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 Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status and Research Perspectives

Celiac disease (CD) affects approximately 1% of the population in Europe and North America, but the number of patients currently undiagnosed is estimated to be far higher than that of diagnosed cases owing to the presence of prevalent forms with nonspecific symptoms. The toxicity of gliadin in children with CD is not destroyed through digestion with gastropancreatic enzymes. An innate immunity to gliadin plays a key role in the development of CD. Autophagy, a physiological catabolic process, plays also a crucial role in the pathogenesis of several inflammatory diseases. Recent studies have described functional involvement of the regulation of autophagy within a pediatric CD cohort. Furthermore, the contribution of autophagy has been highlighted in the degradation and in the reduction of extracellular release of gliadin peptides, thus suggesting novel molecular targets to counteract gliadin-induced toxicity in CD

Development of Artificial Plasma Membranes Derived Nanovesicles Suitable for Drugs Encapsulation

Extracellular vesicles (EVs) are considered as promising nanoparticles theranostic tools in many physiological and pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membranes-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride, the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drugs encapsulation and cell delivery, showing, in particular, similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drugs delivery contexts

Celiac disease and autophagy: new functional perspectives in diagnosis and treatment

Celiac disease (CD) is the most common inflammatory disease of the intestine. It is a chronic systemic autoimmune disorder affecting the small bowel of genetic susceptible individuals. CD is triggered by the ingestion of gluten, a storage protein present in wheat, barley and rye. CD frequency in the general population of Europe and United States is approximately 1% whereas in Finland and Sweden it reaches peaks of respectively 2% to 3%. In Italy, the estimated prevalence is 0.7%, however the number of patients currently undiagnosed seems to be largely superior to known cases. The only effective therapy is a gluten-free diet (GFD). Nevertheless, 7 to 30% of all patients is not responsive to GFD because inadvertent ingestion of gluten. The two major challenges about CD concern diagnosis and treatment. Auto-antibodies in the serum represent a valuable tool for CD diagnosis, but a percentage of patients remains elusive because of the presence of mild-symptoms. Moreover, despite international established guidelines, there is still controversy about the use of endoscopy as the essential step in CD diagnosis. Nowadays, the attention of the researchers has shifted in the identification of new non-invasive diagnostic biomarkers. Because of their characteristics, microRNAs (miRNAs) have been emerged as promising candidate not only in CD field but also for other disorders, such as Crohn disease and ulcerative colitis. The issues concerning the GFD have prompted researchers to investigate for altervative treatments. Currently, two pharmacological agents are investigated in late clinical trials as non-dietary treatments for CD. Autophagy is a cellular process that is implicated in immunity and autoimmunity as well as in the degradation of protein aggregates. Impairment of autophagic flux contributes to the pathogenesis of several disorders characterized by the accumulation of toxic protein aggregates, such as Alzheimer and Parkinson disease. Accordingly, a collaborative study was born with the Pediatric Auxology Unit and the Pediatric Surgery Unit of the Fondazione IRCCS Policlinico San Matteo. Thus, the first resulted aims of this Thesis is to investigate the role of key autophagic genes and of their regulatory miRNAs sequences as new candidate biomarkers in CD. For this purpose, blood and intestinal biopsies were collected by an exploratory cohort of pediatric CD parients and controls matched for sex and age. The obtained results suggest that the investigated autophagy-related genes and miRNAs could have a potential diagnostic power to distinguish between CD patiens and controls. Moreover, specific expression profiles could be use for CD patients’ stratification. The role of autophagy in the metabolism of gliadin was then studied in an in vitro model obtained with Caco-2 cells and modulation of this cellular process was performed in order to counteract the toxicity of these peptides. The results indicate that autophagy is implicated in gliadin degradation and that impairment of this process affects the release of gliadin outside the cells by exocytosis. On the other hand, autophagy induction leads to gliadin degradation, decreases its secretion and confers a proliferative advantage to cells. On the whole, these preliminary results indicate that the study of autophagy could be interesting for the search of new diagnostic biomarkers. The finding that autophagy is implicated in gliadin metabolism will possibly allow the identification of new therapeutical approaches based on the modulation of this cellular process

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

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 much broader than expected so far. In this comprehensive review we discuss the state-of-art and future perspective 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 of scientific attention and well-focused research efforts

