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Glaucoma is a degenerative disease that causes irreversible loss of vision and is characterized by retinal ganglion cell (RGC) loss. Others and we have demonstrated that chronic neuroinflammation mediated by reactive microglial cells plays a role in glaucomatous pathology. Exosomes are extracellular vesicles released by most cells, including microglia, that mediate intercellular communication. The role of microglial exosomes in glaucomatous degeneration remains unknown. Taking the prominent role of microglial exosomes in brain neurodegenerative diseases, we studied the contribution of microglial-derived exosomes to the inflammatory response in experimental glaucoma. Microglial cells were exposed to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure, the main risk factor for glaucoma. Naïve microglia (BV-2 cells or retinal microglia) were exposed to exosomes derived from BV-2 cells under EHP conditions (BV-Exo-EHP) or cultured in control pressure (BV-Exo-Control). We found that BV-Exo-EHP increased the production of pro-inflammatory cytokines, promoted retinal microglia motility, phagocytic efficiency, and proliferation. Furthermore, the incubation of primary retinal neural cell cultures with BV-Exo-EHP increased cell death and the production of reactive oxygen species. Exosomes derived from retinal microglia (MG-Exo-Control or MG-Exo-EHP) were injected in the vitreous of C57BL/6J mice. MG-Exo-EHP sustained activation of retinal microglia, mediated cell death, and impacted RGC number. Herein, we show that exosomes derived from retinal microglia have an autocrine function and propagate the inflammatory signal in conditions of elevated pressure, contributing to retinal degeneration in glaucomatous conditions.

Acute myocardial infarction (AMI) and the heart failure (HF) that often result remain the leading causes of death and disability worldwide. As such, new therapeutic targets need to be discovered to protect the myocardium against acute ischaemia/reperfusion (I/R) injury in order to reduce myocardial infarct (MI) size, preserve left ventricular function and prevent the onset of HF. Mitochondrial dysfunction during acute I/R injury is a critical determinant of cell death following AMI, and therefore, ion channels in the inner mitochondrial membrane, which are known to influence cell death and survival, provide potential therapeutic targets for cardioprotection. In this article, we review the role of mitochondrial ion channels, which are known to modulate susceptibility to acute myocardial I/R injury, and we explore their potential roles as therapeutic targets for reducing MI size and preventing HF following AMI.

New treatments are needed to protect the myocardium against the detrimental effects of acute ischaemia/reperfusion (IR) injury following an acute myocardial infarction (AMI), in order to limit myocardial infarct (MI) size, preserve cardiac function and prevent the onset of heart failure (HF). Given the critical role of mitochondria in energy production for cardiac contractile function, prevention of mitochondrial dysfunction during acute myocardial IRI may provide novel cardioprotective strategies. In this regard, the mitochondrial fusion and fissions proteins, which regulate changes in mitochondrial morphology, are known to impact on mitochondrial quality control by modulating mitochondrial biogenesis, mitophagy and the mitochondrial unfolded protein response. In this article, we review how targeting these inter‐related processes may provide novel treatment targets and new therapeutic strategies for reducing MI size, preventing the onset of HF following AMI.

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents., This work was developed under the scope of the project NORTE‐01‐0145‐FEDER‐000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI‐01‐0145‐FEDER‐007038. OP and BSM are supported by fellowships from the Fundação para a Ciência e Tecnologia (FCT, Portugal) (SFRH/BD/52292/2013 and SFRH/BPD/90533/2012, respectively), info:eu-repo/semantics/publishedVersion

Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and possible recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC-LC-MS and GC-MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), and FA (18:0). Interestingly, PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. J. Cell. Physiol. 231: 2266-2274, 2016. © 2016 Wiley Periodicals, Inc.

