Learning Curve for Flow Diversion of Posterior Circulation Aneurysms: A Long-Term International Multicenter Cohort Study.

BACKGROUND AND PURPOSE: Flow diversion has gradually become a standard treatment for intracranial aneurysms of the anterior circulation. Recently, the off-label use of the flow diverters to treat posterior circulation aneurysms has also increased despite initial concerns of rupture and the suboptimal results. This study aimed to explore the change in complication rates and treatment outcomes across time for posterior circulation aneurysms treated using flow diversion and to further evaluate the mechanisms and variables that could potentially explain the change and outcomes. MATERIALS AND METHODS: A retrospective review using a standardized data set at multiple international academic institutions was performed to identify patients with ruptured and unruptured posterior circulation aneurysms treated with flow diversion during a decade spanning January 2011 to January 2020. This period was then categorized into 4 intervals. RESULTS: A total of 378 procedures were performed during the study period. Across time, there was an increasing tendency to treat more vertebral artery and fewer large vertebrobasilar aneurysms (P = .05). Moreover, interventionalists have been increasingly using fewer overlapping flow diverters per aneurysm (P = .07). There was a trend toward a decrease in the rate of thromboembolic complications from 15.8% in 2011-13 to 8.9% in 2018-19 (P = .34). CONCLUSIONS: This multicenter experience revealed a trend toward treating fewer basilar aneurysms, smaller aneurysms, and increased usage of a single flow diverter, leading to a decrease in the rate of thromboembolic and hemorrhagic complications.

PO-47 - Microparticles derived from ovarian cancer cell line contained genomic and biologically active proteins, including tissue factor involved in coagulation.

INTRODUCTION: The microparticles (MPs) are sized vesicles of less than 1 µm, from different cell types upon activation or subsequent to apoptosis. They are involved in the thrombotic process, particularly in cancer. The role of MPs in ovarian cancer and their involvement in thrombosis being poorly understood. AIM: The aim of this study was to identify in vitro the generation of MPs by an human ovarian adenocarcinoma cell line (OVCAR-3). MATERIALS AND METHODS: OVCAR-3 cells were cultured in three conditions [without stimulation, with protein C (PC), and with activated protein C (APC)]. Then, the MPs present in the supernatant, were isolated by ultracentrifugation and were analyzed for their shape and properties by flow cytometry, electron microscopy, cryofracture analysis, DNA and RNA, and proteomic analysis. The level of tissue factor (TF) on MP was evaluated by TF-induced shortening of Ca(2+) plasma coagulation time. RESULTS: Our results demonstrated that 1) 92% of MPs derived from OVCAR-3 were less than 100 nm. 2) As tested by flow cytometry, the MPs contained b2 microglobulin, annexin, DNA fragments and TF that induces a shortening of Ca(2+) -induced plasma coagulation time. When OVCAR-3 were cultured for 18H with PCA, MPs were generated in greater amount than those generated by OVCAR-3 in its absence and their level of TF was increased of 20%. Curiously, in contrast with intact OVCAR-3 cells, the endothelial protein C receptor (EPCR) was not detected in MPs 3) Proteomic analysis show that the MPs contain proteins involved in cancer progression such as mucins (5A and 5B), keratin type-1, actin, annexin (A1, A2, A4), CD44, glypican, heat shock (70kDa and HS90a) proteins, Agrin associated with heparan sulfate proteoglycan abundant in the tumor-specific basement membrane, Ephrin type A receptor, coronin-1C, catenin α, integrin ß-1 and also p-selectin responsible of platelet activation. They also express several DNA associated proteins includingtranscription factors, various polymerases, nucleases, and histones involved in chromosome packaging and transcription in the cell nucleus. CONCLUSIONS: MPs derived from OVCAR-3 have an apoptotic character. They expressed several biologically active proteins, DNA and their associated proteins. Despite the absence of EPCR expression on MPs that was expressed on intact OVCAR-3 cells, they expressed procoagulant TF activity already found on intact ovarian cancer cells. This activity is greater extent in the presence of APC.

PO-13 - Production of a functional thrombopoietin by a human ovarian cancer cell line.

