Laura Vergani

Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo.

Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis, is characterized by demyelination, inflammation and neurodegeneration of CNS in which free radicals play a role. Recently, the efficacy of murine mesenchimal stem cells (MSCs) as treatment of EAE induced in mice by the encephalitogenic peptide MOG(35–55) was demonstrated. The present study analyzed some markers of oxidative stress, inflammation/degeneration and apoptosis such as metallothioneins (MTs), antioxidant enzymes (superoxide dismutase, catalase and glutathione‐S‐transferase), poly(ADP‐ribose) polymerase‐1 and p53 during EAE progression and following MSC treatment. Expression of the three brain MT isoforms increased significantly in EAE mice compared with healthy controls, but while expression of MT‐1 and MT‐3 increased along EAE course, MT‐2 was up‐regulated at the onset, but returned to levels similar to those of controls in chronic phase. The changes in the transcription and activity of the antioxidant enzymes and in expression of poly(ADP‐ribose) polymerase‐1 and p53 showed the same kinetics observed for MT‐1 and MT‐3 during EAE. Interestingly, i.v. administration of MSCs reduced the EAE‐induced increases in levels/activities of all these proteins. These results support an antioxidant and neuroprotective activity for MSCs that was also confirmed in vitro on neuroblastoma cells exposed to an oxidative insult.

Intravenous mesenchymal stem cells improve survival and motor function in experimental amyotrophic lateral sclerosis.

Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 106) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [3H]d-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase- and glutamate transporter type 1-positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of (3H)d-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.

Mesenchymal Stem Cells Shape Microglia Effector Functions Through the Release of CX3CL1

Mesenchymal stem cells (MSC) display a remarkable ability to modulate the immune response and protect the central nervous system mainly through the release of soluble factors in a paracrine fashion, affecting the functional behavior of cells in the tissues. Here we investigated the effect of the interaction between MSC and microglia in vitro, and we dissected the molecular and cellular mechanisms of this crosstalk. We demonstrated that MSC impair microglia activation by inflammatory cues through the inhibition of the expression and release of inflammatory molecules and stress‐associated proteins. We showed that MSC significantly increase microglial expression and release of molecules associated with a neuroprotective phenotype such as CX3CR1, nuclear receptor 4 family, CD200 receptor, and insulin growth factor 1. Interestingly, MSC can enhance functional changes on microglia as depicted by the increase of intracellular calcium concentration and phagocytic activity. This last event is associated with an increased expression of triggering receptor expressed on myeloid cells‐2, an innate immune receptor involved in phagocytosis in the absence of inflammation. The observed effects on CX3CR1‐expressing microglia are due to the release of CX3CL1 by MSC, driven by inflammatory signals, as demonstrated by the reversal of the observed results when CX3CL1 expression was silenced in MSC or its release was blocked. Finally, we showed that exogenous CX3CL1 induce phenotypic and functional changes of microglia similar to those induced by MSC. These findings demonstrate that MSC instruct, through the release of CX3CL1, microglia responsiveness to proinflammatory signals by modulating constitutive “calming” receptors, typically expressed by “steady‐state microglia” thus switching microglia from a detrimental phenotype to a neuroprotective one. Stem Cells2012;30:2044–2053

Bisphenol-A alters gene expression and functional parameters in molluscan hepatopancreas

Bisphenol-A (BPA) is a well-known xenoestrogen in mammalian systems that can affect reproduction also in aquatic organisms. In this work the possible effects of BPA were investigated in the hepatopancreas of the bivalve mollusc Mytilus galloprovincialis: mussels were injected with different amounts of BPA (3-60ng/g dw tissue) and tissues sampled at 24h post-injection. Expression of different Mytilus genes was evaluated by RT-Q-PCR: BPA exposure increased the expression of MeER2 and induced downregulation of antioxidant genes, catalase and metallothioneins. Moreover, BPA induced changes in activity of catalase, GSH transferase (GST) and GSSG reductase (GSR), and in total glutathione content. A decrease in lysosomal membrane stability and increased neutral lipid accumulation were also observed. The results were compared with those obtained with similar concentrations of 17beta-estradiol. These data demonstrate that BPA can alter gene expression, activities of enzymes involved in redox balance, and lysosomal function in molluscan hepatopancreas, a tissue involved in the control of metabolism and gamete maturation. Overall, these data indicate that BPA, at environmentally relevant concentrations, can have both estrogen-like and distinct effects in invertebrates like in vertebrates.

Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis

In bivalve molluscs, responses to bacterial infection have been largely characterized in terms of both functional responses and gene expression in the immune cells, the hemocytes. The effects of bacterial challenge at the tissue level, where bacterial infection may cause stressful conditions, have not been so far specifically investigated. Biomarkers are widely utilised to evaluate the health status of bivalves, from the molecular to the organism level, in response to both natural and anthropogenic stressors. In this work, the effects of in vivo challenge with heat-killed vibrio species, Vibrio splendidus LGP32 and Vibrio anguillarum (ATCC19264), on different biomarkers in the digestive gland of the marine bivalve Mytilus galloprovincialis were investigated. Mussels were injected with either vibrio and tissues sampled at 3, 6 and 24 h post injection (p.i.). Lysosomal biomarkers, such as lysosomal membrane stability (LMS) and lipofuscin accumulation, as well as specific activities of antioxidant enzymes (catalase and glutathione transferase-GST) were evaluated. Moreover, the expression of antioxidant molecules (catalase, GST-pi and metallothioneins MT10 and MT20) was determined by quantitative RT-PCR. Both V. splendidus and V. anguillarum significantly affected all parameters measured, to a different extent and at different times p.i. Interestingly, whereas both vibrios induced lysosomal membrane destabilisation and increases in the activities of antioxidant enzymes, distinct responses were observed in terms of lysosomal lipofuscin accumulation and expression of antioxidant molecules. In particular, V. splendidus induced a general increase in the transcription of antioxidant genes, indicating that Mytilus digestive gland can mount an efficient antioxidant response towards this vibrio species. On the other hand, a general down-regulation or no effect was observed with V. anguillarum. The lack of this response was reflected in stronger oxidative stress conditions in the digestive gland of mussels challenged with V. anguillarum, as indicated by higher levels of lysosomal lipofuscin observed at longer times p.i. Overall, these data indicate that lysosomal and oxidative stress biomarkers could be usefully applied in order to monitor early changes in the health status of bivalves induced by bacteria. Moreover, the results support the hypothesis that host responses to bacteria may be taken into account when interpreting biomarker data in ecotoxicological studies.

Radical scavenging abilities of fish MT-A and mussel MT-10 metallothionein isoforms: An ESR study

Metallothioneins (MTs) are cysteine-rich proteins involved in homeostasis of essential metals, detoxification of toxic metals and scavenging of free radicals. Scavenging of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured by means of ESR spectroscopy for two recombinant MTs from aquatic species: MT-10 from the sea mussel Mytilus galloprovincialis, and MT-A from the fish Oncorhyncus mykiss. Both the zinc- and the cadmium-loaded forms (Zn(7)-MTs and Cd(7)-MTs) were analysed, using the commercial MT-II (Zn(7)-MT-II and Cd(7)-MT-II, respectively) from rabbit liver as a reference. A decrease in the scavenging ability was observed for all the three MTs passing from the Zn- to the Cd-loaded forms, because of the higher stability of the Cd-mercapto complex. The Zn(7)-MTs from aquatic species were more effective in scavenging DPPH signal than the rabbit Zn(7)-MT-II (2.8 and 4-folds, respectively). Similar results were obtained also for the Cd(7)-MTs, thus confirming the stronger antioxidant power of MTs from aquatic organisms compared with the rabbit MT-II. Moreover, mussel MT-10 was more active in DPPH scavenging than fish MT-A. When the complete release of metals from MTs was obtained by lowering the pH to 3 or, alternatively, by adding the chelating agent diethylenetriaminepentaacetic acid (DTPA), an increase in the scavenging ability of MTs was observed.

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well‐known dumbbell shape, with a β domain that binds three bivalent metal ions and an α domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S‐transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal‐binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7‐MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7‐MT to 4‐(2‐pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.

