Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.

M-TRAP: Safety and performance of metastatic tumor cell trap device in advanced ovarian cancer patients.

OBJECTIVE: Despite radical surgery and chemotherapy, most patients with ovarian cancer die due to disease progression. M-Trap is an implantable medical device designed to capture peritoneal disseminated tumor cells with the aim to focalize the disease. This trial analyzed the safety and performance of the device. METHODS: This first-in-human prospective, multi-center, non-blinded, single-arm study enrolled 23 women with high-grade serous advanced ovarian cancer. After primary or interval debulking surgery, 3 M-Trap devices were placed in the peritoneum of the abdominal cavity. 18-months post-implantation or at disease progression, devices were initially removed by laparoscopy. The primary safety endpoint was freedom from device and procedure-related major adverse events (MAEs) through 6-months post-implantation compared to an historical control. The primary performance endpoint was histopathologic evidence of tumor cells capture. RESULTS: Only one major adverse event was attributable to the device. 18 women were free of device and procedure related MAEs (78.3%). However, the primary safety endpoint was not achieved (p = 0.131), primarily attributable to the greater surgical complexity of the M-Trap patient population. 62% of recurrent patients demonstrated tumor cell capture in at least one device with a minimal tumor cell infiltration. No other long-term device-related adverse events were reported. The secondary performance endpoint demonstrated a lack of disease focalization. CONCLUSIONS: The M-Trap technology failed to meet its primary safety objective, although when adjusted for surgical complexity, the study approved it. Likewise, the devices did not demonstrate the anticipated benefits in terms of tumor cell capture and disease focalization in recurrent ovarian cancer.

Morphological alterations in the hippocampus of the Ts65Dn mouse model for Down Syndrome correlate with structural plasticity markers.

Down syndrome (DS) is the most common chromosomal aneuploidy. Although trisomy on chromosome 21 can display variable phenotypes, there is a common feature among all DS individuals: the presence of intellectual disability. This condition is partially attributed to abnormalities found in the hippocampus of individuals with DS and in the murine model for DS, Ts65Dn. To check if all hippocampal areas were equally affected in 4-5 month adult Ts65Dn mice, we analysed the morphology of dentate gyrus granule cells and cornu ammonis pyramidal neurons using Sholl method on Golgi-Cox impregnated neurons. Structural plasticity has been analysed using immunohistochemistry for plasticity molecules followed by densitometric analysis (Brain Derived Neurotrophic Factor (BDNF), Polysialylated form of the Neural Cell Adhesion Molecule (PSA-NCAM) and the Growth Associated Protein 43 (GAP43)). We observed an impairment in the dendritic arborisation of granule cells, but not in the pyramidal neurons in the Ts65Dn mice. When we analysed the expression of molecules related to structural plasticity in trisomic mouse hippocampus, we observed a reduction in the expression of BDNF and PSA-NCAM, and an increment in the expression of GAP43. These alterations were restricted to the regions related to dentate granule cells suggesting an interrelation. Therefore the impairment in dendritic arborisation and molecular plasticity is not a general feature of all Down syndrome principal neurons. Pharmacological manipulations of the levels of plasticity molecules could provide a way to restore granule cell morphology and function.

Early Social Isolation Stress and Perinatal NMDA Receptor Antagonist Treatment Induce Changes in the Structure and Neurochemistry of Inhibitory Neurons of the Adult Amygdala and Prefrontal Cortex.

The exposure to aversive experiences during early life influences brain development and leads to altered behavior. Moreover, the combination of these experiences with subtle alterations in neurodevelopment may contribute to the emergence of psychiatric disorders, such as schizophrenia. Recent hypotheses suggest that imbalances between excitatory and inhibitory (E/I) neurotransmission, especially in the prefrontal cortex and the amygdala, may underlie their etiopathology. In order to understand better the neurobiological bases of these alterations, we studied the impact of altered neurodevelopment and chronic early-life stress on these two brain regions. Transgenic mice displaying fluorescent excitatory and inhibitory neurons, received a single injection of MK801 (NMDAR antagonist) or vehicle solution at postnatal day 7 and/or were socially isolated from the age of weaning until adulthood (3 months old). We found that anxiety-related behavior, brain volume, neuronal structure, and the expression of molecules related to plasticity and E/I neurotransmission in adult mice were importantly affected by early-life stress. Interestingly, many of these effects were potentiated when the stress paradigm was applied to mice perinatally injected with MK801 ("double-hit" model). These results clearly show the impact of early-life stress on the adult brain, especially on the structure and plasticity of inhibitory networks, and highlight the double-hit model as a valuable tool to study the contribution of early-life stress in the emergence of neurodevelopmental psychiatric disorders, such as schizophrenia.

