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The aim of the present study was to evaluate whether the activation of Cdk5, a protein that has been suggested to participate in higher cognitive functions, is required for the onset of a sensitized anxiety-related behavior induced by stress. The exposure to restraint enhanced both Cdk5 expression in certain subareas of the septohippocampal system, principally in the lateral septum (LS) and septal Cdk5 kinase activity in rats. Behaviorally, restrained wild type mice showed a behavior indicative of enhanced anxiety in the elevated plus maze (EPM). In contrast, unstressed mice and stressed knockout mice, which lacked the p35 protein, the natural activator of Cdk5, displayed similar anxiety-like behavior in the EPM. Finally, the intra-LS infusion of olomoucine - a Cdk5 inhibitor - blocked the enhanced anxiety in the EPM induced by prior stress in rats. All these data provide evidence that septal Cdk5 is required in the emergence of a sensitized emotional process induced by stress.

An important area of the brain aversive circuitry is the lateral septum (LS), together with its principal connections to diverse Hippocampal regions. The aim of this work was to evaluate whether the LS-Hippocampus network participates in the increased anxiety-like behavior produced by a previous defeat experience. The neural activation of different regions of the Hippocampus was assessed by the number of Fos positive cells in animals previously defeated. A notable elevation in the expression of this protein was observed in CA1, CA2, CA3, and Dentate Gyrus, for both dorsal and ventral Hippocampus. The local administration of a glucocorticoid receptor (GR or type II) antagonist, but not of a mineralcorticoid receptor (MR or type II) antagonist, into the LS before the stressful stimuli prevented a rise in the number of Fos positive cells, especially in the ventral portion of the Hippocampus. Furthermore, to evaluate the role of these hippocampal portions in the modulation of the emotional sequelae induced by defeat, the dorsal or the ventral Hippocampus were inactivated by lidocaine at different times following the social confrontation, with the anxiety-like behavior being assessed in the elevated plus maze the next day. Only the inactivation of the ventral region attenuated the excessive anxiety exhibited by defeated animals. The infusion of lidocaine, 1 h after the confrontation, did not affect this behavioral response. These data suggest a preferential participation of the LS and its connections to the ventral Hippocampus in the emotional sequelae induced by the social defeat. Moreover, the GR localized within the LS played an essential role in the modulation of this emotional state.

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.

Retina photoreceptor and ganglion cells isolated from chicks that in vivo were exposed to light have a different phospholipid labeling capacity than those from chicks in the dark. In the light exposed animals, the phospholipid labeling in the ganglion cells is higher (Delta% 45, p

Retinal ganglion cells isolated from chicks that in vivo were exposed to light have a higher phospholipid labeling capacity than those obtained from animals in the dark. Actinomycin D or a mixture of protein synthesis inhibitors or of antisense oligonucleotides to c fos plus c-jun injected intraocularly 1 hr prior to the stimulation period, abolished the light-dark differences for phospholipids but not for gangliosides. Light stimulation induced the formation (and/or stabilization) of c fos mRNA and of the protein c-Fos, indicating that immediate early gene induction, and consequently the synthesis of the protein(s) encoded, is essential to increase the synthesis of phospholipids but not of gangliosides. These results suggest a novel mechanism by which immediate early genes engram neural cells, modifying specifically the metabolism of cell constituents producing long-lasting changes in the cells. © 1996 Wiley-Liss, Inc.

