Toxicity and inflammatory potential of mineral fibres: The contribute of released soluble metals versus cell contact direct effects.

Asbestos fibres have been considered an environmental hazard for decades. However, little is known about the attempts of circulating immune cells to counteract their toxicity. We addressed the early effects of fibre-released soluble factors (i.e. heavy metals) in naïve immune cells, circulating immediately below the alveolar/endothelial cell layer. By comparison, the direct fibre effects on endotheliocytes were also studied since these cells are known to sustain inflammatory processes. The three mineral fibres analysed showed that mainly chrysotile (CHR) and erionite (ERI) were able to release toxic metals in extracellular media respect to crocidolite (CRO), during the first 24 h. Nevertheless, all three fibres were able to induce oxidative stress and genotoxic damage in indirectly challenged naïve THP-1 monocytes (separated by a membrane). Conversely, only CHR-released metal ions induced apoptosis, NF-κB activation, cytokines and CD163 gene overexpression, indicating a differentiation towards the M0 macrophage phenotype. On the other hand, all three mineral fibres in direct contact with HECV endothelial cells showed cytotoxic, genotoxic and apoptotic effects, cytokines and ICAM-I overexpression, indicating the ability of these cells to promote an inflammatory environment in the lung independently from the type of inhaled fibre. Our study highlights the different cellular responses to mineral fibres resulting from both the nature of the cells and their function, but also from the chemical-physical characteristics of the fibres. In conclusion, CHR represented the main pro-inflammatory trigger, able to recruit and activate circulating naïve monocytes, through its released metals, already in the first 24 h after inhalation.

Genetics and Glaucoma: the state of the art.

Glaucoma is the second leading cause of irreversible blindness worldwide. Although genetic background contributes differently to rare early-onset glaucoma (before age 40) or common adult-onset glaucoma, it is now considered an important factor in all major forms of the disease. Genetic and genomic studies, including GWAS, are contributing to identifying novel loci associated with glaucoma or to endophenotypes across ancestries to enrich the knowledge about glaucoma genetic susceptibility. Moreover, new high-throughput functional genomics contributes to defining the relevance of genetic results in the biological pathways and processes involved in glaucoma pathogenesis. Such studies are expected to advance significantly our understanding of glaucoma's genetic basis and provide new druggable targets to treat glaucoma. This review gives an overview of the role of genetics in the pathogenesis or risk of glaucoma.

Citicoline Eye Drops Protect Trabecular Meshwork Cells from Oxidative Stress Injury in a 3D In Vitro Glaucoma Model.

Intraocular pressure (IOP) is considered an important modifiable risk factor for glaucoma, which is known as the second leading cause of blindness worldwide. However, lowering the IOP is not always sufficient to preserve vision due to other non-IOP-dependent mechanisms being involved. To improve outcomes, adjunctive therapies with IOP-independent targets are required. To date, no studies have shown the effect of citicoline on the trabecular meshwork (TM), even though it is known to possess neuroprotective/enhancement properties and multifactorial mechanisms of action. Given that reactive oxygen species seem to be involved in glaucomatous cascade, in this present study, an advanced millifluidic in vitro model was used to evaluate if citicoline could exert a valid TM protection against oxidative stress. To this end, the cellular behavior, in terms of viability, apoptosis, mitochondrial state, senescence and pro-inflammatory cytokines, on 3D human TM cells, treated either with H2O2 alone or cotreated with citicoline, was analyzed. Our preliminary in vitro results suggest a counteracting effect of citicoline eye drops against oxidative stress on TM cells, though further studies are necessary to explore citicoline's potential as a TM-target therapy.

The Acute Toxicity of Mineral Fibres: A Systematic In Vitro Study Using Different THP-1 Macrophage Phenotypes.

Alveolar macrophages are the first line of defence against detrimental inhaled stimuli. To date, no comparative data have been obtained on the inflammatory response induced by different carcinogenic mineral fibres in the three main macrophage phenotypes: M0 (non-activated), M1 (pro-inflammatory) and M2 (alternatively activated). To gain new insights into the different toxicity mechanisms of carcinogenic mineral fibres, the acute effects of fibrous erionite, crocidolite and chrysotile in the three phenotypes obtained by THP-1 monocyte differentiation were investigated. The three mineral fibres apparently act by different toxicity mechanisms. Crocidolite seems to exert its toxic effects mostly as a result of its biodurability, ROS and cytokine production and DNA damage. Chrysotile, due to its low biodurability, displays toxic effects related to the release of toxic metals and the production of ROS and cytokines. Other mechanisms are involved in explaining the toxicity of biodurable fibrous erionite, which induces lower ROS and toxic metal release but exhibits a cation-exchange capacity able to alter the intracellular homeostasis of important cations. Concerning the differences among the three macrophage phenotypes, similar behaviour in the production of pro-inflammatory mediators was observed. The M2 phenotype, although known as a cell type recruited to mitigate the inflammatory state, in the case of asbestos fibres and erionite, serves to support the process by supplying pro-inflammatory mediators.

Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy.

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 µg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

An Innovative In Vitro Open-Angle Glaucoma Model (IVOM) Shows Changes Induced by Increased Ocular Pressure and Oxidative Stress.

Primary Open-Angle Glaucoma (POAG) is a neurodegenerative disease, and its clinical outcomes lead to visual field constriction and blindness. POAG's etiology is very complex and its pathogenesis is mainly explained through both mechanical and vascular theories. The trabecular meshwork (TM), the most sensitive tissue of the eye anterior segment to oxidative stress (OS), is the main tissue involved in early-stage POAG, characterized by an increase in pressure. Preclinical assessments of neuroprotective drugs on animal models have not always shown correspondence with human clinical studies. In addition, intra-ocular pressure management after a glaucoma diagnosis does not always prevent blindness. Recently, we have been developing an innovative in vitro 3Dadvanced human trabecular cell model on a millifluidicplatform as a tool to improve glaucoma studies. Herein, we analyze the effects of prolonged increased pressure alone and, in association with OS, on such in vitro platform. Moreover, we verify whethersuch damaged TM triggers apoptosis on neuron-like cells. The preliminary results show that TM cells are less sensitive to pressure elevation than OS, and OS-damaging effects were worsened by the pressure increase. The stressed TM releases harmful signals, which increase apoptosis stimuli on neuron-like cells, suggesting its pivotal role in the glaucoma cascade.

Risk Factors for Retinal Ganglion Cell Distress in Glaucoma and Neuroprotective Potential Intervention.

Retinal ganglion cells (RGCs) are a population of neurons of the central nervous system (CNS) extending with their soma to the inner retina and with their axons to the optic nerve. Glaucoma represents a group of neurodegenerative diseases where the slow progressive death of RGCs results in a permanent loss of vision. To date, although Intra Ocular Pressure (IOP) is considered the main therapeutic target, the precise mechanisms by which RGCs die in glaucoma have not yet been clarified. In fact, Primary Open Angle Glaucoma (POAG), which is the most common glaucoma form, also occurs without elevated IOP. This present review provides a summary of some pathological conditions, i.e., axonal transport blockade, glutamate excitotoxicity and changes in pro-inflammatory cytokines along the RGC projection, all involved in the glaucoma cascade. Moreover, neuro-protective therapeutic approaches, which aim to improve RGC degeneration, have also been taken into consideration.

Lipoperoxide Nanoemulsion as Adjuvant in Cisplatin Cancer Therapy: In Vitro Study on Human Colon Adenocarcinoma DLD-1 Cells.

Cisplatin is a first-choice chemotherapeutic agent used to treat solid tumors even though the onset of multi-drug resistance and the time-dose side-effects impair its mono-therapeutic application. Therefore, new drug-delivery approaches, based on nanomedicine strategies, are needed to enhance its therapeutic potential in favor of a dose-reduction of cisplatin. Polyunsaturated fatty acids and their metabolism-derived intermediates, as well as lipid peroxidation end-products, are used as adjuvants to improve the effectiveness of chemotherapy. Lipid hydroperoxides, derived from the oxidation of edible oils, can contribute to cell death, generating breakdown products (e.g., reactive aldehydes). In this regard, the aim of this present study was to evaluate an invitro combinatory strategy between a lecithin-based nanoemulsion system of K600, a patented mixture of peroxidated oil and peroxidated cholesterol, and cisplatin on DLD1 human adenocarcinoma cells. Our findings showed that nanoemulsions, acting in synergy with cisplatin, improve cisplatin bioactivity, in terms of enhancing its anti-cancer activity, towards DLD1 cells. Indeed, this combination approach, whilst maintaining cisplatin at low concentrations, induces a significant reduction in DLD1 cell viability, an increase in pro-apoptotic markers, and genotoxic damage. Therefore, K600 nanoemulsions as an efficient targeted delivery system of cisplatin allow for the reduction in the chemotherapeutic agent doses.