Reduced interneuronal dendritic arborization in CA1 but not in CA3 region of mice subjected to chronic mild stress.

INTRODUCTION: Chronic stress induces dendritic atrophy and decreases spine density in excitatory hippocampal neurons, although there is also ample evidence indicating that the GABAergic system is altered in the hippocampus after this aversive experience. Chronic stress causes dendritic remodeling both in excitatory neurons and interneurons in the medial prefrontal cortex and the amygdala. METHODS: In order to know whether it also has an impact on the structure and neurotransmission of hippocampal interneurons, we have analyzed the dendritic arborization, spine density, and the expression of markers of inhibitory synapses and plasticity in the hippocampus of mice submitted to 21 days of mild restrain stress. The analyses were performed in GIN mice, a strain that displays EGFP-labeled interneurons. RESULTS: We observed a significant decrease in the dendritic arborization of interneurons in the CA1 region, which did not occur in those in CA3. We found neither changes in dendritic spine density in these regions nor alterations in the number of EGFP-positive interneurons. Nevertheless, the expression of glutamic acid decarboxylase 67 was reduced in different layers of CA1 and CA3 regions of the hippocampus. No significant changes were found in the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) or synaptophysin. CONCLUSIONS: Chronic stress reduces the interneuronal dendritic arborization in CA1 region of the hippocampus but not in CA3.

MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors.

STUDY QUESTION: Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? SUMMARY ANSWER: This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. WHAT IS ALREADY KNOWN?: An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. STUDY DESIGN, SIZE, DURATION: Case-control study that involved 51 women with endometriosis and 32 women without the disease (controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. LIMITATIONS, REASONS FOR CAUTION: Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. WIDER IMPLICATIONS OF THE FINDINGS: Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

A "double hit" murine model for schizophrenia shows alterations in the structure and neurochemistry of the medial prefrontal cortex and the hippocampus.

Both alterations in neurodevelopment and aversive experiences during childhood and adolescence seem important risk factors for schizophrenia. Animal models reproducing these alterations mimic some of the symptoms, constituting a valid approach to study the etiopathology of this disorder. Among these models, the perinatal injection of N-methyl-d-aspartate receptor antagonists and the postweaning social isolation rearing are among the most widely used. Our aim is to combine them in a "double hit" model, which should produce a wider spectrum of alterations. Lister Hooded rats have been subjected to a single injection of MK-801 at postnatal day 7 and socially isolated from postweaning to adulthood. These animals presented increased body weight gain and volume reductions in their medial prefrontal cortex (mPFC) and hippocampus. They also showed an increased number of activated pyramidal neurons and alterations in the numbers of parvalbumin and calbindin expressing interneurons in the mPFC. The expressions of the polysialylated form of the neural cell adhesion molecule and GAD67 are decreased in the mPFC. The mRNA level of calbindin was decreased, while that of calretinin was increased in the mPFC. The mRNA level of ERbB4, a gene associated to schizophrenia, was also altered in this region. All these structural and neurochemical alterations, specially in cortical inhibitory circuits, are similar to those found in schizophrenic patients and are more numerous than in each of the single models. Consequently, the present "double hit" model may be a better tool to study the neurobiological basis of schizophrenia and to explore new therapeutic approaches.

Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

UNLABELLED: Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. METHODS: Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. RESULTS: Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion , this "in vitro" study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease.

Chronic stress alters inhibitory networks in the medial prefrontal cortex of adult mice.