Some 20 years ago c-Fos was identified as a member of the AP-1 family of inducible transcription factors (Angel and Karin in Biochim Biophys Acta 1072:129–157, 1991). More recently, an additional activity was described for this protein: it associates to the endoplasmic reticulum and activates the biosynthesis of phospholipids (Bussolino et al. in FASEB J 15:556–558, 2001), (Gil et al. in Mol Biol Cell 15:1881–1894, 2004), the quantitatively most important components of cellular membranes. This latter activity of c-Fos determines the rate of membrane genesis and consequently of growth in differentiating PC12 cells (Gil et al. in Mol Biol Cell 15:1881–1894, 2004). In addition, it has been shown that c-Fos is over-expressed both in PNS and CNS tumors (Silvestre et al. in PLoS One 5(3):e9544, 2010). Herein, it is shown that c-Fos-activated phospholipid synthesis is required to support membrane genesis during the exacerbated growth characteristic of brain tumor cells. Specifically blocking c-Fos-activated phospholipid synthesis significantly reduces proliferation of tumor cells in culture. Blocking c-Fos expression also prevents tumor progression in mice intra-cranially xeno-grafted human brain tumor cells. In NPcis mice, an animal model of the human disease Neurofibromatosis Type I (Cichowski and Jacks in Cell 104:593–604, 2001), animals spontaneously develop tumors of the PNS and the CNS, provided they express c-Fos (Silvestre et al. in PLoS One 5(3):e9544, 2010). Treatment of PNS tumors with an antisense oligonucleotide that specifically blocks c-Fos expression also blocks tumor growth in vivo. These results disclose cytoplasmic c-Fos as a new target for effectively controlling brain tumor growth.

c-Fos, a transcription factor that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription factor activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

Withdrawal from chronic ethanol administration facilitated the formation of contextual fear memory. The effect of fear memory retrieval on subsequent ethanol consumption, by employing a two-bottle free-choice procedure with either water or ethanol (2–8% v/v), was investigated in ethanol withdrawn rats. The effect of fear memory extinction with or without d -cycloserine (DCS, 5 mg/kg i.p.) on subsequent ethanol consumption was also evaluated. In addition, we examined c-Fos expression in different brain areas following the fear memory recall. The retrieval of such fear memory induced a significant increase in ethanol consumption in ethanol withdrawn but not in control animals. Regardless of DCS treatment, this increase was attenuated by extinction training. In ethanol withdrawn rats, context-dependent memory retrieval was accompanied by an increased c-Fos expression in the basolateral amygdala, ventrolateral periaqueductal gray, dentate gyrus and dorsomedial periaqueductal gray. Among these brain areas suggested to be implicated in the modulation of motivation and of emotional states, the basolateral amygdala has a crucial role in the emergence of negative affective state during ethanol withdrawal. These data suggest that retrieval of fear memory in ethanol withdrawn rats affected ethanol consumption and that amygdala may be involved in this effect.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in anglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in viro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light ; no increase was observed in preparations obtained from light-exposed animals reated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

The formation of new vessels, (angiogenesis) characterizes body development and is pivotal in some diseases. In particular, angiogenesis is crucial for the growth of tumors and metastasis diffusion. The beginning of tumor angiogenesis is caused by the selection of a cancer cell clone which induces an unbalance between inducers and inhibitors of angiogenesis. The inhibition of this process by natural or chemical compounds increases apoptotic index in the tumors, does not causes drug resistance and has been demonstrated to induce cancer dormancy in many animal models.

Platelet-activating factor (PAF), a mediator of homotypic and heterotypic cell-to-cell communication, activates inflammatory cells and lymphocytes through a seven-spanning transmembrane domain receptor [1, 2]. Following appropriate stimulation, it is produced and released by monocytes, neutrophils, endothelial cells and T lymphocytes [3–8]. It is also produced by neurons and glial cells stimulated by neurotransmitters and tumor necrosis factor (TNF)-α, respectively [9, 10]. Vascular endothelium is a key target for PAF. It modifies the barrier function of a monolayer of endothelial cells in vitro [11, 12], is a powerful vasopermeabilizing molecule in vivo [13, 14], and promotes leukocyte adhesion and transmigration [15–17]. High PAF concentrations are toxic for endothelial cells, causing vacuolization and marked formation of blebs [12, 18].

