The Effects of Silencing PTX3 on the Proteome of Human Endothelial Cells.

The human long pentraxin PTX3 has complex regulatory roles at the crossroad of innate immunity, inflammation, and tissue repair. PTX3 can be produced by various cell types, including vascular endothelial cells (ECs), in response to pro-inflammatory cytokines or bacterial molecules. PTX3 has also been involved in the regulation of cardiovascular biology, even if ambiguous results have been so far provided in both preclinical and clinical research. In this study, we compared the proteomic profiles of human ECs (human umbilical vein ECs, HUVECs), focusing on differentially expressed proteins between the control and PTX3-silenced ECs. We identified 19 proteins that were more abundant in the proteome of control ECs and 23 proteins that were more expressed in PTX3-silenced cells. Among the latter, proteins with multifunctional roles in angiogenesis, oxidative stress, and inflammation were found, and were further validated by assessing their mRNAs with RT-qPCR. Nevertheless, the knock down of PTX3 did not affect in vitro angiogenesis. On the contrary, the lack of the protein induced an increase in pro-inflammatory markers and a shift to the more oxidative profile of PTX3-deficient ECs. Altogether, our results support the idea of a protective function for PTX3 in the control of endothelial homeostasis, and more generally, in cardiovascular biology.

Involvement of the IL-6 Signaling Pathway in the Anti-Anhedonic Effect of the Antidepressant Agomelatine in the Chronic Mild Stress Model of Depression.

Neuroinflammation has emerged as an important factor in the molecular underpinnings of major depressive disorder (MDD) pathophysiology and in the mechanism of action of antidepressants. Among the inflammatory mediators dysregulated in depressed patients, interleukin (IL)-6 has recently been proposed to play a crucial role. IL-6 activates a signaling pathway comprising the JAK/STAT proteins and characterized by a specific negative feedback loop exerted by the cytoplasmic protein suppressor of cytokine signalling-3 (SOCS3). On these bases, here, we explored the potential involvement of IL-6 signaling in the ability of the antidepressant drug agomelatine to normalize the anhedonic-like phenotype induced in the rat by chronic stress exposure. To this aim, adult male Wistar rats were subjected to the chronic mild stress (CMS) paradigm and chronically treated with vehicle or agomelatine. The behavioral evaluation was assessed by the sucrose consumption test, whereas molecular analyses were performed in the prefrontal cortex. We found that CMS was able to stimulate IL-6 production and signaling, including SOCS3 gene and protein expression, but the SOCS3-mediated feedback-loop inhibition failed to suppress the IL-6 cascade in stressed animals. Conversely, agomelatine treatment normalized the stress-induced decrease in sucrose consumption and restored the negative modulation of the IL-6 signaling via SOCS3 expression and activity. Our results provide additional information about the pleiotropic mechanisms that contribute to agomelatine's therapeutic effects.

Multicellular 3D Models to Study Tumour-Stroma Interactions.

Two-dimensional (2D) cell cultures have been the standard for many different applications, ranging from basic research to stem cell and cancer research to regenerative medicine, for most of the past century. Hence, almost all of our knowledge about fundamental biological processes has been provided by primary and established cell lines cultured in 2D monolayer. However, cells in tissues and organs do not exist as single entities, and life in multicellular organisms relies on the coordination of several cellular activities, which depend on cell-cell communication across different cell types and tissues. In addition, cells are embedded within a complex non-cellular structure known as the extracellular matrix (ECM), which anchors them in a three-dimensional (3D) formation. Likewise, tumour cells interact with their surrounding matrix and tissue, and the physical and biochemical properties of this microenvironment regulate cancer differentiation, proliferation, invasion, and metastasis. 2D models are unable to mimic the complex and dynamic interactions of the tumour microenvironment (TME) and ignore spatial cell-ECM and cell-cell interactions. Thus, multicellular 3D models are excellent tools to recapitulate in vitro the spatial dimension, cellular heterogeneity, and molecular networks of the TME. This review summarizes the biological significance of the cell-ECM and cell-cell interactions in the onset and progression of tumours and focuses on the requirement for these interactions to build up representative in vitro models for the study of the pathophysiology of cancer and for the design of more clinically relevant treatments.

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Hormone-deprived serum impairs angiogenic properties in human endothelial cells regardless of estrogens.

AIMS: In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs). METHODS: Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay. RESULTS: When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-ß2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs. CONCLUSIONS: Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

A multidisciplinary consensus document on follow-up strategies for patients treated with percutaneous coronary intervention.

The number of percutaneous coronary interventions (PCI) is increasing worldwide. Follow-up strategies after PCI are extremely heterogeneous and can greatly affect the cost of medical care. Of note, clinical evaluations and non-invasive exams are often performed to low risk patients. In the present consensus document, practical advises are provided with respect to a tailored follow-up strategy on the basis of patients' risk profile. Three strategies follow-up have been defined and types and timing of clinical and instrumental evaluations are reported. Clinical and interventional cardiologists, cardiac rehabilitators, and general practitioners, who are in charge to manage post-PCI patients, equally contributed to the creation of the present document.

Silencing of Eps8 blocks migration and invasion in human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors.

Lost in HELLS: Disentangling the mystery of SALNR existence in senescence cellular models.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence, Senescence Associated Long Non-coding RNA (SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015, SALNR has not been annotated in any database or public repository, and no other experimental data have been published. The SALNR sequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3' end of the HELLS gene. This investigation helped to unravel the mystery of the existence of SALNR by analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression of HELLS has been studied in cellular models of replicative senescence, both in silico and in vitro. Our findings, while not supporting the actual existence of SALNR as an independent transcript in the analyzed experimental models, demonstrate the expression of a predicted HELLS isoform entirely covering the SALNR genomic region. Furthermore, we observed a strong down-regulation of HELLS in senescent cells versus proliferating cells, supporting its role in the senescence and aging process.

