Eva Ercolino

Erythroid cell differentiation is characterized by nuclear matrix localization and phosphorylation of protein kinases C (PKC) α, δ, and ζ

Protein kinases C (PKC) ζ expression and phosphoryation at nuclear level during dimethyl sulfoxide (DMSO)‐induced differentiation in Friend erythroleukemia cells have been previously reported, suggesting a possible role of this PKC isoform in the DMSO‐related signaling. In order to shed more light on this tantalizing topic, we investigated PKC intracellular and sub‐cellular localization and activity during DMSO‐induced erythroid differentiation. Results indicated that at least PKC α, ζ, and δ are strongly and temporally involved in the DMSO‐induced differentiation signals since their expression and phosphorylation, though at different extents, were observed during treatments. Intriguingly, while PKC α and ζ associate to the nuclear matrix during the differentiation event, PKC δ appears to be residentially associated to the nuclear matrix. Furthermore, an evident downregulation of the β‐globin gene transcription (differentiation hallmark) was detected upon a progressive inhibition of these PKC isoforms by means of specific inhibitors, indicating, therefore, that PKC α, ζ, and δ phosphorylation play a crucial role in the control of erythroid differentiation.

Human Mesenchymal Stem Cells Reendothelialize Porcine Heart Valve Scaffolds: Novel Perspectives in Heart Valve Tissue Engineering

Abstract Heart valve diseases are usually treated by surgical intervention addressed for the replacement of the damaged valve with a biosynthetic or mechanical prosthesis. Although this approach guarantees a good quality of life for patients, it is not free from drawbacks (structural deterioration, nonstructural dysfunction, and reintervention). To overcome these limitations, the heart valve tissue engineering (HVTE) is developing new strategies to synthesize novel types of valve substitutes, by identifying efficient sources of both ideal scaffolds and cells. In particular, a natural matrix, able to interact with cellular components, appears to be a suitable solution. On the other hand, the well-known Whartons jelly mesenchymal stem cells (WJ-MSCs) plasticity, regenerative abilities, and their immunomodulatory capacities make them highly promising for HVTE applications. In the present study, we investigated the possibility to use porcine valve matrix to regenerate in vitro the valve endothelium by WJ-MSCs differentiated along the endothelial lineage, paralleled with human umbilical vein endothelial cells (HUVECs), used as positive control. Here, we were able to successfully decellularize porcine heart valves, which were then recellularized with both differentiated-WJ-MSCs and HUVECs. Data demonstrated that both cell types were able to reconstitute a cellular monolayer. Cells were able to positively interact with the natural matrix and demonstrated the surface expression of typical endothelial markers. Altogether, these data suggest that the interaction between a biological scaffold and WJ-MSCs allows the regeneration of a morphologically well-structured endothelium, opening new perspectives in the field of HVTE.

Enhanced release of acid sphingomyelinase-enriched exosomes generates a lipidomics signature in CSF of Multiple Sclerosis patients

Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Proteomic insights in extracellular microvesicles from multiple sclerosis patients

To date the most important biomarkers for Multiple Sclerosis (MuS) diagnosis are the oligoclonal bands (OCBs) in CSF and Link Index. CSF is the body fluid that might better provide information about the pathological processes occurring in the CNS, because of its proximity. Anyway, it is obtained through an invasive procedure, thus tears, may represent an useful alternative source of biomarkers. Emerging evidences showed that distinct types of brain cells release high number of Extracellular Vesicles (EVs), that play important roles in the CNS, and represent a relevant source of biomarkers, relative free from confounding factors. In the present study, we analysed EVs from MuS patients obtained from tears and CSF samples. In details, 50μl of CSF or 50 μl of tears/sample were processed by a common flow cytometry no-lyse and no-wash method, in order to identify EVs. Exosomes and microvesicles (MVs) were sorted (70 μm nozzle, FACSAria III cell sorter, BD) from pooled CSF samples on the basis of their positivity to specific tetraspainins (for exosomes) or markers identifying each MV subset. Fractions were analysed by electron microscopy and Dynamic Light Scattering. Purified MV fractions undergone to FASP tryptic digestion and nanoLC-ESI-QTOF-MS/MS based shotgun proteomic approach. Identified MVs proteins were processed by Ingenuity Pathway Analysis (IPA) and PANTHER - Gene List Analysis. Our data shows the presence of subpopulations of extracellular MVs of neuronal and microglia origins in tears , indicating a cross talk between the two compartment. We also identified 55 proteins (FDR<2.38) for the MVs fraction. To uncover the molecular events underlying these proteins profiles, we studied the Gene Ontology (GO) information in terms of biological process and molecular function by using PANTHER software and we observed that about 70% of the identified proteins resulted were involved in binding processes, while 40% of them were related to cell communication. Ingenuity Pathway Analysis (IPA) of the identified MVs proteins revealed that the top network associated to them are “Cellular Movement, Hematological Disease, Immunological Disease”, well matching with MS. Among the upstream regulators, the most significant one is PRDM with a p-value of 6.68E-07. The remaining upstream regulators, including APOE (the most relevant lipid carrier protein in the brain involved in brain development and repair), well related to neurological disease. These data underlined that MVs form neuronal and microcglial origin are detectable not only in the CSF, but also in tears from MuS patients. Of note, MVs of CSF origin carry relevant targets involved in immune responses in MuS patients, therefore they might be proposed as useful tools in MuS diagnosis and characterization.

