Role of Luteolin as Potential New Therapeutic Option for Patients with Glioblastoma through Regulation of Sphingolipid Rheostat.

Glioblastoma (GBM) is the most aggressive brain tumor, still considered incurable. In this study, conducted on primary GBM stem cells (GSCs), specifically selected as the most therapy-resistant, we examined the efficacy of luteolin, a natural flavonoid, as an anti-tumoral compound. Luteolin is known to impact the sphingolipid rheostat, a pathway regulated by the proliferative sphingosine-1-phosphate (S1P) and the proapoptotic ceramide (Cer), and implicated in numerous oncopromoter biological processes. Here, we report that luteolin is able to inhibit the expression of SphK1/2, the two kinases implicated in S1P formation, and to increase the expression of both SGPL1, the lyase responsible for S1P degradation, and CERS1, the ceramide synthase 1, thus shifting the balance toward the production of ceramide. In addition, luteolin proved to decrease the expression of protumoral signaling as MAPK, RAS/MEK/ERK and PI3K/AKT/mTOR and cyclins involved in cell cycle progression. In parallel, luteolin succeeded in upregulation of proapoptotic mediators as caspases and Bcl-2 family and cell cycle controllers as p53 and p27. Furthermore, luteolin determined the shutdown of autophagy contributing to cell survival. Overall, our data support the use of luteolin as add-on therapy, having demonstrated a good ability in impairing GSC viability and survival and increasing cell sensitivity to TMZ.

Effects of Metformin as Add-On Therapy against Glioblastoma: An Old Medicine for Novel Oncology Therapeutics.

BACKGROUND: Glioblastoma is the most aggressive primary brain malignancy in adults, with a poor prognosis of about 14 months. Recent evidence ascribed to metformin (MET), an antihyperglycemic drug, the potential to reduce cancer incidence and progression, but the molecular mechanisms underlying these effects need to be better investigated. METHODS: Here, we tested the efficacy of MET on n = 10 primary glioblastoma endothelial cells (GECs), by viability and proliferation tests, as MTT and Live/Dead assays, apoptosis tests, as annexin V assay and caspase 3/7 activity, functional tests as tube-like structure formation and migration assay and by mRNA and protein expression performed by quantitative real-time PCR analysis (qRT-PCR) and Western Blot, respectively. RESULTS: Data resulting revealed a time- and µ-dependent ability of MET to decrease cell viability and proliferation, increasing pro-apoptotic mechanisms mediated by caspases 3/7. Also, MET impacted GEC functionality with a significant decrease of angiogenesis and invasiveness potential. Mechanistically, MET was able to interfere with sphingolipid metabolism, weakening the oncopromoter signaling promoted by sphingosine-1-phosphate (S1P) and shifting the balance toward the production of the pro-apoptotic ceramide. CONCLUSIONS: These observations ascribed to MET the potential to serve as add-on therapy against glioblastoma, suggesting a repurposing of an old, totally safe and tolerable drug for novel oncology therapeutics.

Inflammatory interactions between degenerated intervertebral discs and microglia: Implication of sphingosine-1-phosphate signaling.

The etiology of intervertebral disc degeneration is largely unknown, but local neuroinflammation may exert a crucial role through activation of cells as microglia and pro-inflammatory cytokines production. We aimed to compare the effect of degenerated and normal intervertebral disc microenvironment on microglial cells and the potential role of sphingosine-1-phosphate, a pro-inflammatory sphingolipid, in their crosstalk. Human degenerated intervertebral discs (Pfirrmann grade IV) were obtained at surgery for spondylolisthesis. Normal intervertebral discs were collected from cadaveric normal lumbar spines. Normal and degenerated-intervertebral discs were kept in culture to obtain media conditioning. Then, microglial cells were cocultured with conditioned media and viability, proliferation, migration, chemotaxis, and inflammatory gene expression were evaluated. The results demonstrate that conditioned media from degenerated intervertebral discs activate microglial cells, increasing chemotaxis, migration, and pro-inflammatory mediators release to a great extent than normal discs. In addition, we show that the administration of sphingosine-1-phosphate to normal intervertebral disc/microglia coculture mimicked degenerative effects. Interestingly, sphingosine-1-phosphate content in conditioned media from degenerated discs was significantly higher than that from normal ones. In addition, FTY720, a functional antagonist of sphingosine-1-phosphate, potently inhibited the effect of degenerated intervertebral discs on microglial inflammatory factor transcription and migration. Our data report, for the first time, that sphingosine-1-phosphate is involved as signal in the microenvironment of human degenerated intervertebral discs. Sphingosine-1-phosphate signaling modulation by FTY720 may induce beneficial effects in counteracting microglial activation during intervertebral disc degeneration.

