Áurea F. Castilho

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H2O2). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H2O2 and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H2O2 and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

High glucose and oxidative/nitrosative stress conditions induce apoptosis in retinal endothelial cells by a caspase-independent pathway.

Diabetic retinopathy (DR) is a leading cause of vision loss among working-age adults. Retinal endothelial cell apoptosis is an early event in DR, and oxidative stress is known to play an important role in this pathology. Recently, we found that high glucose induces apoptosis in retinal neural cells by a caspase-independent mechanism. Here, we investigated the mechanisms underlying retinal endothelial cell apoptosis induced by high glucose and oxidative/nitrosative stress conditions. Endothelial cells (TR-iBRB2 rat retinal endothelial cell line) were exposed to high glucose (long-term exposure, 7 days), or to NOC-18 (nitric oxide donor; 250microM) or H(2)O(2) (100microM) for 24h. Cell viability was assessed by the MTT assay and cell proliferation by [methyl-(3)H]-thymidine incorporation into DNA. Apoptotic cells were detected with Hoechst or Annexin V staining. Active caspases were detected by an apoptosis detection kit. Active caspase-3 and apoptosis-inducing factor (AIF) protein levels were assessed by Western blot or immunohistochemistry. High glucose, NOC-18 and H(2)O(2) increased apoptosis in retinal endothelial cells. High glucose and mannitol decreased cell proliferation, but mannitol did not induce apoptosis. Caspase activation did not increase in high glucose- or NOC-18-treated cells, but it increased in cells exposed to H(2)O(2). However, the protein levels of AIF decreased in mitochondrial fractions and increased in nuclear fractions, in all conditions. These results are the first demonstrating that retinal endothelial cell apoptosis induced by high glucose is independent of caspase activation, and is correlated with AIF translocation to the nucleus. NOC-18 and H(2)O(2) also activate a caspase-independent apoptotic pathway, although H(2)O(2) can also induce caspase-mediated apoptosis.

Diabetes differentially affects the content of exocytotic proteins in hippocampal and retinal nerve terminals

Diabetes has been associated with cognitive and memory impairments, and with alterations in color and contrast perception, suggesting that hippocampus and retina are particularly affected by this disease. A few studies have shown that diabetes differentially affects neurotransmitter release in different brain regions and in retina, and induces structural and molecular changes in nerve terminals in both hippocampus and retina. We now detailed the impact over time of diabetes (2, 4 and 8 weeks of diabetes) on a large array of exocytotic proteins in hippocampus and retina.The exocytotic proteins density was evaluated by immunoblotting in purified synaptosomes and in total extracts of hippocampus and retina from streptozotocin-induced diabetic and age-matched control animals. Diabetes affected differentially the content of synaptic proteins (VAMP-2, SNAP-25, syntaxin-1, synapsin-1 and synaptophysin) in hippocampal and retinal nerve terminals. Changes were more pronounced and persistent in hippocampal nerve terminals. In general, the alterations in retina occurred earlier, but were transitory, with the exception of synapsin-1, since its content decreased at all time points studied. The content of synaptotagmin-1 and rabphilin 3a in nerve terminals of both tissues was not affected. In total extracts, no changes were detected in the retina, whereas in hippocampus SNAP-25 and syntaxin-1 content was decreased, particularly when more drastic changes were also detected in nerve terminals. These results show that diabetes affects the content of several exocytotic proteins in hippocampus and retina, mainly at the presynaptic level, but hippocampus appears to be more severely affected. These changes might influence neurotransmission in both tissues and may underlie, at least partially, previously detected physiological changes in diabetic humans and animal models. Since diabetes differentially affects exocytotic proteins, according to tissue and insult duration, functional studies will be required to assess the physiological impairment induced by diabetes on the exocytosis in central synapses.

