VEGF and ELAVL1/HuR protein levels are increased in dry and wet AMD patients. A new tile in the pathophysiologic mechanisms underlying RPE degeneration?

Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula's retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD.

Dimethyl Fumarate and Intestine: From Main Suspect to Potential Ally against Gut Disorders.

Dimethyl fumarate (DMF) is a well-characterized molecule that exhibits immuno-modulatory, anti-inflammatory, and antioxidant properties and that is currently approved for the treatment of psoriasis and multiple sclerosis. Due to its Nrf2-dependent and independent mechanisms of action, DMF has a therapeutic potential much broader than expected. In this comprehensive review, we discuss the state-of-the-art and future perspectives regarding the potential repurposing of DMF in the context of chronic inflammatory diseases of the intestine, such as inflammatory bowel disorders (i.e., Crohn's disease and ulcerative colitis) and celiac disease. DMF's mechanisms of action, as well as an exhaustive analysis of the in vitro/in vivo evidence of its beneficial effects on the intestine and the gut microbiota, together with observational studies on multiple sclerosis patients, are here reported. Based on the collected evidence, we highlight the new potential applications of this molecule in the context of inflammatory and immune-mediated intestinal diseases.

Chiral 2-phenyl-3-hydroxypropyl esters as PKC-alpha modulators: HPLC enantioseparation, NMR absolute configuration assignment, and molecular docking studies.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. In our previous work, we identified in racemate 1-2, based on the 2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα and PKCδ ligands, targeting the C1 domain of these two kinases. Herein, we report the resolution of the racemates by enantioselective semi-preparative HPLC. The attribution of the absolute configuration (AC) of homochirals 1 was performed by NMR, via methoxy-α-trifluoromethyl-α-phenylacetic acid derivatization (MTPA or Mosher's acid). Moreover, the match between the experimental and predicted electronic circular dichroism (ECD) spectra confirmed the assigned AC. These results proved that Mosher's esters can be properly exploited for the determination of the AC also for chiral primary alcohols. Lastly, homochiral 1 and 2 were assessed for binding affinity and functional activity against PKCα. No significative differences in the Ki of the enantiopure compounds was observed, thus suggesting that chirality does not seem to play a significant role in targeting PKC C1 domain. These results are in accordance with the molecular docking studies performed using a new homology model for the human PKCαC1B domain.

Increased intraocular pressure alters the cellular distribution of HuR protein in retinal ganglion cells - A possible sign of endogenous neuroprotection failure.

The RNA-binding protein, HuR, modulates mRNA processing and gene expression of several stress response proteins i.e. Hsp70 and p53 that have been postulated to be involved in the pathogenesis of glaucoma, a chronic optic neuropathy leading to irreversible blindness. We evaluated HuR protein expression in retinas and optic nerves of glaucomatous rats and human primary open angle glaucoma patients and its possible impact on stress response mechanisms. We found that the cytoplasmic content of HuR was reduced more extensively in glaucomatous retinas than in optic nerves and this was linked with a declined cytoplasmic Hsp70 level and p53 nuclear translocation. In the optic nerve, the p53 content was decreased as a feature of reactive gliosis. Based on our findings, we conclude that the alteration in the HuR content, observed both in rat glaucoma model and human glaucoma samples, affects post-transcriptionally the expression of genes crucial for maintaining cell homeostasis; therefore, we postulate that HuR may be involved in the pathogenesis of glaucoma.

Targeting VEGF in eye neovascularization: What's new?: A comprehensive review on current therapies and oligonucleotide-based interventions under development.

Roughly ten years ago the FDA approved most of the presently used anti-VEGF drugs for the treatment of neovascular AMD and other eye pathologies characterized by ocular neoangiogenesis. However, the recent findings on the physiologic activities of VEGF isoforms impose to reconsider the inhibitory effects of pan-VEGF antagonists and the concept that to face pathological alterations at ocular level is possible only through the full block of all VEGF isoforms. In fact, although pan-VEGF agents rapidly and effectively contrast ocular neovascularization, vascular leakage, and other pathological changes, in the long-term the inhibition of all VEGF isoforms likely may result in the loss of the physiologic effects exerted by VEGF121 and the anti-angiogenic VEGF165b. Notably, selective inhibitors of VEGF165a, such as pegaptanib, spare these targets. Moreover, preclinical and clinical evidence suggests that also systemic side effects, secondary to intraocular treatment with non-selective anti-VEGF drugs, may be reinterpreted in light of these recent findings, which may be useful to clinicians for the choice of the most appropriate anti-VEGF agent. Another aspect that should be considered is the involvement of VEGF-independent pathways in ocular neovascularization, therefore a combined therapy can represent a more effective pharmacological approach that might help also to counteract tachyphylaxis, an important issue in anti-VEGF treatment. This complex picture and the recent findings on current anti-VEGF drugs should be therefore taken into account to guide the development of novel agents targeting VEGF and/or other key factors involved in the pathogenesis of neovascular ocular diseases along the signaling pathways stimulated by the various isoforms. Accordingly, this review also reports on novel pharmacological molecules targeting VEGF at ocular level and currently under development, with a special attention to oligonucleotide-based interventions.

