Adipose Tissue-Derived Mesenchymal Stem Cell Concentrated Conditioned Medium Alters the Expression Pattern of Glutamate Regulatory Proteins and Aquaporin-4 in the Retina after Mild Traumatic Brain Injury.

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 µL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

Activated microglia-derived macrophage-like cells exacerbate brain edema after ischemic stroke correlate with astrocytic expression of aquaporin-4 and interleukin-1 alpha release.

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3-7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema.

Oxytocin Reduces Brain Injury and Maintains Blood-Brain Barrier Integrity After Ischemic Stroke in Mice.

The present study was designed to determine the effect of different doses of oxytocin (OXT) on neuronal injury, spatial memory, blood-brain barrier (BBB) integrity and to explore possible underlying molecular mechanisms in the early stage of stroke in mice. Stroke model was generated by middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion in mice. OXT at doses of 1, 2, 4 and 8 IU/per mouse was administrated intranasally at the beginning of brain ischemia. Brain injury, BBB integrity, and spatial memory were evaluated by standard methods. Changes in the expression of nuclear factor-kappa B (NF-κB), and TUNEL positive cell were detected by immunohistochemistry. The levels of vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4) and brain-derived neurotrophic factor (BDNF) proteins were determined by western blotting and ELISA methods. OXT at doses of 4 and 8 IU/per mouse reduced the infarct size by 42% and 52%, respectively, and improved spatial memory function (p < 0.001). OXT (8 IU/per mouse) significantly reduced brain edema, BBB disruption and upregulated the AQP4 expression (p < 0.001). Finally, OXT significantly diminished the number of apoptotic, NF-κB positive cells and enhanced the expression of BDNF and VEGF proteins in the brain tissue (p < 0.001). These findings provide important evidences that OXT significantly suppresses neuronal damage in the early stage of stroke by inhibiting apoptotic and NF-κB signaling pathway, increasing the expression of VEGF, AQP4 and BDNF proteins and reducing the BBB leakage.

Reduced sarcolemmal aquaporin 4 expression can support the differential diagnosis of neuromyelitis optica spectrum disorders.

This study aimed to investigate the underlying pathological muscle damage in neuromyelitis optica spectrum disorder (NMOSD) patients without muscular symptoms. We prospectively enrolled 15 patients with aquaporin 4 (AQP4) antibody seropositive NMOSD and 16 patients with non-NMOSD diseases as a control group. Biceps biopsy samples from 18 patients were examined. Six NMOSD patients exhibited inflammatory lesions/edema in lower muscles on muscle MRI. On histopathological examination, NMOSD samples showed significantly decreased IgG-targeting AQP4 expression on sarcolemma compared with non-NMOSD samples in terms of the area of positive staining and integrated optical density. Muscle biopsy can support the differential diagnosis of NMOSD.

Aquaporin 4 regulation by ginsenoside Rb1 intervenes with oxygen-glucose deprivation/reoxygenation-induced astrocyte injury.

BACKGROUND: Spinal cord ischemia-reperfusion injury (SCII) is a common complication of spinal surgery as well as thoracic and abdominal surgery. Acute cytotoxic edema is the key pathogenic alteration. Therefore, avoiding or decreasing cellular edema has become the major target for SCII treatment. METHODS: The antiedema activity of ginsenoside Rb1 on aquaporin (AQP) 4, nerve growth factor (NGF), and brain-derived neurotrophic factor expression was detected by western blot and real-time polymerase chain reaction under conditions of oxygen-glucose deprivation/reoxygenation (OGD/R) in a rat astrocyte model in vitro. In addition, the cellular membrane permeability of AQP4 overexpressing cells or AQP4 small interfering RNA-transfected cells was detected. RESULTS: Ginsenoside Rb1 significantly prevented OGD/R-induced AQP4 downregulation in rat astrocytes. In addition, ginsenoside Rb1 treatment or AQP4 overexpression in rat astrocytes significantly attenuated the OGD/R-induced increase of cellular membrane permeability. Moreover, ginsenoside Rb1 obviously prevented the OGD/R-induced decrease of NGF and BDNT expression in rat astrocytes. CONCLUSION: These findings demonstrate that ginsenoside Rb1 can relieve spinal cord edema and improve neurological function by increasing AQP4 expression.

