Phenomena of Intussusceptive Angiogenesis and Intussusceptive Lymphangiogenesis in Blood and Lymphatic Vessel Tumors.

Intussusceptive angiogenesis (IA) and intussusceptive lymphangiogenesis (IL) play a key role in the growth and morphogenesis of vessels. However, there are very few studies in this regard in vessel tumors (VTs). Our objective is to assess the presence, characteristics, and possible mechanisms of the formation of intussusceptive structures in a broad spectrum of VTs. For this purpose, examples of benign and malignant blood and lymphatic VTs were studied via conventional procedures, semithin sections, and immunochemistry and immunofluorescence microscopy. The results demonstrated intussusceptive structures (pillars, meshes, and folds) in benign (lobular capillary hemangioma or pyogenic granuloma, intravascular papillary endothelial hyperplasia or Masson tumor, sinusoidal hemangioma, cavernous hemangioma, glomeruloid hemangioma, angiolipoma, and lymphangiomas), low-grade malignancy (retiform hemangioendothelioma and Dabska tumor), and malignant (angiosarcoma and Kaposi sarcoma) VTs. Intussusceptive structures showed an endothelial cover and a core formed of connective tissue components and presented findings suggesting an origin through vessel loops, endothelialized thrombus, interendothelial bridges, and/or splitting and fusion, and conditioned VT morphology. In conclusion, the findings support the participation of IA and IL, in association with sprouting angiogenesis, in VTs, and therefore in their growth and morphogenesis, which is of pathophysiological interest and lays the groundwork for in-depth molecular studies with therapeutic purposes.

A follicular volume of >0.56 cm3 at trigger is the cutoff to predict oocyte maturity: a starting point for novel volume-based triggering criteria.

OBJECTIVE: To determine the minimum follicular volume on the day of trigger that will correspond to a mature oocyte at egg retrieval by individualized follicular puncture and to calculate the mean follicular growth from ovulation induction to egg retrieval using SonoAVCfollicle. DESIGN: A prospective observational study of 53 women undergoing in vitro fertilization, in which it was possible to identify unequivocally one or more follicles at trigger and egg retrieval using three-dimensional ultrasound. SETTING: University-affiliated private in vitro fertilization center. PATIENTS: The final sample included 206 follicles from 14 oocyte donors and 39 patients. INTERVENTIONS: A three-dimensional ultrasound with SonoAVCfollicle was performed at trigger and egg retrieval. The same operator selected follicles that were identified easily on both scans and verified that they were apt to be aspirated individually. Follicles were punctured individually, recording the real aspirated volume and the maturity stage of the oocyte. MAIN OUTCOME MEASURES: The primary outcome was the relationship between follicular volume on the day of the trigger and the oocyte maturity stage. The secondary outcome was the rate of follicular growth from the day of trigger to the day of oocyte retrieval, as measured using SonoAVCfollicle. RESULTS: On the day of trigger 206, follicles were selected. Of these, 5 could not be identified on the day of oocyte retrieval, probably because of follicular rupture (mean volume: 4 cm3, range: 2-7 cm3), and in 48, an oocyte was not obtained. The relationship between follicular volume and oocyte maturity was studied in 153 follicles: 125 (82%) contained mature and 28 (18%) contained immature oocytes. Receiver operating characteristic curves showed an area under the curve value of 0.73 (95% confidence interval: 0.65-0.80). A follicular volume of >0.56 cm3 is the cutoff point, with the highest Youden index having a sensitivity of 85% and a specificity of 64% to predict oocyte maturity. The mean follicular growth from trigger to egg retrieval was 26%-50% in 53% of cases. CONCLUSION: A follicular volume of >0.56 cm3 at trigger is the cutoff point with the optimal balance between sensitivity and specificity for oocyte maturity. Follicles of >2-3 cm3 may undergo spontaneous rupture before egg retrieval. Given these findings, we propose new volume-based criteria for trigger: 70% of follicles of >0.6 cm3 and dominant follicles between 2 and 3 cm3. These findings need validation by randomized controlled trials.