Chronic stress in experimental animals induces dendritic atrophy and decreases spine density in principal neurons of the medial prefrontal cortex (mPFC). This structural plasticity may play a neuroprotective role and underlie stress-induced behavioral changes. Different evidences indicate that the prefrontocortical GABA system is also altered by stress and in major depression patients. In the amygdala, chronic stress induces dendritic remodeling both in principal neurons and in interneurons. However, it is not known whether similar structural changes occur in mPFC interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) may mediate these changes, because it is known to influence the dendritic organization of adult cortical interneurons. We have analyzed the dendritic arborization and spine density of mPFC interneurons in adult mice after 21 days of restraint stress and have found dendritic hypertrophy in a subpopulation of interneurons identified mainly as Martinotti cells. This aversive experience also decreases the number of glutamate decarboxylase enzyme, 67 kDa isoform (GAD67) expressing somata, without affecting different parameters related to apoptosis, but does not alter the number of interneurons expressing PSA-NCAM. Quantitative retrotranscription-polymerase chain reaction (qRT-PCR) analysis of genes related to general and inhibitory neurotransmission and of PSA synthesizing enzymes reveals increases in the expression of NCAM, synaptophysin and GABA(A)α1. Together these results show that mPFC inhibitory networks are affected by chronic stress and suggest that structural plasticity may be an important feature of stress-related psychiatric disorders where this cortical region, specially their GABAergic system, is altered.

Alterations in the expression of PSA-NCAM and synaptic proteins in the dorsolateral prefrontal cortex of psychiatric disorder patients.

Alterations in the structure and physiology of the prefrontal cortex (PFC) have been found in different psychiatric disorders and some of them involve inhibitory networks, especially in schizophrenia and major depression. Changes in the structure of these networks may be mediated by the polysialylated neural cell adhesion molecule (PSA-NCAM), a molecule related to neuronal structural plasticity, expressed in the PFC exclusively by interneurons. Different studies have found that PSA-NCAM expression in the hippocampus and the amygdala is altered in schizophrenia, major depression and animal models of these disorders, in parallel to changes in the expression of molecules related to inhibitory neurotransmission and synaptic plasticity. We have analyzed post-mortem sections of the dorsolateral PFC from the Stanley Neuropathology Consortium, which includes controls, schizophrenia, bipolar and major depression patients, to check whether similar alterations occur. PSA-NCAM was found in neuronal somata and neuropil puncta, many of which corresponded to interneurons. PSA-NCAM expression was only reduced significantly in schizophrenic patients, in parallel to a decrease in glutamic acid-decarboxylase-67 (GAD67) and to an increased expression of vesicular glutamate transporter 1 (VGLUT1) in the white matter. Depressed patients showed significant decreases in synaptophysin (SYN) and VGLUT1 expression. Whereas in bipolar patients, decreases in VGLUT1 expression have also been found, together with a reduction of GAD67. These results indicate that the expression of synaptic proteins is altered in the PFC of patients suffering from these disorders and that, particularly in schizophrenia, abnormal PSA-NCAM and GAD67 expression may underlie the alterations observed in inhibitory neurotransmission.

microRNAs related to angiogenesis are dysregulated in endometrioid endometrial cancer.

STUDY QUESTION: Which is the role of microRNAs (miRNAs) related to several angiogenesis regulators such as VEGF-A (Vascular endothelial growth factor-A) and TSP-1 (Thrombospondin-1) in endometrial cancer? SUMMARY ANSWER: A dysregulated expression of miRNAs related to angiogenesis and an increase in the VEGF-A levels were observed in endometrial cancer in comparison with control. The different expression of miRNAs could modulate the expression of angiogenic and antiangiogenic factors, which may play an important role in the pathogenesis of endometrial cancer. WHAT IS KNOWN ALREADY: Dysregulated miRNA expression has been previously evaluated in endometrial adenocarcinoma. To the best of our knowledge, there are no studies on the relationship between angiogenic factors and miRNAs in endometrial cancer. STUDY DESIGN, SIZE, DURATION: Case-control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNAs isolated from tissue samples were analyzed using the GeneChip miRNA 2.0 Array platform (Affymetrix). TaqMan qRT-PCR was used to assess the expression of the selected miRNAs related to angiogenesis (miR-15b, -16, -17-5p, -20a, -21, -125a, -200b, -210, -214*, -221, -222 and -424), and VEGF-A and TSP-1 mRNAs were assessed by qRT-PCR using SYBR Green. Protein levels were quantified by ELISAs. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with the miRNAs in the control endometrium, eight miRNAs (miR-15b, -17-5p, -20a, -125a, -214*, -221, -222 and -424) were significantly down-regulated and two miRNAs (miR-200b and -210) were significantly up-regulated in the cancerous endometrium. A significant increase in VEGF-A mRNA and protein expression and in TSP-1 protein levels (P <0.01) was observed in endometrial cancer. Moreover, significant inverse correlations between VEGF-A protein levels and miR-20a, -125a, -214*, -221, -222 and -424 were detected. In contrast, a positive correlation was observed between VEGF-A and miR-200b and -210. Furthermore, stage IB endometrial cancer was associated with a higher VEGF-A protein/mRNA ratio and lower miR-214*, -221 and -222 expression in comparison with stage IA. LIMITATIONS, REASONS FOR CAUTION: Future functional studies (e.g. miRNA inhibition or ectopic overexpression) in cell culture models are needed to confirm the VEGF targeting by the miRNAs found in the present study. WIDER IMPLICATIONS OF THE FINDINGS: The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, PI080185, PI0110091) and Red RECAVA (RD06/0014/0004), by Consellería de Sanidad (AP-141/11) and Consellería de Educación (PROMETEO/2011/027), Generalitat Valenciana, by Beca Fibrinolisis 2009 and Becario 2010, 2011 from Fundación Española de Trombosis y Hemostasia and by the Fundación Investigación Hospital La Fe, Spain. None of the authors have any conflicts of interest.