Regulation of aquaporin 4 expression by lipoxin A4 in astrocytes stimulated by lipopolysaccharide.

Aquaporin (AQP4) could be associated with inflammation, common in central nervous system diseases. We investigated the effect of lipoxin A4 (LXA4) on the activation of astrocytes, AQP4 expression, and inflammatory response induced by lipopolysaccharide (LPS). Astrocytes were cultured in vitro and changes in transcript and protein levels of AQP4, interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX-2), and protein levels of P38 and phospho-P38 were determined. The LPS group showed increased AQP4, IL-1ß, TNF-α, and COX-2 levels, whereas they decreased in the LPS + LXA4 group, suggesting that LXA4 inhibits AQP4 expression. Furthermore, levels of phospho-P38 increased in the LPS group, but decreased in the LPS + LXA4 group. In conclusion, LXA4 alleviated the LPS-induced increase in AQP4 expression and inflammatory cytokine secretion by astrocytes, possibly by inhibiting P38 phosphorylation. For the first time, we found that LXA4 may inhibit the expression of inflammatory factors by regulating the expression of AQP4. AQP4 on astrocytes is likely to be the target of anti-inflammatory effect of LXA4.

Cerebral aquaporin-4 expression is independent of seizures in tuberous sclerosis complex.

Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC.

Hydrogen­rich solution against myocardial injury and aquaporin expression via the PI3K/Akt signaling pathway during cardiopulmonary bypass in rats.

Myocardial ischemia, hypoxia and reperfusion injury are induced by aortic occlusion, cardiac arrest and resuscitation during cardiopulmonary bypass (CPB), which can severely affect cardiac function. The aim of the present study was to investigate the effects of hydrogen­rich solution (HRS) and aquaporin (AQP) on cardiopulmonary bypass (CPB)­induced myocardial injury, and determine the mechanism of the phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague Dawley rats were divided into a sham operation group, a CPB surgery group and a HRS group. A CPB model was established, and the hemodynamic parameters were determined at the termination of CPB. The myocardial tissues were observed by hematoxylin and eosin, and Masson staining. The levels of myocardial injury markers [adult cardiac troponin I (cTnI), lactate dehydrogenase (LDH), creatine kinase MB (CK­MB) and brain natriuretic peptide (BNP)], inflammatory factors [interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α (TNF­α)] and oxidative stress indicators [superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO)] were determined by ELISA. Furthermore, H9C2 cells were treated with HRS following hypoxia/reoxygenation. Cell viability and cell apoptosis were investigated. The expression of apoptosis regulator Bcl­2 (Bcl­2), apoptosis regulator Bax (Bax), caspase 3, AQP­1, AQP­4, phosphorylated (p)­Akt, heme oxygenase 1 (HO­1) and nuclear factor erythroid 2­related factor 2 (Nrf2) were investigated using western blotting and quantitative­polymerase chain reaction of tissues and cells. Following CPB, myocardial cell arrangement was disordered, myocardial injury markers (cTnI, LDH, CK­MB and BNP), inflammatory cytokines (IL­1ß, IL­6 and TNF­α) and MDA levels were significantly increased compared with the sham group; whereas the SOD levels were significantly downregulated following CPB compared with the sham group. HRS attenuated myocardial injury, reduced the expression levels of cTnI, LDH, CK­MB, BNP, IL­1ß, IL­6, TNF­α, MDA and MPO, and increased SOD release. Levels of Bcl­2, AQP­1, AQP­4, p­Akt, HO­1 and Nrf2 were significantly increased following HRS; whereas Bax and caspase­3 expression levels were significantly reduced following CPB. HRS treatment significantly increased the viability of myocardial cells, reduced the rate of myocardial cell apoptosis and the release of MDA and LDH compared with the CPB group. A PI3K inhibitor (LY294002) was revealed to reverse the protective effect of HRS treatment. HRS was demonstrated to attenuate CPB­induced myocardial injury, suppress AQP­1 and AQP­4 expression following CPB treatment and protect myocardial cells via the PI3K/Akt signaling pathway.