A 3D Model of Human Trabecular Meshwork for the Research Study of Glaucoma.

Glaucoma is a multifactorial syndrome in which the development of pro-apoptotic signals are the causes for retinal ganglion cell (RGC) loss. Most of the research progress in the glaucoma field have been based on experimentally inducible glaucoma animal models, which provided results about RGC loss after either the crash of the optic nerve or IOP elevation. In addition, there are genetically modified mouse models (DBA/2J), which make the study of hereditary forms of glaucoma possible. However, these approaches have not been able to identify all the molecular mechanisms characterizing glaucoma, possibly due to the disadvantages and limits related to the use of animals. In fact, the results obtained with small animals (i.e., rodents), which are the most commonly used, are often not aligned with human conditions due to their low degree of similarity with the human eye anatomy. Although the results obtained from non-human primates are in line with human conditions, they are little used for the study of glaucoma and its outcomes at cellular level due to their costs and their poor ease of handling. In this regard, according to at least two of the 3Rs principles, there is a need for reliable human-based in vitro models to better clarify the mechanisms involved in disease progression, and possibly to broaden the scope of the results so far obtained with animal models. The proper selection of an in vitro model with a "closer to in vivo" microenvironment and structure, for instance, allows for the identification of the biomarkers involved in the early stages of glaucoma and contributes to the development of new therapeutic approaches. This review summarizes the most recent findings in the glaucoma field through the use of human two- and three-dimensional cultures. In particular, it focuses on the role of the scaffold and the use of bioreactors in preserving the physiological relevance of in vivo conditions of the human trabecular meshwork cells in three-dimensional cultures. Moreover, data from these studies also highlight the pivotal role of oxidative stress in promoting the production of trabecular meshwork-derived pro-apoptotic signals, which are one of the first marks of trabecular meshwork damage. The resulting loss of barrier function, increase of intraocular pressure, as well the promotion of neuroinflammation and neurodegeneration are listed as the main features of glaucoma. Therefore, a better understanding of the first molecular events, which trigger the glaucoma cascade, allows the identification of new targets for an early neuroprotective therapeutic approach.

Can Polyphenols in Eye Drops Be Useful for Trabecular Protection from Oxidative Damage?

Polyphenols, with anti-oxidant properties, counteract oxidative stress effects. Increasing evidence has found oxidative stressto be the main risk factor for trabecular meshwork (TM) damage, leading to high-tension glaucoma. Topical anti-oxidants could represent a new target for glaucoma treatment. Our aim is to investigate the protective mechanisms on a human TM culture of a patented polyphenol and fatty acid (iTRAB®)formulation in response to oxidative stress using an advanced invitromodel consisting of 3D-human TM cells, embedded in a natural hydrogel, and a milli-scaled multi-organ device model for constantdynamic conditions. The 3D-human TM cells(3D-HTMCs) were treated daily with 500 µM H2O2or 500 µM H2O2and 0.15% iTRAB®(m/v) for 72 h, and molecular differences in the intracellular reactive oxygen species (iROS), state of the cells, activation of the apoptosis pathway and NF-kB and the expression ofinflammatory and fibrotic markers wereanalyzed at different time-points.Concomitant exposure significantly reduced iROS and restored TM viability, iTRAB® having a significant inhibitory effect on the apoptotic pathway, activation of NF-κB, induction of pro-inflammatory (IL-1α, IL-1ß and TNFα) and pro-fibrotic (TGFß) cytokines and the matrix metalloproteinase expressions. It is clear that this specific anti-oxidant provides a valid TM protection, suggesting iTRAB® could be an adjuvant therapy in primary open-angle glaucoma (POAG).

Neuroinflammation in Primary Open-Angle Glaucoma.

Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Increasing evidence suggests oxidative damage and immune response defects are key factors contributing to glaucoma onset. Indeed, both the failure of the trabecular meshwork tissue in the conventional outflow pathway and the neuroinflammation process, which drives the neurodegeneration, seem to be linked to the age-related over-production of free radicals (i.e., mitochondrial dysfunction) and to oxidative stress-linked immunostimulatory signaling. Several previous studies have described a wide range of oxidative stress-related makers which are found in glaucomatous patients, including low levels of antioxidant defences, dysfunction/activation of glial cells, the activation of the NF-κB pathway and the up-regulation of pro-inflammatory cytokines, and so on. However, the intraocular pressure is still currently the only risk factor modifiable by medication or glaucoma surgery. This present review aims to summarize the multiple cellular processes, which promote different risk factors in glaucoma including aging, oxidative stress, trabecular meshwork defects, glial activation response, neurodegenerative insults, and the altered regulation of immune response.