Androgen deficiency is associated with a better prognosis in glioblastoma.

BACKGROUND: The androgen receptor (AR) has been demonstrated to play a role in the pathogenesis of glioblastoma; however, the implications of circulating testosterone levels in the biology of glioblastoma remain unknown. AIM: This study aimed to analyze the association between circulating testosterone levels and the prognosis of patients with glioblastoma. METHODS: Forty patients with primary glioblastoma were included in the study. The main prognostic endpoint was progression-free survival (PFS). Circulating testosterone levels were used to determine the state of androgen deficiency (AD). AR expression was analyzed by reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence. Survival analysis was performed using the log-rank test and univariate and multivariate Cox regression analysis. RESULTS: Most of the patients showed AR expression, and it was mainly located in the cytoplasm, as well as in the nucleus of tumor cells. Patients with AD presented a better PFS than those patients with normal levels (252.0 vs. 135.0 days; p = 0.041). Furthermore, normal androgenic status was an independent risk factor for progression in a multivariate regression model (hazard ratio = 6.346; p = 0.004). CONCLUSION: Circulating testosterone levels are associated with the prognosis of glioblastoma because patients with AD show a better prognosis than those with normal androgenic status.

Evaluation of Zonulin Expression and Its Potential Clinical Significance in Glioblastoma.

Glioblastoma, the deadliest adult brain tumor, poses a significant therapeutic challenge with a dismal prognosis despite current treatments. Zonulin, a protein influencing tight junctions and barrier functions, has gained attention for its diverse roles in various diseases. This study aimed to preliminarily analyze the circulating and tumor zonulin levels, evaluating their impact on disease prognosis and clinical-radiological factors. Additionally, we investigated in vitro zonulin expression in different glioblastoma cell lines under two different conditions. The study comprised 34 newly diagnosed glioblastoma patients, with blood samples collected before treatment for zonulin and haptoglobin analysis. Tumor tissue samples from 21 patients were obtained for zonulin expression. Clinical, molecular, and radiological data were collected, and zonulin protein levels were assessed using ELISA and Western blot techniques. Furthermore, zonulin expression was analyzed in vitro in three glioblastoma cell lines cultured under standard and glioma-stem-cell (GSC)-specific conditions. High zonulin expression in glioblastoma tumors correlated with larger preoperative contrast enhancement and edema volumes. Patients with high zonulin levels showed a poorer prognosis (progression-free survival [PFS]). Similarly, elevated serum levels of zonulin were associated with a trend of shorter PFS. Higher haptoglobin levels correlated with MGMT methylation and longer PFS. In vitro, glioblastoma cell lines expressed zonulin under standard cell culture conditions, with increased expression in tumorsphere-specific conditions. Elevated zonulin levels in both the tumor and serum of glioblastoma patients were linked to a poorer prognosis and radiological signs of increased disruption of the blood-brain barrier. In vitro, zonulin expression exhibited a significant increase in tumorspheres.

Genetic Profiling of Glucocorticoid (NR3C1) and Mineralocorticoid (NR3C2) Receptor Polymorphisms before Starting Therapy with Androgen Receptor Inhibitors: A Study of a Patient Who Developed Toxic Myocarditis after Enzalutamide Treatment.

Enzalutamide is a nonsteroidal inhibitor of the androgen receptor (AR) signaling pathway and is used to treat patients with metastatic castration-resistant prostate cancer. However, the risk of cardiovascular-related hospitalization in patients with no contraindications for the use of enzalutamide is about 1-2%. To date, the underlying molecular basis of this has not been established. The androgen receptor, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are nuclear receptors that share structural similarities and have closely related DNA-binding sites and coregulators. In non-epithelial cells, a fine balance of the activities of these receptors is essential to ensure correct cellular function. In this study, we present a molecular characterization of these nuclear receptors in a prostate cancer patient who developed congestive heart failure after enzalutamide treatment. White cell RNAseq revealed a homozygous rs5522 MR polymorphism and both the rs143711342 and rs56149945 GR polymorphisms, carried in different alleles. No different specific splice isoforms were detected. Recent research suggests that AR inhibition by enzalutamide makes available a coregulator that specifically interacts with the rs5522-mutated MR, increasing its activity and producing adverse effects on cardiovascular health. We suggest an evaluation of the MR rs5522 polymorphism before starting therapy with AR inhibitors.

