ERK1 and ERK2 are involved in recruitment and maturation of human mesenchymal stem cells induced to adipogenic differentiation

Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation

Mesenchymal stem cells protect sensory neurons, but not cortical neurons, from the chemotherapeutics-induced neurotoxicity

Mesenchymal stem cells (MSCs) have been often proposed for the therapy of several neurological diseases, due to their manifold peculiar properties. In particular, since it has been previously demonstrated that these cells are able to increase the survival of untreated sensory neurons [1], in this work we evaluated their possible protective effect on sensory neurons previously exposed to toxic agents. This could be particularly relevant to design a supportive therapy to counteract the peripheral neuropathy, a very common side effect of several chemotherapeutic agents, such as platinum and taxanes compounds, which often represents their dose limiting factor [2]. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging [3]. Here we demonstrated that Cisplatin (CDDP) and Paclitaxel-treated sensory neurons are protected by the co-culture with MSCs, but in two different manners: through a direct contact able to block apoptosis for CDDP-treated neurons, and by the release of trophic factors (including glutathione) for Paclitaxel-treated ones. In addition, the MSCs’ effectiveness was also verified on cortical neurons, since the recent advances in targeted drug delivery allowed to drive chemotherapeutic drugs also to the central nervous system. We verified that cortical neurons are more vulnerable to the toxic action of the drugs, and overall that MSCs fail at all to protect them. All these data demonstrated that MSCs are potentially useful to limit the peripheral neuropathy onset for their protective effect on injured-sensory neurons, but they also identified for the first time a different susceptibility of cortical and sensory neurons to MSC action

Effects of valproic Acid, berberin and resveratrol on human mesenchymal stem cells adipogenic differentiation

Nowadays obesity and its related diseases represent a major health problem with an increasing worldwide prevalence. Hyperplasia and hypertrophy of adipocytes lead to an excessive fat accumulation that is not efficiently prevented by current pharmacological treatments. So the research on anti-obesity drugs with good efficacy and tolerability able both to prevent and to reduce fat accumulation is of pivotal interest. In the present study we evaluated in vitro the effects of Valproic Acid, Berberin and Resveratrol on adipogenesis. Our experimental model was represented by human Mesenchymal Stem Cells (hMSCs), physiological precursors of adipocytes that can differentiate into adipocytes also in vitro. Preliminary cytotoxicity assays were performed in order to choose non-toxic doses of the three drugs. hMSCs were induced to adipogenic differentiation and treated with Valproic Acid, Berberin and Resveratrol at the selected doses. Controls were represented by hMSCs treated for adipogenesis in absence of the drugs. At different time points intracellular lipid droplets accumulation, a typical feature of adipogenesis, was assessed by Oil Red O staining. Valproic Acid, Berberin and Resveratrol inhibited hMSCs adipogenic differentiation in a dose dependent manner as demonstrated by the reduction of the lipid droplets accumulation. To understand the molecular mechanisms of the drugs-induced adipogenesis inhibition, we focused our attention on the effects of the drugs treatment on cell cycle progression, known to be altered by many antiadipogenic drugs, and on the MAP Kinases ERK1 and ERK2, involved in the adipogenesis control. We evaluated the expression of cyclins and CDKs by immunoblotting and flow-cytometry analyses, demonstrating that Valproic Acid, Berberin and Resveratrol interfere on cell cycle progression. The expression and the phosphorylation status of the two kinases ERK1 and ERK2 were assessed by immunoblotting demonstrating an increase of ERK1 phosphorylation (i.e. activation) in hMSCs treated with Berberin and a reduction in hMSCs treated with Valproic Acid and Resveratrol compared to control cells. No changes in phosphorylation and expression of ERK2 were observed. Our study demonstrate that Valproic Acid, Berberin and Resveratrol exert an anti-adipogenic effect in our experimental model. The mechanisms of action of these drugs involve the alteration of cell cycle progression and, at least in part, ERK1/2 modulation. However other molecular pathways are likely implicated and other studies are required to identify them

Air pollution: a study of citizen's attitudes and behaviors using different information sources