Effect of infusion speed of 7.5% hypertonic saline on brain edema in patients with craniocerebral injury: An experimental study.

This study firstly used a rat traumatic brain injury model to compare the therapeutic effects of different intravenous infusion speed of 7.5% hypertonic saline (HS). Then the authors applied different delivery rate of 7.5% HS to two groups of patients to figure out the optimal infusion rates. A total of 100 rats were randomly divided into control group, group A (7.5% HS 6 mL/h), group B (7.5% HS 3 mL/h), and group C (7.5% HS 2 mL/h). All rats were established for the brain injury model. A total of 30 patients were selected and randomly divided into group A (250 mL/h) and group B (125 mL/h), with 15 cases in each group. Urine amount was recorded per hour; furthermore, blood was extracted from the patients to measure the levels of AQP4, NKCC1, tumour necrosis factor-α (TNF-a), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Compared with other groups, the expression levels of NKCC1 and AQP4 mRNA in group A was the lowest (P < 0.05). NKCC1 and AQP4 protein expression levels were the lowest in all the groups (P < 0.05). On the aspect of patients, group A displayed more significant difference compared with B group in terms of AQP4, NKCC1, TNF-a, IL-1ß, and IL-6 (P < 0.05). In the two groups, a significant difference was noted in the urine amount at 4 h after administration (P < 0.05). In our study, infusion of hypertonic saline (250 mL/h) at the optimal rate of 7.5% HS decreased the intracranial pressure, brain tissue edema, and inflammatory cytokine expression; moreover, it can promote brain tissue protection.

Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner.

BACKGROUND: Spinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS: The post-natal day 1-2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett's test. RESULTS: The OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated-at least in part-via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes. CONCLUSIONS: HMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.

Discovery of Aquaporin-1 and Aquaporin-4 Expression in an Intramedullary Spinal Cord Ependymal Cyst: Case Report.

BACKGROUND: Intramedullary ependymal cysts of the spinal cord are rare, benign, fluid-filled cysts usually situated along the ventral surface of the spinal cord. Only 32 cases have been reported since they were first described. Thus, owing to the rarity at which these cysts are encountered, their management and pathogenesis remain controversial. Whereas some investigators have advocated for cystosubarachnoid shunt placement for symptomatic ependymal cysts, others have argued for complete cyst resection or simple fenestration. Here we report the case of a 56-year-old female with a T11-T12 ependymal cyst that was successfully managed with cyst fenestration. We further investigated a potential pathological mechanism of cyst formation by performing immunohistochemistry to detect aquaporin expression in the cyst lining. CASE DESCRIPTION: A 56-year-old female was found to harbor an enlarging cystic lesion of the conus that was discovered on workup of progressive paraparesis and urinary incontinence. She had lower extremity weakness and progressive myelopathy. Thoracic laminectomy with cyst fenestration arrested her neurologic deterioration. Pathological analysis revealed an intramedullary ependymal cyst. Immunohistochemistry was subsequently performed for expression of aquaporin-1 and aquaporin-4. There was dense staining of the underlying neuropil with concurrent membranous staining pattern of the cyst lining. CONCLUSIONS: Intramedullary ependymal cysts are rare, cystic lesions of the spinal cord. Early cyst fenestration decompresses the cyst and prevents neurologic deterioration. Here we report for the first time that aquaporins are expressed in the cyst wall, which is consistent with a passive, osmotic pathogenic mechanism of cyst formation.

Cortical Aquaporin-4 in relation to brain oedema and neurological function of cortical cryo-injured mice.