Angiogenesis is required for the growth of solid tumors which, in avascular condition, are limited to 2-3 mm3 volume. The switch to the angiogenic phenotype allows new vessels to converge upon the tumor, growth to proceed at an exponential rate and metastasis diffusion. The evaluation of tumoral angiogenesis has been proposed to be an independent prognostic marker of behaviour of some solid tumors: it has been demonstrated that, in some types of carcinoma (breast, prostate, lung, etc...), an intense vascular proliferation correlates with the aggressiveness of the disease. However in malignant melanomas and colorectal carcinomas, there are conflicting results on the correlation between angiogenesis and the progression of the cancer. There are also conflicting data on the role of microvessels count (MC) in the management of head and neck cancer. Despite a large number of studies, at the present, there are not biological or molecular markers available to predict consistently the outcome of the patients with laryngeal squamous cell carcinoma (LSCC). In fact the prognosis of this tumor has been mainly based, up to now, on a number of clinico-pathological parameters, especially localization, tumor extent and nodal involvement. The aim of this study has been to compare MC inside LSCC with disease free survival, grading, pT, pN and pathological stage. We investigated the relevance of the number of microvessels in the peritumoral stroma of 68 LSCC (only Caucasian males, age 35-70 years), classified according to UICC/1987 randomly selected (33 classified in clinical stage I e II and 35 in clinical stage III-IV). All patients have been surgically treated and pN + cases have been also submitted to radiotherapy. The follow-up was 60-84 months. The vascular density was assessed according to Horak et Al. with an immunohistochemical method using JC70 monoclonal antibody (CD31; Dako, Astrup, Denmark). Univariate analysis showed that MC, pT, pN, Pathological Stage and grading were correlated with the disease-free survival. A MC120/mm2 was predictive for a high survival index; in contrast a MC150 mm2 were associated with relapse. Furthermore, multivariate analysis demonstrated that MC was the only independent predictor for the disease free survival. Our findings demonstrate that in LSCC, MC is the first measurable biological parameter which is significant for evaluating the disease free-survival. Therefore, MC in LSCC is crucial in the prognosis and in the choice of a more aggressive management of the disease, including the possible treatment with antiangiogenic compound.

Background We have previously shown that the transcription factor c-Fos is also capable of associating to endoplasmic reticulum membranes (ER) and activating phospholipid synthesis. Herein we examined phospholipid synthesis status in brain tumors from human patients and from NPcis mice, an animal model of the human disease Neurofibromatosis Type 1 (NF1). Principal Findings In human samples, c-Fos expression was at the limit of detection in non-pathological specimens, but was abundantly expressed associated to ER membranes in tumor cells. This was also observed in CNS of adult tumor-bearing NPcis mice but not in NPcis fos(−/−) KO mice. A glioblastoma multiforme and a malignant PNS tumor from a NF1 patient (MPNST) showed a 2- and 4- fold c-Fos-dependent phospholipid synthesis activation, respectively. MPNST samples also showed increased cell proliferation rates and abundant c-Fos expression. Conclusions Results highlight a role of cytoplasmic c-Fos as an activator of phospholipid synthesis in events demanding high rates of membrane biogenesis as occurs for the exacerbated growth of tumors cells. They also disclose this protein as a potential target for controlling tumor growth in the nervous system.

Murine endothelial cell lines containing middle T antigen provide a good model to study the biology of capillary cells of different anatomical districts. These cell lines obtained from skin (sEnd.1), brain (bEnd.4), thymus (tEnd-1) and embryo tissue (eEnd.1) are activated by platelet-activating factor (PAF). PAF, but not lyso-PAF or the enantiomer (S)-PAF, induces the rapid translocation of protein kinase C (PKC) from the cytosol to the membrane. Maximal translocation of the enzyme is achieved after 5 min in all cell lines tested. The ED50 of PAF able to promote PKC translocation has a value of 5 nM in sEnd.1, bEnd.4 and eEnd.1, and 20 nM in tEnd.1, suggesting a different sensitivity between these cell lines. The data indicate that PAF activates capillary cells, thus explaining its activation of microvascular beds.

Platelet-activating-factor (PAF) is a potent, biologically active lipid mediator produced by several tissues, including brain. Its role in the central nervous system (CNS) is still unknown, even if its involvement in brain damage and neurotoxicity has been postulated. Its production by neural cells has been demonstrated in different species, but not in man. This paper provides evidence that PAF can be produced by human fetal neurons and/or glial cells in culture. Its synthesis dramatically increased upon stimulation with acetylcholine (ACh), and it was significantly lowered by the cholinergic receptor antagonist atropine. Almost no PAF was detected in the incubation medium, which indicated no release of PAF from cultured cells. Characterization of the cells in culture with specific monoclonal antibodies excluded the presence of endothelial cells or macrophages, which also produce PAF.