INTRODUCTION: Hemostatic abnormalities are frequently noticed in patients with malignant diseases. These complications include platelets disorders. The role of platelets in cancer extends beyond thrombocytosis and thrombosis, and also platelets promote cancer growth and metastatic dissemination. In the physiology, platelet production is regulated by thrombopoietin, which is mainly secreted by the liver. We, previously, reported that thrombopoietin could be secreted by the ovarian adenocarcinoma cell line, OVCAR-3. AIM: Our main purpose is to analyze the gene expression of thrombopoietin in ovarian cancer cells and to assess its functionality. MATERIALS AND METHODS: The thrombopoietin gene expression in ascitic cells from patients with ovarian carcinomatosis, as well as, in three cancer cell lines, including OVCAR-3 cells, performed using reverse transcription PCR, real-time PCR and gene sequencing, Normal human ovary and liver tissues are used as controls. The functionality of thrombopoietin on the basis of the viability of a thrombopoietin-dependent cell line (Ba/F3) using a co-culture method. RESULTS: Similarly to liver and ovary tissues, all cancer cells lines express the three TPO-1 (full length TPO), TPO-2 (12bp deletion) and TPO-3 (116pb deletion) variants. By flow cytometry, we show that thrombopoietin production by OVCAR-3 could be increased when cells are stimulated by activated protein C. Lastly, Our results confirm that activated protein C may act, in a paracrine fashion, to boost thrombopoietin production. CONCLUSIONS: We report, for the first time, that thrombopoietin secreted by ovarian cancer cells is functional. Hence, thrombopoietin produced by tumor cells may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

PO-23 - Expression of heparanase in cancer as biomarker of malignancies: overexpression in an aggressive, poor survival gastric cancer "gastric signet ring cell carcinoma" compared with that of other gastric cancers.

INTRODUCTION: Gastric signet ring cell carcinoma (GSRC) is a distinct entity among of other gastric cancer. With unknown etiopathology, their incidence is increasing and it presents a low sensitivity to chemotherapy. AIM: Here, we studied the expression of the heparanase (HPA) in cancer tissues from GSRC patients and several cancer cell lines. HPA is an endo-ß-D-glucuronidase, capable of cleaving the lateral chains of heparan sulfate on cell surfaces and the extracellular matrix at acidic pH. Apart from its well-characterized enzyme activity, HPA also has independent enzymatic functions, such as up-regulation of vascular endothelial growth factor (VEGF) -A and VEGF-C and activation of intracellular signaling pathways involved in, survival, migration and proliferation of tumor cells. It can also induce an hypercoagulability by a non-enzymatic manner. MATERIALS AND METHODS: HPA was tested in several cancer cell lines from ovaries (OVCAR-3, SKOV-3), breast (MDAMB231, MCF7) colon (LS-174), lung (A549), uterus (HELA) and gastric (adenocarcinoma (AGS) and GSRC (KATO-III) using several techniques such as RT-PCR, Q-PCR, immunocytochemistry, flow cytometry and degradation of Fondaparinux at pH 5, evaluated by its anti Xa activity evaluated by Factor Xa amidolytic activity. The amount of HPA mRNA in the biopsy simples of gastric adenocarcinoma (n=10) and GSRC (n=11) in tumors and their environment were analyzed. RESULTS: HPA gene is expressed in all cancer cell lines, but its level varies depending on the tumor cell line. In biopsies of gastric cancer, the HPA gene is more expressed in the tumor regions (p=0.0002) and tumor environment (p=0.015) in GSRC than in gastric adenocarcinoma. B) The activity of HPA, evaluated by degradation of Fondaparinux at pH 5, 1) in the supernatants of 10(6) cancer cells: the residual activity of Fondaparinux after 2 hours incubation at 37°C with OVCAR-3 supernatant was of 70% of control value, and of 80% with KATO-III cell supernatants. 2) in patient's plasmas, this technique cannot be used because the site of degradation of fondaparinux by heparanase is masked by AT present in plasma. CONCLUSIONS: Heparanase was found in in many cancer cell lines and its level depends on origin of tumor cells and on its aggressivity. Taking into account the pro-metastatic functions, proangiogenic and procoagulant activity of HPA and its overexpression in gastric signet ring cell carcinoma of poor prognosis and its cell line, HPA can be considered as a biomarker of malignancy and as a therapeutic target in GSRC patients.

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines.

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the alpha subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5'-AGGCTACCACAGTCTCCCTG-3' and 5'-GCAGTGTAAAATCTCCAGTC-3' (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5'-CTGCCmATG GATGATGGT-3' (sense) and 5'-GTTCAGCTCGAAGAAGA-3' (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells.

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries.

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.

Defective cell migration in an ovarian cancer cell line is associated with impaired urokinase-induced tyrosine phosphorylation.

The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.

Immunopurification of an S-antigen-like protein from human platelets.

