Patrizia Proia

Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles

We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood‐brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well‐known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain β1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

The HLA locus and multiple sclerosis in Sicily.

The authors report the analysis of HLA-class II allelic heterogeneity in a well characterized multiple sclerosis (MS) Sicilian dataset. Family-based association analysis revealed evidence for excess transmission to affected individuals for alleles HLA-DRB1*1501, DRB1*04, and DQB1*0302. When analyzed as haplotypes, the authors observed excess transmission for the DRB1*0400-DQB1*0302 haplotype. Sicilian patients share the HLA-DRB1*1501 susceptibility allele with affecteds living in continental Italy, but also display the allelic heterogeneity that characterizes Mediterranean populations.

Semaphorin 4D cooperates with VEGF to promote angiogenesis and tumor progression.

The semaphorins and plexins comprise a family of cysteine-rich proteins implicated in control of nerve growth and development and regulation of the immune response. Our group and others have found that Semaphorin 4D (SEMA4D) and its receptor, Plexin-B1, play an important role in tumor-induced angiogenesis, with some neoplasms producing SEMA4D in a manner analogous to vascular endothelial growth factor (VEGF) in order to attract Plexin-B1-expressing endothelial cells into the tumor for the purpose of promoting growth and vascularity. While anti-VEGF strategies have been the focus of most angiogenesis inhibition research, such treatment can lead to upregulation of pro-angiogenic factors that can compensate for the loss of VEGF, eventually leading to failure of therapy. Here, we demonstrate that SEMA4D cooperates with VEGF to promote angiogenesis in malignancies and can perform the same function in a setting of VEGF blockade. We also show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor–resistant tumors, suggesting that this may represent a novel treatment for some cancers.

Plexin-B1 Activates NF-κB and IL-8 to Promote a Pro-Angiogenic Response in Endothelial Cells

Background The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium. Methods/Principal Findings Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma. Conclusion/Significance These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.

Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes

Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their messages to the target cells, we labeled oligodendroglioma cells with radioactive methionine and then added the labeled vesicles shed from tumor cells to unlabeled astrocytes in culture. Here we report that labeled proteins were delivered to the test cells. In order to investigate whether astrocytes, like neurons, are sensitive to oligodendroglioma-derived vesicles, MVs were prepared from media conditioned by G26/24 oligodendroglioma cells and added to primary cultures of rat cortical astrocytes. These cells were clearly more resistant than neurons to microvesicle-induced damage: a high dose (40 µg) of shed MVs induced cell death in only about 40% of astrocytes. Finally, we demonstrated that Hsp70 is specifically enriched in MVs which also contain, even if at lower level, the Hsc70 constitutive chaperone.

Lactate as a Metabolite and a Regulator in the Central Nervous System

More than two hundred years after its discovery, lactate still remains an intriguing molecule. Considered for a long time as a waste product of metabolism and the culprit behind muscular fatigue, it was then recognized as an important fuel for many cells. In particular, in the nervous system, it has been proposed that lactate, released by astrocytes in response to neuronal activation, is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to be used for the tricarboxylic acid cycle. More recently, in addition to this metabolic role, the discovery of a specific receptor prompted a reconsideration of its role, and lactate is now seen as a sort of hormone, even involved in processes as complex as memory formation and neuroprotection. As a matter of fact, exercise offers many benefits for our organisms, and seems to delay brain aging and neurodegeneration. Now, exercise induces the production and release of lactate into the blood which can reach the liver, the heart, and also the brain. Can lactate be a beneficial molecule produced during exercise, and offer neuroprotection? In this review, we summarize what we have known on lactate, discussing the roles that have been attributed to this molecule over time.

Plexin-B1 and Semaphorin 4D Cooperate to Promote Perineural Invasion in a RhoA/ROK-Dependent Manner

Perineural invasion (PNI) is a tropism of tumor cells for nerve bundles located in the surrounding stroma. It is a pathological feature observed in certain tumors, referred to as neurotropic malignancies, that severely limits the ability to establish local control of disease and results in pain, recurrent growth, and distant metastases. Despite the importance of PNI as a prognostic indicator, its biological mechanisms are poorly understood. The semaphorins and their receptors, the plexins, compose a family of proteins originally shown to be important in nerve cell adhesion, axon migration, and proper central nervous system development. Emerging evidence has demonstrated that these factors are expressed in tissues outside of the nervous system and represent a widespread signal transduction system that is involved in the regulation of motility and adhesion in different cell types. We believe that the plexins and semaphorins, which are strongly expressed in both axons and many carcinomas, play a role in PNI. In this study, we show that plexin-B1 is overexpressed in tissues and cell lines from neurotropic malignancies and is attracted to nerves that express its ligand, semaphorin 4D, in a Rho/Rho kinase-dependent manner. We also demonstrate that nerves are attracted to tumors through this same system of proteins, suggesting that both plexin-B1 and semaphorin 4D are important in the promotion of PNI.

