review the emerging role of ELS and lymphoid chemokines in driving extranodal B cell lymphomagenesis in SS and we focus on recent evidence suggesting that ELS identify subsets of SS patients at increased risk of developing systemic manifestations and lymphoma.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in osteoblast differentiation and in supporting osteoclastogenesis.

Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.

Over-expression of cyclooxygenase-2 in endoscopic biopsies of ectopic gastric mucosa

Ectopic gastric mucosa (EGM) is considered to be a congenital condition. Rare cases of adenocarcinoma have been described. There are no data justifying regular biopsies or follow-up. Cyclooxygenase-2 (COX-2) is a protein involved in gastrointestinal tumor development by inhibiting apoptosis and regulating angiogenesis. The aim of this prospective study was to evaluate COX-2 expression in EGM and compare it with normal tissue and Barrett's esophagus. We evaluated 1327 patients. Biopsies were taken from the inlet patch for histological evaluation and from the gastric antrum to assess Helicobacter pylori infection. Biopsies taken from normal esophageal, gastric antrum and body mucosa and Barrett's esophagus were retrieved from a tissue bank. EGM biopsies were evaluated with respect to type of epithelium, presence of H. pylori, and inflammation. COX-2 was detected by immunohistochemistry using the avidin-biotin complex. EGM islets were found in 14 patients (1.1 percent). Histological examination revealed fundic type epithelium in 58.3 percent of cases, H. pylori was present in 50 percent and chronic inflammation in 66.7 percent. Expression of COX-2 was negative in normal distal esophagus, normal gastric antrum and normal gastric body specimens (10 each). In contrast, EGM presented over-expression of COX-2 in 41.7 percent of cases and Barrett's esophagus in 90 percent of cases (P = 0.04 and 0.03, respectively). COX-2 immunoexpression in EGM was not related to gender, age, epithelium type, presence of inflammation or intestinal metaplasia, H. pylori infection, or any endoscopic finding. Our results demonstrate up-regulation of COX-2 in EGM, suggesting a possible malignant potential of this so-called harmless mucosa.

Over-expression of cyclooxygenase-2 in endoscopic biopsies of ectopic gastric mucosa.

Ectopic gastric mucosa (EGM) is considered to be a congenital condition. Rare cases of adenocarcinoma have been described. There are no data justifying regular biopsies or follow-up. Cyclooxygenase-2 (COX-2) is a protein involved in gastrointestinal tumor development by inhibiting apoptosis and regulating angiogenesis. The aim of this prospective study was to evaluate COX-2 expression in EGM and compare it with normal tissue and Barrett's esophagus. We evaluated 1327 patients. Biopsies were taken from the inlet patch for histological evaluation and from the gastric antrum to assess Helicobacter pylori infection. Biopsies taken from normal esophageal, gastric antrum and body mucosa and Barrett's esophagus were retrieved from a tissue bank. EGM biopsies were evaluated with respect to type of epithelium, presence of H. pylori, and inflammation. COX-2 was detected by immunohistochemistry using the avidin-biotin complex. EGM islets were found in 14 patients (1.1%). Histological examination revealed fundic type epithelium in 58.3% of cases, H. pylori was present in 50% and chronic inflammation in 66.7%. Expression of COX-2 was negative in normal distal esophagus, normal gastric antrum and normal gastric body specimens (10 each). In contrast, EGM presented over-expression of COX-2 in 41.7% of cases and Barrett's esophagus in 90% of cases (P = 0.04 and 0.03, respectively). COX-2 immunoexpression in EGM was not related to gender, age, epithelium type, presence of inflammation or intestinal metaplasia, H. pylori infection, or any endoscopic finding. Our results demonstrate up-regulation of COX-2 in EGM, suggesting a possible malignant potential of this so-called harmless mucosa.

Immunohistochemical detection of heparanase-1 expression in eutopic and ectopic endometrium from women with endometriosis.