Disruption of a Neural Microcircuit in the Rod Pathway of the Mammalian Retina by Diabetes Mellitus

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca2+ imaging in rat retinal slices, we investigated diabetes-evoked changes in a microcircuit consisting of rod bipolar cells and their dyad postsynaptic targets, AII and A17 amacrine cells, which play an essential role in processing scotopic visual signals. AII amacrines forward their signals to ON- and OFF-cone bipolar cells and A17 amacrines provide GABAergic feedback inhibition to rod bipolar cells. Whereas Ca2+-permeable AMPA receptors mediate input from rod bipolar cells to both AII and A17 amacrines, diabetes changes the synaptic receptors on A17, but not AII amacrine cells. This was expressed as a change in pharmacological properties and single-channel conductance of the synaptic receptors, consistent with an upregulation of the AMPA receptor GluA2 subunit and reduced Ca2+ permeability. In addition, two-photon imaging revealed reduced agonist-evoked influx of Ca2+ in dendritic varicosities of A17 amacrine cells from diabetic compared with normal animals. Because Ca2+-permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca2+ permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.

Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

Long-term exposure to high glucose increases the content of several exocytotic proteins and of vesicular GABA transporter in cultured retinal neural cells

Diabetic retinopathy is a leading cause of vision loss and blindness. Increasing evidence has shown that the neuronal components of the retina are affected even before the detection of vascular lesions. Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. Nevertheless, other factors like neuroinflammation, might also contribute for neural changes. To clarify whether hyperglycemia can be the main trigger of synaptic changes, we evaluated whether prolonged elevated glucose per se, mimicking chronic hyperglycemia, is able to change the content and distribution of several exocytotic proteins and vesicular glutamate and GABA transporters in retinal neurons. Moreover, we also tested the hypothesis that an inflammatory stimulus (interleukin-1β) could exacerbate the effects induced by exposure to elevated glucose, contributing for changes in synaptic proteins in retinal neurons. Rat retinal neural cells were cultured for 9 days. Cells were exposed to elevated D-glucose (30 mM) or D-mannitol (osmotic control), for 7 days, or were exposed to interleukin-1β (10 ng/ml) or LPS (1 μg/ml) for 24 h. The protein content and distribution of SNARE proteins (SNAP-25, syntaxin-1, VAMP-2), synapsin-1, synaptotagmin-1, rabphilin 3a, VGluT-1 and VGAT, were evaluated by western blotting and immunocytochemistry. The protein content and immunoreactivity of syntaxin-1, synapsin-1, rabphilin 3a and VGAT increased in retinal neural cells exposed to high glucose. No changes were detected when cells were exposed to interleukin-1β, LPS or mannitol per se. Particularly, exposure to interleukin-1β for 24 h did not exacerbate the effect of high glucose on the content and immunoreactivity of exocytotic proteins, suggesting the primordial role of hyperglycemia for neuronal changes. In summary, prolonged exposure to elevated glucose alters the total content of several proteins involved in exocytosis, suggesting that hyperglycemia per se is a fundamental factor for neuronal changes caused by diabetes.

Diabetic hyperglycemia reduces Ca2+ permeability of extrasynaptic AMPA receptors in AII amacrine cells

There is increasing evidence that diabetic retinopathy is a primary neuropathological disorder that precedes the microvascular pathology associated with later stages of the disease. Recently, we found evidence for altered functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in A17, but not AII, amacrine cells in the mammalian retina, and the observed changes were consistent with an upregulation of the GluA2 subunit, a key determinant of functional properties of AMPA receptors, including Ca(2+) permeability and current-voltage (I-V) rectification properties. Here, we have investigated functional changes of extrasynaptic AMPA receptors in AII amacrine cells evoked by diabetes. With patch-clamp recording of nucleated patches from retinal slices, we measured Ca(2+) permeability and I-V rectification in rats with ∼3 wk of streptozotocin-induced diabetes and age-matched, noninjected controls. Under bi-ionic conditions (extracellular Ca(2+) concentration = 30 mM, intracellular Cs(+) concentration = 171 mM), the reversal potential (Erev) of AMPA-evoked currents indicated a significant reduction of Ca(2+) permeability in diabetic animals [Erev = -17.7 mV, relative permeability of Ca(2+) compared with Cs(+) (PCa/PCs) = 1.39] compared with normal animals (Erev = -7.7 mV, PCa/PCs = 2.35). Insulin treatment prevented the reduction of Ca(2+) permeability. I-V rectification was examined by calculating a rectification index (RI) as the ratio of the AMPA-evoked conductance at +40 and -60 mV. The degree of inward rectification in patches from diabetic animals (RI = 0.48) was significantly reduced compared with that in normal animals (RI = 0.30). These results suggest that diabetes evokes a change in the functional properties of extrasynaptic AMPA receptors of AII amacrine cells. These changes could be representative for extrasynaptic AMPA receptors elsewhere in AII amacrine cells and suggest that synaptic and extrasynaptic AMPA receptors are differentially regulated.