Gluten Exorphins Promote Cell Proliferation through the Activation of Mitogenic and Pro-Survival Pathways

Celiac disease (CD) is a chronic and systemic autoimmune disorder that affects preferentially the small intestine of individuals with a genetic predisposition. CD is promoted by the ingestion of gluten, a storage protein contained in the endosperm of the seeds of wheat, barley, rye, and related cereals. Once in the gastrointestinal (GI) tract, gluten is enzymatically digested with the consequent release of immunomodulatory and cytotoxic peptides, i.e., 33mer and p31-43. In the late 1970s a new group of biologically active peptides, called gluten exorphins (GEs), was discovered and characterized. In particular, these short peptides showed a morphine-like activity and high affinity for the δ-opioid receptor (DOR). The relevance of GEs in the pathogenesis of CD is still unknown. Recently, it has been proposed that GEs could contribute to asymptomatic CD, which is characterized by the absence of symptoms that are typical of this disorder. In the present work, GEs cellular and molecular effects were in vitro investigated in SUP-T1 and Caco-2 cells, also comparing viability effects with human normal primary lymphocytes. As a result, GEs treatments increased tumor cell proliferation by cell cycle and Cyclins activation as well as by induction of mitogenic and pro-survival pathways. Finally, a computational model of GEs interaction with DOR is provided. Altogether, the results might suggest a possible role of GEs in CD pathogenesis and on its associated cancer comorbidities

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

Development of Berberine-Loaded Nanoparticles for Astrocytoma Cells Administration and Photodynamic Therapy Stimulation

Berberine (BBR) is known for its antitumor activity and photosensitizer properties in anti-cancer photodynamic therapy (PDT), and it has previously been favorably assayed against glioblastoma multiforme (GBM)-derived cells. In this work, two BBR hydrophobic salts, dodecyl sulfate (S) and laurate (L), have been encapsulated in PLGA-based nanoparticles (NPs), chitosan-coated by the addition of chitosan oleate in the preparation. NPs were also further functionalized with folic acid. All the BBR-loaded NPs were efficiently internalized into T98G GBM established cells, and internalization increased in the presence of folic acid. However, the highest mitochondrial co-localization percentages were obtained with BBR-S NPs without folic acid content. In the T98G cells, BBR-S NPs appeared to be the most efficient in inducing cytotoxicity events and were therefore selected to assess the effect of photodynamic stimulation (PDT). As a result, PDT potentiated the viability reduction for the BBR-S NPs at all the studied concentrations, and a roughly 50% reduction of viability was obtained. No significant cytotoxic effect on normal rat primary astrocytes was observed. In GBM cells, a significant increase in early and late apoptotic events was scored by BBR NPs, with a further increase following the PDT scheme. Furthermore, a significantly increased depolarization of mitochondria was highlighted following BBR-S NPs’ internalization and mostly after PDT stimulation, compared to untreated and PDT-only treated cells. In conclusion, these results highlighted the efficacy of the BBR-NPs-based strategy coupled with photoactivation approaches to induce favorable cytotoxic effects in GBM cells

Trehalose Modulates Autophagy Process to Counteract Gliadin Cytotoxicity in an In Vitro Celiac Disease Model

Celiac disease (CD) is a chronic systemic autoimmune disorder that is triggered by the ingestion of gliadin peptides, the alcohol-soluble fraction of wheat gluten. These peptides, which play a key role in the immune response that underlies CD, spontaneously form aggregates and exert a direct toxic action on cells due to the increase in the reactive oxygen species (ROS) levels. Furthermore, peptic-tryptic digested gliadin peptides (PT-gliadin) lead to an impairment in the autophagy pathway in an in vitromodel based on Caco-2 cells. Considering these premises, in this studywe have analyzed differentmTOR-independent inducers, reporting that the disaccharide trehalose, amTOR-independent autophagy activator, rescued the autophagy flux in Caco-2 cells treated with digested gliadin, as well as improved cell viability. Moreover, trehalose administration to Caco-2 cells in presence of digested gliadin reduced the intracellular levels of these toxic peptides. Altogether, these results showed the beneficial effects of trehalose in a CD in vitro model as well as underlining autophagy as a molecular pathway whose modulation might be promising in counteracting PT-gliadin cytotoxicity