OBJECTIVE: To investigate the expression of heparanase-1, an endoglycosidase that degrades heparan sulfate proteoglycans, in eutopic and ectopic endometrial tissues from women with endometriosis. DESIGN: An immunohistochemical study. SETTING: Academic research laboratory and a private infertility clinic affiliated with a university medical center. PATIENT(S): Premenopausal women undergoing laparoscopy for endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Expression of heparanase-1 analyzed by immunohistochemical staining in 91 eutopic and 14 ectopic endometrial specimens. RESULT(S): We found that 17% (4/24) of the eutopic endometrial specimens in the early proliferative phase and 32% (9/28) of the samples in the midproliferative phase were heparanase-1 positive. However, >or=80% of eutopic endometrial specimens at late proliferate phase and at luteal phase were heparanase-1 positive. Twelve of 14 ectopic endometriotic specimens stained heparanase-1 positive. Comparison of heparanase-1 expression in paired eutopic and ectopic endometrial tissues revealed that 5 of 6 ectopic specimens in the early proliferative phase were heparanase-1 positive, whereas only 1 eutopic specimen was heparanase-1 positive. In comparison with our recent study of heparanase-1 expression in normal endometrium, we found that there was no significant difference in heparanase-1 expression in the eutopic endometrium from women with or without endometriosis. CONCLUSION(S): Heparanase-1 was differentially expressed in the eutopic endometrium in the different menstrual phases. Heparanase-1 was highly expressed in the ectopic endometriotic lesions regardless of their menstrual phases, suggesting that the local environment is responsible for increased heparanase-1 expression.

Expression and regulation of estrogen-converting enzymes in ectopic human endometrial tissue.

OBJECTIVE: To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. DESIGN: Animal study. SETTING: Academic medical center. ANIMAL(S): Sixty female nude mice with implanted human endometrial tissue. PATIENT(S): Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. INTERVENTION(S): Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. MAIN OUTCOME MEASURE(S): Transcript levels of steroid hormone receptors, 17beta-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. RESULT(S): Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17beta-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. CONCLUSION(S): Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

Abnormal neocortical development in mice lacking cGMP-dependent protein kinase I.

Cyclic GMP-dependent protein kinase type I (cGKI) is a key signaling intermediate important for synaptic potentiation in the hippocampus and cerebellum, but its expression and function in cortical development have not been elucidated. The expression of cGKI in the developing mouse neocortex was evaluated by immunofluorescence labeling, and effect of cGKI deletion on cortical development was studied in adult cGKI knockout mice. cGKI was expressed at highest levels at embryonic stages in young neurons and radial glial fibers, corresponding to the major period of radial migration and laminar development of pyramidal neurons (embryonic day E13.5-E14.5), declining upon maturation (E17.5-postnatal day P28). The cerebral cortex of homozygous null mutant mice lacking cGKI exhibited heterotopic collections of neurons in the upper cortical layers and abnormal invaginations of layer I, in accord with a neuronal migration or positioning defect. Some cGKI mutant mice displayed defects in midline development resulting in partial fusion of cerebral hemispheres with adjacent neuronal heterotopias. Apical dendrites of cortical pyramidal neurons were misoriented in the cerebral cortex of cGKI null mutants, as shown in reporter mice expressing yellow fluorescent protein in layer V pyramidal neurons and by Golgi impregnation. These results demonstrate a role for cGKI signaling in cortical development related to neuronal migration/positioning that is important for dendritic orientation and connectivity.

Characterization of metaplastic and heterotopic epithelia in the human gastrointestinal tract by the expression pattern of acyl-CoA synthetase 5.