FKBP51, AmotL2 and IQGAP1 Involvement in Cilastatin Prevention of Cisplatin-Induced Tubular Nephrotoxicity in Rats.

The immunophilin FKBP51, the angiomotin AmotL2, and the scaffoldin IQGAP1 are overexpressed in many types of cancer, with the highest increase in leucocytes from patients undergoing oxaliplatin chemotherapy. Inflammation is involved in the pathogenesis of nephrotoxicity induced by platinum analogs. Cilastatin prevents renal damage caused by cisplatin. This functional and confocal microscopy study shows the renal focal-segmental expression of TNFα after cisplatin administration in rats, predominantly of tubular localization and mostly prevented by co-administration of cilastatin. FKBP51, AmotL2 and IQGAP1 protein expression increases slightly with cilastatin administration and to a much higher extent with cisplatin, in a cellular- and subcellular-specific manner. Kidney tubule cells expressing FKBP51 show either very low or no expression of TNFα, while cells expressing TNFα have low levels of FKBP51. AmotL2 and TNFα seem to colocalize and their expression is increased in tubular cells. IQGAP1 fluorescence increases with cilastatin, cisplatin and joint cilastatin-cisplatin treatment, and does not correlate with TNFα expression or localization. These data suggest a role for FKBP51, AmotL2 and IQGAP1 in cisplatin toxicity in kidney tubules and in the protective effect of cilastatin through inhibition of dehydropeptidase-I.

AmotL2, IQGAP1, and FKBP51 Scaffold Proteins in Glioblastoma Stem Cell Niches.

Glioma stem cells (GSCs) live in a continuous process of stemness reprogramming to achieve specific cell commitment within the so-called GSC niches, specifically located in periarteriolar regions. In this review, we analyze the expression levels, cellular and subcellular location, and role of three scaffold proteins (IQGAP1, FKBP51, and AmotL2) in GSC niches. Scaffold proteins contribute to cell differentiation, migration, and angiogenesis in glioblastoma. It could be of diagnostic interest for establishing stages, for therapeutic targets, and for improving glioblastoma prognosis, which is still at the experimental level.

Celastrol and Melatonin Modify SIRT1, SIRT6 and SIRT7 Gene Expression and Improve the Response of Human Granulosa-Lutein Cells to Oxidative Stress.

An excess of oxidative stress (OS) may affect several physiological processes fundamental to reproduction. SIRT1, SIRT6 and SIRT7 are involved in protection stress systems caused by OS, and they can be activated by antioxidants such as celastrol or melatonin. In this study, we evaluate SIRT1, SIRT6 and SIRT7 gene expression in cultured human granulosa-lutein (hGL) cells in response to OS inductors (glucose or peroxynitrite) and/or antioxidants. Our results show that celastrol and melatonin improve cell survival in the presence and absence of OS inductors. In addition, melatonin induced SIRT1, SIRT6 and SIRT7 gene expression while celastrol only induced SIRT7 gene expression. This response was not altered by the addition of OS inductors. Our previous data for cultured hGL cells showed a dual role of celastrol as a free radical scavenger and as a protective agent by regulating gene expression. This study shows a direct effect of celastrol on SIRT7 gene expression. Melatonin may protect from OS in a receptor-mediated manner rather than as a scavenger. In conclusion, our results show increased hGL cells survival with melatonin or celastrol treatment under OS conditions, probably through the regulation of nuclear sirtuins' gene expression.

