Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels.

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 µM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

Hemoxygenase-1 Promotes Head and Neck Cancer Cell Viability.

Head and neck squamous cell carcinoma (HNSCC) is a remarkably heterogeneous disease with around 50% mortality, a fact that has prompted researchers to try new approaches to improve patient survival. Hemoxygenase-1 (HO-1) is the rate-limiting step for heme degradation into carbon monoxide, free iron and biliverdin. We have previously reported that HO-1 protein is upregulated in human HNSCC samples and that it is localized in the cytoplasmic and nuclear compartments; additionally, we have demonstrated that HO-1 nuclear localization is associated with malignant progression. In this work, by using pharmacological and genetic experimental approaches, we begin to elucidate the mechanisms through which HO-1 plays a role in HNSCC. We found that high HO-1 mRNA was associated with decreased patient survival in early stages of HNSCC. In vitro experiments have shown that full-length HO-1 localizes in the cytoplasm, and that, depending on its enzymatic activity, it increases cell viability and promotes cell cycle progression. Instead, HO-1 does not alter migration capacity. Furthermore, we show that C-terminal truncated HO-1 localizes into the nucleus, increases cell viability and promotes cell cycle progression. In conclusion, we herein demonstrate that HO-1 displays protumor activities in HNSCC that depend, at least in part, on the nuclear localization of HO-1.

New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease.

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into "epithelial" type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.

Canonical phospholipase D isoforms in visual function and ocular response to stress.

Classical phospholipase D (PLD) isoforms, PLD1 and PLD2, catalyze the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) which can be further dephosphorylated to diacylglycerol (DAG). Through the generation of these lipid messengers, the PLD pathway can modulate several cellular events, such as proliferation, membrane trafficking, autophagy and the inflammatory response, among many others. This review summarizes the participation of canonical PLD isoforms in physiological and pathological responses in the eye. Although the role of the PLD pathway in ocular and retinal response to stress has not been fully elucidated, pharmacological inhibition of these signaling enzymes seems to be a promising therapeutic tool to avoid inflammatory processes in the retina, abnormal cellular proliferation on the ocular surface and pathological neovascularization. On the contrary, the modulation of classical PLDs may potentiate corneal healing. In summary, the knowledge of the role of PLD1 and PLD2 in the molecular basis of ocular inflammatory and degenerative diseases opens new avenues for potential therapeutic exploration.

Glucocorticoid and progesterone mechanisms in photoreceptor survival.

Death of retinal photoreceptors is the basis of prevalent blinding diseases. Since steroids might have a therapeutic role in retinal degenerations, we compared the protective effects of dexamethasone and progesterone on photoreceptor death induced by mifepristone and light exposure. Therefore, we studied the effective protection doses for each steroid in the two models. In addition, we analyzed changes in the levels of pro- and antiapoptotic molecules, glucocorticoid receptors α and ß (GRα and GRß), and rhodopsin under conditions of successful protection and photoreceptor survival. Mifepristone and light exposure selectively damaged photoreceptors. In light exposed retinas, photoreceptors mainly disappeared in the dorsotemporal region, while mifepristone produced a uniform damage. Dexamethasone and progesterone, at the same dose of 4 mg/kg/day for 2 days, preserved over 88% photoreceptor nuclei in both models. Assessment of cell death regulators showed that, in control retinas, both steroids activated BCL-XL, a prosurvival molecule, and decreased BID, a proapoptotic regulator. After steroid treatment of damaged retinas, BCL-XL, BCL2 and BAX showed characteristic patterns depending on the use of dexamethasone or progesterone on mifepristone or light exposed retinas. By contrast, BID decreased with any injury-steroid combination. Changes in GRα or GRß levels did not correlate with survival but were consistent with a mechanism of ligand induced downregulation of receptor expression. GRß might be upregulated by progesterone. Both dexamethasone and progesterone increased retinal rhodopsin stores, suggesting a link between photoreceptor protection and transduction pathways. Results show that dexamethasone and progesterone induced comparable but not identical protection responses in each model.

Lipid Signaling in Retinal Pigment Epithelium Cells Exposed to Inflammatory and Oxidative Stress Conditions. Molecular Mechanisms Underlying Degenerative Retinal Diseases.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells whose function is essential for the integrity of the retina and for visual function. Retinal diseases that eventually end in vision loss and blindness involve inflammation, oxidative stress (OS), and alterations in the RPE-photoreceptor cellular partnership. This chapter summarizes the role of lipid signaling pathways and lipidic molecules in RPE cells exposed to inflammatory and OS conditions. The modulation of these pathways in the RPE, through either enzyme inhibitors or receptor stimulation or blockage, could open new therapeutic strategies for retinal degenerative diseases.

Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway.

Inflammation and oxidative stress are common factors involved in the pathogenesis of retinal diseases, such as aged-related macular degeneration (AMD) and diabetic retinopathy (DR). Autophagy is a catabolic process essential to cell survival in response to stress. This process is highly active in retinal pigment epithelium (RPE) cells. Our previous findings demonstrated that lipopolysaccharide (LPS) induces an inflammatory response of RPE cells that implies classical phospholipases D (PLD1 and 2) activation, cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production and reduced cell viability. In this work, we studied the autophagic process and its modulation by the PLD pathway in D407 and ARPE-19 RPE cells exposed to LPS. LPS (10 µg/ml or 25 µg/ml) exposure for 24 h increased light chain 3B-II (LC3B-II) content (an autophagy marker) and LC3B-positive punctate structures in both RPE cell lines studied. Next, the drug bafilomycin A1 (BAF, 50 nM) was used to block the autophagic flux. In cells pre-incubated with BAF, LC3B-II and sequestosome 1 (SQSTM1/p62) levels and autophagosome-like structures were increased by LPS, demonstrating that the inflammatory injury increases the autophagic process in RPE cells. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595) or PLD2 (VU0285655-1) inhibitors prior to LPS addition. Under control condition, LC3B-positive punctate structures were increased in cells pre-incubated with PLD2 inhibitor while with PLD1 inhibitor were increased in cells exposed to LPS. MTT reduction assays showed that early autophagy inhibitors, 3-methyladenin (3-MA) or LY294002, enhanced the loss in cell viability induced by LPS exposure for 48 h. On the contrary, the inhibition of PLD1 and PLD2 prevented the loss in cell viability induced by LPS. In conclusion, our results show that even though LPS treatment promotes an inflammatory response in RPE cells, it also triggers the activation of the autophagic process which in turn may serve as a protective mechanism for the cells. In addition, we demonstrate that the PLD pathway modulates the autophagic process in RPE cells. Our findings contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases and open new avenues for potential therapeutic exploration.

High glucose-induced phospholipase D activity in retinal pigment epithelium cells: New insights into the molecular mechanisms of diabetic retinopathy.

Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Early ciliary and prominin-1 dysfunctions precede neurogenesis impairment in a mouse model of type 2 diabetes.

Diabetes mellitus (DM) is reaching epidemic conditions worldwide and increases the risk for cognition impairment and dementia. Here, we postulated that progenitors in adult neurogenic niches might be particularly vulnerable. Therefore, we evaluated the different components of the mouse subventricular zone (SVZ) during the first week after chemical induction of type 1 and type 2 diabetes-like (T1DM and T2DM) conditions. Surprisingly, only T2DM mice showed SVZ damage. The initial lesions were localized to ependymal cilia, which appeared disorientated and clumped together. In addition, they showed delocalization of the ciliary membrane protein prominin-1. Impairment of neuroprogenitor proliferation, neurogenic marker abnormalities and ectopic migration of neuroblasts were found at a later stage. To our knowledge, our data describe for the first time such an early impact of T2DM on the SVZ. This is consistent with clinical data indicating that brain damage in T2DM patients differs from that in T1DM patients.

Mifepristone, a blocker of glucocorticoid receptors, promotes photoreceptor death.

PURPOSE: Glucocorticoids are best known by their protective effect on retinal photoreceptor damage. However, they could also be involved in photoreceptor homeostasis under basal, nonstressful conditions. Therefore, we aimed to study glucocorticoid-induced changes of survival-related molecules in male mice retinas under standard illumination conditions (12 hours light, ≤ 60 lux/12 h dark). METHODS: Male Balb-c mice were injected with dexamethasone (DEX), a selective glucocorticoid receptor α (GRα) agonist, its antagonist mifepristone (MFP), or both drugs (D+M) at noon. A group of mice was subjected to surgical adrenalectomy (AdrX). Retinas were studied by histology, immunohistochemistry, TUNEL procedure, and Western blotting at different periods after pharmacological or surgical intervention (6 hours, 48 hours, or 7 days). RESULTS: The antiapoptotic molecule Bcl-X(L) significantly increased 6 hours after DEX injection. By contrast, this molecule could no longer be found after MFP injection. At the same time, high levels of cleaved caspase-3 (CC-3) and Bax appeared in retinal extracts, and TUNEL(+) nuclei selectively showed in the outer nuclear layer (ONL). After MFP, retinal extracts also contained phosphorylated histone H2AX (p-H2AX), a marker of DNA breakage and repair. Loss of ONL nuclear rows and decrease of rhodopsin levels were evident 7 days after MFP administration. These changes were minimized when DEX was given together with MFP (D+M). In the absence of MFP, DEX increased Bcl-X(L) in every retinal layer, with a marked intensification in photoreceptor inner segments. Numerous TUNEL(+) nuclei rapidly appeared in the ONL after AdrX. CONCLUSIONS: A single dose of MFP induced selective photoreceptor damage in the absence of other environmental stressors. Because damage was prevented by DEX, and was reproduced by AdrX, our findings suggest that glucocorticoids play a critical role in photoreceptor survival.