Frontiers in Anti-Cancer Drug Discovery: Challenges and Perspectives of Metformin as Anti-Angiogenic Add-On Therapy in Glioblastoma.

Glioblastoma is the most common primitive tumor in adult central nervous system (CNS), classified as grade IV according to WHO 2016 classification. Glioblastoma shows a poor prognosis with an average survival of approximately 15 months, representing an extreme therapeutic challenge. One of its distinctive and aggressive features is aberrant angiogenesis, which drives tumor neovascularization, representing a promising candidate for molecular target therapy. Although several pre-clinical studies and clinical trials have shown promising results, anti-angiogenic drugs have not led to a significant improvement in overall survival (OS), suggesting the necessity of identifying novel therapeutic strategies. Metformin, an anti-hyperglycemic drug of the Biguanides family, used as first line treatment in Type 2 Diabetes Mellitus (T2DM), has demonstrated in vitro and in vivo antitumoral efficacy in many different tumors, including glioblastoma. From this evidence, a process of repurposing of the drug has begun, leading to the demonstration of inhibition of various oncopromoter mechanisms and, consequently, to the identification of the molecular pathways involved. Here, we review and discuss metformin's potential antitumoral effects on glioblastoma, inspecting if it could properly act as an anti-angiogenic compound to be considered as a safely add-on therapy in the treatment and management of glioblastoma patients.

Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19.

The severity of coronavirus disease 2019 (COVID-19) is a crucial problem in patient treatment and outcome. The aim of this study is to evaluate circulating level of sphingosine-1-phosphate (S1P) along with severity markers, in COVID-19 patients. One hundred eleven COVID-19 patients and forty-seven healthy subjects were included. The severity of COVID-19 was found significantly associated with anemia, lymphocytopenia, and significant increase of neutrophil-to-lymphocyte ratio, ferritin, fibrinogen, aminotransferases, lactate dehydrogenase (LDH), C-reactive protein (CRP), and D-dimer. Serum S1P level was inversely associated with COVID-19 severity, being significantly correlated with CRP, LDH, ferritin, and D-dimer. The decrease in S1P was strongly associated with the number of erythrocytes, the major source of plasma S1P, and both apolipoprotein M and albumin, the major transporters of blood S1P. Not last, S1P was found to be a relevant predictor of admission to an intensive care unit, and patient's outcome. Circulating S1P emerged as negative biomarker of severity/mortality of COVID-19 patients. Restoring abnormal S1P levels to a normal range may have the potential to be a therapeutic target in patients with COVID-19.

Personalized and translational approach for malignant brain tumors in the era of precision medicine: the strategic contribution of an experienced neurosurgery laboratory in a modern neurosurgery and neuro-oncology department.

Personalized medicine (PM) aims to optimize patient management, taking into account the individual traits of each patient. The main purpose of PM is to obtain the best response, improving health care and lowering costs. Extending traditional approaches, PM introduces novel patient-specific paradigms from diagnosis to treatment, with greater precision. In neuro-oncology, the concept of PM is well established. Indeed, every neurosurgical intervention for brain tumors has always been highly personalized. In recent years, PM has been introduced in neuro-oncology also to design and prescribe specific therapies for the patient and the patient's tumor. The huge advances in basic and translational research in the fields of genetics, molecular and cellular biology, transcriptomics, proteomics, and metabolomics have led to the introduction of PM into clinical practice. The identification of a patient's individual variation map may allow to design selected therapeutic protocols that ensure successful outcomes and minimize harmful side effects. Thus, clinicians can switch from the "one-size-fits-all" approach to PM, ensuring better patient care and high safety margin. Here, we review emerging trends and the current literature about the development of PM in neuro-oncology, considering the positive impact of innovative advanced researches conducted by a neurosurgical laboratory.

Sphingosine-1-Phosphate in the Tumor Microenvironment: A Signaling Hub Regulating Cancer Hallmarks.

As a key hub of malignant properties, the cancer microenvironment plays a crucial role intimately connected to tumor properties. Accumulating evidence supports that the lysophospholipid sphingosine-1-phosphate acts as a key signal in the cancer extracellular milieu. In this review, we have a particular focus on glioblastoma, representative of a highly aggressive and deleterious neoplasm in humans. First, we highlight recent advances and emerging concepts for how tumor cells and different recruited normal cells contribute to the sphingosine-1-phosphate enrichment in the cancer microenvironment. Then, we describe and discuss how sphingosine-1-phosphate signaling contributes to favor cancer hallmarks including enhancement of proliferation, stemness, invasion, death resistance, angiogenesis, immune evasion and, possibly, aberrant metabolism. We also discuss the potential of how sphingosine-1-phosphate control mechanisms are coordinated across distinct cancer microenvironments. Further progress in understanding the role of S1P signaling in cancer will depend crucially on increasing knowledge of its participation in the tumor microenvironment.

