A Vicious NGF-p75NTR Positive Feedback Loop Exacerbates the Toxic Effects of Oxidative Damage in the Human Retinal Epithelial Cell Line ARPE-19.

In spite of its variety of biological activities, the clinical exploitation of human NGF (hNGF) is currently limited to ocular pathologies. It is therefore interesting to test the effects of hNGF in preclinical models that may predict their efficacy and safety in the clinical setting of ocular disorders and compare the effects of hNGF with those of its analogs. We used a human retinal pigment cell line, ARPE-19 cells, to investigate the effects of hNGF and its analogs, mouse NGF (mNGF) and painless NGF (pNGF), on cell viability under basal conditions and after exposure to oxidative stimuli, i.e., hydrogen peroxide (H2O2) and ultraviolet (UV)-A rays. The effects of hNGF and pNGF were also tested on the gene expression and protein synthesis of the two NGF receptor subtypes, p75 neurotrophic receptors (p75NTR) and tyrosine kinase A (TrkA) receptors. We drew the following conclusions: (i) the exposure of ARPE-19 cells to H2O2 or UV-A causes a dose-dependent decrease in the number of viable cells; (ii) under baseline conditions, hNGF, but not pNGF, causes a concentration-dependent decrease in cell viability in the range of doses 1-100 ng/mL; (iii) hNGF, but not pNGF, significantly potentiates the toxic effects of H2O2 or of UV-A on ARPE-19 cells in the range of doses 1-100 ng/mL, while mNGF at the same doses presents an intermediate behavior; (iv) 100 ng/mL of hNGF triggers an increase in p75NTR expression in H2O2-treated ARPE-19 cells, while pNGF at the same dose does not; (v) pNGF, but not hNGF (both given at 100 ng/mL), increases the total cell fluorescence intensity for TrkA receptors in H2O2-treated ARPE-19 cells. The present findings suggest a vicious positive feedback loop through which NGF-mediated upregulation of p75NTR contributes to worsening the toxic effects of oxidative damage in the human retinal epithelial cell line ARPE-19. Looking at the possible clinical relevance of these findings, one can postulate that pNGF might show a better benefit/risk ratio than hNGF in the treatment of ocular disorders.

mTOR Inhibition Is Effective against Growth, Survival and Migration, but Not against Microglia Activation in Preclinical Glioma Models.

Initially introduced in therapy as immunosuppressants, the selective inhibitors of mTORC1 have been approved for the treatment of solid tumors. Novel non-selective inhibitors of mTOR are currently under preclinical and clinical developments in oncology, attempting to overcome some limitations associated with selective inhibitors, such as the development of tumor resistance. Looking at the possible clinical exploitation in the treatment of glioblastoma multiforme, in this study we used the human glioblastoma cell lines U87MG, T98G and microglia (CHME-5) to compare the effects of a non-selective mTOR inhibitor, sapanisertib, with those of rapamycin in a large array of experimental paradigms, including (i) the expression of factors involved in the mTOR signaling cascade, (ii) cell viability and mortality, (iii) cell migration and autophagy, and (iv) the profile of activation in tumor-associated microglia. We could distinguish between effects of the two compounds that were overlapping or similar, although with differences in potency and or/time-course, and effects that were diverging or even opposite. Among the latter, especially relevant is the difference in the profile of microglia activation, with rapamycin being an overall inhibitor of microglia activation, whereas sapanisertib was found to induce an M2-profile, which is usually associated with poor clinical outcomes.

Rutin Protects Fibroblasts from UVA Radiation through Stimulation of Nrf2 Pathway.

This study explores the photoprotective effects of rutin, a bioflavonoid found in some vegetables and fruits, against UVA-induced damage in human skin fibroblasts. Our results show that rutin increases cell viability and reduces the high levels of ROS generated by photo-oxidative stress (1 and 2 h of UVA exposure). These effects are related to rutin's ability to modulate the Nrf2 transcriptional pathway. Interestingly, activation of the Nrf2 signaling pathway results in an increase in reduced glutathione and Bcl2/Bax ratio, and the subsequent protection of mitochondrial respiratory capacity. These results demonstrate how rutin may play a potentially cytoprotective role against UVA-induced skin damage through a purely antiapoptotic mechanism.

The effects of painless nerve growth factor on human microglia polarization.

Previous studies in the rat suggest that microglial cells represent a potential druggable target for nerve growth factor (NGF) in the brain. The painless human Nerve Growth Factor (hNGFp) is a recombinant mutated form of human nerve growth factor (hNGF) that shows identical neurotrophic and neuroprotective properties of wild-type NGF but displays at least 10-fold lower algogenic activity. From the pharmacological point of view, hNGFp is a biased tropomyosin receptor kinase A (TrkA) agonist and displays a significantly lower affinity for the p75 neurotrophin receptor (p75NTR). This study aimed to evaluate the expression of TrkA and p75NTR NGF receptors in two different human microglia cell lines, and to investigate the effects of hNGFp and wild-type NGF (NGF) on L-arginine metabolism, taken as a marker of microglia polarization. Both NGF receptors are expressed in human microglia cell lines and are effective in transducing signals triggered by NGF and hNGFp. The latter and, to a lesser extent, NGF inhibit cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these cells. Conversely NGF but not hNGFp stimulates arginase-mediated urea production.

