Erika Conforti

NEU3 Sialidase Strictly Modulates GM3 Levels in Skeletal Myoblasts C2C12 Thus Favoring Their Differentiation and Protecting Them from Apoptosis

Membrane-bound sialidase NEU3, often referred to as the “ganglioside sialidase,” has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.

Isolation and Characterization of 2 New Human Rotator Cuff and Long Head of Biceps Tendon Cells Possessing Stem Cell–Like Self-Renewal and Multipotential Differentiation Capacity:

Background: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Hypothesis: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Study Design: Controlled laboratory study. Methods: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Results: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types—namely, osteoblasts, adipocytes, and skeletal muscle cells. Conclusion: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Clinical Relevance: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

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.

Background: NEU3 sialidase removes sialic acid from gangliosides on adjacent cells. Results: NEU3 is up-regulated upon exposure of skeletal myoblasts to hypoxic stress, and it stimulates the EGFR signaling cascade ultimately activating HIF-1α. Conclusion: NEU3 plays a physiological role in protecting myoblasts from hypoxic stress. Significance: NEU3 role in cell response to hypoxia may suggest new therapeutic approaches to ischemic diseases. 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.

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 3H-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.

The synthetic purine reversine selectively induces cell death of cancer cells

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de‐differentiation of adult cells, including fibroblasts, into stem‐cell‐like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti‐cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. J. Cell. Biochem. 113: 3207–3217, 2012.

Acquired coronary artery disease in adult patients with congenital heart disease: a true or a false problem?

Background The population of adults with congenital heart disease (ACHD) is increasing and aging, and a large percentage of this population is now over 65 years of age. For this reason, it is probable that acquired coronary artery disease (CAD) will become an important issue that needs to be addressed also in these patients. We retrospectively analyzed all ACHD patients who underwent surgery in our Institution with the aim to investigate the incidence of associated CAD and the results of surgical treatment. Methods From January 2000 to December 2015, a total of 1154 ACHD underwent surgery in our center. Fifty patients (4.3%) were diagnosed with acquired CAD and required coronary artery bypass grafting. The mean age at surgery was 66 years (range 41–78 years). The primary diagnosis were atrial septal defect (n = 40 patients), Tetralogy of Fallot (n = 4 patients), ventricular septal defect (n = 2 patients), partial AV canal (n = 1), partial anomalous pulmonary venous drainage (n = 1), Ebsteins anomaly (n = 1), and subaortic stenosis (n = 1). Results Hospital mortality was 2% (one patient). During a mean follow-up of 9 years (maximum follow-up: 15 years), seven patients died (14%). The actuarial survival was 83% at 5 years and 77% at 10 years. Freedom from reoperation for coronary artery bypass grafting or percutaneous coronary intervention was 88% at 5 years and 82% at 10 years. Conclusion Acquired CAD may coexist with congenital heart defects but the association is quite rare. It typically occurs later during adulthood and it is usually associated with atrial septal defect. Acquired CAD and congenital heart defects can be treated contemporarily with good early and late results.

NEU3 sialidase role in activating HIF-1α in response to chronic hypoxia in cyanotic congenital heart patients

BACKGROUND Hypoxia is a common feature of many congenital heart defects (CHDs) and significantly contributes to their pathophysiology. Thus, understanding the mechanism underlying cell response to hypoxia is vital for the development of novel therapeutic strategies. Certainly, the hypoxia inducible factor (HIF) has been extensively investigated and it is now recognized as the master regulator of cell defense machinery counteracting hypoxic stress. Along this line, we recently discovered and reported a novel mechanism of HIF activation, which is mediated by sialidase NEU3. Thus, aim of this study was to test whether NEU3 played any role in the cardiac cell response to chronic hypoxia in congenital cyanotic patients. METHODS Right atrial appendage biopsies were obtained from pediatric patients with cyanotic/non-cyanotic CHDs and processed to obtain mRNA and proteins. Real-Time PCR and Western Blot were performed to analyze HIF-1α and its downstream targets expression, NEU3 expression, and the NEU3 mediated effects on the EGFR signaling cascade. RESULTS Cyanotic patients showed increased levels of HIF-1α, NEU3, EGFR and their downstream targets, as compared to acyanotic controls. The same patients were also characterized by increased phosphorylation of the EGFR signaling cascade proteins. Moreover, we found that HIF-1α expression levels positively correlated with those recorded for NEU3 in both cyanotic and control patients. CONCLUSIONS Sialidase NEU3 plays a central role in activating cell response to chronic hypoxia inducing the up-regulation of HIF-1α, and this represent a possible novel tool to treat several CHD pathologies.

S100B maternal blood levels are gestational age- and gender-dependent in healthy pregnancies

Abstract Background: S100B is a well-established biomarker of central nervous system (CNS) development and damage in the perinatal period. Because the fetal CNS induces an overproduction of S100B measurable in the maternal bloodstream we evaluated S100B protein in healthy pregnancies in order to provide a reference curve of the protein in the second and third trimesters and to provide information on CNS development when standard monitoring procedures could be silent or unavailable. Methods: Between July 2012 and December 2014 we conducted a prospective study in 1213 healthy pregnancies delivering healthy newborns. Maternal blood samples were collected for standard monitoring procedures and S100B assessment. S100B correlations with selected outcomes (gestational age at sampling, gender of fetus, gestational age and weight at birth, delivery mode) were calculated using multiple forward stepwise regression analysis. Results: S100B concentrations in the second and third trimesters of pregnancy were found to be gestational age-, gender- and delivery mode-dependent (p<0.05, for all). Multiple forward stepwise regression analysis with S100B as the dependent variable and gestational age at sampling, gender, delivery mode, gestational age and weight at birth as independent variables, showed a significant correlation between S100B and gestational age at sampling (R=0.13; p<0.001). Conclusions: The present findings offering a S100B protein reference curve in maternal blood suggest that non-invasive fetal CNS monitoring is becoming feasible and open the way to further research in neuro-biomarker assessment in the maternal bloodstream.

Circulating S100B and Adiponectin in Children Who Underwent Open Heart Surgery and Cardiopulmonary Bypass

Background. S100B protein, previously proposed as a consolidated marker of brain damage in congenital heart disease (CHD) newborns who underwent cardiac surgery and cardiopulmonary bypass (CPB), has been progressively abandoned due to S100B CNS extra-source such as adipose tissue. The present study investigated CHD newborns, if adipose tissue contributes significantly to S100B serum levels. Methods. We conducted a prospective study in 26 CHD infants, without preexisting neurological disorders, who underwent cardiac surgery and CPB in whom blood samples for S100B and adiponectin (ADN) measurement were drawn at five perioperative time-points. Results. S100B showed a significant increase from hospital admission up to 24 h after procedure reaching its maximum peak (P < 0.01) during CPB and at the end of the surgical procedure. Moreover, ADN showed a flat pattern and no significant differences (P > 0.05) have been found all along perioperative monitoring. ADN/S100B ratio pattern was identical to S100B alone with the higher peak at the end of CPB and remained higher up to 24 h from surgery. Conclusions. The present study provides evidence that, in CHD infants, S100B protein is not affected by an extra-source adipose tissue release as suggested by no changes in circulating ADN concentrations.

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the “hypoxic niches” present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.