The mature product of the c-met proto-oncogene is a putative tyrosine kinase receptor of 190 kd with an alpha beta heterodimeric structure. The c-met protein is phosphorylated in vivo on the beta subunit in the gastric carcinoma cell line GTL-16 (Giordano et al., 1988). Western blots with phosphotyrosine antibodies show that tyrosine phosphorylation of the beta subunit is reduced by treatment of GTL-16 cells with protein kinase C activators (tumor promoting phorbol esters such as phorbol 12-myristate 13-acetate, TPA, and beta-phorbol 12,13-dibutyrate, PdBu, or membrane permeable synthetic diacylglycerol 1-oleyl-2-acetyl-sn-glycerol, OAG). The inactive analog alpha-phorbol 12,13-didecanoate has no effect. The inhibition induced by TPA is dose dependent and maximal after 1 h. Depletion of protein kinase-C by prolonged treatment with TPA (18-48 h) increases the phosphorylation on tyrosine of the beta subunit. Phospho-amino acid analysis of the c-met protein immunoprecipitated from [32P]orthophosphate-labelled GTL-16 cells shows that protein kinase-C activation leads to an increase in serine phosphorylation and to concomitant decrease in tyrosine phosphorylation. These results suggest that, similar to the EGF and insulin receptor, the putative receptor encoded by the c-met proto-oncogene may be negatively modulated by protein kinase-C phosphorylation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for three days (8 micrograms/kg/day) to 14 subjects who had solid tumors and normal hemopoiesis. The treatment induced a rapid 3- to 5-fold increase in the number of circulating neutrophils, eosinophils and monocytes. Lymphocytes, platelets and reticulocytes were unmodified during treatment. Activation of circulating neutrophils during GM-CSF treatment was demonstrated by a significant, increased release of neutrophil-derived platelet-activating factor after stimulation with N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha or phagocytosis. The granulomonocytosis was dependent on increased bone marrow production of mature cells. Using the thymidine suicide technique, we observed that GM-CSF more than doubled the percentage of granulocyte-macrophage and megakaryocyte colony-forming units (CFU-gm and CFU-meg) and erythroid burst-forming units (BFU-e) in the S phase of the cell cycle. However, at the level of morphologically recognizable cells with autoradiography, we observed that GM-CSF increased the labeling index of the granulo-monopoietic cells, whereas that of the erythroblasts was unchanged. These data suggest that in accordance with in vitro observations, GM-CSF exerts its activity through all granulo-monopoietic lineages, whereas other cytokines (erythropoietin, thrombopoiesis-stimulating factors) may be needed to fully exploit the proliferative stimulus of GM-CSF on BFU-e and CFU-meg. After treatment discontinuation, the proliferative activity drops to values lower than before treatment, suggesting a period of relative refractoriness of marrow progenitors to the cytocidal effect of cell cycle-specific antineoplastic agents. This hypothesis is under evaluation in a controlled clinical trial where GM-CSF is given prior to chemotherapy.

9021 Background. Despite intensive chemotherapy and surgery treatment, lung and bone metastases develop in about 30% of OS patients. Mechanisms for this preferential metastatic behaviour are largely unknown. The CXCR4/SDF-1 system was shown to play a role in the homing of neoplastic cells in breast cancer as well as in rabdhomyosarcoma. This hypothesis was tested in vitro and in vivo in a mouse model. Methods. Human OS cell lines MG-63, SJSA, U2-OS and HOS were studied in standard colture conditions. Cell lines were evaluated for: CXCR4 expression by FACS analysis (mAb anti-CXCR4, BD Pharmingen); SDF-1 levels in conditioned medium (antibody sandwich ELISA, RD modulation of CXCR4 expression on OS cells by exposure to recombinant human SDF-1 (rhSDF-1). The adhesion and migration of SJSA cells were tested on a monolayer of bone marrow stromal cells in response to rhSDF-1. We evaluated lung metastasis development in mouse model (Balb-c nu/nu) with and without monoclonal antibody anti-CXCR4 (T134) t...