The hypoxia-inducible factor-responsive proteins semaphorin 4D and vascular endothelial growth factor promote tumor growth and angiogenesis in oral squamous cell carcinoma

Growth and metastasis of solid tumors requires induction of angiogenesis to ensure the delivery of oxygen, nutrients and growth factors to rapidly dividing transformed cells. Through either mutations, hypoxia generated by cytoreductive therapies, or when a malignancy outgrows its blood supply, tumor cells undergo a change from an avascular to a neovascular phenotype, a transition mediated by the hypoxia-inducible factor (HIF) family of transcriptional regulators. Vascular endothelial growth factor (VEGF) is one example of a gene whose transcription is stimulated by HIF. VEGF plays a crucial role in promoting tumor growth and survival by stimulating new blood vessel growth in response to such stresses as chemotherapy or radiotherapy-induced hypoxia, and it therefore has become a tempting target for neutralizing antibodies in the treatment of advanced neoplasms. Emerging evidence has shown that the semaphorins, proteins originally associated with control of axonal growth and immunity, are regulated by changes in oxygen tension as well and may play a role in tumor-induced angiogenesis. Through the use of RNA interference, in vitro and in vivo angiogenesis assays and tumor xenograft experiments, we demonstrate that expression of semaphorin 4D (SEMA4D), which is under the control of the HIF-family of transcription factors, cooperates with VEGF to promote tumor growth and vascularity in oral squamous cell carcinoma (OSCC). We use blocking antibodies to show that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of OSCC and other solid tumors.

Oligodendroglioma cells synthesize the differentiation-specific linker histone H1˚ and release it into the extracellular environment through shed vesicles

Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1° linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1° expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1° expression in other brain cells. Even less is known relating to tumor glial cells. In this study we report that, like in maturing brain and isolated neurons, H1° synthesis sharply increases in differentiating astrocytes growing in a serum-free medium, while the corresponding mRNA decreases. Unexpectedly, in tumor glial cells both H1° RNA and protein are highly expressed, in spite of the fact that H1° is considered a differentiation-specific histone variant. Persistence of H1° mRNA in oligodendroglioma cells is accompanied by high levels of H1° RNA-binding activities which seem to be present, at least in part, also in actively proliferating, but not in differentiating, astrocytes. Finally, we report that oligodendroglioma cells, but not astrocytes, release H1° protein into the culture medium by shedding extracellular vesicles. These findings suggest that deregulation of H1° histone expression can be linked to tumorigenesis.

Rho-mediated activation of PI(4)P5K and lipid second messengers is necessary for promotion of angiogenesis by Semaphorin 4D

Phosphatidylinositol 4-phosphate 5-kinase (PI(4)P5K) is a type I lipid kinase that generates the lipid second messenger phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and functions downstream of RhoA in actin organization. It is known to play an essential role in neurite remodeling, yielding a phenotype identical to that seen in cells treated with Semaphorin 4D (Sema4D), a protein that regulates proliferation, adhesion and migration in many different cell types. Plexin-B1, the receptor for Sema4D, activates RhoA in order to generate a pro-angiogenic signal in endothelial cells. Therefore, we looked in human umbilical vein endothelial cells (HUVEC) to determine if Plexin-B1 exerted control over the cytoskeleton by regulation of PI(4)P5K activity. Here we demonstrate the Rho/Rho Kinase (ROK)-dependent generation of PI(4,5)P2 upon treatment of HUVEC with Sema4D, as well as co-localization of PI(4)P5Kα with Plexin-B1. Formation of PI(4,5)P2 was necessary for cytoskeletal polymerization, as expression of the phosphatase synaptojanin blocked this effect. We noted phosphorylation and activation of PLCγ and an increase in intracellular calcium upon treatment of HUVEC with Sema4D, responses that were necessary for a pro-angiogenic phenotype observed in vitro. Taken together, these results suggest that Plexin-B1 promotes angiogenesis in endothelial cells by signaling through PI(4)P5Kα and generating lipid second messengers.