Effect of resistance exercise training on plasma neurofilaments in multiple sclerosis: a proof of concept for future designs.

Multiple sclerosis (MS) is a dysimmune and neurodegenerative disease of the central nervous system that continues to be one of the main causes of non-traumatic disability in young people despite the recent availability of highly effective drugs. Exercise-based interventions seem to have a positive impact on the course of the disease although pathophysiological mechanisms responsible for this benefit remain unclear. This is a longitudinal study to examine the effects of a short-term training program on neurofilament plasma levels, a biomarker of axonal destruction, measured using the ultrasensitive single molecule array (SiMoA). Eleven patients completed a 6-week supervised resistance-training program of 18 sessions that consisted of 3 sets of 8-10 repetitions of 7 exercises. Median plasma neurofilament levels significantly decreased from baseline (6.61 pg/ml) to 1 week after training intervention (4.44 pg/ml), and this effect was maintained after 4 weeks of detraining (4.38 pg/ml). These results suggest a neuroprotective effect of resistance training in this population and encourage us to investigate further the beneficial impact of physical exercise and to emphasize the importance of lifestyle in MS.

Treatment with the Olive Secoiridoid Oleacein Protects against the Intestinal Alterations Associated with EAE.

Multiple sclerosis (MS) is a CNS inflammatory demyelinating disease. Recent investigations highlight the gut-brain axis as a communication network with crucial implications in neurological diseases. Thus, disrupted intestinal integrity allows the translocation of luminal molecules into systemic circulation, promoting systemic/brain immune-inflammatory responses. In both, MS and its preclinical model, the experimental autoimmune encephalomyelitis (EAE) gastrointestinal symptoms including "leaky gut" have been reported. Oleacein (OLE), a phenolic compound from extra virgin olive oil or olive leaves, harbors a wide range of therapeutic properties. Previously, we showed OLE effectiveness preventing motor defects and inflammatory damage of CNS tissues on EAE mice. The current studies examine its potential protective effects on intestinal barrier dysfunction using MOG35-55-induced EAE in C57BL/6 mice. OLE decreased EAE-induced inflammation and oxidative stress in the intestine, preventing tissue injury and permeability alterations. OLE protected from EAE-induced superoxide anion and accumulation of protein and lipid oxidation products in colon, also enhancing its antioxidant capacity. These effects were accompanied by reduced colonic IL-1ß and TNFα levels in OLE-treated EAE mice, whereas the immunoregulatory cytokines IL-25 and IL-33 remained unchanged. Moreover, OLE protected the mucin-containing goblet cells in colon and the serum levels of iFABP and sCD14, markers that reflect loss of intestinal epithelial barrier integrity and low-grade systemic inflammation, were significantly reduced. These effects on intestinal permeability did not draw significant differences on the abundance and diversity of gut microbiota. However, OLE induced an EAE-independent raise in the abundance of Akkermansiaceae family. Consistently, using Caco-2 cells as an in vitro model, we confirmed that OLE protected against intestinal barrier dysfunction induced by harmful mediators present in both EAE and MS. This study proves that the protective effect of OLE in EAE also involves normalizing the gut alterations associated to the disease.

Anti-Neuroinflammatory Potential of a Nectandra angustifolia (Laurel Amarillo) Ethanolic Extract.

Microglia, the resident macrophage-like population in the CNS, plays an important role in the pathogenesis of many neurodegenerative disorders. Nectandra genus is known to produce different metabolites with anti-inflammatory, anti-oxidant and analgesic properties. Although the species Nectandra angustifolia is popularly used for the treatment of different types of inflammatory processes, its biological effects on neuroinflammation have not yet been addressed. In this study, we have investigated the role of a Nectandra angustifolia ethanolic extract (NaE) in lipopolysaccharide (LPS)-induced neuroinflammation in vitro and in vivo. In LPS-activated BV2 microglial cells, NaE significantly reduced the induced proinflammatory mediators TNF-α, IL-1ß, IL-6, COX-2 and iNOS, as well as NO accumulation, while it promoted IL-10 secretion and YM-1 expression. Likewise, reduced CD14 expression levels were detected in microglial cells in the NaE+LPS group. NaE also attenuated LPS-induced ROS and lipid peroxidation build-up in BV2 cells. Mechanistically, NaE prevented NF-κB and MAPKs phosphorylation, as well as NLRP3 upregulation when added before LPS stimulation, although it did not affect the level of some proteins related to antioxidant defense such as Keap-1 and HO-1. Additionally, we observed that NaE modulated some activated microglia functions, decreasing cell migration, without affecting their phagocytic capabilities. In LPS-injected mice, NaE pre-treatment markedly suppressed the up-regulated TNF-α, IL-6 and IL-1ß mRNA expression induced by LPS in brain. Our findings indicate that NaE is beneficial in preventing the neuroinflammatory response both in vivo and in vitro. NaE may regulate microglia homeostasis, not only restraining activation of LPS towards the M1 phenotype but promoting an M2 phenotype.