The catastrophe theory evolved by Thom and Zeeman proposes a mathematical definition for the abrupt or 'catastrophic' changes that can suddenly occur in normally well-ordered and smooth-running systems. We have integrated this theory with our own PAF/cytokine feedback network hypothesis to explain the control and dysfunction of the inflammatory response. This process involves the activation of cells and factors such as proteases, and is coordinated by mediators such as PAF, cytokines and growth factors, minute amounts of which can prime cells to respond in an enhanced manner to subsequent agonistic stimuli. PAF and certain cytokines also possess the unique property of being able to induce the release of each other and their own generation in vivo. This 'singularity' may enable a self-generating feedback network to become established. The priming ability of these mediators indicates the extreme sensitivity of the inflammatory process and importance of a homeostatic equilibrium between the vectors involved in the priming and feedback processes and internal suppressive mechanisms. In pathological conditions, one can consider the phenomenon of PAF and cytokine autogeneration as a 'fold' in the feedback network and an expression of the singularity characteristic of the catastrophe hypothesis. This may lead to systemic toxicity and microcirculatory collapse, a characteristic feature of shock, sepsis, asthma, ischemia and graft rejection. A combination of drugs antagonizing the various feedback components may inhibit this catastrophic process and thus provide more successful therapy of these conditions.

In previous work we showed that platelet-activating factor (PAF) was produced upon stimulation of the chick retina with acetylcholine (ACh) and dopamine (DA), via a dithiothreitol-insensitive cholinephosphotransferase (DTT-CPT). Other neurotransmitters were ineffective. Since ACh and DA stimulated PAF production at different stages of development, we advanced the hypothesis that selected cholinergic and dopaminergic receptors were involved. In this paper we demonstrate that only muscarinic or D2 (and not D1 and nicotinic) receptor stimulation induced PAF production in the chick retina either before or after hatching. Moreover, our data show that PAF production was completely prevented by blockage of these receptors by specific antagonists. These data further substantiate the hypothesis that PAF synthesis could be physiologically associated with synapse stimulation.

It has been shown that human endothelial cells (HEC) are stimulated to migrate and proliferate by granulocyte (G)- and granulocyte-macrophage (GM)-colony-stimulating factor (CSF) (Bussolino, F., Wang, J. M., Defilipii, P. Turrini, F., Sanavio, F., Edgell, C.-J. S., Aglietta, M., Arese, P., and Mantovani, A. (1989) Nature 337, 471-473). The rapid intracellular events initiated by these cytokines on binding to their receptors on HEC are not defined. Addition of G- or GM-CSF to HEC produced a rapid activation of Na+/H+ exchanger resulting in an increase in intracellular pH (pHi). Both cytokines induced an alkaline displacement in the pHi dependence of the exchanger without affecting the affinity for external Na+ (Nao) and the rate of exchanger. Ethylisopropylamiloride, a selective inhibitor of the Na+/H+ exchanger, inhibited the intracellular alkalinization, the migration, and proliferation induced by G- and GM-CSF. The data indicate that G- and GM-CSF initiate a rapid exchange of Na+ and H+ by means of the Na+/H+ exchanger and that this ethylisopropylamiloride-sensitive ions flux is important to the biological effects of these cytokines on HEC.

Oligomerization of band 3 protein has been recently indicated as an early event in senescent or damaged red cell membrane followed by specific deposition of anti-band 3 antibodies and binding of complement C3 fragments. The band 3-anti-band 3-C3b complex is recognized by homologous monocytes, and phagocytosis ensues. This study shows that recognition of the anti-band 3-C3b complex by the monocyte C3b receptor type one (CR1) plays a crucial role in the process of removal of damaged red cells. Indeed, blocking of monocyte CR1 with an anti-CR1 monoclonal antibody abrogated phagocytosis of diamide-treated red cells. Platelet-activating factor (PAF) is a phospholipid mediator involved in inflammatory processes. Nanomolar (R)-PAF enhanced the CR1-dependent phagocytosis of diamide-treated human red cell and of sheep red cells coated with C3b, induced the fast translocation of protein kinase C to monocyte membrane compartment, and stimulated the phosphorylation of monocyte CR1. The biologically inert lyso-PAF and the enantiomer (S)-PAF were inactive. PAF receptor antagonists and inhibitors of protein kinase C blocked the enhancement of phagocytosis induced by PAF. Protein kinase C translocation, phosphorylation of CR1, and stimulation of this receptor to an active state capable of mediating phagocytosis represent a novel pathway by which PAF interferes with red cell homeostasis and possibly modulates inflammatory reactions and host mechanisms against infections.

