Role of Chaperone-Mediated Autophagy in Ageing Biology and Rejuvenation of Stem Cells

What lies at the basis of the mechanisms that regulate the maintenance and self-renewal of pluripotent stem cells is still an open question. The control of stemness derives from a fine regulation between transcriptional and metabolic factors. In the last years, an emerging topic has concerned the involvement of Chaperone-Mediated Autophagy (CMA) as a key mechanism in stem cell pluripotency control acting as a bridge between epigenetic, transcriptional and differentiation regulation. This review aims to clarify this new and not yet well-explored horizon discussing the recent studies regarding the CMA impact on embryonic, mesenchymal, and haematopoietic stem cells. The review will discuss how CMA influences embryonic stem cell activity promoting self-renewal or differentiation, its involvement in maintaining haematopoietic stem cell function by increasing their functionality during the normal ageing process and its effects on mesenchymal stem cells, in which modulation of CMA regulates immunosuppressive and differentiation properties. Finally, the importance of these new discoveries and their relevance for regenerative medicine applications, from transplantation to cell rejuvenation, will be addressed

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo

Relation between TLR4/NF-kB signaling pathway activation by 27-hydroxycholesterol and 4-hydroxynonenal, and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll-like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low-density lipoproteins, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high-risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27-OH and HNE were found to enhance cell release of IL-8, IL-1β, and TNF-α and to upregulate matrix metalloproteinase-9 (MMP-9) via TLR4/NF-κB-dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP-9 upregulation, thus enhancing the release of this matrix-degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP-9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF-κB downstream signaling

A Dietary Mixture of Oxysterols Induces In Vitro Intestinal Inflammation through TLR2/4 Activation: The Protective Effect of Cocoa Bean Shells

Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play a role in inflammatory bowel diseases. This report deals with the ability of a mixture of oxysterols widely present in cholesterol-rich foods to induce in vitro intestinal inflammation through TLR up-regulation. The anti-inflammatory action of four cocoa bean shell (CBS) extracts with different polyphenol content, was tested. Methods: Differentiated intestinal CaCo-2 cells were treated with a dietary oxysterol mixture (Oxy-mix) (60 µM). The expression and activation of TLR2 and TLR4, as well as the production of their downstream signaling effectors IL-8, IFNβ and TNFα were analyzed in the presence or absence of TLR antibodies. Honduras CBS extracts were characterized for their polyphenol contents; their anti-inflammatory action was analyzed in CaCo-2 cells treated with Oxy-mix. Results: Oxysterol-dependent TLR-2 and TLR4 over-expression and activation together with cytokine induction were abolished by blocking TLRs with specific antibodies. Polyphenol-rich CBS extracts consisting of high quantities of (−)-epicatechin and tannins also prevented TLR induction. Conclusions: TLR2 and TLR4 mainly contribute to inducing oxysterol-dependent intestinal inflammation. The fractionation method of CBS allowed the recovery of fractions rich in (−)-epicatechin and tannins able to counteract oxysterol-induced inflammation, thus highlighting the beneficial biological potential of specific CBS extracts

Protective Effect of Cocoa Bean Shell against Intestinal Damage: An Example of Byproduct Valorization

Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of components such as polyphenols and methylxanthines, which have been associated with a reduced risk of several diseases. Therefore, CBS has potential application as a food ingredient. Intestinal mucosa is exposed to immune and inflammatory responses triggered by dietary agents, such as oxysterols, which derive from cholesterol oxidation and are pro-oxidant compounds able to affect intestinal function. We aimed at investigating the capability of the Forastero cultivar CBS, added or not added to ice cream, to protect against the intestinal barrier damage induced by a dietary oxysterol mixture. Methods: Composition and antioxidant capacity of in vitro digested CBS and CBS-enriched ice cream were analyzed by high-performance liquid chromatography and 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay, respectively. CaCo-2 cells differentiated into enterocyte-like monolayer were incubated with 60 µM oxysterol mixture in the presence of CBS formulations. Results: The oxysterol mixture induced tight junction impairment, interleukin-8 and monocyte chemoattractant protein-1 cell release, and oxidative stress-related nuclear factor erythroid 2 p45-related factor 2 response Nrf2. Both CBSs protected cells from these adverse effects, probably thanks to their high phenolic content. CBS-enriched ice cream showed the highest antioxidant capacity. Theobromine, which is in high concentrations of CBS, was also tested. Although theobromine exerted no effect on Nrf2 expression, its anti-inflammatory cooperating activity in CBS effect cannot be excluded. Conclusions: Our findings suggest that CBS-enriched ice cream may be effective in the prevention of gut integrity damage associated with oxidative/inflammatory reactionsThis work was funding by the University of Turin, Italy [grant numbers BIAF_RILO_17_01; BIAF_RILO_18_01]; the Italian Ministry of University and Research [BIAF_FFABR_17_01]; the project COVALFOOD “Valorisation of high added-value compounds from cocoa industry by-products as food ingredients and additives” has received funding from the European Union’s Seventh Framework programme for research and innovation under the Marie Skłodowska-Curie grant agreement No 609402-2020 researchers: Train to Move (T2M); Spanish Ministry of Science, Innovation and Universities “Juan de la Cierva—Incorporación” Grant (Agreement No. IJCI-2017-31665)S

Inhibition of herpes simplex-1 virus replication by 25-hydroxycholesterol and 27-hydroxycholesterol

Oxysterols are known pleiotropic molecules whose antiviral action has been recently discovered. Here reported is the activity of a panel of oxysterols against HSV-1 with the identification of a new mechanism of action. A marked antiviral activity not only of 25HC but also of 27HC against HSV-1 was observed either if the oxysterols were added before or after infection, suggesting an activity unrelated to the viral entry inhibition as proposed by previous literature. Therefore, the relation between the pro-inflammatory activity of oxysterols and the activation of NF-kB and IL-6 induced by HSV-1 in the host cell was investigated. Indeed, cell pre-incubation with oxysterols further potentiated IL-6 production as induced by HSV-1 infection with a consequent boost of the interleukin's total cell secretion. Further, a direct antiviral effect of IL-6 administration to HSV-1 infected cells was demonstrated, disclosing an additional mechanism of antiviral action by both 25HC and 27HC. Keywords: Oxysterols, 27-hydroxycholesterol, 25-hydroxycholesterol, Herpes simplex-1, Viral inhibition, Interleukin-