To estimate the spatial and temporal expression of Aquaporin-4 (AQP-4) in a murine model of automated cerebral cryoinjury and correlate AQP-4 expression with development of brain oedema and neurological function. AQP-4 levels were determined quantitatively by Western blots at site of injury and at sites adjacent to and distant from injury in brains of cryoinjured (experimental) (n=18), sham injured (n=18) & normal mice at 24, 48, 72h post injury. AQP-4 expression was correlated with percentage water content of brain, Neurological Severity Score (NSS) and rotarod scores. We found a 1.4-fold increase in expression of AQP-4 at the site of injury and at sites distant from injury at 24h when compared to normal mice (p=0.05). The increase in expression of AQP-4 24h post injury was significantly higher in experimental group at the site of injury and at the site adjacent to the injury in the ipsilateral hemisphere when compared to the sham injured mice (p=0.05). At 24h post injury the median NSS score in the experimental group was 9 (interquartile range 7.25-10) and that in the sham group was 0.5 (interquartile range 0.0-1.0) (p<0.001). At 48 and 72h, AQP-4 expression remained elevated in the experimental group when compared to normal brain, but the levels were not significantly different from that in sham group. AQP-4 expression was significantly elevated in the ipsilateral hemisphere in the first 24h following cerebral cortical injury in mice and this could be correlated with worsening of neurological function. Over the next 48h, there was a trend towards decrease in AQP-4 expression that was associated with partial recovery of neurological function.

Down-Regulation of AQP4 Expression via p38 MAPK Signaling in Temozolomide-Induced Glioma Cells Growth Inhibition and Invasion Impairment.

Glioma is the most common and lethal central nervous system tumors. Temozolomide (TMZ) is an effective drug for malignant glioma, however, the intracellular and molecular mechanisms behind this anti-cancer effect have yet to be fully understood. The aim of the present study was to determine whether TMZ inhibits proliferation, invasion of glioma cells in vitro and whether these effects can be mediated through modulation of aquaporin 4 (AQP4) and phosphorylation of the MAPK pathway. The viability of U87 and U251 human glioma cells was evaluated using MTT assay. The cell cycle distribution was detected with flow cytometry. Migration ability and invasion ability were tested by scratch assays and transwell assays, respectively. The levels of AQP4 and MAPK were measured using immunoblot analyses. Our results showed that TMZ inhibited proliferation, migration and invasion, and induced G2/M arrest in U87 and U251 glioma cell lines. These changes were associated with a decrease in the levels of AQP4 expression as well as activation phosphorylated level of p38. Treatment with a p38 chemical activator (anisomycin) resulted in similar effects as TMZ treatment on glioma cells. And p38 chemical inhibitor (SB203580) could block these effects in glioma treated with TMZ, suggesting a direct up-regulation of the p38 signaling pathway. Therefore, we identified that TMZ might have therapeutic potential for controlling proliferation, invasion of malignant glioma by inhibiting AQP4 expression through activation of p38 signal transduction pathway. J. Cell. Biochem. 118: 4905-4913, 2017. © 2017 Wiley Periodicals, Inc.

Implication of VEGF and aquaporin 4 mediating Müller cell swelling to diabetic retinal edema.

PURPOSE: Aquaporin 4 (AQP4), a water channel protein, is known to be expressed in retinal Müller cells. The purpose of this study was to determine the effects of VEGF and AQP4 channels on the volumetric changes in Müller cells. METHODS: Retinas from diabetic rats and a cultured Müller cell line, TR-MUL5, were used in this study. Intravitreal injections of VEGF or PBS were performed on either streptozotocin (STZ)-induced diabetic or normoglycemic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. VEGF protein levels from collected retinas were determined by western blot analysis. Volumetric changes and nitric oxide (NO) levels in cultured Müller cells were determined using flow cytometry (FACS), in the presence or absence of VEGF and TGN-020, a selective AQP4 inhibitor. RESULTS: In the diabetic rat retina, VEGF immunoreactivity was concentrated in the internal retinal layers, and AQP4 immunoreactivity was higher than controls. The expressions of AQP4 were colocalized with GFAP. Protein levels of VEGF in the hyperglycemic rat retina were significantly higher than controls. FACS analyses showed that exposure to VEGF enlarged Müller cells, while exposure to TGN-020 suppressed the enlargement. Intracellular levels of NO were increased after exposure to VEGF, which was suppressed following the addition of TGN-020. CONCLUSION: The observed Müller cell swelling is mediated by VEGF and AQP4.