Disproportion in Pericyte/Endothelial Cell Proliferation and Mechanisms of Intussusceptive Angiogenesis Participate in Bizarre Vessel Formation in Glioblastoma.

Glioblastoma (GBM) is the most malignant tumor in the brain. In addition to the vascular pattern with thin-walled vessels and findings of sprouting angiogenesis, GBM presents a bizarre microvasculature (BM) formed by vascular clusters, vascular garlands, and glomeruloid bodies. The mechanisms in BM morphogenesis are not well known. Our objective was to assess the role of pericyte/endothelial proliferation and intussusceptive angiogenic mechanisms in the formation of the BM. For this purpose, we studied specimens of 66 GBM cases using immunochemistry and confocal microscopy. In the BM, the results showed (a) transitional forms between the BM patterns, mostly with prominent pericytes covering all the abluminal endothelial cell (EC) surface of the vessels, (b) a proliferation index high in the prominent pericytes and low in ECs (47.85 times higher in pericytes than in ECs), (c) intravascular pillars (hallmark of intussusceptive angiogenesis) formed by transcapillary interendothelial bridges, endothelial contacts of opposite vessel walls, and vessel loops, and (d) the persistence of these findings in complex glomeruloid bodies. In conclusion, disproportion in pericyte/EC proliferation and mechanisms of intussusceptive angiogenesis participate in BM formation. The contributions have morphogenic and clinical interest since pericytes and intussusceptive angiogenesis can condition antiangiogenic therapy in GBM.

Molecular-Morphological Relationships of the Scaffold Protein FKBP51 and Inflammatory Processes in Knee Osteoarthritis.

Knee osteoarthritis (OA) is one of the most prevalent chronic conditions affecting the adult population. OA is no longer thought to come from a purely biomechanical origin but rather one that has been increasingly recognized to include a persistent low-grade inflammatory component. Intra-articular corticosteroid injections (IACSI) have become a widely used method for treating pain in patients with OA as an effective symptomatic treatment. However, as the disease progresses, IACSI become ineffective. FKBP51 is a regulatory protein of the glucocorticoid receptor function and have been shown to be dysregulated in several pathological scenario's including chronic inflammation. Despite of these facts, to our knowledge, there are no previous studies of the expression and possible role of FKBP51 in OA. We investigated by double and triple immunofluorescence confocal microscopy the cellular and subcellular expression of FKBP51 and its relations with inflammation factors in osteoarthritic knee joint tissues: specifically, in the tibial plateau knee cartilage, Hoffa's fat pad and suprapatellar synovial tissue of the knee. Our results show co-expression of FKBP51 with TNF-α, IL-6, CD31 and CD34 in OA chondrocytes, synovial membrane cells and adipocytes in Hoffa's fat pad. FKBP51 is also abundant in nerve fibers within the fat pad. Co-expression of FKBP51 protein with these markers may be indicative of its contribution to inflammatory processes and associated chronic pain in OA.

Celastrol Prevents Oxidative Stress Effects on FSHR, PAPP, and CYP19A1 Gene Expression in Cultured Human Granulosa-Lutein Cells.

Regulation of oxidative stress (OS) is important to prevent damage to female reproductive physiology. While normal OS levels may have a regulatory role, high OS levels may negatively affect vital processes such as folliculogenesis or embryogenesis. The aim of this work was to study OS induced by glucose, a reactive oxygen species generator, or peroxynitrite, a reactive nitrogen species generator, in cultured human granulosa-lutein (hGL) cells from oocyte donors, analyzing expression of genes involved in oocyte maturation (FSHR, PAPP, and CYP19A1) and OS damage response (ALDH3A2). We also evaluated the effect of celastrol as an antioxidant. Our results showed that although both glucose and peroxynitrite produce OS increments in hGL cells, only peroxynitrite treatment increases ALDH3A2 and PAPP gene expression levels and decreases FSHR gene expression levels. Celastrol pre-treatment prevents this effect of peroxynitrite. Interestingly, when celastrol alone was added, we observed a reduction of the expression of all genes studied, which was independent of both OS inductors. In conclusion, regulation of OS imbalance by antioxidant substances such as celastrol may prevent negative effects of OS in female fertility. In addition to the antioxidant activity, celastrol may well have an independent role on regulation of gene expression in hGL cells.