Platelet-activating factor (PAF) is a soluble factor that induces platelet aggregation and release reaction (1–4), independently from ADP- and Arachidonic Acid/Thromboxane A2 -mediated pathways (2, 4). PAF is alow molecular weight phospholipid (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine)(5,6). PAF was initially obtained from antigen-stimulated, IgE-sensitized rabbit basophils (2). In man PAF release from basophils still awaits confirmatory evidence, mainly because in man basophils constitute a much smaller fraction of the total leukocytes than in the rabbit. Lewis recovered a PAF-like activity from purified leukemic basophils in the presence of Calcium Ionophore (7). Later, we have shown that immune complexes (IC), the specific antigen, anaphylatoxin and neutrophil cationic proteins stimulate PAF release from human leukocytes (8). Although basophil degranulation was coincident with PAF release, the cell populations used in those experiments were relatively impure. More recently, ultrastructural evidence of platelet interaction only with degranulated basophils but not with either neutrophils or monocytes was obtained when basophils were degranulated by the specific antigen, anti-IgE or Synachten (9). However, PAF release occurred independently from basophil degranulation during phagocytosis by polymorphonuclear neutrophils (PMN) and monocytes (9,10). Lotner et al. demonstrated that purified human PMN are capable of releasing PAF (11). The purpose of the present report was to comparatively study the mechanism of PAF release from PMN and monocytes under phagocytic stimuli.

Inflammatory processes are often associated with pathological alteration of the vessel wall and sometimes with local or disseminated thrombotic phenomena. Interleukin-1 (IL-1), a monokine produced by activated cells of the monocyte/macrophage lineage and responsible of most of the changes associated with the inflammatory acute phase response, appears to dramatically' modify several endothelial cell (EC) functions. Some groups including ours (for review 1) have shown that IL-1 stimulates prostacyclin (PGI2), platelet activating factor (PAF), plasminogen activator inhibitor (PAi), thromboplastin (PCA) synthesis by cultured human EC in vitro. In addition IL-1 can act directly on EC to increase neuthophil and other leukocyte adhesion on their surface (2). All these effects, in contrast to previously described inducers, require a long time of interaction (30 min to 4 hours) of IL-1 with EC to be apparent and then last for several hours (4 to 12 hours). The IL-1 effects are concentration dependent (minimal active concentration being about 1 unit/ml) and require protein and RNA synthesis. To better define the structural requirement for IL-1 induced modification of EC functions we compared the activity of different IL-1 molecular species. Our approach is based on the observation that IL-1 is indeed a family of polypeptides biochemically different(3). At least two dissimilar gene products have been cloned with very limited homology (denominated α and β). These molecules, though biochemically different, share common activities and possibly the same receptor in different cell types. On EC we investigated whether the αand β IL-1 forms have similar biological activities (4). All the IL-1 preparations used were active on thymocyte costimulatory assay and comparison was made on the basis of the concentrations of these agents equally active on this assay.Human recombinant IL- αandβ (hr IL-1 α and hr IL-1 β) were both active in stimulating PGI2, PCA, PAi production and in increasing neutrophil adhesion to EC. In contrast PAF synthesis was Stimulated by hr IL-1 α but not by hr IL-1 β. Murine recombinant IL-1 (mr IL-1 α highly homologous with hr IL-1 < α, at concentrations able to maximally activate thymocytes was inactive on PGI2, PCA and in increasing neutrophil adhesion to EC. In contrast, mr IL-1 α was equally effective on PAF production as hr IL-1 α. A short peptide fragment of hr IL-1β (fragment 167-171) was synthesized on the basis of its predicted exposure on the surface of the molecule (5). This peptide is also located in a region (150-186) of high homology between hr IL-1α and β sequences. While the peptide showed high thymocyte activation capacity it was inactive on EC activities. Overall these results indicate that the α and β forms of human IL-1 elicit largely but not completely overlapping patterns of response in EC. In addition they suggest that the structural requirement for activation by IL-1 is not identical for thymocytes and EC. These results might provide some clues to novel strategies for modulation of IL-1 vascular and immunological activities.1. Mantovani A. and E. Dejana (1987) Biochem. Pharm. 36:301.2. Bevilacqua M. et al. (1985) J. Clin. Invest. 76:20033. Dinarello C.A. (1985) J. Clin. Immunol. 5:287.4. Dejana E. et al. (1987) Blood 69:695.5. Antoni G. et al. (1987) J. Immunol, (in press).