S-antigen (also named arrestin or 48K protein) is a protein abundant in photoreceptor cells of vertebrates and invertebrates. The presently known function of this protein in retina is to arrest the enzymatic cascade of phototransduction in retinal rods, through its binding to photoactivated and phosphorylated rhodopsin. Proteins closely related to S-antigen were recently demonstrated in several non photosensitive cells. In this work, we demonstrated the presence of a protein similar to retinal S-antigen with regards to its immunoreactivity with a panel of monoclonal antibodies and its molecular weight in soluble extracts of human platelets. This protein was purified by immunoaffinity chromatography using a rabbit antibody to retinal S-antigen. This S-antigen-like protein could have a regulatory function in G-protein-mediated transduction of chemical signals in platelets, similar to arrestin function in phototransduction.

Comparative study of fibrinolytic activity on 937 line after stimulation by interferon gamma, 1,25 dihydroxyvitamin D3 and their combination.

Previous study showed that the secretion of urokinase (UK) by monoblastic cell line U 937 and the number of binding sites for urokinase and for plasminogen (Plg) on the cell surfaces were augmented by interferon gamma (INF tau). This induction led to an increase in fibrinolytic activity on cell surfaces. A similar increase was also observed when treating the U 937 cells with 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3. Here we report that the combination of these two agents induced a 2.7 fold increase in the plasminogen activator activity on U 937 cell surfaces in comparison with 1 fold increase induced by INF tau and 1.3 fold increase by 1,25(OH)2D3. As evaluated by a flow cytometer, the increased fibrinolytic activity induced by the combination of INF tau and 1,25(OH)2D3 could be attributed to the increase of the number of binding sites both for UK (3.7 x 10(4) vs 1.2 x 10(4) per cell) and for Plg (16.2 x 10(4) vs 3.6 x 10(4) per cell), accompanied by an increased expression of CD 14, which is an antigen of differentiation on cell surfaces. These results suggest that the expression of urokinase receptors and plasminogen receptors may be coupled together by unknown intracellular mechanisms during cell differentiation, and support the idea that the concomitant regulation of these two receptors for UK and Plg is an important aspect in cell associated-fibrinolytic activity.

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Evolutionary stability of fibrinogen epitopes implicated in fibrin polymerization and in fibrinolysis.

In this work, fibrinogen evolution was analysed by testing the reactivity of fibrinogen from different species with monoclonal antibodies against human fibrinogen fragment D. One epitope concerning the fibrin polymerization site 'a' and two epitopes responsible for tPA binding to fibrin were conserved in all mammalian fibrogens tested but not in crab coagulogen or pleurodella fibrinogen. In these two species, some epitopes which are not implicated in fibrinogen function were conserved. Therefore, we can conclude that polymerizing site 'a' and tPA binding sites have not been modified for at least 80 million years.

Potent activity of peripheral blood lymphocytes in inducing hepatocyte stimulating factor and urokinase in monocytes.

As fibrinogen is an independent risk factor for arterial thrombosis we were interested in analysing the mechanism controlling fibrinogen biosynthesis. In this work, we showed that incubation of monocytes with lymphocytes increased hepatocyte stimulating factor (HSF) production. Different mechanisms are involved and our results demonstrated that this effect is in part mediated by an increase in interleukin 6 (IL-6) production. However, IL-6 cannot account for the whole effect and other cytokines could be implicated. In addition, we observed a stimulation of urokinase-type plasminogen activator (u-PA) associated with monocytes when these cells were incubated with lymphocytes for 18 h at 37 degrees C. By producing fragment D (fibrinogen degradation product) and D-dimer (fibrin degradation product) this fibrinolytic activity might also contribute to fibrinogen biosynthesis by hepatocytes.

Incubation of monocytes with adriamycin increases secretion of hepatocyte stimulating factor for fibrinogen biosynthesis.

This work provides evidence that the production by monocytes of hepatocyte stimulating factor(s) for fibrinogen biosynthesis was dramatically increased when monocytes were exposed to Adriamycin. This effect was related to an increased production of leukaemia inhibiting factor (LIF), a cytokine known to stimulate fibrinogen biosynthesis by hepatic cells. Adriamycin also induces an increase in membrane-associated urokinase on monocytes. These results are consistent with the clinical observation in patients with ovarian cancer that when the CA-125 tumour marker decreases during chemotherapy, an increased level of D-dimer is a marker of good prognosis.

Analysis of endothelial protein C receptor functionality on living cells'.