Metaplastic and heterotopic epithelia are frequently found in the human intestine. The recently cloned human acyl-CoA synthetase 5 (ACS5) is a key enzyme in providing cytosolic acyl-CoA thioesters. The aim of the study was to identify and to locate the expression of ACS5 in the gastric body and the small intestine with metaplasia or heterotopia by different methods. In the normal gastrointestinal tract, ACS5 was predominantly found in the villus epithelium of the small intestine, but not in the gastric mucosa. Of note, strong expression of ACS5 was also detectable in intestinal metaplasia of the stomach. Inversely, ACS5 expression could neither be detected in heterotopic gastric mucosa of the corpus type nor in gastric, pseudopyloric, or antral metaplasia of the small intestine. In conclusion, our data implicate that ACS5 is a suitable differentiating marker molecule in the gastrointestinal tract.

Incidentally detected giant oncocytoma arising in retroperitoneal heterotopic adrenal tissue.

A nonfunctional retroperitoneal oncocytoma incidentally discovered in a 40-year-old woman is described. The tumor, which was 17 cm in largest dimension, was completely separated from the kidneys and adrenal glands and consisted of nests of polygonal cells with large, granular, eosinophilic cytoplasm. Significant nuclear atypia, necrosis, and mitosis were absent. Ultrastructural analysis confirmed the oncocytic nature of the neoplastic cells. Since neoplastic cells were not immunoreactive for chromogranin and did not contain dense-core secretory granules, the diagnosis of oncocytic paraganglioma was excluded. Cells immunoreactive for 3beta-hydroxysteroid dehydrogenase, the enzyme catalyzing the conversions of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione, were identified in the tumor, thus strongly indicating adrenocortical tissue origin. Multiple nests of 3beta-hydroxysteroid dehydrogenase-positive cells were detected in the loose retroperitoneal connective tissue. These findings strongly support the origin of the tumor from heterotopic retroperitoneal rests of the adrenal gland. To our knowledge, only 1 similar case has been described in the literature to date.

Xanthine oxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis.

OBJECTIVE: To investigate the expression of xanthine oxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis. DESIGN: Immunohistochemical identification of xanthine oxidase in endometrial tissues by using polyclonal antibody. SETTING: University hospital. PATIENT(S): Thirty-four women with endometriosis, 34 women with adenomyosis, and 44 fertile control women. INTERVENTION(S): Biopsy samples were obtained from the endometrium throughout the menstrual cycle. MAIN OUTCOME MEASURE(S): Semiquantitative immunostaining (evaluation nomogram) score of endometrial cells. RESULT(S): The level of xanthine oxidase expression in the glandular epithelium of control varied according to menstrual phase, but no such variation in expression was seen in endometriosis. Variation in xanthine oxidase expression was observed during the menstrual cycle in patients with adenomyosis; this variation differed completely from that in controls. Xanthine oxidase expression was found in ectopic endometrial tissue in all cases. The mean evaluation nomogram levels in the glandular epithelium in adenomyosis tissue were as high as those in the early secretory phase in the eutopic endometrium. CONCLUSION(S): Aberrant expression of xanthine oxidase in eutopic and ectopic endometrium appears to play a pathologic role in endometriosis and adenomyosis.

Heterogeneous carbamoylphosphate synthetase I expression in testicular transplants of fetal mouse liver.

Expression of carbamoylphosphate synthetase I (CPSI; EC 6.3.4.16) was examined immunohistochemically in normal development of the mouse liver, and in testicular transplants of fetal liver fragments. CPSI started to be expressed in all hepatocytes around 15 days of gestation, and became heterogeneous (i.e. absent from pericentral hepatocytes) around 2 weeks after birth. Most hepatocytes in fetal liver fragments placed for 2 months under the testicular capsule expressed this enzyme except for the pericentral ones, most of which were positively stained with anti-glutamine synthetase (GS; EC 6.3.1.2) antiserum. This distribution resembled that in the adult liver. The steep change in CPSI immunostaining in liver lobules suggests that the microenvironment tightly connected to the central veins plays an important role in the suppression of CPSI expression in the pericentral hepatocytes. Some pericentral hepatocytes were also negative for both enzymes. Thus, control mechanisms of CPSI expression may be different from those of GS expression in pericentral hepatocytes.