Nanosystems based on siRNA silencing HuR expression counteract diabetic retinopathy in rat.

We evaluated whether specifically and directly targeting human antigen R (HuR), a member of embryonic lethal abnormal vision (ELAV) proteins family, may represent a new potential therapeutic strategy to manage diabetic retinopathy. Nanosystems loaded with siRNA silencing HuR expression (lipoplexes), consisting of solid lipid nanoparticles (SLN) and liposomes (SUV) were prepared. Photon correlation spectroscopy analysis, Zeta potential measurement and atomic force microscopy (AFM) studies were carried out to characterize the complexation of siRNA with the lipid nanocarriers. Nanosystems were evaluated by using AFM and scanning electron microscopy. The lipoplexes were injected into the eye of streptozotocin (STZ)-induced diabetic rats. Retinal HuR and VEGF levels were detected by Western blot and ELISA, respectively. Retinal histology was also carried out. The results demonstrated that retinal HuR and VEGF are significantly increased in STZ-rats and are blunted by HuR siRNA treatment. Lipoplexes with a weak positive surface charge and with a 4:1 N/P (cationic lipid nitrogen to siRNA phosphate) ratio exert a better transfection efficiency, significantly dumping retinal HuR and VEGF levels. In conclusion, we demonstrated that siRNA can be efficiently delivered into the rat retina using lipid-based nanocarriers, and some of the lipoplexes loaded with siRNA silencing HuR expression are potential candidates to manage retinal diseases.

Induction of VEGFA mRNA translation by CoCl2 mediated by HuR.

Vascular endothelial growth factor (VEGF) A is a master regulator of neovascularization and angiogenesis. VEGFA is potently induced by hypoxia and by pathological conditions including diabetic retinopathy and tumorigenesis. Fine-tuning of VEGFA expression by different stimuli is important for maintaining tissue vascularization and organ homeostasis. Here, we tested the effect of the hypoxia mimetic cobalt chloride (CoCl2) on VEGFA expression in human cervical carcinoma HeLa cells. We found that CoCl2 increased the levels of VEGFA mRNA and VEGFA protein without affecting VEGFA mRNA stability. Biotin pulldown analysis to capture the RNA-binding proteins (RBPs) bound to VEGFA mRNA followed by mass spectrometry analysis revealed that the RBP HuR [human antigen R, a member of the embryonic lethal abnormal vision (ELAV) family of proteins], interacts with VEGFA mRNA. VEGFA mRNA-tagging experiments showed that exposure to CoCl2 increases the interaction of HuR with VEGFA mRNA and promoted the colocalization of HuR and the distal part of the VEGFA 3'-untranslated region (UTR) in the cytoplasm. We propose that under hypoxia-like conditions, HuR enhances VEGFA mRNA translation.

Pre-exposure of neuroblastoma cell line to pulsed electromagnetic field prevents H2 O2 -induced ROS production by increasing MnSOD activity.

Electromagnetic fields (EMFs) have been linked to increased risk of cancers and neurodegenerative diseases; however, EMFs can also elicit positive effects on biological systems, and redox status seems crucially involved in EMF biological effects. This study aimed to assess whether a short and repeated pulsed EMF (PEMF) could trigger adaptive responses against an oxidative insult in a neuronal cellular model. We found that a 40 min overall (four times a week, 10 min each) pre-exposure to PEMF did not affect major physiological parameters and led to a significant increase of Mn-dependent superoxide dismutase activity in the human neuroblastoma SH-SY5Y cell line. In addition, we found PEMF-pre-exposed cells exhibited decreased reactive oxygen species production following a 30 min H2 O2 challenge, with respect to non pre-exposed cells. Our findings might provide new insights on the role played by short and repeated PEMF stimulations in the enhancement of cellular defenses against oxidative insults. Although studies in normal neuronal cells would be useful to further confirm our hypothesis, we suggest that specific PEMF treatments may have potential biological repercussions in diseases where oxidative stress is implicated.