Post-weaning social isolation rearing influences the expression of molecules related to inhibitory neurotransmission and structural plasticity in the amygdala of adult rats.

Several lines of evidence indicate that alterations in the structure of neural circuits and inhibitory neurotransmission underlie the physiopathogenesis of schizophrenia. Most of the studies on these parameters have been focused on cortical regions and, despite the crucial role of the amygdala in this psychiatric disorder, there is less information on this region. In order to expand this knowledge, we have studied the expression of molecules related to inhibitory neurotransmission and structural plasticity in rats subjected to post-weaning isolation rearing, an animal model that reproduces several core symptoms of schizophrenia. We have analyzed, using qRT-PCR and immunohistochemistry, the expression of synaptophysin, GAD65, GAD67, the neural cell adhesion molecule (NCAM), its polysialylated form (PSA-NCAM) and its synthesizing enzymes (St8siaII and St8SiaIV). Isolation-reared rats showed significant increases in the expression of GAD67 protein in the centromedial, medial and basolateral amygdaloid nuclei, but no significant changes in GAD65 or synaptophysin expression were found in these regions. The expression of PSA-NCAM and NCAM was significantly increased in the basolateral and medial nuclei respectively. Our results indicate that isolation-rearing influences positively inhibitory neurotransmission and neuronal structural plasticity in the amygdala, probably through PSA-NCAM. These findings are in contrast to reports describing decreased expression of molecules related to inhibitory neurotransmission in the amygdala of schizophrenic patients. Consequently, although the social isolation rearing model can reproduce some of the behavioral traits of schizophrenics it may fail to reproduce some of the neurobiological features of this disorder, particularly in the amygdala.

Expression of PSA-NCAM and synaptic proteins in the amygdala of psychiatric disorder patients.

Neuroimaging has revealed structural abnormalities in the amygdala of different psychiatric disorders. The polysialylated neural cell adhesion molecule (PSA-NCAM), a molecule related to neuronal structural plasticity, which expression is altered in schizophrenia, major depression and in animal models of these disorders, may participate in these changes. However, PSA-NCAM has not been studied in the human amygdala. To know whether its expression and that of presynaptic markers, was affected in psychiatric disorders, we have analyzed post-mortem sections from the Stanley Neuropathology Consortium, which includes controls, schizophrenia, bipolar and major depression patients. PSA-NCAM was expressed in neuronal somata and neuropil puncta, many of which corresponded to interneurons. Depressed patients showed decreases in PSA-NCAM expression in the basolateral and basomedial amygdala; synaptophysin and GAD67 were also decreased, while VGLUT-1 was increased, in different nuclei. Increases in PSA-NCAM expression were found in the lateral nucleus of bipolar patients; synaptophysin and GAD67 were reduced, and VGLUT-1 increased, in their basolateral and lateral nuclei. The expression of synaptophysin and GAD67 was downregulated in the basolateral nucleus of schizophrenics. These results indicate that inhibitory and excitatory amygdaloid circuits are affected in these disorders and that abnormal PSA-NCAM expression in depressive and bipolar patients may underlie these alterations.

Plasminogen activator inhibitor-1 (PAI-1) 4 G/5 G polymorphism and endometrial cancer. Influence of PAI-1 polymorphism on tissue PAI-1 antigen and mRNA expression and tumor severity.