Water Channels Aquaporin 4 and -1 Expression in Subependymoma Depends on the Localization of the Tumors.

BACKGROUND: We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior. METHODS AND RESULTS: Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels. CONCLUSIONS: Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1.

Genistein inhibits rotavirus replication and upregulates AQP4 expression in rotavirus-infected Caco-2 cells.

Rotavirus (RV) is the primary cause of severe dehydrating gastroenteritis and acute diarrheal disease in infants and young children. Previous studies have revealed that genistein can inhibit the infectivity of enveloped or nonenveloped viruses. Although the biological properties of genistein are well studied, the mechanisms of action underlying their anti-rotavirus properties have not been fully elucidated. Here, we report that genistein significantly inhibits RV-Wa replication in vitro by repressing viral RNA transcripts, and possibly viral protein synthesis. Interestingly, we also found that aquaporin 4 (AQP4) mRNA and protein expression, which was downregulated in RV-infected Caco-2 cells, can be upregulated by genistein in a time- and dose-dependent manner. Further experiments confirmed that genistein triggers CREB phosphorylation through PKA activation and subsequently promotes AQP4 gene transcription. These findings suggest that the pathophysiological mechanism of RV infection involves decreased expression of AQP4 and that genistein may be a useful candidate for developing a new anti-RV strategy by inhibiting rotavirus replication and upregulating AQP4 expression via the cAMP/PKA/CREB signaling pathway. Further studies on the effect of genistein on RV-induced diarrhea are warranted.

Retinal vasculopathy is reduced by dietary salt restriction: involvement of Glia, ENaCα, and the renin-angiotensin-aldosterone system.

OBJECTIVE: Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy. APPROACH AND RESULTS: Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression. CONCLUSIONS: An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system.

Osmotic stress decreases aquaporin-4 expression in the human retinal pigment epithelial cell line, ARPE-19.

The regulation of water movement is of utmost importance for normal retinal function. Under physiological conditions, water is transported, dependent on the osmotic gradient, through the retinal pigment epithelial cell layer from the subretinal space to the choroid. The osmotic gradient has been found to be modified in eye diseases, thus leading to water accumulation in the subretinal space and the sensory retina, and subsequently contributing to the formation of macular oedema. Understanding the regulation of aquaporin expression is therefore crucial. In this study, we investigated the effects of hyperosmolarity on aquaporin-4 (AQP4) protein expression in the human retinal pigment epithelial cell line, ARPE­19. AQP4 expression was examined by PCR, western blot analysis and immunofluorescence. Ubiquitinylation was examined by immunoprecipitation. The results revealed that hyperosmotic stress rapidly decreased AQP4 expression in the ARPE-19 cells. The effect remained unmodified by lysosomal or mitogen-activated protein kinase inhibitors, but was reversed by proteasome inhibitors. However, no ubiquitinylation of AQP4 was detected. Our results suggest that hyperosmotic stress markedly reduces AQP4 expression possibly through a proteasome ubiquitinylation-independent pathway. This may represent an adaptation to hyperosmotic stress. The results presented in this study contribute to our understanding of the formation of macular oedema.

Paraneoplastic neuromyelitis optica spectrum disorder associated with metastatic carcinoid expressing aquaporin-4.

IMPORTANCE: Reports of neuromyelitis optica spectrum disorder (NMOSD) occurring in the setting of neoplasia suggest that aquaporin-4 autoimmunity may in some cases have a paraneoplastic basis. OBSERVATIONS: In this case report, we describe a patient with NMOSD whose test results were seropositive for aquaporin-4 IgG and who had a hepatic metastasis from a small-bowel neuroendocrine tumor. The tumor cells expressed aquaporin-4 immunoreactivity. She presented to the Neurology Department at Wayne State University with bilateral leg weakness, ascending paresthesias, and decreased sensation. CONCLUSIONS AND RELEVANCE: This case extends the context of NMOSD as a paraneoplastic disorder.