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1− resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth. Cancer Res; 72(24); 6371–81. ©2012 AACR.

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

Marco Soster,1,* Riccardo Juris,2,* Sara Bonacchi,2 Damiano Genovese,2 Marco Montalti,2 Enrico Rampazzo,2 Nelsi Zaccheroni,2 Paolo Garagnani,2 Federico Bussolino,3,4 Luca Prodi,2 Serena Marchiò1,4 1Institute for Cancer Research and Treatment, Laboratory of Tumor Microenvironment and University of Torino, Department of Oncological Sciences, Candiolo, 2University of Bologna, Department of Chemistry "G.Ciamician", Bologna, 3Institute for Cancer Research and Treatment, Laboratory of Vascular Oncology and University of Torino, Department of Oncological Sciences, Candiolo, 4APAvadis Biotechnologies, BioIndustry Park S Fumero, Colleretto Giacosa, Italy*These authors equally contributed to the workBackground and methods: Despite the recent introduction of targeted bio-drugs, the scarcity of successful therapeutic options for advanced colorectal cancer remains a limiting factor in patient management. The efficacy of curative surgical interventions can only be extended through earlier detection of metastatic foci, which is dependent on both the sensitivity and specificity of the diagnostic tools.Results: We propose a high-performance imaging platform based on silica-poly(ethylene glycol) nanoparticles doped with rhodamine B and cyanine 5. Simultaneous detection of these dyes is the basis for background subtraction and signal amplification, thus providing high-sensitivity imaging. The functionalization of poly(ethylene glycol) tails on the external face of the nanoparticles with metastasis-specific peptides guarantees their homing to and accumulation at target tissues, resulting in specific visualization, even of submillimetric metastases.Conclusions: The results reported here demonstrate that our rationally designed modular nanosystems have the ability to produce a breakthrough in the detection of micrometastases for subsequent translation to clinics in the immediate future.Keywords: colorectal cancer, imaging platform, luminescent targeting, micrometastasis, peptide targeting

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system we provide evidence that epithelial cell polarization is a chemical phase separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase separation scenario is likely common to many processes of cell polarity formation., 7 pages, 6 figures

Platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a mediator of inflam¬mation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) generated from 1-O-alkyl-2-acyl-¬sn-glycero-3-phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid-¬induced anti-(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen-induced anti-inflammatory protein, SV-IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV-IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl-CoA: lyso-PAF acetyltransfe

Many eukaryotic cell types share the ability to migrate directionally in response to external chemoattractant gradients. The binding of chemoattractants to specific receptors leads to a wide range of biochemical responses that become highly localized as cells polarize and migrate by chemotaxis. This ability is central in the development of complex organisms, and is the result of a billion,years of evolution. Cells exposed to shallow gradients in chemoattractant concentration respond with strongly asymmetric accumulation of several factors, including the phosphoinositides PIP(3) and PIP(2), the PI 3-kinase PI3K, and phosphatase PTEN. This early symmetry-breaking stage is believed to trigger effector pathways leading to cell movement. Although many signaling factors implied in directional sensing have been recently discovered the physical mechanism of signal amplification is not yet well understood. We propose that directional sensing is the consequence of a phase ordering process mediated by phosphoinositide diffusion and driven by the distribution of the chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we have shown that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system towards phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally.

The ability of eukaryotic cells to navigate along spatial gradients of extracellular guidance cues is crucial for embryonic development, tissue regeneration, and cancer progression. One proposed model for chemotaxis is a phosphoinositide-based phase separation process, which takes place at the plasma membrane upon chemoattractant stimulation and triggers directional motility of eukaryotic cells. Here, we make available virtual-cell software that allows the execution and spatiotemporal analysis of in silico chemotaxis experiments, in which the user can control physical and chemical parameters as well as the number and position of chemoattractant sources.

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data., Comment: 4 pages, 11 eps figures