Circulating endothelial cells (CECs) in peripheral blood

CECs as well as bone-marrow-derived endothelial precursor cells (EPCs) are very rare events in the peripheral blood that have a high potential diagnostic value in different diseases which are characterized by cardiovascular problems and/or angiogenesis, e.g. cancer, ischemia and diabetes. Flow cytometry analysis of CECs is difficult because CECs are often discriminated using a combination of antigens with low, dull, or a continuum of cell surface expression. Since CECs can’t be characterized by a single marker, a combination of at least two markers is necessary. Therefore different combination of several endothelial markers (CD31, CD34, CD146, KDR and CD144) was used in order to get a more accurate discrimination of CECs. Such a test evidenced that KDR and CD144 were very weakly expressed on the CEC cell surface and could not be reliably analysed, while CD31, CD34 and CD146 were largely detected and therefore chosen for the panel. Dead cells, microparticles [1] and platelets were excluded from the analysis by using a DNA stain (Syto16) and a live/dead marker (NiRed). Leucocytes were excluded by gating CD45- cells. CD106 is expressed on endothelial cells after stimulation with cytokines and allows analysis of activated subsets of CECs

Subcellular localization and phosphorylation of phosphoinositide-phospholipase C γ1 correlate with breast cancer invasiveness

Activation of the enzyme phosphoinositide-phospholipase C γ1 (PLCγ1) is thought to play a critical role in both cytoskeletal changes and migration associated with the metastatic process. Activation of PLCγ1 by phosphorylation can occur downstream of many tyrosine kinase receptors including epidermal growth factor receptor, vascular endothelial growth factor receptor-2, c-MET, platelet-derived growth factor receptor, and also certain integrins. Activation induces hydrolysis of phosphatidylinositol 4,5-biphosphate to form the second messengers diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate a number of signalling pathways. PLCγ1 is highly expressed in several tumours, including breast carcinomas in which the enzyme has been shown to be required for epidermal growth factor induced migration of breast cancer cells. In order to establish the significance of PLCγ1 subcellular localization and phosphorylation (PLCγ1-pY783 and PLCγ1-pY1253) in breast cancer, we compared, through the use of different methods, two different breast cancer models: the low-tumorigenic BT-474 cell line and MDA-MB-231 cell line which represents a more aggressiveness de-differentiated cell type, obtained from a pleural effusion from a patient

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

Plasma from pre-pubertal obese children impairs insulin stimulated Nitric Oxide (NO) bioavailability in endothelial cells: Role of ER stress.

Childhood obesity is commonly associated with early signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular Nitric Oxide (NO) availability. However, the underlying mechanisms remain to be established. Hence, we tested the hypothesis that endothelial insulin-stimulated NO production and availability was impaired and related to Endoplasmic Reticulum (ER) in human umbilical vein endothelial cells (HUVECs) cultured with plasma obtained from pre-pubertal obese (OB) children. OB children (N = 28, age: 8.8 ± 2.2; BMI z-score: 2.15 ± 0.39) showed impaired fasting glucose, insulin and HOMA-IR than normal weight children (CTRL; N = 28, age: 8.8 ± 1.7; BMI z-score: 0.17 ± 0.96). The in vitro experiments showed that OB-plasma significantly impaired endothelial insulin-stimulated NO production and bioavailability compared to CTRL-plasma. In parallel, in HUVECs OB-plasma increased GRP78 and activated PERK, eIF2α, IkBα and ATF6 (all ER stress markers). Moreover, OB-plasma increased NF-κB activation and its nuclear translocation. Notably, all these effects proved to be significantly restored by using PBA and TUDCA, known ER stress inhibitors. Our study demonstrate for the first time that plasma from obese children is able to induce in vitro endothelial insulin resistance, which is characterized by reduced insulin-stimulated NO production and bioavailability, endothelial ER stress and increased NF-κB activation

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

Cryopreservation influence in the WJCs Proteome

Cryopreservation is the only mode of long-term storage of viable cells and tissues for cellular therapy, stem cell transplantation and/or tissue engineering. However the freeze-thaw process strongly contributes to cell and tissue damage with several mechanism, including oxidative stress, intracellular ice formation (IIF) dependent cell injury and altered physical cellular properties, i.e. osmotic and ion homeostasis. Our previous proteomics investigation was carried on Wharton’s jelly cells (WJCs), fibroblastlike cells with similar properties to mesenchymal stem cells, therefore a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJCs proteome at different culture conditions (freshly and post-frozen cell preparations) and to elucidate possible mechanism involved in maintaining active proliferation and maximal cellular plasticity in order to optimize in vitro culturing procedure. To analyze changes in protein expression of WJCs we performed a comparative proteomic analysis (2DE followed by MALDI–TOF MS) between fresh and post-frozen cell culturing. WJCs postfrozen showed a qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defense mechanism and metabolism, that ensure freeze-thaw survival. However, further investigations are needed to clarify the biological mechanisms involved in maintaining active proliferation, plasticity, multipotency cell during in vitro expansio

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

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 (FD

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Stima precoce dei consumi privati di contabilit nazionale mediante modelli strutturali dinamici

Consiglio Nazionale delle Ricerche (CNR). Biblioteca Centrale / CNR - Consiglio Nazionale delle RichercheSIGLEITItal