Molecular changes in glaucomatous trabecular meshwork. Correlations with retinal ganglion cell death and novel strategies for neuroprotection.

Glaucoma is a chronic neurodegenerative disease characterized by retinal ganglion cell loss. Although significant advances in ophthalmologic knowledge and practice have been made, some glaucoma mechanisms are not yet understood, therefore, up to now there is no effective treatment able to ensure healing. Indeed, either pharmacological or surgical approaches to this disease aim in lowering intraocular pressure, which is considered the only modifiable risk factor. However, it is well known that several factors and metabolites are equally (if not more) involved in glaucoma. Oxidative stress, for instance, plays a pivotal role in both glaucoma onset and progression because it is responsible for the trabecular meshwork cell damage and, consequently, for intraocular pressure increase as well as for glaucomatous damage cascade. This review at first shows accurately the molecular-derived dysfunctions in antioxidant system and in mitochondria homeostasis which due to both oxidative stress and aging, lead to a chronic inflammation state, the trabecular meshwork damage as well as the glaucoma neurodegeneration. Therefore, the main molecular events triggered by oxidative stress up to the proapoptotic signals that promote the ganglion cell death have been highlighted. The second part of this review, instead, describes some of neuroprotective agents such as polyphenols or polyunsaturated fatty acids as possible therapeutic source against the propagation of glaucomatous damage.

Corrigendum to An advanced in vitro model to assess glaucoma onset.

In this manuscript, which appeared in ALTEX 37, 265-274 (doi: 10.14573/altex.1909262), the affiliation of Stefania Vernazza should read: Stefania Vernazza 5# 5 IRCCS-Fondazione Bietti, Rome, Italy and the address for correspondence should read: Stefania Vernazza, PhD, IRCCS, Fondazione Bietti via Livenza 3, 00198 Rome, Italy (stefania.vernazza@yahoo.it).

An advanced in vitro model to assess glaucoma onset.

Glaucoma is the second leading cause of blindness worldwide. Currently, glaucoma treatments aim to lower intraocular pressure by decreasing aqueous humor production or increasing aqueous humor outflow through pharmacological approaches or trabeculectomy. The lack of an effective cure requires new therapeutic strategies. We compared the bio­logical responses of a three-dimensional trabecular meshwork model with or without perfusion bioreactor technology to better understand the early molecular changes induced by prolonged oxidative stress conditions induced by repeated daily peroxide exposure. We used standard 3D cultures of trabecular meshwork cells in Matrigel cultured under either static and dynamic conditions for one week. We studied changes in F-actin expression and organization in the cells, cellular metabolic activity, proinflammatory gene expression, expression of pro- and anti-apoptotic proteins, PARP-1 cleavage, and NFκB activation in the model. We demonstrate that the dynamic conditions improve the adaptive behavior of 3D trabecular meshwork cultures to chronic oxidative stress via offsetting pathway activation.

2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study.

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

Simulated microgravity induces nuclear translocation of Bax and BCL-2 in glial cultured C6 cells.

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed.

Conscientious Objection to Animal Testing: A Preliminary Survey Among Italian Medical and Veterinary Students.

The use of animals for educational and research purposes is common in both veterinary and human medicine degree courses, and one that involves important ethical considerations. The aim of this study was to assess the extent of differences between the knowledge and attitudes of veterinary students and medical students on animal bioethics, on alternative strategies and on their right to conscientiously object to animal experimentation. To this end, a questionnaire was completed by 733 students (384 human medicine students (HMS) and 349 veterinary medicine students (VMS)). VMS were more aware than HMS (72.2% and 59.6%, respectively) of the existence of an Italian law on the right to conscientiously object to animal experimentation. However, very few of them had exercised this right. Many VMS (43.3%) felt that animal bioethics courses should be mandatory (only 17.4% of HMS felt the same way). More VMS than HMS (81.7% and 59.1%, respectively) expressed an interest in attending a course on alternatives to animal experimentation. The data suggest the need for appropriate educational interventions, in order to allow students to make choices based on ethical principles. Fostering close collaborations between departments of human medicine and veterinary medicine, for example, through shared study modules, could promote the development of ethical competence as a basic skill of students of both veterinary and human medicine courses.