Generation of Periventricular Reactive Astrocytes Overexpressing Aquaporin 4 Is Stimulated by Mesenchymal Stem Cell Therapy.

Aquaporin-4 (AQP4) plays a crucial role in brain water circulation and is considered a therapeutic target in hydrocephalus. Congenital hydrocephalus is associated with a reaction of astrocytes in the periventricular white matter both in experimental models and human cases. A previous report showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into the lateral ventricles of hyh mice exhibiting severe congenital hydrocephalus are attracted by the periventricular astrocyte reaction, and the cerebral tissue displays recovery. The present investigation aimed to test the effect of BM-MSC treatment on astrocyte reaction formation. BM-MSCs were injected into the lateral ventricles of four-day-old hyh mice, and the periventricular reaction was detected two weeks later. A protein expression analysis of the cerebral tissue differentiated the BM-MSC-treated mice from the controls and revealed effects on neural development. In in vivo and in vitro experiments, BM-MSCs stimulated the generation of periventricular reactive astrocytes overexpressing AQP4 and its regulatory protein kinase D-interacting substrate of 220 kDa (Kidins220). In the cerebral tissue, mRNA overexpression of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF1α), and transforming growth factor beta 1 (TGFß1) could be related to the regulation of the astrocyte reaction and AQP4 expression. In conclusion, BM-MSC treatment in hydrocephalus can stimulate a key developmental process such as the periventricular astrocyte reaction, where AQP4 overexpression could be implicated in tissue recovery.

Expression patterns of aquaporin 1 in vascular tumours.

BACKGROUND: Aquaporins (AQPs) are a family of water channels expressed in various body tissues. Beyond osmotic balance, AQPs have recently been confirmed to be involved in processes related to cancer (tumour proliferation, angiogenesis, etc.). OBJECTIVES: To analyse the presence of these proteins in the endothelium of several vascular tumours, both benign and malignant, in order to establish whether AQPs may be used as a marker or future therapeutic target. MATERIALS AND METHODS: We studied AQP1 expression in 39 patients with vascular tumours, classified into six groups according to ISSVA classification: haemangiomas, benign vascular tumours different from infantile haemangiomas, angiosarcomas, classic Kaposi's sarcoma (KS), and epidemic KS. RESULTS: AQP1 expression was present in 28 of 39 patients, representing 92.9% benign lesions, whereas no expression was found in 72% of malignant lesions. AQP1 expression was associated with benign lesions with an OR of 34.5 (95% CI: 5-250); p<0.0005, and was most frequently identified with a focal endothelial pattern (38%). A kappa index of 0.823 (95% CI: 0.678-0.971) was determined regarding the patterns of expression overall. CONCLUSION: The expression of AQP1 was greater in benign lesions than malignant lesions and this difference was statistically significant, thus AQP1 expression could serve as a marker for benignity of vascular tumours. In addition, the expression pattern of AQP1 was different according to the type of vascular tumour.

Expression Pattern of Aquaporin 1 and Aquaporin 3 in Melanocytic and Nonmelanocytic Skin Tumors.

OBJECTIVES: Study of aquaporin 1 (AQP1) and aquaporin 3 (AQP3) expression to understand its potential role in the pathophysiology of skin cancer. METHODS: Analysis of AQP1 and AQP3 expression by immunohistochemistry of 72 skin biopsy specimens from melanocytic skin tumors, nonmelanocytic tumors, or healthy samples. RESULTS: AQP1 showed strong labeling in 100% of benign common melanocytic nevi. Small blood vessels, stroma, and melanophages surrounding different types of melanomas tumors also were positive. Tumoral melanocytes in atypical nevi and melanomas were negative for AQP1. AQP3 showed strong labeling in 100% of melanocytic nevi, 100% of atypical melanocytic nevi, and 100% of melanomas. In all basal cell carcinomas and squamous cell carcinomas, staining for AQP3 was positive. CONCLUSIONS: To our knowledge, this work represents the first demonstration of AQP1/AQP3 expression in human melanocytic skin tumors. More studies are needed to understand the underlying molecular mechanisms of expression of both AQPs in melanocytic tumors and their potential as molecular therapeutic targets.

Aquaporin-1 plays important role in proliferation by affecting cell cycle progression.

Aquaporin-1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild-type (AQP1(+/+) ) and knock out (AQP1(-/-) ) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU(+) and TH-BrdU(+) cells were observed in AQP1(-/-) mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild-type cells. In the AQP1-overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation-related molecules, including, Zeb 2, Jun, NF-kß, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors.

Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kß, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

Expression of aquaporins in bronchial tissue and lung parenchyma of patients with chronic obstructive pulmonary disease.