Background: From November 2015 to January 2016, the routine air monitoring showed a peak of air pollution (in particular of PM10) that caused alarm in many Italian cities and was widely reported by mass media. After some weeks from this alarm, we tried to evaluate the citizen awareness and interest towards air pollution together with their positive behaviors, using different information sources. Methods: From September 2015 to March 2016, in parallel with the number of exceedances of the PM10 in Italy we evaluated the press coverage, the average monthly searches on Google and the interest on Twitter. Moreover, a qualitative content analysis on daily newspapers was conducted and a self-compiled questionnaire on the attitudes and behaviors about environmental issues and their determinants was administered to 598 parents involved in the project MAPEC_LIFE (LIFE12 ENV/IT/00614). Results: The media coverage of the theme of air pollution was very high from the end of 2015 to the beginning of 2016, as well as internet searches and twitter messages. Our qualitative analysis highlighted that only a small portion of articles included information about positive behaviors and environmental awareness. Despite the high media coverage and the satisfactory self-perceived knowledge, the majority of respondents judged negatively the received information (as untrue and incomplete) and declared a limited adoption of pro-environmental behaviors.  Conclusion: The parallel study of mass media information and people’s attitudes and behaviors seem to indicate that the high media coverage was not followed by a very high motivation towards pro-environmental behaviors

Improving awareness of health hazards associated with air pollution in primary school children: Design and test of didactic tools

One of the objectives of the MAPEC-Life project is raising children’s awareness on air quality and its health effects. To achieve this goal, we designed didactic tools for primary school students, including leaflets with more information for teachers, a cartoon, and three educational videogames. The tools were then tested with 266 children who attended six primary schools in four Italian cities. A control group of 51 children received only explanations from teachers. An improvement in knowledge after using the audiovisual package was demonstrated, with higher efficacy compared with the control group. In addition, the use of videogames was greatly appreciated

Human Mesenchymal Stem Cells and Endothelial Progenitor Cells exert a neuroprotective effect on rat cortical neurons injured by oxygen and glucose deprivation

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs

Effects induced by particles derived from two anthropogenic sources on respiratory, cardiovascular and central nervous systems

Air pollution represents a well-known environmental problem related to public health. Particulate matter (PM) is a heterogeneous mixture of chemicals, metals and soils. Its adverse effects have been correlated with particles size, being smaller particles more likely to cause a worst damage, so their study deserves more attention. Ultrafine particles (UFPs, dae < 100 nm) are short-lived particles dispersed in the environment. In Lombardy, diesel combustion and solid biomass burning are the most relevant contributors to primary UFPs emissions (15-30 nm in diameter). Toxicological studies, mainly in vitro, indicate specific effects for particles of different origin but comparative in vivo studies are scarce. PM exposure has been primarily associated to pulmonary and cardiovascular diseases through oxidative stress and inflammatory response, but recently it has been postulated that PM exposure could also be an important risk factor for neurotoxicity and could have a role in neurodegenerative diseases. In this study we analysed in BALB/c mice the effect of single and repeated intratracheal instillation of diesel (DEP) and biomass (BC) particles on respiratory, cardiovascular and central nervous systems, comparing the two different UFPs sources. The study was performed at biochemical and histopathological level. Different pro-inflammatory, cytotoxic, pro-coagulant and oxidative stress markers were measured. For the histopathological evaluation, sections of lung, heart and different parts of the central nervous system (CNS) were examined at light microscope, using standard staining tecniques and immunohistochemical methods. Inflammation was also monitored in living mice following BC or DEP intratracheal repeated administration using the FMT 1500 fluorescence tomography imaging system and the MMPSense 750 Fast probe.  Our results indicate that even a single instillation of both the sources of UFPs induces a wide range of biochemical changes in the respiratory and cardiovascular systems, then confirmed by repeated instillation. In the CNS similar modifications were observed, although these were much more evident after repeated instillations. Histological examination demonstrated the presence of macrophages containing particles in the lungs after UFPs single and, more abundantly, repeated administration. However, significant changes were not observed in sections of heart and CNS. DEP was more effective in inducing oxidative stress and inflammation compared to BC

Differentiation of Mesenchymal Stem Cells towards an insulin-releasing phenotype after co-culture with Pancreatic Islets

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001