Autophagy is modulated in human neuroblastoma cells through direct exposition to low frequency electromagnetic fields.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aß peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aß peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.

Extracellular vesicles in degenerative retinal diseases: A new therapeutic paradigm.

Nanoscale extracellular vesicles (EVs), consisting of exomers, exosomes and microvesicles/ectosomes, have been extensively investigated in the last 20 years, although their biological role is still something of a mystery. EVs are involved in the transfer of lipids, nucleic acids and proteins from donor to recipient cells or distant organs as well as regulating cell-cell communication and signaling. Thus, EVs are important in intercellular communication and this is not limited to sister cells, but may also mediate the crosstalk between different cell types even over long distances. EVs play crucial functions in both cellular homeostasis and the pathogenesis of diseases, and since their contents reflect the status of the donor cell, they represent an additional valuable source of information for characterizing complex biological processes. Recent advances in isolation and analytical methods have led to substantial improvements in both characterizing and engineering EVs, leading to their use either as novel biomarkers for disease diagnosis/prognosis or even as novel therapies. Due to their capacity to carry biomolecules, various EV-based therapeutic applications have been devised for several pathological conditions, including eye diseases. In the eye, EVs have been detected in the retina, aqueous humor, vitreous body and also in tears. Experiences with other forms of intraocular drug applications have opened new ways to use EVs in the treatment of retinal diseases. We here provide a comprehensive summary of the main in vitro, in vivo, and ex vivo literature-based studies on EVs' role in ocular physiological and pathological conditions. We have focused on age-related macular degeneration, diabetic retinopathy, glaucoma, which are common eye diseases leading to permanent blindness, if not treated properly. In addition, the putative use of EVs in retinitis pigmentosa and other retinopathies is discussed. Finally, we have reviewed the potential of EVs as therapeutic tools and/or biomarkers in the above-mentioned retinal disorders. Evidence emerging from experimental disease models and human material strongly suggests future diagnostic and/or therapeutic exploitation of these biological agents in various ocular disorders with a good possibility to improve the patient's quality of life.

Posttranscriptional regulation of SOD1 gene expression under oxidative stress: Potential role of ELAV proteins in sporadic ALS.

Increased levels of SOD1 mRNA have been observed in sporadic ALS patients (SALS) compared to controls. Hence, the understanding of the mechanisms by which SOD1 gene expression is modulated may shed new light on SOD1 involvement in ALS. Of interest, some adenine/uracil-rich elements (AREs) in SOD1 3'-untranslated region have been identified. These sequences represent the docking sites for several RNA-binding proteins such as ELAV proteins (ELAVs), positive regulators of gene expression. We first investigated in SH-SY5Y cells whether SOD1 mRNA represents a target of ELAVs. Results from RNA Electrophoretic Mobility Shift and RNA-immunoprecipitation assays showed a molecular interaction between ELAVs and SOD1 mRNA. We also observed that the treatment with H2O2 induced a significant increase of the amount of SOD1 mRNA bound by ELAVs and an up-regulation of SOD1 protein levels. We found a specific increase in ELAV/HuR phosphorylation, suggesting activation of this protein, in peripheral blood mononuclear cells from SALS patients compared to controls. Finally, we found increased levels of ELAV proteins in the motor cortex and spinal cord from SALS patients compared to controls, in parallel with SOD1 up-regulation in the same areas. This study suggests, for the first time, that ELAVs are involved in the regulation of SOD1 gene expression at post-transcriptional level and that these proteins are more activated in ALS pathology. The link between ELAVs and SOD1 may open novel perspectives for ALS research, paving the way for new therapeutic options.

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20µM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10µM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.

Novel potential pharmacological applications of dimethyl fumarate-an overview and update.

Dimethyl fumarate (DMF) is an FDA-approved drug for the treatment of psoriasis and multiple sclerosis. DMF is known to stabilize the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. It has also been shown that DMF influences autophagy and participates in the transcriptional control of inflammatory factors by inhibiting NF-κB and its downstream targets. DMF is receiving increasing attention for its potential to be repurposed for several diseases. This versatile molecule is indeed able to exert beneficial effects on different medical conditions through a pleiotropic mechanism, in virtue of its antioxidant, immunomodulatory, neuroprotective, anti-inflammatory, and anti-proliferative effects. A growing number of preclinical and clinical studies show that DMF may have important therapeutic implications for chronic diseases, such as cardiovascular and respiratory pathologies, cancer, eye disorders, neurodegenerative conditions, and systemic or organ specific inflammatory and immune-mediated diseases. This comprehensive review summarizes and highlights the plethora of DMF's beneficial effects and underlines its repurposing opportunities in a variety of clinical conditions.