BACKGROUND: Aquaporins AQP1 and AQP5 are highly expressed in the lung. Recent studies have shown that the expression of these proteins may be mechanistically involved in the airway inflammation and in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the expression of AQP1 and AQP5 in the bronchial tissue and the lung parenchyma of patients with COPD and COPD-resistant smokers. METHODS: Using a case-control design, we selected a group of 15 subjects with COPD and 15 resistant smokers (smokers without COPD) as a control, all of whom were undergoing lung resection surgery due to a lung neoplasm. We studied the expression of AQP1 and AQP5 in the bronchial tissue and the lung parenchyma by means of immunohistochemistry and reverse-transcription real-time polymerase chain reaction. Tissue expression of AQP1 and AQP5 was semi-quantitatively assessed in terms of intensity and expression by immunohistochemistry using a 4-point scale ranging from 0 (none) to 3 (maximum). RESULTS: There were no significant differences in gene expression between COPD patients and resistant smokers both in the bronchial tissue and in the lung parenchyma. However, AQP1 gene expression was 2.41-fold higher in the parenchyma of smokers with COPD compared to controls, whereas the AQP5 gene showed the opposite pattern, with a 7.75-fold higher expression in the bronchus of smokers with COPD compared with controls. AQP1 and AQP5 proteins were preferentially expressed in endothelial cells, showing a higher intensity for AQP1 (66.7% of cases with an intensity of 3, and 93.3% of subjects with an extension of 3 among patients with COPD). Subtle interstitial disease was associated with type II pneumocyte hyperplasia and an increased expression of AQP1. CONCLUSIONS: This study provides pilot observations on the differences in AQP1 and AQP5 expression between COPD patients and COPD-resistant smokers. Our findings suggest a potential role for AQP1 in the pathogenesis of COPD.

Functional inhibition of aquaporin-3 with a gold-based compound induces blockage of cell proliferation.

AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 µM) by counting number of viable cells and analyzing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's antiproliferative effect correlates with its ability to inhibit AQP3 Pgly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression.

Cellular overexpression of aquaporins slows down the natural HIF-2α degradation during prolonged hypoxia.

Overexpression of cell membrane aquaporins (AQPs) has recently been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the hypoxia inducible factor (HIF) family has been extensively implicated in tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic hypoxia, characteristic of a growing tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different proteins (AQP1, AQP3, AQP5, G6PD, and GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16 h at 1% O2, in wt-PC12 and in PC12 overexpressing non-AQP proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to cobalt chloride or dimethyloxallyl glycine. Normal proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3, VEGF, and PGK1) were 2-4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more tumor-like phenotype.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Overexpression of Aquaporin-1 in lung adenocarcinomas and pleural mesotheliomas.

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuro-pulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung adenocarcinomas (55%, P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong expression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuro-pulmonary neoplasms.

Development of cytosolic hypoxia and hypoxia-inducible factor stabilization are facilitated by aquaporin-1 expression.

O(2) is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O(2) permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O(2) channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O(2) transporter, because this transmembrane protein appears to be CO(2)-permeable and is highly expressed in cells with rapid O(2) turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O(2), as well as stabilization of the O(2)-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O(2) permeability and thus could facilitate O(2) diffusion across the cell membrane.

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells.

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO(2). Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes.

Desarrollo de ensayos inmunoenzimáticos para la detección de anticuerpos antiplaquetarios de las clases IgG, IgA e IgM / Development of immunoenzymatic assays to detect IgG and IgM antiplatelet antibodies

Se desarrollaron tres métodos para la detección de inmunoglobulina IgG, IgA e IgM asociada a la membrana plaquetaria. Los valores de densidad óptica (DO) de 50 donantes obtenidos por estas técnicas fueron de 0,124-0,236, 0,07-0,129 y 0,06-0,112 para las IgG, IgA e IgM, respectivamente. Se aceptaron como positivas las muestras con una DO mayor de tres desviaciones estándar de la media de los controles negativos. Se obtuvieron resultados positivos en 10 de los 12 pacientes (83,3 por ciento) con púrpura trombocitopénica autoinmune crónica (PTI-c). En todos los casos en remisión los resultados fueron negativos. La IgG fue la clase de inmunoglobulina más frecuentemente detectada tanto en la PTI-a como en la PTI-, seguida por la IgM y la IgA. La frecuencia con que se detectó una sola clase de inmunoglobulina fue similar a la frecuencia con que se detectaron combinaciones de más de un isotipo, en ambas formas de PTI. En 11 de los 23 pacientes con trombocitopenias y otros diagnósticos de enfermedades hematológicas o autoinmunes se encontraron resultados positivos. En estos casos de trombocitopenias autoinmunes secundarias fue frecuente encontrar la coexistencia de más de un isotipo de inmunoglobulina. Los métodos desarrollados permitirán realizar estudios acerca de la significación clínica del espectro de clases de inmunoglobulinas en las trombocitopenias autoinmunes.

Confección de un panel de eritrocitos de prueba para la detección y caracterización de anticuerpos / Preparation of a testing erythrocyte panel for detection and characterization of antibodies

Se estudiaron 73 personas pertenecientes al grupo sanguíneo O y aptas para donar sangre, para conformar un panel de eritrocitos de prueba para la detección y clasificación de anticuerpos antieritrocitarios. Se determinaron los siguientes antígenos de grupo sanguíneo. C, c, E, e, D, C(w), K, Kp(a), Kp(b), Fy(a), Fy(b), M, N, S, s, Pl, Le(a), Le(b), Lu(b), Jk(a), Jk(b) y se realizaron pruebas de control de calidad. Se confeccionó un panel de 10 donantes con el que se pueden detectar y clasificar todos los anticuerpos antieritrocitarios de importancia clínica