Mesenchymal stem cell extracellular vesicles: potential use in regenerative medicine

Complex biological systems are composed of great amount of cells with sophisticated mechanisms for information exchange, involving the molecular as well as the cellular level. Vesicle release results in a process highly conserved in prokaryotes as well as in eukaryotes, therefore representing an evolutionary link and suggesting that such a dynamic extracellular vesicular compartment may play a key role in remote organ and tissue regulation. Microparticles serve as a vehicle to transfer proteins and messenger RNA and microRNA (miRNA) to distant cells, which alters the gene expression, proliferation, and differentiation of the recipient cells. Microparticles released from mesenchymal stem cells have the potential to be exploited in novel therapeutic approaches in regenerative medicine to repair damaged tissues, as an alternative to stem cell-based therapy.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted sub- populations of peripheral blood (PB), cord blood (CB) and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving the endothe- lial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been so far published, none of them have reached consist- ent outcomes, therefore consensus guidelines with respect to CEC and EPC identifi- cation and quantification need to be established. Here, we have carried out a deep investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/ CD146neg). This approach, combined to a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and CEC counts were consistent with previous reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies are needed to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Reendothelization of porcine heart valve scaffolds with WJ-MSC: a new approach in the heart valve tissue engineering.

Heart valve substitution, based on biosynthetic or mechanical prosthesis replacement, is one of the most frequent surgical approach to treat heart valve diseases. Even if the prosthesis implantation gives a good life quality for patients, there are many long-term disadvantages related to the substitution, such as structural deterioration, non-structural dysfunction and re-intervention. The heart valve tissue engineering (HVTE), a novel branch of regenerative medicine, is developing innovative models and testing new methods to overcome the above reported limitations. In the present study, we investigated the possibility to reendothelize a porcine heart valve scaffold, previously decellularized, by using two cell types: Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and human umbilical vein endothelial cells (HUVEC), the last used as control cells for the reendothelialization process. Both cell types showed, by fluorescence microscopy, that they were able to reconstitute a valid and functional monolayer of neo-endothelium, characterized by the surface expression of typical endothelial markers (i.e. CD144 and CD146). All together, these data suggest that both HUVEC and WJ-MSC are suitable for in vitro autologous endothelium regeneration, opening new perspectives in the field of HVTE.

A standardized multi-colour flow cytometry approach to characterize the many faces of peripheral circulating microparticles

Microparticles (MP) are small vescicles (<1 µm of diameter) released by several cell types and characterized by an integral plasma membrane expressing the phenotype of the cell from which they originate (Jayachandran et al., 2012). MP play a crucial role in a multitude of pathologies, Including inflammatory and autoimmune disease, diabetes, atherosclerosis, malignancies and cardiovascular disease. The role, as potential biomarker, attributed to circulating MP has been emphasized by the recent literature. In such context, multicolour flow cytometry has great potential In the MP studies (Lanuti et al., 2012). Unfortunately, consensus guidelines on MP identification and enumeration has not been reached yet, due to their small sizes. We purpose to identify, characterize and count MP from whole blood by a seven-colors flow cytometry protocol, with the aim to standardize such method and to allow the identification of both the whole compartment and different MP subpopulations (i.e. endothelial-, platelet- and leukocyte-derived MP). We optimized a novel flow cytometry protocol, using peripheral whole blood stained by a combinations of seven different antigens/probes. Different panel combinations, anticoagulants and storage conditions were evaluated to set the best protocol with the aim to obtain reliable and reproducible MP counts. MP enumeration was carried out by a single platform method by using TrueCount (BD Biosciences). Results demonstrated that the application of the newly optimized flow cytometry method here described, allows to obtain high reproducibility of MP enumeration, pointing out different MP subpopulations both in healthy donors and in different patients. This method may open new routes for the monitoring of MP numbers and phenotypes in different clinical setting.