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution.

The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR M4 segment (the one in closer contact with lipids) was reconstituted into "raft"-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fluorescence studies, and lipid analyses were performed on each sample. The influence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft-like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The gammaM4 peptide displays a marked preference for raft-like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a significant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specific membrane domain.

Puericultura prenatal: Importancia en la promoción de la lactancia materna / Prenatal puericulture: importance in the promotion of breastfeeding

Se realizó estudio de intervención de tipo cuasi experimental en 10 consultorios urbanos pertenecientes al Policlínico Docente de Remedios, entre diciembre de 2000 y diciembre de 2001, con el objetivo de demostrar la importancia de una labor educativa sistemática en la consulta de Puericultura Prenatal en la promoción de la lactancia materna. El grupo de estudio quedó integrado por 35 embarazadas con fecha probable de parto en los meses de mayo, junio y julio de 2001, a las cuales se aplicaron estrategias de intervención; otro grupo, con igual número de madres, recibió puericultura prenatal tradicional (control). Se encontró una duración mayor de la lactancia materna en el grupo de estudio, y se comprobó que las madres que recibieron la puericultura prenatal con labor educativa especial lactaron por encima de los cuatro meses. El tiempo de duración de la lactancia materna exclusiva comprueba la positividad del trabajo educativo prenatal; se recomienda la aplicación del método de intervención educativa en la consulta municipal de lactancia materna (AU)

Puericultura prenatal: Importancia en la promoción de la lactancia materna / Prenatal puericulture: importance in the promotion of breastfeeding

Se realizó estudio de intervención de tipo cuasi experimental en 10 consultorios urbanos pertenecientes al Policlínico Docente de Remedios, entre diciembre de 2000 y diciembre de 2001, con el objetivo de demostrar la importancia de una labor educativa sistemática en la consulta de Puericultura Prenatal en la promoción de la lactancia materna. El grupo de estudio quedó integrado por 35 embarazadas con fecha probable de parto en los meses de mayo, junio y julio de 2001, a las cuales se aplicaron estrategias de intervención; otro grupo, con igual número de madres, recibió puericultura prenatal tradicional (control). Se encontró una duración mayor de la lactancia materna en el grupo de estudio, y se comprobó que las madres que recibieron la puericultura prenatal con labor educativa especial lactaron por encima de los cuatro meses. El tiempo de duración de la lactancia materna exclusiva comprueba la positividad del trabajo educativo prenatal; se recomienda la aplicación del método de intervención educativa en la consulta municipal de lactancia materna(AU)

Um plano de ação para o controle da hipertensão arterial, na Estratégia Saúde da Família Ermida II, Divinópolis/MG

A hipertensão arterial sistêmica (HAS) é hoje uma epidemia dentro das doenças crônicas, atingindo populações no mundo inteiro com uma frequência muito alta. A hipertensão arterial sistêmica e suas complicações reduzem a qualidade de vida dos afetados, pois as pessoas que possuem essa doença, com pouco ou nenhum controle, tem risco maior de cardiopatia isquêmica, retinopatia hipertensiva, acidentes vasculares encefálicos (AVC), de insuficiência cardíaca, insuficiência renal crônica, aneurismas e, da até pouco conhecida, insuficiência arterial crônica. Na Estratégia de Saúde da Família Ermida II foram diagnosticados altos índices de hipertensos descontrolados evoluindo para as complicações já citadas. Tornou-se necessário construir um plano de ação que permita prolongar a qualidade de vida desta população além de ensiná-la a prevenir a doença ou, pelo menos, controlá-la. Foi realizada uma revisão bibliográfica nas bases de dados LILACS e SciELO e o plano de ação proposto e desenvolvido priorizou ações de informação para população geral e hipertensa sobre fatores de risco modificáveis como o sedentarismo, a obesidade, o mau hábito de fumar, o consumo em excesso de bebidas alcoólicas, consumo indevido de medicação e automedicação, controle de outras doenças envolvidas como a Diabetes Mellitus e ainda, ações de individualização dos protocolos de atuação e encaminhamento oportuno dos casos refratários. A integração de atividade no plano comunitário, a educação em saúde, em conjunto com a equipe mostrou melhora nos índices de controle e adesão terapêutica dos pacientes afetados e gerando conhecimentos úteis na população.