High glucose and interleukin-1β downregulate interleukin-1 type I receptor (IL-1RI) in retinal endothelial cells by enhancing its degradation by a lysosome-dependent mechanism

Diabetic retinopathy has been considered a low-grade chronic inflammatory disease. The production of interleukin-1beta (IL-1beta) in the retina is increased, and this finding has been correlated with an increase in blood-retinal barrier permeability, suggesting that IL-1beta might have an important role in the pathogenesis of diabetic retinopathy. However, in this context, no attention has been given to interleukin-1 type I receptor (IL-1RI), which is the receptor responsible for IL-1beta triggered effects. Therefore, we investigated the effect of high glucose and IL-1beta on the IL-1RI regulation in retinal endothelial cells. A time-dependent downregulation of IL-1RI protein levels was detected in retinal endothelial cells exposed (1-24h) to high glucose, mannitol or IL-1beta. Long-term exposure (7days) to high glucose or mannitol also decreased IL-1RI protein content. IL-1RI downregulation was due to its activation by IL-1beta, since it was inhibited by the presence of anti-IL-1RI or anti-IL-1beta antibodies. Moreover, IL-1RI downregulation was prevented by lysosome inhibitors, chloroquine and ammonium chloride, but not by proteasome inhibitors, MG132 and lactacystin. We also found that IL-1RI translocates to the nucleus after high glucose or IL-1beta treatment. In conclusion, our results indicate that high glucose, probably due to osmotic stress, and IL-1beta downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation. High glucose and IL-1beta also enhance the translocation of IL-1RI to the nucleus.

AMPA receptors at ribbon synapses in the mammalian retina: kinetic models and molecular identity

In chemical synapses, neurotransmitter molecules released from presynaptic vesicles activate populations of postsynaptic receptors that vary in functional properties depending on their subunit composition. Differential expression and localization of specific receptor subunits are thought to play fundamental roles in signal processing, but our understanding of how that expression is adapted to the signal processing in individual synapses and microcircuits is limited. At ribbon synapses, glutamate release is independent of action potentials and characterized by a high and rapidly changing rate of release. Adequately translating such presynaptic signals into postsynaptic electrical signals poses a considerable challenge for the receptor channels in these synapses. Here, we investigated the functional properties of AMPA receptors of AII amacrine cells in rat retina that receive input at spatially segregated ribbon synapses from OFF-cone and rod bipolar cells. Using patch-clamp recording from outside-out patches, we measured the concentration dependence of response amplitude and steady-state desensitization, the single-channel conductance and the maximum open probability. The GluA4 subunit seems critical for the functional properties of AMPA receptors in AII amacrines and immunocytochemical labeling suggested that GluA4 is located at synapses made by both OFF-cone bipolar cells and rod bipolar cells. Finally, we used a series of experimental observables to develop kinetic models for AII amacrine AMPA receptors and subsequently used the models to explore the behavior of the receptors and responses generated by glutamate concentration profiles mimicking those occurring in synapses. These models will facilitate future in silico modeling of synaptic signaling and processing in AII amacrine cells.

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.