[Do critical pathways improve outcomes of patients with cardiac failure?]. / I percorsi assistenziali migliorano gli outcome dei pazienti affetti da scompenso cardiaco?

UNLABELLED: Cardiac failure represents an important public health problem and despite recent clinical, diagnostic and therapeutic advances, the incidence and prevalence of this syndrome show a steady increase. In view of this, the authors conducted a meta-analysis to evaluate the effect of critical pathways in the management of patients with cardiac failure when compared with standard care. The impact of critical pathways on the following outcomes were evaluated: hospital mortality, mortality at six months, mean length of hospital stay, direct costs, readmission rates at one, three and six months. METHODS: The following databases were consulted: Medline, Embase, CINAHL, Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews. The research was limited to articles published between January 1975 and June 2010. Methodological quality of studies was evaluated by the Jadad method (for RCTs, cRCT, CCT) and the New Castle Ottawa Scale for case-control and cohort studies. Data analysis was performed by using the statistical methods described in the Cochrane Collaboration guidelines. Meta-analyses were performed using RevMan software version 5. RESULTS: Eleven studies were included in the meta-analysis (5,460 patients). A lower mortality (hospital mortality and mortality at 6 months) was observed in the critical pathways group compared to the group treated with standard care. A positive impact of critical pathways was also observed in length of stay, direct costs, readmission after one, three and six months. CONCLUSIONS: Critical pathways can improve the quality of care provided to patients with cardiac failure. Further studies are needed to evaluate which mechanisms within the care pathways can truly improve the quality of care.

Sex-dependent differences in the secretome of human endothelial cells.

BACKGROUND: Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and "omics" approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. METHODS: We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. RESULTS: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. CONCLUSIONS: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism.

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.

Autophagy in the Regulation of Tissue Differentiation and Homeostasis.

Autophagy is a constitutive pathway that allows the lysosomal degradation of damaged components. This conserved process is essential for metabolic plasticity and tissue homeostasis and is crucial for mammalian post-mitotic cells. Autophagy also controls stem cell fate and defective autophagy is involved in many pathophysiological processes. In this review, we focus on established and recent breakthroughs aimed at elucidating the impact of autophagy in differentiation and homeostasis maintenance of endothelium, muscle, immune system, and brain providing a suitable framework of the emerging results and highlighting the pivotal role of autophagic response in tissue functions, stem cell dynamics and differentiation rates.

Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells.

Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release.

Metabolism of Stem and Progenitor Cells: Proper Methods to Answer Specific Questions.

Stem cells can stay quiescent for a long period of time or proliferate and differentiate into multiple lineages. The activity of stage-specific metabolic programs allows stem cells to best adapt their functions in different microenvironments. Specific cellular phenotypes can be, therefore, defined by precise metabolic signatures. Notably, not only cellular metabolism describes a defined cellular phenotype, but experimental evidence now clearly indicate that also rewiring cells towards a particular cellular metabolism can drive their cellular phenotype and function accordingly. Cellular metabolism can be studied by both targeted and untargeted approaches. Targeted analyses focus on a subset of identified metabolites and on their metabolic fluxes. In addition, the overall assessment of the oxygen consumption rate (OCR) gives a measure of the overall cellular oxidative metabolism and mitochondrial function. Untargeted approach provides a large-scale identification and quantification of the whole metabolome with the aim to describe a metabolic fingerprinting. In this review article, we overview the methodologies currently available for the study of in vitro stem cell metabolism, including metabolic fluxes, fingerprint analyses, and single-cell metabolomics. Moreover, we summarize available approaches for the study of in vivo stem cell metabolism. For all of the described methods, we highlight their specificities and limitations. In addition, we discuss practical concerns about the most threatening steps, including metabolic quenching, sample preparation and extraction. A better knowledge of the precise metabolic signature defining specific cell population is instrumental to the design of novel therapeutic strategies able to drive undifferentiated stem cells towards a selective and valuable cellular phenotype.

Cross-talk between sphingosine-1-phosphate and EGFR signaling pathways enhances human glioblastoma cell invasiveness.

We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum.

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-ß estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS.

Sex-specific eNOS activity and function in human endothelial cells.

Clinical and epidemiological data show that biological sex is one of the major determinants for the development and progression of cardiovascular disease (CVD). Impaired endothelial function, characterized by an imbalance in endothelial Nitric Oxide Synthase (eNOS) activity, precedes and accelerates the development of CVD. However, whether there is any sexual dimorphism in eNOS activity and function in endothelial cells (ECs) is still unknown. Here, by independently studying human male and female ECs, we found that female ECs expressed higher eNOS mRNA and protein levels both in vitro and ex vivo. The increased eNOS expression was associated to higher enzymatic activity and nitric oxide production. Pharmacological and genetic inhibition of eNOS affected migratory properties only in female ECs. In vitro angiogenesis experiments confirmed that sprouting mostly relied on eNOS-dependent migration in female ECs. At variance, capillary outgrowth from male ECs was independent of eNOS activity but required cell proliferation. In this study, we found sex-specific differences in the EC expression, activity, and function of eNOS. This intrinsic sexual dimorphism of ECs should be further evaluated to achieve more effective and precise strategies for the prevention and therapy of diseases associated to an impaired endothelial function such as CVD and pathological angiogenesis.

Expression studies in gliomas and glial cells do not support a tumor suppressor role for LGI1.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

Deregulated human glioma cell motility: inhibitory effect of somatostatin.

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Silencing of Eps8 inhibits in vitro angiogenesis.

AIMS: Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis. MAIN METHODS: Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence. KEY FINDINGS: We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs. SIGNIFICANCE: Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.