TP53 mutations and polymorphisms have been widely related to many cancers as long as these alterations may impair its capacity to induce cell cycle arrest, DNA repair mechanisms, and apoptosis. Although TP53 alterations have been studied in astrocytic tumors, there is a lack of analysis considering specific TP53 mutations and their associations with p53 immunostainning, polymorphisms and their significance among the histological grades. Thus, we analyzed TP53 alterations in exons 2-11, including the codon 72 polymorphism, using DNA sequencing in 96 astrocytic gliomas (18 grade I, 20 grade II, 14 grade III, and 44 grade IV). Also, immunohistochemistry was assessed to evaluate the p53 protein expression. In this study, we found that the higher histological grades were statistically associated with TP53 mutations. Some of these mutations, such as TP53 P98T and TP53 G244S, seemed to be a specific marker for the higher grades, and the TP53 E286K mutation appears to be a World Health Organization grade III-IV progression marker. Also, the TP53 P98T mutation, in exon 4, is very likely to be important on the stabilization of the p53 protein, leading to its immunopositivity and it is potentially associated with the TP53 72Pro/Pro genotype.

Astrocytomas represent the most frequent primary tumors of the central nervous system. Recently, the determination of the proliferative index of astrocytic tumors by different methods has been proposed as a valuable tool for tumor grading and also as a prognostic marker. The aim of the present study was to evaluate the expression of cell proliferation-related proteins in human astrocytic tumors of different histopathological grades (WHO). An immunohistochemical study of the Ki-67, Topoisomerase IIalpha (Topo IIalpha) and c-MYC proteins using the avidin-biotin-peroxidase method was performed in 55 astrocytomas (13 grade I, 14 grade II, 7 grade III and 21 grade IV) and five samples of non-tumor brain tissue (control group). Ki-67, Topo IIalpha and c-MYC positive indices tended to increase according to malignant progression, were absent in non-tumor brain tissue and showed maximum values in high-grade astrocytomas (III and IV). A gradual increase in Ki-67 antigen expression was observed in agreement with mitotic index and histopathological classification. The same was not observed for Topo IIalpha and c-MYC. Ki-67 antigen detection in more than 8.0% of the tumor cells distinguished astrocytoma grade IV, while a labeling index between 1.5 and 8.0% characterized astrocytomas grade III and values below 1.5% discriminated low-grade tumors (I and II). These results indicate that Topo IIalpha and c-MYC expression is associated with cell proliferation in astrocytomas, although not in an exclusive way. Moreover, Ki-67 antigen was found to be the best marker of cellular proliferation, and its expression predicts the grade of astrocytic tumors.

Purpose Age-related Macular Degeneration (AMD) is the leading cause of blindness in people older than 50 in developed countries, and is associated with formation of subretinal deposits (drusen) and damage to Bruch’s membrane (BM) basal to the retinal pigment epithelium (RPE). It has been reported that activities of the ubiquitin-proteasome pathway (UPP) in retinal pigment epithelium decrease upon aging. Previous studies demonstrated that expression of ubiquitin in which lysine 6 is replaced by tryptophan (K6W-Ub) impairs the function of UPP. The main objective of this work was to investigate the hypothesis that chronically impairment of UPP in RPE contributes to some of the cardinal features of AMD, including drusens. Methods To address this question ARPE-19 cells were infected with empty vector, K6W Ubiquitin mutant and Wild Type Ubiquitin. The drusen formation and accumulation in basal matrix was evaluated by confocal microscopy, using specific drusen markers such as APOE, while exosome realease was determined by western blot (such as CD63 and Tsg101) and flow cytometry following exosomes isolation by ultracentrifugation. Results The results obtained showed that expression of K6W mutant Ub leads to an impairment of proteasome activity and an increased amount of exosomes release and drusen accumulation. Conclusion In this study the data might provide a vaulable tool to eluciadte the biological determinats of age-related proteolytic stress and its impacts on proteostasis in the retina.