CD34+ Stromal Cells/Telocytes as a Source of Cancer-Associated Fibroblasts (CAFs) in Invasive Lobular Carcinoma of the Breast.

Several origins have been proposed for cancer-associated fibroblasts (CAFs), including resident CD34+ stromal cells/telocytes (CD34+SCs/TCs). The characteristics and arrangement of mammary CD34+SCs/TCs are well known and invasive lobular carcinoma of the breast (ILC) is one of the few malignant epithelial tumours with stromal cells that can express CD34 or αSMA, which could facilitate tracking these cells. Our objective is to assess whether tissue-resident CD34+SCs/TCs participate in the origin of CAFs in ILCs. For this purpose, using conventional and immunohistochemical procedures, we studied stromal cells in ILCs (n:42) and in normal breasts (n:6, also using electron microscopy). The results showed (a) the presence of anti-CD34+ or anti-αSMA+ stromal cells in varying proportion (from very rare in one of the markers to balanced) around nests/strands of neoplastic cells, (b) a similar arrangement and location of stromal cells in ILC to CD34+SCs/TCs in the normal breast, (c) both types of stromal cells coinciding around the same nest of neoplastic cells and (d) the coexpression of CD34 and αSMA in stromal cells in ILC. In conclusion, our findings support the hypothesis that resident CD34+SCs/TCs participate as an important source of CAFs in ILC. Further studies are required in this regard in other tumours.

Over-Production of Therapeutic Growth Factors for Articular Cartilage Regeneration by Protein Production Platforms and Protein Packaging Cell Lines.

This review article focuses on the current state-of-the-art cellular and molecular biotechnology for the over-production of clinically relevant therapeutic and anabolic growth factors. We discuss how the currently available tools and emerging technologies can be used for the regenerative treatment of osteoarthritis (OA). Transfected protein packaging cell lines such as GP-293 cells may be used as "cellular factories" for large-scale production of therapeutic proteins and pro-anabolic growth factors, particularly in the context of cartilage regeneration. However, when irradiated with gamma or x-rays, these cells lose their capacity for replication, which makes them safe for use as a live cell component of intra-articular injections. This innovation is already here, in the form of TissueGene-C, a new biological drug that consists of normal allogeneic primary chondrocytes combined with transduced GP2-293 cells that overexpress the growth factor transforming growth factor ß1 (TGF-ß1). TissueGene-C has revolutionized the concept of cell therapy, allowing drug companies to develop live cells as biological drug delivery systems for direct intra-articular injection of growth factors whose half-lives are in the order of minutes. Therefore, in this paper, we discuss the potential for new innovations in regenerative medicine for degenerative diseases of synovial joints using mammalian protein production platforms, specifically protein packaging cell lines, for over-producing growth factors for cartilage tissue regeneration and give recent examples. Mammalian protein production platforms that incorporate protein packaging eukaryotic cell lines are superior to prokaryotic bacterial expression systems and are likely to have a significant impact on the development of new humanized biological growth factor therapies for treating focal cartilage defects and more generally for the treatment of degenerative joint diseases such as OA, especially when injected directly into the joint.

Physiological Effects of the Electrogenic Current Generated by the Na+/K+ Pump in Mammalian Articular Chondrocytes.