Deficiency of the RNA-binding protein ELAVL1/HuR leads to the failure of endogenous and exogenous neuroprotection of retinal ganglion cells.

Introduction: ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of hur-silenced RGC in the rat glaucoma model. Methods: The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. Results: Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram. Conclusions: Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR's key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.

Dimethyl Fumarate Triggers the Antioxidant Defense System in Human Retinal Endothelial Cells through Nrf2 Activation.

Dimethyl fumarate (DMF) is a well-known activator of Nrf2 (NF-E2-related factor 2), used in the treatment of psoriasis and multiple sclerosis. The mechanism of action consists in the modification of the cysteine residues on the Nrf2-inhibitor Keap1, thus leading to the dissociation of these two proteins and the consequent activation of Nrf2. Considering the paucity of evidence of DMF effects in the context of retinal endothelium, this in vitro study investigated the role of DMF in human retinal endothelial cells (HREC). Here, we show for the first time in HREC that DMF activates the Nrf2 pathway, thus leading to an increase in HO-1 protein levels and a decrease in intracellular ROS levels. Furthermore, this molecule also shows beneficial properties in a model of hyperglucose stress, exerting cytoprotective prosurvival effects. The overall collected results suggest that DMF-mediated activation of the Nrf2 pathway may also be a promising strategy in ocular diseases characterized by oxidative stress. This study opens a new perspective on DMF and suggests its potential repositioning in a broader therapeutical context.

The Challenge of Dimethyl Fumarate Repurposing in Eye Pathologies.

Dimethyl fumarate (DMF) is a small molecule currently approved and used in the treatment of psoriasis and multiple sclerosis due to its immuno-modulatory, anti-inflammatory, and antioxidant properties. As an Nrf2 activator through Keap1 protein inhibition, DMF unveils a potential therapeutical use that is much broader than expected so far. In this comprehensive review we discuss the state-of-art and future perspectives regarding the potential repositioning of this molecule in the panorama of eye pathologies, including Age-related Macular Degeneration (AMD). The DMF's mechanism of action, an extensive analysis of the in vitro and in vivo evidence of its beneficial effects, together with a search of the current clinical trials, are here reported. Altogether, this evidence gives an overview of the new potential applications of this molecule in the context of ophthalmological diseases characterized by inflammation and oxidative stress, with a special focus on AMD, for which our gene-disease (KEAP1-AMD) database search, followed by a protein-protein interaction analysis, further supports the rationale of DMF use. The necessity to find a topical route of DMF administration to the eye is also discussed. In conclusion, the challenge of DMF repurposing in eye pathologies is feasible and worth scientific attention and well-focused research efforts.

Nature-Inspired Hybrids (NIH) Improve Proteostasis by Activating Nrf2-Mediated Protective Pathways in Retinal Pigment Epithelial Cells.

Antioxidant systems play key roles in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, autophagy impairment and inflammation are well-described in AMD, especially in retinal pigment epithelial (RPE) cells. The master regulator of antioxidant defense Nrf2 has been linked to AMD, autophagy and inflammation. In this study, in human ARPE-19 cells, some nature-inspired hybrids (NIH1-3) previously shown to induce Nrf2-mediated protection against oxidative stress were further investigated for their potential against cellular stress caused by dysfunction of protein homeostasis. NIH1-3 compounds increased the expression of two Nrf2-target genes coding defense proteins, HO-1 and SQSTM1/p62, in turn exerting beneficial effects on intracellular redox balance without modification of the autophagy flux. NIH1-3 treatments predisposed ARPE-19 cells to a better response to following exposure to proteasome and autophagy inhibitors, as revealed by the increase in cell survival and decreased secretion of the pro-inflammatory IL-8 compared to NIH-untreated cells. Interestingly, NIH4 compound, through an Nrf2-independent pathway, also increased cell viability and decreased IL-8 secretion, although to a lesser extent than NIH1-3, suggesting that all NIHs are worthy of further investigation into their cytoprotective properties. This study confirms Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress, such as AMD.

NGF and heart: Is there a role in heart disease?