Tumor-Educated Platelets and Angiogenesis in Glioblastoma: Another Brick in the Wall for Novel Prognostic and Targetable Biomarkers, Changing the Vision from a Localized Tumor to a Systemic Pathology.

: Circulating platelets (PLTs) are able to affect glioblastoma (GBM) microenvironment by supplying oncopromoter and pro-angiogenic factors. Among these mediators, sphingosine-1-phophate (S1P) has emerged as a potent bioactive lipid enhancing cell proliferation and survival. Here, we investigated the effect of "tumor education", characterizing PLTs from GBM patients in terms of activation state, protein content, and pro-angiogenic potential. PLTs from healthy donors (HD-PLTs) and GBM patients (GBM-PLTs) were collected, activated, and analyzed by flow cytometry, immunofluorescence, and Western blotting. To assess the pro-angiogenic contribution of GBM-PLTs, a functional cord formation assay was performed on GBM endothelial cells (GECs) with PLT-releasate. GBM-PLTs expressed higher positivity for P-selectin compared to HD-PLTs, both in basal conditions and after stimulation with adenosine triphosphate (ADP) and thrombin receptor activating peptide (TRAP). PLTs showed higher expression of VEGFR-1, VEGFR-2, VWF, S1P, S1PR1, SphK1, and SPNS. Interestingly, increased concentrations of VEGF and its receptors VEGFR1 and VEGFR2, VWF, and S1P were found in GBM-PLT-releasate with respect to HD-PLTs. Finally, GBM-PLT-releasate showed a pro-angiogenic effect on GECs, increasing the vascular network's complexity. Overall, our results demonstrated the contribution of PLTs to GBM angiogenesis and aggressiveness, advancing the potential of an anti-PLT therapy and the usefulness of PLT cargo as predictive and monitoring biomarkers.

Increased VEGF levels in a case of papillary tumor of the pineal region with intracranial hemorrhage: a potential surrogate indicator of tumor angiogenesis and aggressiveness?

Pineal tumors are rare, about 1% of all intracranial tumors. At variance with pineocytomas, usually characterized by a good prognosis, papillary tumors behave more aggressively. Owing to their rarity, little is known about their biology and clinical behavior, moreover conflicting data on prognosis have been reported. Here we present an unusual case of papillary neuroepithelial tumor of the pineal region in a 40-year-old man who was admitted in a state of unconsciousness due to the presence of intracranial hemorrhage. After 21 days from admission, he underwent third ventriculostomy for hydrocephalus and biopsy of the lesion. Since bleeding manifestations are uncommonly associated with this kind of tumors, we performed some additional non routine laboratory tests in order to identify biological indicators of disease course and abnormal angiogenesis. Coagulation screening tests were performed to rule out the presence of coagulopathy and vascular endothelial growth factor (VEGF ) levels were measured in plasma as marker of tumor angiogenic potential. Histologic evaluation confirmed the diagnosis of a papillary tumor of the pineal region with the presence of tiny vessel lumens that may account for increased angiogenesis Coagulation screening was normal and VEGF levels were extremely high if compared to healthy individuals. After 20 months of follow-up the tumor mass, radiotherapy treated, appeared dramatically reduced at MRI evaluation, and, interestingly, VEGF levels, although still higher than in healthy individuals, resulted significantly decreased as compared to those measured at time of first hospital admission suggesting a role for VEGF as indicator of tumor aggressiveness. In conclusion, measurement of angiogenesis circulating soluble markers could have an additional feedback in the diagnosis, therapy and monitoring the disease in patients with very rare CNS tumors as papillary tumors of pineal region that have non univocal clinical behavior and prognosis.

Significance and Prognostic Value of The Coagulation Profile in Patients with Glioblastoma: Implications for Personalized Therapy.