Activated protein C (APC) is a major control system of blood coagulation. APC prevents coagulation pathway by degrading Va and VIIIa plasma's coagulation factors. Protein C activation requires its binding to specific endothelial cell receptor (EPCR). APC binding to EPCR also activates a wide range of defense mechanisms (anti-inflammatory, antiapoptosis...). EPCR expression by cells can be detected by various methods, including immunoanalysis and molecular biology. However, no assays evaluate its functionality. A method, inspired of a standard fibrinoformation time assay, was developed to estimate EPCR ability to bind APC on living cell surface in vitro. Endothelial cells were incubated with APC and fibrinoformation on cells was followed by spectrophotometry (plasma absorbance increases with fibrin polymerization). Membrane-bound EPCR retain APC, thus prolonging fibrinoformation time in a dose-dependent manner. Control was realized with EPCR-negative cells. This new method can be used on any cell type to study the expression of other coagulation receptors.

[Mitogenic activity of vasculotropin for peripheral human lymphocytes]. / Activité mitogène de la vasculotropine pour les lymphocytes périphériques humains.

Vasculotropin is a growth factor with a unique specificity for vascular derived endothelial cells. We report that normal human peripheral lymphocytes represent another target for vasculotropin. The mitogenic activity of the medium conditioned by these cells cultured in the presence of Concanavalin A is potentiated by vasculotropin. This effect is exerted more likely through interactions with the soluble mediators rather than through the VAS receptors since VAS binds equally to Concanavalin A stimulated and to unstimulated lymphocytes.

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line.

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.

Down-regulation of fibrinogen biosynthesis by IL-4, IL-10 and IL-13.

High levels of fibrinogen are recognized as an important vascular risk factor; however, it is not known if the increase of plasma fibrinogen is directly responsible for this risk, or is only a marker of vascular inflammation. To support this second hypothesis, Oncostatin M (OSM) is a potent stimulator of fibrinogen biosynthesis and induces smooth muscle cell proliferation. In the same way, we analysed whether interleukin-4 (IL-4), interleukin-10 (IL-10) or interleukin-13 (IL-13), which protect vessel walls from monocytes injuries leading to atherosclerosis, could influence fibrinogen biosynthesis. The two levels of regulation of fibrinogen biosynthesis were tested: firstly, the direct effect of these cytokines on fibrinogen production by the hepatoma cell line Hep G2, and secondly their effect on the secretion of hepatocyte stimulating factor (HSF) activity in the supernatant of lipopolysaccharide (LPS)-activated monocytes. IL-4 and IL-13 added to Hep G2 cells down-regulated both the increase of fibrinogen secretion induced by IL-6 and fibrinogen mRNA levels, this effect being more pronounced when Hep G2 were preincubated with the two cytokines before IL-6 addition. The effect of IL-10 was evidenced only on mRNA expression. IL-10 and IL-13 dose-dependently decrease HSF activity secreted by LPS-activated monocytes, whereas IL-4 had no effect. However, the three cytokines decreased HSF activity when monocytes were incubated with the cytokines before LPS activation. The effects of these cytokines on HSF activity are related to variations of IL-6 and OSM secretion. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since cytokines which have a protective vascular effect down-regulate fibrinogen production.

Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1∶2560 4 weeks after immunization, and this bleed was used in all subsequent experiments. Immunoblotting with the polyclonal anti-alphaENaC serum, revealed a major band of 82-86 kDa in extracts prepared from whole bovine or rat retina; a minor component of 92 kDa in the extract from bovine ciliary body may represent a glycosylated species. Immunohistochemistry, using the alphaENaC-specific antiserum, revealed strong fluorescence in specific areas of the rat and human eye. Pronounced labelling was observed in the epithelial cell layer of the retina, the lens, as well as both the pigmented and the nonpigmented epithelium of the ciliary body and the iris. All of the cell layers (epithelium, endothelium and fibroblasts) in the cornea, the blood vessels in the iris, and iris epithelium, were also strongly immunopositive. The somatic body of the photoreceptor cells (cones and rods) in the inner and outer segments could be traced to forming a synapse in both the internal and external portions of the internal nuclear layer. The bipolar cells and ganglia in the neuronal compartment also exhibited occasional immunofluorescence. The method of fixation and the source of the tissue were important parameters for the immunochemical localization of the ENaC. The resolution was very poor when rat eye was fixed in Bouin's solution but this method was satisfactory for human tissues. For rat eye, optimum resolution was obtained with AMeX fixation. This widespread distribution of the ENaC generally colocalizes with the previously observed immunopositivity for the mineralocorticoid receptor such that steroid hormone-mediated ion regulation would appear to add a new parameter to the functional expression of ocular tissues.