The review emphasizes the role of NGF, the most representative member of the neurotrophins family, in cardiac physiopathology with a particular focus on healing and sprouting processes occurring after tissue damage. Cardiac and circulating NGF levels dramatically increase following myocardial injury (MI). A very early rise of this neurotrophin is indeed observed soon after MI (hours). Such a rise may lead to sympathetic nerve sprouting which may underlie the later genesis of arrhythmias but may also favor the healing process. At later times (months after), when heart failure develops, the opposite is detected and NGF tissue levels are below the normal range, an event that may in turn participate to defective innervation and cardiac failure. Through a careful analysis of preclinical and clinical studies, this review proposes that time is the key variable when studying these opposite changes in NGF expression observed following MI and attempting to interpret and correlate them with cardiac physiopathology. The examination of the results leads to the speculation that NGF modulation may be a pharmacological target for interventions in specific stages of heart dysfunction following MI.

A Nature-Inspired Nrf2 Activator Protects Retinal Explants from Oxidative Stress and Neurodegeneration.

Oxidative stress (OS) plays a key role in retinal dysfunctions and acts as a major trigger of inflammatory and neurodegenerative processes in several retinal diseases. To prevent OS-induced retinal damage, approaches based on the use of natural compounds are actively investigated. Recently, structural features from curcumin and diallyl sulfide have been combined in a nature-inspired hybrid (NIH1), which has been described to activate transcription nuclear factor erythroid-2-related factor-2 (Nrf2), the master regulator of the antioxidant response, in different cell lines. We tested the antioxidant properties of NIH1 in mouse retinal explants. NIH1 increased Nrf2 nuclear translocation, Nrf2 expression, and both antioxidant enzyme expression and protein levels after 24 h or six days of incubation. Possible toxic effects of NIH1 were excluded since it did not alter the expression of apoptotic or gliotic markers. In OS-treated retinal explants, NIH1 strengthened the antioxidant response inducing a massive and persistent expression of antioxidant enzymes up to six days of incubation. These effects resulted in prevention of the accumulation of reactive oxygen species, of apoptotic cell death, and of gliotic reactivity. Together, these data indicate that a strategy based on NIH1 to counteract OS could be effective for the treatment of retinal diseases.

Eye-Light on Age-Related Macular Degeneration: Targeting Nrf2-Pathway as a Novel Therapeutic Strategy for Retinal Pigment Epithelium.

Age-related macular degeneration (AMD) is a common disease with a multifactorial aetiology, still lacking effective and curative therapies. Among the early events triggering AMD is the deterioration of the retinal pigment epithelium (RPE), whose fundamental functions assure good health of the retina. RPE is physiologically exposed to high levels of oxidative stress during its lifespan; thus, the integrity and well-functioning of its antioxidant systems are crucial to maintain RPE homeostasis. Among these defensive systems, the Nrf2-pathway plays a primary role. Literature evidence suggests that, in aged and especially in AMD RPE, there is an imbalance between the increased pro-oxidant stress, and the impaired endogenous detoxifying systems, finally reverberating on RPE functions and survival. In this in vitro study on wild type (WT) and Nrf2-silenced (siNrf2) ARPE-19 cells exposed to various AMD-related noxae (H2O2, 4-HNE, MG132 + Bafilomycin), we show that the Nrf2-pathway activation is a physiological protective stress response, leading downstream to an up-regulation of the Nrf2-targets HO1 and p62, and that a Nrf2 impairment predisposes the cells to a higher vulnerability to stress. In search of new pharmacologically active compounds potentially useful for AMD, four nature-inspired hybrids (NIH) were individually characterized as Nrf2 activators, and their pharmacological activity was investigated in ARPE-19 cells. The Nrf2 activator dimethyl-fumarate (DMF; 10 µM) was used as a positive control. Three out of the four tested NIH (5 µM) display both direct and indirect antioxidant properties, in addition to cytoprotective effects in ARPE-19 cells under pro-oxidant stimuli. The observed pro-survival effects require the presence of Nrf2, with the exception of the lead compound NIH1, able to exert a still significant, albeit lower, protection even in siNrf2 cells, supporting the concept of the existence of both Nrf2-dependent and independent pathways mediating pro-survival effects. In conclusion, by using some pharmacological tools as well as a reference compound, we dissected the role of the Nrf2-pathway in ARPE-19 stress response, suggesting that the Nrf2 induction represents an efficient defensive strategy to prevent the stress-induced damage.