Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

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.

Inhibition of Sphingolipid Synthesis as a Phenotype-Modifying Therapy in Cystic Fibrosis.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.

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.

Regenerating the human heart: direct reprogramming strategies and their current limitations.

Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

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.

Association between postoperative thromboembolism prophylaxis and complications following urological surgery.

Thromboembolism represents the most significant complication and cause of non-surgical mortality in major urological surgery. The aim of the present study was to assess the association between the type of pharmacological thromboembolism prophylaxis and the postoperative complication rate in a cohort of patients undergoing major urological surgery. All consecutive patients treated with major urological surgery between December 2011 and March 2013 were evaluated. For each patient, clinical and demographic data, as well as information on the post-surgical complications and the type of pharmacological thromboembolism prophylaxis, were collected. In total, 453 patients (mean age, 63.36±12.05 years) were recruited (43.5% for prostate surgery, 33.1% for renal surgery, 12.1% for bladder surgery and 11.3% for other surgery). Postoperative blood transfusions were required in 50 cases (11.0%). A total of 32 patients (7.1%) underwent re-intervention due to the occurrence of grade ≥3 complications, with a readmission rate of 2.0%. According to the Clavien-Dindo Classification, the complications were grade 1 in 36.0% of the cases, grade 2 in 19.4%, grade 3 in 6.0%, grade 4 in 2.0% and grade 5 (mortality) in 0.7%. Only 1 case of deep venous thrombosis not associated with pulmonary thromboembolism was observed. Univariate analyses showed a significant negative association (higher risk of complications) between the use of >4,000 IU enoxaparin as the thromboembolism prophylaxis and postoperative blood transfusion rate (P=0.045), re-intervention rate (P=0.001) and the occurrence of grade ≥3 complications (P<0.001). Multivariate analysis confirmed the significant association between the use of >4,000 IU enoxaparin and both re-intervention rate (P=0.013) and occurrence of grade ≥3 complications (P=0.002). High doses of enoxaparin (>4,000 IU) may lead to an increased risk of re-intervention and severe postoperative complications following major urological surgery. Randomised, controlled trials comparing the effect of different types of pharmacological thromboembolism prophylaxis on postoperative complications following major urological surgery are required.

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.

Application of direct HPTLC-MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: the case of NEU3 overexpressing C2C12 murine myoblasts.

Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.

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.

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.

Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration.

RATIONALE: Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. OBJECTIVE: Insulin-like growth factor (IGF)-1, IGF-2, and angiotensin II (Ang II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in c-kit(+) hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. METHODS AND RESULTS: C-kit(+) hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation, and attenuated apoptosis. In addition to IGF-1, IGF-1R(+) hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R(+) hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit(+) hCSCs. IGF-1R(+) hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R(+) hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. CONCLUSIONS: hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage, and myocyte differentiation, which points to this hCSC subset as the ideal candidate cell for the management of human heart failure.

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.