BACKGROUND: Coagulation is an important aspect of the vascular microenvironment in which brain tumors evolve. Patients with tumor often show aberrant coagulation and fibrinolysis activation. In particular, glioblastoma (GBM), the most aggressive primary brain tumor, is associated with a state of hypercoagulability, and venous thromboembolism is a common complication of this cancer and its treatment. Our study aims to investigate the clinical and prognostic significance of routine laboratory tests to assess the coagulative state of patients with brain tumors, to identify potential new prognostic factors and targets for personalized therapy. METHODS: Blood samples were collected from patients with GBM (n = 58) and patients with meningioma (MNG, n = 22), before any treatment. The parameters analyzed were prothrombin time (PT), activated partial thromboplastin time (aPTT), D dimer (DD), fibrinogen, von Willebrand factor (VWF), leukocyte count, and hemoglobin levels. RESULTS: Plasma levels of PT and aPTT were significantly reduced in GBMs compared with MNGs (P < 0.05), whereas DD, VWF:Ag levels, and leukocyte count were significantly higher in GBMs than in MNGs (P < 0.01). Furthermore, we observed that patients with GBM with reduced PT and aPTT and high levels of DD and VWF, defined as hypercoagulable patients, showed reduced overall survival (P < 0.05) compared with nonhypercoagulable patients. CONCLUSIONS: Our data support the assumption that patients with GBM show a plasma hypercoagulable profile and that coagulation profile is related to adverse outcome in patients with GBM. If confirmed, hypercoagulability could play an important role as a prognostic factor of the disease and in the decision of an antithrombotic prophylaxis.

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins.

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

Aspirin Affects Tumor Angiogenesis and Sensitizes Human Glioblastoma Endothelial Cells to Temozolomide, Bevacizumab, and Sunitinib, Impairing Vascular Endothelial Growth Factor-Related Signaling.

BACKGROUND: Glioblastoma (GBM) is the most common and fatal human brain tumor, with the worst prognosis. The aberrant microenvironment, enhanced by the activation of proangiogenic mediators such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and their downstream effectors, sustain GBM malignancy. Proangiogenic signaling represents an attractive chemotherapeutic target. Recent evidence suggests a therapeutic benefit from aspirin (acetylsalicylic acid, or ASA) intake in reducing risk and cancer progression. METHODS: In the present study, human primary GBM-endothelial cells (ECs) were used to ascertain whether ASA could inhibit angiogenesis and improve cell sensitivity to drugs. The impact of ASA was observed by measuring cell viability, tube-like structure formation, migration, VEGF production, and proliferative, proangiogenic, and apoptotic modulators expression, such as HIF-1α/VEGF/vascular endothelial growth factor receptor/(VEGFR)-1/VEGFR-2, Ras/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/AKT signaling axis, and Bcl-2-associated X protein/B-cell lymphoma 2 (BCL-2) ratio. Furthermore, we evaluated the effect of ASA alone or in combination with temozolomide (TMZ), bevacizumab (BEV), and sunitinib (SUN). RESULTS: Our data reported that ASA affected GBM-EC viability, tube-like structure formation, cell migration, and VEGF releasing in a dose-dependent manner and that combined treatments with TMZ, BEV, and SUN synergized to counteract proangiogenic cell ability. mRNA expression analysis displayed a marked effect of ASA in reducing VEGF, VEGFR-1, HIF-1α, RAS, mitogen-activated protein kinase kinase, AKT, and BCL-2, as well a combined anticancer effect of ASA together with TMZ, BEV, and SUN. Levels of HIF-1α, VEGFR-2, Bcl-2-associated X protein, and BCL-2 protein expression confirmed a positive trend. CONCLUSIONS: ASA and antiangiogenic therapies showed synergetic anticancer efficacy in human primary GBM-ECs. Thus, the combination of conventional chemotherapy with ASA may offer a new strategy to counteract tumor malignancy.

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment.

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P1 and S1P3, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.

Angiogenesis in human brain tumors: screening of drug response through a patient-specific cell platform for personalized therapy.

Gliomas are the most common brain tumors, with diverse biological behaviour. Glioblastoma (GBM), the most aggressive and with the worst prognosis, is characterized by an intense and aberrant angiogenesis, which distinguishes it from low-grade gliomas (LGGs) and benign expansive lesions, as meningiomas (MNGs). With increasing evidence for the importance of vascularization in tumor biology, we focused on the isolation and characterization of endothelial cells (ECs) from primary GBMs, LGGs and MNGs. Gene expression analysis by Real-Time PCR, immunofluorescence and flow cytometry analysis, tube-like structures formation and vascular permeability assays were performed. Our results showed a higher efficiency of ECs to form a complex vascular architecture, as well as a greater impairment of a brain blood barrier model, and an overexpression of pro-angiogenic mediators in GBM than in LGG and MNG. Furthermore, administration of temozolomide, bevacizumab, and sunitinib triggered a different proliferative, apoptotic and angiogenic response, in a dose and time-dependent manner. An increased resistance to temozolomide was observed in T98G cells co-cultured in GBM-EC conditioned media. Therefore, we developed a novel platform to reproduce tumor vascularization as "disease in a dish", which allows us to perform screening of sensitivity/resistance to drugs, in order to optimize targeted approaches to GBM therapy.

