Regulation of Satellite Cells Functions during Skeletal Muscle Regeneration: A Critical Step in Physiological and Pathological Conditions.

Skeletal muscle regeneration is a complex process involving the generation of new myofibers after trauma, competitive physical activity, or disease. In this context, adult skeletal muscle stem cells, also known as satellite cells (SCs), play a crucial role in regulating muscle tissue homeostasis and activating regeneration. Alterations in their number or function have been associated with various pathological conditions. The main factors involved in the dysregulation of SCs' activity are inflammation, oxidative stress, and fibrosis. This review critically summarizes the current knowledge on the role of SCs in skeletal muscle regeneration. It examines the changes in the activity of SCs in three of the most common and severe muscle disorders: sarcopenia, muscular dystrophy, and cancer cachexia. Understanding the molecular mechanisms involved in their dysregulations is essential for improving current treatments, such as exercise, and developing personalized approaches to reactivate SCs.

Effect of Chemically Induced Hypoxia on Osteogenic and Angiogenic Differentiation of Bone Marrow Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in Direct Coculture.

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)-the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)-a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.

Single-shot morpho-functional and structural characterization of the left-ventricle in a mouse model of acute ischemia-reperfusion injury with an optimized 3D IntraGate cine FLASH sequence at 7T MR.

Preclinical cardiac MR is challenging and time-consuming. A fast and comprehensive acquisition protocol and standardized image post-processing may improve preclinical research, reducing acquisition time, costs and variability of results. In the present study, we evaluated the feasibility of a contrast-enhanced 3D IntraGate steady-state cine sequence (ce-3D-IG-cine) with short acquisition time (11 min) for a single-shot combined characterization of left ventricle (LV) remodeling and infarct size (IS) in a mouse model of acute ischemia-reperfusion injury. Sixteen male C57BL/6N mice underwent 7T cardiac MR (Bruker, BioSpec 70/30) including optimized ce-3D-IG-cine (total scan time 11 min) at day 1, 5 and 28 after surgery. LV end-diastolic volume (EDVMR) and ejection fraction (EFMR) extracted from MR were compared to ones from short-axis (SA-EDVecho, SA-EFecho) and parasternal long-axis (LA-EDVecho, LA-EFecho) echocardiography. IS was manually and semiautomatically segmented from ce-3D-IG-cine using different standard deviation (SD +2, +3, +4, +5, +6 in respect to a reference tissue). Mice were sacrificed at day 28, immediately after imaging. IS at day 28 was compared to injury burden at histology. MR and echocardiographic morpho-functional parameters were compared, as IS from MR and histology. Bland-Altman plots were used to assess the agreement in ischemic burden segmentation. Volumetric and functional parameters measured on ce-3D-IG-cine correlated to the correspondent echocardiographic parameter (EDVMR vs SA-EDVecho: ρ = 0.813; EDVMR vs LA-EDVecho: ρ = 0.845; EFMR vs SA-EFecho ρ = 0.612; EFMR vs LA-EFecho ρ = 0.791; p < 0.001 in all cases). Manually segmented IS strongly correlated with the scar at histology (ρ = 0.904, p < 0.001). A threshold of +3SD showed the highest performance for semiautomatic assessment of IS compared to manual segmentation (ρ = 0.965, p < 0.001), with an overall reproducibility of 73%, and a peak reproducibility of 80% at day 1. The ce-3D-IG-cine sequence, manually or semiautomatically segmented using 3SD threshold, allows fast and comprehensive LV morpho-functional and structural characterization in myocardial ischemia-reperfusion injury model.

Reversine: A Synthetic Purine with a Dual Activity as a Cell Dedifferentiating Agent and a Selective Anticancer Drug.

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.

Cell-Based Therapies for Cardiac Regeneration: A Comprehensive Review of Past and Ongoing Strategies.

Despite considerable improvements in the treatment of cardiovascular diseases, heart failure (HF) still represents one of the leading causes of death worldwide. Poor prognosis is mostly due to the limited regenerative capacity of the adult human heart, which ultimately leads to left ventricular dysfunction. As a consequence, heart transplantation is virtually the only alternative for many patients. Therefore, novel regenerative approaches are extremely needed, and several attempts have been performed to improve HF patients' clinical conditions by promoting the replacement of the lost cardiomyocytes and by activating cardiac repair. In particular, cell-based therapies have been shown to possess a great potential for cardiac regeneration. Different cell types have been extensively tested in clinical trials, demonstrating consistent safety results. However, heterogeneous efficacy data have been reported, probably because precise end-points still need to be clearly defined. Moreover, the principal mechanism responsible for these beneficial effects seems to be the paracrine release of antiapoptotic and immunomodulatory molecules from the injected cells. This review covers past and state-of-the-art strategies in cell-based heart regeneration, highlighting the advantages, challenges, and limitations of each approach.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes.

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1α.

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.

NEU4L sialidase overexpression promotes ß-catenin signaling in neuroblastoma cells, enhancing stem-like malignant cell growth.

Neuroblastoma (NB) is a frequently lethal tumor that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the deregulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch and Wnt/ß-catenin (CTNNB1) signaling. In this article, we demonstrate that sialidase NEU4 long (NEU4L) influences the differentiation/proliferation behavior of NB SK-N-BE cells by determining hyperactivation of the Wnt/ß-catenin signaling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, nonphosphorylated ß-catenin content, ß-catenin/TCF transcriptional activity and ß-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2) and CDC25A. In turn, these molecular features strongly modified the behavior of NEU4L SK-N-BE overexpressing cells, promoting the following: (1) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; (2) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133 and NES (nestin); (3) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/ß-catenin signaling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment.

Glycoglycerolipid analogues inhibit PKC translocation to the plasma membrane and downstream signaling pathways in PMA-treated fibroblasts and human glioblastoma cells, U87MG.

Glycoglycerolipid analogues, derived from 2-O-ß-D-galactosylglycerol, have been synthesized on the base of the structure of natural glycoglycerolipids showing anti-tumor and anti-inflammatory efficacy. These compounds have been previously demonstrated to inhibit phorbol 12-myristate-13-acetate (PMA) induced tumor promotion in mouse skin, but their mechanism of action has never been elucidated. In this work, we studied the effects of glycoglycerolipid analogues on PKC activation induced by PMA and its downstream target molecules, in human fibroblasts. Our results proved that: a) the tested compounds were able to block PKC translocation to the plasma membrane, promoted by PMA, in a dose-dependent manner (IC50: 0.48 µM for the most active compound 2); b) the efficacy of these compounds was strongly connected to their acyl chain linked to galactose; in particular, the addition of hexanoyl and branched chains enhanced PKC inhibition, the presence of a cyclohexane ring and an excessive length of the acyl chain, or its lack, exerted a negative effect; c) the inhibition of PKC translocation blocked enzyme activation and downstream signaling pathways, MAPK and FAK, involved in proliferation and adhesion/migration control. In addition, the branched glycoglycerolipid (compound 2) was able to inhibit PKC translocation and activation in naturally highly PKC activating glioblastoma cells, U87MG. As consequence, U87MG cell proliferation and, especially, migration potential resulted to be markedly reduced (-30% and -84%, respectively). Thus, these results reveal the role of a PKC-dependent mechanism in glycoglycerolipid analogues mediated protective effects and highlight their possible employment in the field of prevention/treatment of cancer.