Purpose It has been suggested that aging affects the cells ability to protect protein integrity, which is essential for cellular homeostasis. On cell aging there is impaired protein homeostasis, which can lead to protein insolubilization that is toxic to cells. The carboxyl terminus of Hsp70-interacting protein (CHIP) is an important ubiquitin ligase for protein quality control, and particularly in selective degradation of aggregation prone proteins. The objective of this study was to characterize the transgenic mice that overexpress CHIP/Ubc5 in lens epithelial cells and investigate the role of these proteins in the process that maintains proteins in solution and therefore participates in a number of age-related diseases, including cataract. Methods To overexpress CHIP, the α-crystallin promoter was used to drive the expression of CHIP-Ubc5 in lens epithelial cells of transgenic mice. The CHIP levels of transgenic mice (Tg) were determined with western blotting analysis. Cataract was induced by single subcutaneous injection of sodium selenite and the lens transparency was assessed by slit lamp examination. The soluble and insoluble protein content in all groups was evaluated. Results The data show that the transgenic mice only overexpress the transgene in lens epithelial cells.The slit lamp analysis showed that transgenic mice appears developed less cataract compared with wild type, with concomitantly increase in ratio of the soluble and insoluble proteins. Conclusion Together the data suggest that overexpression of CHIP/Ubc5 in lens epithelial cells could prevent cataract formation by promoting reduction of protein insolubilization.

Purpose Cholesterol oxides was implicated in the development of cataract. The purpose of this study is to elucidate the molecular mechanisms underlying cholesterol oxide-induced cell dysfunction in the lens. Methods Lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-KC) and 25-hydroxycholesterol (25-OH). The effects of cholesterol oxides on cytoskeleton were evaluated by immunofluorescence microscopy. Activation of p38 MAPK and Akt was detected by Western Blot. LC3 processing was detected by Western Blot and immunofluorescence confocal microscopy and autophagy was assessed by the LC3 I/LC3 II ratio. Results Cells treated with 25-OH show increased migration and formation of lamellipodia. These effects are reversed by inhibitors of PI3K and p38 MAPK. Consistently, 25-OH induces activation of Akt and p38 MAPK in LEC. On the other hand, treatment of LEC with 7-KC decreases Akt levels and up-regulates autophagy. The increase in autophagy following treatment with 7-KC is prevented by overexpressing a constitutive active Akt. Chemical inhibition of PI3K or overexpression of a dominant negative Akt also leads to stimulation of autophagy. Conclusion 7-KC stimulates autophagy in LEC by promoting degradation of Akt and activation of mTOR. The two different cholesterol oxides (7-KC, 25-OH) have opposite effects in the regulation of Akt, resulting in increased autophagy (7-KC) and increased cell migration (25-OH). Accumulation of cholesterol oxides in lens may thus account for loss of cell transparency by interfering with cell migration and by deregulating autophagy in LEC. Supported by FCT grants POCI/SAU-MMO/57216/2004 and POCI/SAU-OBS/57772/2004.

Purpose Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration (AMD). Lysosomal impairment in RPE cells is one of the traits present in the pathology of AMD. Authophagy, a process whereby proteins and subcellular organelles are degraded in the lysosome is activated by several stress stimuli. RPEs internalize large amounts of oxidized lipids including 7-ketocholesterol, the most cytotoxic of oxysterols. The purpose of this study is to determine if exposing RPE to 7-ketocholesterol can act as a stimulus to induce autophagy and thus account to the dysfunction of the RPE. Methods RPE cells where incubated with 7-ketocholesterol (20 ug/ml) for 6 or 12 h, or deprived from nutrients for 12h. To assess Macroautophagy cells were transfected with EGFP-LC3 plasmid. Formation of autophagic vesicles was assessed by confocal microscopy. LC3 processing was measured by western blot with antibodies against GFP. Chaperone mediated autophagy (CMA) was assessed by determining if chaperones interacted with Lamp2, performing Lamp2 immunoprecipitation. Results 7-ketocholesterol induces the formation of autophagic vesicles and the processing of the LC3-I into LC3-II, both hallmarks of Macroautophagy, Moreover treatment with 7-ketocholesterol increases the interaction between Lamp2 and Hsc70, Hsp90 and Hsp40, suggesting that this cholesterol oxide also stimulates CMA. Conclusion RPE cells undergo Macroautophagy and CMA activation when exposed to 7-ketocholesterol. At early stages both may act as a cell survival mechanism whereas prolonged exposure may result in macroautophagic cell death.