Background: Although the chondrocyte is a nonexcitable cell, there is strong interest in gaining detailed knowledge of its ion pumps, channels, exchangers, and transporters. In combination, these transport mechanisms set the resting potential, regulate cell volume, and strongly modulate responses of the chondrocyte to endocrine agents and physicochemical alterations in the surrounding extracellular microenvironment. Materials and Methods: Mathematical modeling was used to assess the functional roles of energy-requiring active transport, the Na+/K+ pump, in chondrocytes. Results: Our findings illustrate plausible physiological roles for the Na+/K+ pump in regulating the resting membrane potential and suggest ways in which specific molecular components of pump can respond to the unique electrochemical environment of the chondrocyte. Conclusion: This analysis provides a basis for linking chondrocyte electrophysiology to metabolism and yields insights into novel ways of manipulating or regulating responsiveness to external stimuli both under baseline conditions and in chronic diseases such as osteoarthritis.

IQGAP1, AmotL2, and FKBP51 Scaffoldins in the Glioblastoma Microenvironment.

Glioblastoma (GB) is the most frequently occurring and aggressive primary brain tumor. Glioma stem cells (GSCs) and astrocytoma cells are the predominant malignant cells occurring in GB besides a highly heterogeneous population of migrating, neovascularizing and infiltrating myeloid cells that forms a complex tumor microenvironment (TME). Cross talk between the TME cells is pivotal in the biology of this tumor and, consequently, adaptor proteins at critical junctions of signaling pathways may be crucial. Scaffold proteins (scaffolins or scaffoldins) integrate external and internal stimuli to regulate various signaling pathways, interacting simultaneously with multiple proteins involved. We investigated by double and triple immunofluorescence the localization of IQGAP1, AmotL2, and FKBP51, three closely related scaffoldins, in malignant cells and TME of human GB tumors. We found that IQGAP1 is preferentially expressed in astrocytoma cells, AmotL2 in GSCs, and FKBP51 in white blood cells in human GB tumors. As GSCs are specially the target for novel therapies, we will investigate in further studies whether AmotL2 inhibition is effective in the treatment of GB.

The chondrocyte channelome: A narrative review.

Chondrocytes are the main cells in the extracellular matrix (ECM) of articular cartilage and possess a highly differentiated phenotype that is the hallmark of the unique physiological functions of this specialised load-bearing connective tissue. The plasma membrane of articular chondrocytes contains a rich and diverse complement of membrane proteins, known as the membranome, which defines the cell surface phenotype of the cells. The membranome is a key target of pharmacological agents and is important for chondrocyte function. It includes channels, transporters, enzymes, receptors, and anchors for intracellular, cytoskeletal and ECM proteins and other macromolecular complexes. The chondrocyte channelome is a sub-compartment of the membranome and includes a complete set of ion channels and porins expressed in these cells. Many of these are multi-functional proteins with "moonlighting" roles, serving as channels, receptors and signalling components of larger molecular assemblies. The aim of this review is to summarise our current knowledge of the fundamental aspects of the chondrocyte channelome, discuss its relevance to cartilage biology and highlight its possible role in the pathogenesis of osteoarthritis (OA). Excessive and inappropriate mechanical loads, an inflammatory micro-environment, alternative splicing of channel components or accumulation of basic calcium phosphate crystals can result in an altered chondrocyte channelome impairing its function. Alterations in Ca2+ signalling may lead to defective synthesis of ECM macromolecules and aggravated catabolic responses in chondrocytes, which is an important and relatively unexplored aspect of the complex and poorly understood mechanism of OA development.

Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women.