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in endometrial cancer patients are associated with a poor prognosis. The objective of this study was to evaluate the PAI-1 4G/5G polymorphism in women with and without endometrial cancer and to analyze the influence of this polymorphism on PAI-1 expression in endometrial tissue. In 423 women (212 patients with endometrial cancer and 211 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. Quantitative real-time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 protein levels were quantified by ELISA in tissue extracts from 33 patients with endometrial cancer and from 70 endometrial tissues from control women. The frequency of PAI-1 4G/4G genotype (P=0.010) and the PAI-1 4G allele (P=0.009) was significantly higher in patients than in controls. The frequency of PAI-1 4G allele was significantly higher in patients with stage IB than in those with stage IA (P=0.03). Control women with the 4G/4G genotype had higher endometrial PAI-1 protein (P=0.018) and mRNA (P=0.004) levels than those with the 5G/5G genotype. A significant increase in PAI-1 protein and mRNA was observed in endometrial cancer tissue in comparison with the endometrial tissue from control women (P<0.01). In conclusion, frequencies of the PAI-1 4G allele and 4G/4G genotype were found significantly more often in women with endometrial cancer than in controls. PAI-1 levels in endometrial tissue seem to be associated with PAI-1 4G/5G polymorphism. These findings suggest that the PAI-1 4G/4G genotype may be associated with the risk of endometrial cancer in a Caucasian population. Further studies with a larger number of patients are needed to clarify the influence of this PAI-1 polymorphism in endometrial cancer.

Chronic stress induces changes in the structure of interneurons and in the expression of molecules related to neuronal structural plasticity and inhibitory neurotransmission in the amygdala of adult mice.

Chronic stress in experimental animals, one of the most accepted models of chronic anxiety and depression, induces structural remodeling of principal neurons in the amygdala and increases its excitation by reducing inhibitory tone. These changes may be mediated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a molecule related to neuronal structural plasticity and expressed by interneurons in the adult CNS, which is downregulated in the amygdala after chronic stress. We have analyzed the amygdala of adult mice after 21 days of restraint stress, studying with qRT-PCR the expression of genes related to general and inhibitory neurotransmission, and of PSA synthesizing enzymes. The expression of GAD67, synaptophysin and PSA-NCAM was also studied in specific amygdaloid nuclei using immunohistochemistry. We also analyzed dendritic arborization and spine density, and cell activity, monitoring c-Fos expression, in amygdaloid interneurons. At the mRNA level, the expression of GAD67 and of St8SiaII was significantly reduced. At the protein level there was an overall reduction in the expression of GAD67, synaptophysin and PSA-NCAM, but significant changes were only detected in specific amygdaloid regions. Chronic stress did not affect dendritic spine density, but reduced dendritic arborization in interneurons of the lateral and basolateral amygdala. These results indicate that chronic stress modulates inhibitory neurotransmission in the amygdala by regulating the expression of molecules involved in this process and by promoting the structural remodeling of interneurons. The addition of PSA to NCAM by St8SiaII may be involved in these changes.

microRNAs expression in endometriosis and their relation to angiogenic factors.

BACKGROUND: Endometriosis is a common, multifactorial disease in which angiogenesis may be involved in the growth of endometrium outside the uterus. microRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. The objective of this study was to analyze several miRNAs related to angiogenesis and the angiogenic factors, vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), in endometriotic lesions (ovarian endometrioma, peritoneal lesion and rectovaginal nodule) and eutopic endometrium from women with endometriosis. METHODS: TaqMan real-time PCR was used to assess the expression of the miRNAs (miR-15b, -16, -17-5p, -20a, -21, -125a, -221 and -222), while VEGF-A and TSP-1 mRNA were assessed by real-time PCR, with SYBR Green I and VEGF-A and TSP-1 protein levels were quantified by ELISA. Included in the study were 58 women with endometriosis and 38 control women. RESULTS: In paired samples, ovarian endometrioma showed significantly lower VEGF-A mRNA (P = 0.02) and protein (P = 0.002) expression than eutopic endometrium and higher expression of miR-125a (P = 0.003) and miR-222 (P <0.001). However, ovarian endometrioma had significantly higher expression of the angiogenic inhibitor TSP-1 and lower expression of miR-17-5p than eutopic endometrium (P < 0.001). Moreover, a significant inverse correlations between miR-222 and VEGF-A protein levels (-0.267, P = 0.018) and between miR-17-5p and TSP-1 protein levels (-0.260, P=0.022) were observed. Peritoneal lesions showed a significant increase in VEGF-A in comparison with ovarian endometrioma (P < 0.01). CONCLUSIONS: Expression levels of miRNAs related to angiogenesis were different in eutopic endometrium from that observed in ovarian endometrioma. This could influence the expression of angiogenic factors and play a role in the pathogenesis of endometriosis.