Enhanced phosphorylation of sphingosine and ceramide sustains the exuberant proliferation of endothelial progenitors in Kaposi sarcoma.

Endothelial colony-forming cells (ECFCs), a unique endothelial stem cell population, are highly increased in the blood of Kaposi sarcoma (KS) patients. KS-derived ECFCs (KS-ECFCs) are also endowed with increased proliferative and vasculogenic potential, thus suggesting that they may be precursors of KS spindle cells. However, the mechanisms underlying the increased proliferative activity of KS-ECFCs remain poorly understood. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are metabolically interconnected sphingoid mediators crucial to cell proliferation. Here, we investigated the metabolism, release, and proliferative effects of S1P and C1P in KS-ECFCs compared with control ECFCs (Ct-ECFCs). Metabolic studies by cell labeling, chromatographic analyses, and digital autoradiography revealed that S1P and C1P biosynthesis and S1P secretion are all efficient processes in KS-ECFCs, more efficient in KS-ECFCs than Ct-ECFCs. Quantitative PCR analyses demonstrated a significantly higher ceramide kinase and sphingosine kinase-2 expression in KS-ECFCs. Notably, also the expression of S1P1 and S1P3 receptors was augmented in KS-ECFCs. Accordingly, treatment with exogenous C1P or S1P induced a significant, concentration-dependent stimulation of KS-ECFC proliferation, but was almost completely ineffective in Ct-ECFCs. Hence, we identified C1P and S1P as autocrine/paracrine proliferative signals in KS-ECFCs. A better understanding of the mechanisms that enhance S1P/C1P formation in KS-ECFCs may yield effective therapeutic modalities.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells.

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+ oscillations in bEND5 cells, 300 µM being the most effective dose to generate a prolonged Ca2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+ release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Modulation of Neuroinflammation in the Central Nervous System: Role of Chemokines and Sphingolipids.

Neuroinflammation is a process involved in the pathogenesis of different disorders, both autoimmune, such as neuropsychiatric systemic lupus erythematosus, and degenerative, such as Alzheimer's and Parkinson's disease. In the central nervous system, the local milieu is tightly regulated by different mediators, among which are chemoattractant cytokines, also known as chemokines. These small molecules are able to modulate trafficking of immune cells in the course of nervous system development or in response to tissue damage, and different patterns of chemokine molecule and receptor expression have been described in several neuroinflammatory disorders. In recent years, a number of studies have highlighted a pivotal role of sphingolipids in regulating neuroinflammation. Sphingolipids have different functions, among which are the control of leukocyte egress from lymphonodes into inflamed tissues, the expression of various mediators of inflammation and a direct effect on the cells of the central nervous system as regulators of neuroinflammation. In the future, a better knowledge of these two groups of mediators could provide insight into the pathogenesis of neuroinflammatory disorders and could help develop novel diagnostic tools and therapeutic strategies.

Platelets from glioblastoma patients promote angiogenesis of tumor endothelial cells and exhibit increased VEGF content and release.

Glioblastoma multiforme (GBM) is the most common and fatal intracranial cancer in humans and exhibits intense and aberrant angiogenesis that sustains its malignancy and involves several angiogenic signals. Among them, vascular endothelial growth factor (VEGF) plays a key role and is overexpressed in GBM. Different cells appear to act as triggers of the aberrant angiogenesis, and, among them, platelets act as key participants. In order to provide further insights into the platelet features and angiogenic role in GBM, this study investigated the effects of platelet releasate on GBM-derived endothelial cells (GECs) and the levels of VEGF and endostatin, as pro- and anti-angiogenic components of platelet releasate from GBM patients. We demonstrate for the first time that: 1) platelet releasate exerts powerful pro-angiogenic effect on GECs, suggesting it might exert a role in the aberrant angiogenesis of GBM; 2) ADP and thrombin stimulation leads to significantly higher level of VEGF, but not of endostatin, in the releasate of platelets from GBM patients than those from healthy subjects; and 3) the intraplatelet concentrations of VEGF were significantly elevated in GBM patients as compared to controls. Moreover, we found a direct correlation between platelet-released VEGF and overall survival in our patient cohort. Although preliminary, these findings prompt further investigations to clarify the biologic relevance of platelet VEGF in GBM and prospective studies for screening GBM patients for anti-VEGF therapy and/or to optimize this treatment.