Sirtuins are a family of deacetylases that modify structural proteins, metabolic enzymes, and histones to change cellular protein localization and function. In mammals, there are seven sirtuins involved in processes like oxidative stress or metabolic homeostasis associated with aging, degeneration or cancer. We studied gene expression of sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL) from IVF patients in different infertility diagnostic groups and in oocyte donors (OD; control group). Study 1: sirtuins genes' expression levels and correlations with age and IVF parameters in women with no ovarian factor. We found significantly higher expression levels of SIRT1, SIRT2 and SIRT5 in patients ≥40 years old than in OD and in women between 27 and 39 years old with tubal or male factor, and no ovarian factor (NOF). Only SIRT2, SIRT5 and SIRT7 expression correlated with age. Study 2: sirtuin genes' expression in women poor responders (PR), endometriosis (EM) and polycystic ovarian syndrome. Compared to NOF controls, we found higher SIRT2 gene expression in all diagnostic groups while SIRT3, SIRT5, SIRT6 and SIRT7 expression were higher only in PR. Related to clinical parameters SIRT1, SIRT6 and SIRT7 correlate positively with FSH and LH doses administered in EM patients. The number of mature oocytes retrieved in PR is positively correlated with the expression levels of SIRT3, SIRT4 and SIRT5. These data suggest that cellular physiopathology in PR's follicle may be associated with cumulative DNA damage, indicating that further studies are necessary.

Extracellular multivesicular bodies in tissues affected by inflammation/repair and tumors.

Extracellular vesicles (EVs) are a heterogeneous population involved in intercellular communication. Little attention has been paid to a peculiar EV type with the appearance of a multivesicular body: extracellular multivesicular body (EMVB), also termed matrix vesicle cluster/multivesicular cargo. The aim of this work is to assess the ultrastructural characteristics, participation, and tissue location of EMVBs in inflammation/repair and tumors (with physiopathological processes involving intense intercellular communication), for which representative specimens were used. The results showed several forms of EMVBs: a) mature EMVBs, made up of clusters of vesicles surrounded by a plasma membrane, b) pre-EMVBs, with protruding grouped vesicles under the cell membrane, and c) post-EMVBs, releasing their vesicles. In tissues with inflammation/repair, EMVBs were observed in vessel lumens, interstitial spaces of vessel walls (between endothelial cells, pericytes, and smooth muscle cells) and between inflammatory and stromal cells. In tumors, such as basal cell carcinoma, craniopharyngioma, syringocystoadenoma, fibrous histiocytoma, alveolar rhabdomyosarcoma, lymphomas, neuroblastoma, astrocytomas, meningiomas, and hydatiform mole, EMVBs were present in tumor gland lumens and between tumor cells. In conclusion, in numerous physiopathological processes, we contribute EMVB ultrastructural characteristics (including different forms of mature, pre- and post-EMVBs, suggesting a more efficient EV transport), location and relationship with different types of cells. Further studies are required to assess the role of EMVBs in these physiopathological conditions.

Increased expression of ATP12A proton pump in cystic fibrosis airways.

Proton secretion mediated by ATP12A protein on the surface of the airway epithelium may contribute to cystic fibrosis (CF) lung disease by favoring bacterial infection and airway obstruction. We studied ATP12A in fresh bronchial samples and in cultured epithelial cells. In vivo, ATP12A expression was found almost exclusively at the apical side of nonciliated cells of airway epithelium and in submucosal glands, with much higher expression in CF samples. This could be due to bacterial infection and inflammation, since treating cultured cells with bacterial supernatants or with IL-4 (a cytokine that induces goblet cell hyperplasia) increased the expression of ATP12A in nonciliated cells. This observation was associated with upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A. ATP12A function was evaluated by measuring the pH of the apical fluid in cultured epithelia. Under resting conditions, CF epithelia showed more acidic values. This abnormality was minimized by inhibiting ATP12A with ouabain. Following treatment with IL-4, ATP12A function was markedly increased, as indicated by strong acidification occurring under bicarbonate-free conditions. Our study reveals potentially novel aspects of ATP12A and remarks its importance as a possible therapeutic target in CF and other respiratory diseases.

The Na, K-ATPase ß-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the ß-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase ß subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, ß1, ß2/AMOG (Adhesion Molecule On Glia) and ß3, were found to be expressed in GBM samples. Generally, ß1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. ß2/AMOG and ß3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, ß2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the ß3 subunit was more intense. These changes in ß subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (ß2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase ß subunits as adaptor proteins and transcription factors.