Short-term effects of environmentally relevant concentrations of EDC mixtures on Mytilus galloprovincialis digestive gland

Endocrine disrupting compounds (EDCs), including both natural estrogens and estrogenic chemicals, are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus galloprovincialis different estrogenic compounds, both individually and in mixtures, were shown to affect the immune function both in vitro and in vivo. Moreover, individual estrogens, the natural estrogen 17beta-estradiol (E(2)) and the xenoestrogen bisphenol A (BPA), have been recently demonstrated to alter functional parameters and gene expression in mussel digestive gland, a tissue that plays a central role in metabolism and in nutrient distribution to the gonad during gamete maturation, with possible consequences on gametogenesis. In this work, the possible effects of a synthetic mixture of EDCs on the digestive gland were evaluated. The mixture contained seven estrogenic chemicals (17beta-estradiol, 17alpha-ethynyl estradiol, mestranol (MES), nonylphenol, nonylphenol monoethoxylate carboxylate (NP1EC), BPA, benzophenone (BP)), in proportions similar to those previously found in water samples of a coastal lagoon. Mussels were injected with different concentrations of the mixture (approximate nominal concentrations of total EDCs: 0.0177, 0.177, 1.77 and 177 ng/g dw) and tissues sampled 24 h post-injection. The mixture induced significant changes in lysosomal biomarkers (lysosomal membrane stability (LMS), neutral lipid (NL) and lipofuscin (LF) accumulation) as well as in the activities of catalase, glutathione transferase (GST), and of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). Moreover, downregulation of the gene transcription for the Mytilus estrogen receptor MeER1 isoform and for catalase, as evaluated by quantitative RT-PCR, were observed. Significant changes in lysosomal biomarkers, enzyme activities and gene transcription were also recorded at 72 h post-injection. The results demonstrate that short-term exposure to environmentally relevant concentrations of EDC mixtures can interfere with the lysosomal function, redox-related enzyme activities and gene transcription of mussel digestive gland.

3,5-Diiodo-l-thyronine modulates the expression of genes of lipid metabolism in a rat model of fatty liver

Recent reports demonstrated that 3,5-diiodo-l-thyronine (T(2)) was able to prevent lipid accumulation in the liver of rats fed a high-fat diet (HFD). In this study, we investigated how the rat liver responds to HFD and T(2) treatment by assessing the transcription profiles of some genes involved in the pathways of lipid metabolism: oxidation, storage and secretion. The mRNA levels of the peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ), and of their target enzymes acyl-CoA oxidase and stearoyl-CoA desaturase were evaluated by real-time RT-PCR. Moreover, the expression of the adipose triglyceride lipase involved in lipid mobilisation, of the main PAT proteins acting in lipid droplet (LD) turnover, and of apoprotein B (apo B), the major protein component of very low-density lipoproteins (VLDLs) were analysed. Overall, our data demonstrated that T(2) administration to HFD rats counteracts most of the hepatic transcriptional changes that occurred in response to the excess exogenous fat. In particular, our results suggest that T(2) may prevent the pathways leading to lipid storage in LDs, promote the processes of lipid mobilisation from LDs and secretion as VLDL, in addition to the stimulation of pathways of lipid oxidation. In conclusion, our findings might give an insight into the mechanisms underlying the anti-steatotic ability of T(2) and help to define the potential therapeutic role of T(2) for preventing or treating liver steatosis.

Effects of 3,5-diiodo-L-thyronine administration on the liver of high fat diet-fed rats.

In rats fed a high fat diet (HFD), long-term administration of 3,5-diiodo-L-thyronine (T2), a naturally occurring iodothyronine, was shown to reduce body-weight gain, fat mass, and hepatic lipid accumulation. This work was aimed at investigating the mechanisms of T2 action in the liver of HFD rats. The results show that HFD induces liver lipid peroxidation and stimulates the activity of enzymes involved in hydrogen peroxide (H2O2) metabolism, catalase in particular. Moreover, quantitative RT-PCR revealed HFD-induced upregulation of the transcription factor PPARα, as well as of metallothionein isoforms (MT-1 and MT-2). T2 administration prevented the HDF-induced lipid peroxidation, as well as the increase in H2O2 metabolism, and reduced the upregulation of both PPARα and MT-2. These data demonstrate that in the liver of HFD rats, T2 prevents both lipid accumulation and oxidative stress associated with increased fat metabolism.