Green tea is neuroprotective in diabetic retinopathy.

PURPOSE: Green tea (GT), widely studied for its beneficial properties in protecting against brain ischemia, is a rich source of polyphenols, particularly (-)-epigallocatechin gallate (EGCG). The results presented here demonstrate the beneficial effects of GT in diabetic retinas and in retinal cells under diabetic conditions. METHODS: Diabetes was induced in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats. Treatment animals received GT orally for 12 weeks. A vehicle was administered orally to the control animals. The protective effects of GT were also evaluated in Müller and in ARPE-19 cells. RESULTS: In diabetic rats, there was an increase in the expression of glial fibrillary acidic protein (GFAP), oxidative retinal markers, and glutamine synthetase levels. In addition, there was a decrease in occludin and glutamate transporter and receptor. Diabetic SHR also demonstrated blood-retinal barrier breakdown and impaired electroretinography results. Müller cells exposed to high-glucose medium produced higher levels of reactive oxygen species (ROS) and glutamine synthetase but reduced levels of glutathione, glutamate transporter, and glutamate receptor. Similarly, ARPE-19 cells exhibited increased ROS production accompanied by decreased expression of claudin-1 and glutamate transporter. Treatment with GT fully restored all the above-mentioned alterations in diabetic animals as well as in retinal cells. CONCLUSIONS: GT protected the retina against glutamate toxicity via an antioxidant mechanism. These findings reveal a novel mechanism by which GT protects the retina against neurodegeneration in disorders such as diabetic retinopathy.

p38{alpha} MAP kinase controls IL-17 synthesis in vogt-koyanagi-harada syndrome and experimental autoimmune uveitis.

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38alpha mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4(+)IL-17(+) cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4(+) lymphocytes from MSK-1/2-deficient mice, human CD4(+) cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38alpha MAPK inhibitor and then assayed for IL-17, IFN-gamma, and IL-4 production. RESULTS. The inhibition of p38alpha MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38alpha MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4(+) T cells and that inhibition of p38alpha MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU.

Connexin-mediated communication controls cell proliferation and is essential in retinal histogenesis.

Connexin (Cx) channels and hemichannels are involved in essential processes during nervous system development such as apoptosis, propagation of spontaneous activity and interkinetic nuclear movement. In the first part of this study, we extensively characterized Cx gene and protein expression during retinal histogenesis. We observed distinct spatio-temporal patterns among studied Cx and an overriding, ubiquitous presence of Cx45 in progenitor cells. The role of Cx-mediated communication was assessed by using broad-spectrum (carbenoxolone, CBX) and Cx36/Cx50 channel-specific (quinine) blockers. In vivo application of CBX, but not quinine, caused remarkable reduction in retinal thickness, suggesting changes in cell proliferation/apoptosis ratio. Indeed, we observed a decreased number of mitotic cells in CBX-injected retinas, with no significant changes in the expression of PCNA, a marker for cells in proliferative state. Taken together, our results pointed a pivotal role of Cx45 in the developing retina. Moreover, this study revealed that Cx-mediated communication is essential in retinal histogenesis, particularly in the control of cell proliferation.

Rod bipolar cells in the retina of the capuchin monkey (Cebus apella): characterization and distribution.

Rod bipolar cells in Cebus apella monkey retina were identified by an antibody against the alpha isoform of protein kinase C (PKCalpha), which has been shown to selectively identify rod bipolars in two other primates and various mammals. Vertical sections were used to confirm the identity of these cells by their characteristic morphology of dendrites and axons. Their topographic distribution was assessed in horizontal sections; counts taken along the dorsal, ventral, nasal, and temporal quadrants. The density of rod bipolar cells increased from 500 to 2900 cells/mm2 at 1 mm from the fovea to reach a peak of 10,000-12,000 cells/mm2 at 4 mm, approximately 5 deg of eccentricity, and then gradually decreased toward retinal periphery to values of 5000 cells/mm2 or less. Rod to rod bipolar density ratio remained between 10 and 20 across most of the retinal extension. The number of rod bipolar cells per retina was 6,360,000 +/- 387,433 (mean +/- s.d., n = 6). The anti-PKCalpha antibody has shown to be a good marker of rod bipolar cells of Cebus, and the cell distribution is similar to that described for other primates. In spite of the difference in the central retina, the density variation of rod bipolar cells in the Cebus and Macaca as well as the convergence from rod to rod bipolar cells are generally similar, suggesting that both retinae stabilize similar sensitivity (as measured by rod density) and convergence.

Distribution of small Rho GTPases in the developing rat submandibular gland.

During the rat submandibular gland (SMG) development, organogenesis and cytodifferentiation depend on the actin cytoskeleton, which is regulated by small Rho GTPases. These proteins link cell surface receptors to pathways that regulate cell motility, polarity, gene expression, vesicular trafficking, proliferation and apoptosis. The aim of this study was to evaluate, by immunohistochemistry, the distribution pattern of RhoA, RhoB, RhoC, Rac1 and Cdc42 during cytodifferentiation of the rat SMG and in male adults. All GTPases were found in epithelial and mesenchymal tissues throughout gland development. Rac1 appeared to be important for parenchyma expansion at the beginning of cytodifferentiation, while RhoC, Cdc42 and the inactive phosphorylated form of Rac1 seemed associated with lumen formation and cell polarization in terminal tubules. RhoA and RhoB labeling was evident throughout development. All GTPases were differentially expressed in the adult gland, suggesting that they play specific roles during differentiation and function of the rat SMG.

Small Rho GTPases are important for acinus formation in a human salivary gland cell line.

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.

Light-Induced photoreceptor degeneration in the mouse involves activation of the small GTPase Rac1.

PURPOSE: Rho GTPases play a central role in actin-based cytoskeleton reorganization, and they participate in signaling pathways that regulate gene transcription, cell cycle entry, and cell survival. This study verifies the role of Rac1 during light-induced retinal degeneration. METHODS: BALB/c mice were exposed to degenerative light stimulus, and their eyes were enucleated immediately or after the mice were kept in the dark for 6, 24, and 48 hours. Retinas were fixed and processed for immunohistochemical analysis. The distribution of Rac1 and its effectors-p21-activated kinases (PAKs) 1, 2, and 3-was studied by immunohistochemistry, whereas the expression of PAKs 3, 4, and 5 mRNA was analyzed by real-time PCR. Rac1 activity was measured using a pull-down assay. RESULTS: In control retinas, Rac1 was mostly observed in photoreceptors, plexiform layers, and Müller glial cells. In light-damaged retinas, some TUNEL-positive photoreceptors upregulated Rac1 expression. Conversely, most of the Rac1-positive cells were TUNEL-positive, mainly in early stages of retinal degeneration. The increase in Rac1 expression was preceded by enhanced Rac1 activity, detectable at the end of the light stimulus and still present 48 hours later. The distribution patterns of PAK1, PAK2, and PAK3 did not change in light-damaged retinas. However, there was a marked increase in PAK3 and PAK4 gene expression, whereas that of PAK5 mRNA remained the same. CONCLUSIONS: Rac1 may play a role in the apoptosis of light-damaged photoreceptors. The increased expression of PAK4 after light stimulus possibly functions as a protective mechanism against apoptosis.