Cytoprotective Effect of Idebenone through Modulation of the Intrinsic Mitochondrial Pathway of Apoptosis in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress Induced by Hydrogen Peroxide.

Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of Leber's hereditary optic neuropathy. Mitochondria of retinal pigment epithelium (RPE) are particularly vulnerable to oxidative damage associated with cellular senescence. Therefore, the aim of this study was to explore idebenone's cytoprotective effect and its underlying mechanism. We used a human-RPE cell line (ARPE-19) exposed to idebenone pre-treatment for 24 h followed by conditions inducing H2O2 oxidative damage for a further 24 h. We found that idebenone: (a) ameliorated H2O2-lowered cell viability in the RPE culture; (b) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation; (c) increased Bcl-2 protein levels, leaving unmodified those of Bax, thereby reducing the Bax/Bcl-2 ratio; (d) maintained the mitochondrial membrane potential (ΔΨm) at physiological levels, preserving the functionality of mitochondrial respiratory complexes and counteracting the excessive production of ROS; and (e) reduced mitochondrial cytochrome C-mediated caspase-3 activity. Taken together, our findings show that idebenone protects RPE from oxidative damage by modulating the intrinsic mitochondrial pathway of apoptosis, suggesting its possible role in retinal epitheliopathies associated with mitochondrial dysfunction.

Cytoprotective Effects of Punicalagin on Hydrogen-Peroxide-Mediated Oxidative Stress and Mitochondrial Dysfunction in Retinal Pigment Epithelium Cells.

The retinal pigment epithelium (RPE) is a densely pigmented, monostratified epithelium that provides metabolic and functional support to the outer segments of photoreceptors. Endogenous or exogenous oxidative stimuli determine a switch from physiological to pathological conditions, characterized by an increase of intracellular levels of reactive oxygen species (ROS). Accumulating evidence has elucidated that punicalagin (PUN), the major ellagitannin in pomegranate, is a potent antioxidant in several cell types. The present study aimed to investigate the protective effect of PUN on mitochondrial dysfunction associated with hydrogen peroxide (H2O2)-induced oxidative stress. For this purpose, we used a human RPE cell line (ARPE-19) exposed to H2O2 for 24 h. The effects of PUN pre-treatment (24 h) were examined on cell viability, mitochondrial ROS levels, mitochondrial membrane potential, and respiratory chain complexes, then finally on caspase-3 enzymatic activity. The results showed that supplementation with PUN: (a) significantly increased cell viability; (b) kept the mitochondrial membrane potential (ΔΨm) at healthy levels and limited ROS production; (c) preserved the activity of respiratory complexes; (d) reduced caspase-3 activity. In conclusion, due to its activity in helping mitochondrial functions, reducing oxidative stress, and subsequent induction of cellular apoptosis, PUN might be considered a useful nutraceutical agent in the treatment of oxidation-associated disorders of RPE.

Phospho-mTOR expression in human glioblastoma microglia-macrophage cells.

The glioblastoma (GBM) immune microenvironment is highly heterogeneous, and microglia may represent 30-70% of the entire tumor. However, the role of microglia and other specific immune populations is poorly characterized. Activation of mTOR signaling occurs in numerous human cancers and has roles in microglia-glioma cell interactions. We now show in human tumor specimens (42 patients), that 39% of tumor-associated microglial (TAM) cells express mTOR phosphorylated at Ser-2448; and similar mTOR activation is observed using a human microglia-glioma interaction paradigm. In addition, we confirm previous studies that microglia express urea and ARG1 (taken as M2 marker) in the presence of glioma cells, and this phenotype is down-regulated in the presence of a mTOR inhibitor. These results suggest that mTOR suppression in GBM patients might induce a reduction of the M2 phenotype expression in up to 40% of all TAMs. Since the M2 profile of microglial activation is believed to be associated with tumor progression, reductions in that phenotype may represent an additional anti-tumor mechanism of action of mTOR inhibitors, along with direct anti-proliferative activities.

Interactions between integrase inhibitors and human arginase 1.

The neuro-pathogenic mechanism(s) underlying HIV-associated neurocognitive disorders are mostly unknown. HIV-infected macrophages and microglial cells play a crucial role and the metabolic fate of l-arginine may be highly relevant to microglia activation. In this context, arginase (ARG), which uses l-arginine as substrate, can be on the same time a target and source of oxidative stress and inflammation. In this study, we investigated whether integrase strand transfer inhibitors share with the other antiretroviral drugs the ability to inhibit ARG activity. We used the previously validated cell model, namely the human microglia cell line, as well as the computational chemistry approach. Furthermore, here we characterized the activity of purified human ARG in a cell-free in vitro system, and investigated the effects of integrase strand transfer inhibitors in this newly validated model. Overall evidence shows that Dolutegravir, Raltegravir and Elvitegravir inhibit ARG activity.