The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed in a subpopulation of mature cortical interneurons characterized by reduced structural features and connectivity.

Principal neurons in the adult cerebral cortex undergo synaptic, dendritic, and spine remodeling in response to different stimuli, and several reports have demonstrated that the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) participates in these plastic processes. However, there is only limited information on the expression of this molecule on interneurons and on its role in the structural plasticity of these cells. We have found that PSA-NCAM is expressed in mature interneurons widely distributed in all the extension of the cerebral cortex and have excluded the expression of this molecule in most principal cells. Although PSA-NCAM expression is generally considered a marker of immature neurons, birth-dating analyses reveal that these interneurons do not have an adult or perinatal origin and that they are generated during embryonic development. PSA-NCAM expressing interneurons show reduced density of perisomatic and peridendritic puncta expressing different synaptic markers and receive less perisomatic synapses, when compared with interneurons lacking this molecule. Moreover, they have reduced dendritic arborization and spine density. These data indicate that PSA-NCAM expression is important for the connectivity of interneurons in the adult cerebral cortex and that its regulation may play an important role in the structural plasticity of inhibitory networks.

Vascular endothelial growth factor polymorphisms (-460C/T, +405G/C, and 936C/T) and endometriosis: their influence on vascular endothelial growth factor expression.

OBJECTIVE: To analyze three functional vascular endothelial growth factor (VEGF) polymorphisms (-460C/T, +405G/C, and 936C/T) in women with and without endometriosis and their correlation with VEGF expression in endometrial tissue and peritoneal fluid (PF). DESIGN: Case-control study. SETTING: University-based hospital. PATIENT(S): One hundred eighty-six women with endometriosis and 180 controls without the disease. INTERVENTION(S): Endometrial biopsies were performed by aspiration and PF samples were obtained at laparoscopy. MAIN OUTCOME MEASURE(S): VEGF polymorphisms (-460C/T, +405G/C, and 936C/T) were determined using a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. Quantitative real-time reverse transcriptase (RT)-PCR assay was used to quantify VEGF-A messenger RNA (mRNA) and VEGF-A antigen levels were quantified by ELISA. RESULT(S): Patients with endometriosis showed a higher VEGF 936T allele frequency than controls. However, the distribution of genotypes and allele frequencies in the other two VEGF (-460C/T, +405G/C) polymorphisms was similar in the endometriosis and control groups. Endometrium and PF from women with endometriosis showed an increase in VEGF levels, but no association was found between the VEGF polymorphisms studied and VEGF expression in endometrial tissue and PF. CONCLUSION(S): These findings suggest that the VEGF 936C/T polymorphism may be associated with the risk of endometriosis in a Caucasian population, but the increased VEGF levels observed in endometriosis do not appear to be associated with this polymorphism.

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and endometriosis. Influence of PAI-1 polymorphism on PAI-1 antigen and mRNA expression.

INTRODUCTION: Endometriosis is a benign gynecologic disease with a high prevalence. It is a multifactorial and polygenic entity in which the fibrinolytic system may be implicated. The objective of this study was to evaluate the plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in a group of women with and without endometriosis and to analyze the influence of this polymorphism in PAI-1 expression in endometrial tissue and peritoneal fluid. MATERIAL AND METHODS: In 389 women (170 patients with endometriosis and 219 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. Quantitative real-time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 antigen (ag) levels were quantified by ELISA. RESULTS: The genotype and allele frequencies of PAI-1 4G/5G polymorphism did not differ significantly between patients and controls. Control women with the 4G/4G genotype had higher endometrial PAI-1ag (P=0.026) and mRNA (P=0.014) levels than those with the 5G/5G genotype. Control carrying the 4G/4G genotype tended to have higher peritoneal fluid PAI-1ag levels than those carrying the 5G/5G genotype. Moreover, PAI-1ag levels in peritoneal fluid were higher in patients than in controls (P=0.003). CONCLUSIONS: The PAI-1 genotype distribution was similar in patients and controls. PAI-1 levels in endometrial tissue and peritoneal fluid seem to be associated with PAI-1 4G/5G polymorphism in controls. The increased PAI-1ag levels observed in peritoneal fluid from patients could contribute to increase the peritoneal adhesions observed in endometriosis.