Oleacein Attenuates the Pathogenesis of Experimental Autoimmune Encephalomyelitis through Both Antioxidant and Anti-Inflammatory Effects.

Oxidative stress and proinflammatory cytokines are factors affecting multiple sclerosis (MS) disease progression. Oleacein (OLE), an olive secoiridoid, possesses powerful antioxidant and anti-inflammatory activities, which suggests its potential application to treat neuroinflammatory disorders. Herein, we investigated the impact of OLE on the main clinic-pathological features of experimental autoimmune encephalomyelitis (EAE), an animal model for MS, including paralysis, demyelination, central nervous system (CNS) inflammation/oxidative stress and blood-brain barrier (BBB) breakdown. METHODS: Mice were immunized with the myelin oligodendrocyte glycoprotein peptide, MOG35-55, to induce EAE, and OLE was administrated from immunization day. Serum, optic nerve, spinal cord and cerebellum were collected to evaluate immunomodulatory activities at a systemic level, as well as within the CNS. Additionally, BV2 microglia and the retinal ganglion cell line RGC-5 were used to confirm the direct effect of OLE on CNS-resident cells. RESULTS: We show that OLE treatment effectively reduced clinical score and histological signs typical of EAE. Histological evaluation confirmed a decrease in leukocyte infiltration, demyelination, BBB disruption and superoxide anion accumulation in CNS tissues of OLE-treated EAE mice compared to untreated ones. OLE significantly decreased expression of proinflammatory cytokines (IL-13, TNFα, GM-CSF, MCP-1 and IL-1ß), while it increased the anti-inflammatory cytokine IL-10. Serum levels of anti-MOG35-55 antibodies were also lower in OLE-treated EAE mice. Further, OLE significantly diminished the presence of oxidative system parameters, while upregulated the ROS disruptor, Sestrin-3. Mechanistically, OLE prevented NLRP3 expression, phosphorylation of p65-NF-κB and reduced the synthesis of proinflammatory mediators induced by relevant inflammatory stimuli in BV2 cells. OLE did not affect viability or the phagocytic capabilities of BV2 microglia. In addition, apoptosis of RGC-5 induced by oxidative stressors was also prevented by OLE. CONCLUSION: Altogether, our results show that the antioxidant and anti-inflammatory OLE has neuroprotective effects in the CNS of EAE mice, pointing out this natural product as a candidate to consider for research on MS treatments.

Oleanolic acid ameliorates intestinal alterations associated with EAE.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease affecting the CNS. Recent studies have indicated that intestinal alterations play key pathogenic roles in the development of autoimmune diseases, including MS. The triterpene oleanolic acid (OA), due to its anti-inflammatory properties, has shown to beneficially influence the severity of the experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. We herein investigate EAE-associated gut intestinal dysfunction and the effect of OA treatment. METHODS: Mice with MOG35-55-induced EAE were treated with OA or vehicle from immunization day and were daily analyzed for clinical deficit. We performed molecular and histological analysis in serum and intestinal tissues to measure oxidative and inflammatory responses. We used Caco-2 and HT29-MTX-E12 cells to elucidate OA in vitro effects. RESULTS: We found that OA protected from EAE-induced changes in intestinal permeability and preserved the mucin-containing goblet cells along the intestinal tract. Serum levels of the markers for intestinal barrier damage iFABP and monocyte activation sCD14 were consistently and significantly reduced in OA-treated EAE mice. Beneficial OA effects also included a decrease of pro-inflammatory mediators both in serum and colonic tissue of treated-EAE mice. Moreover, the levels of some immunoregulatory cytokines, the neurotrophic factor GDNF, and the gastrointestinal hormone motilin were preserved in OA-treated EAE mice. Regarding oxidative stress, OA treatment prevented lipid peroxidation and superoxide anion accumulation in intestinal tissue, while inducing the expression of the ROS scavenger Sestrin-3. Furthermore, short-chain fatty acids (SCFA) quantification in the cecal content showed that OA reduced the high iso-valeric acid concentrations detected in EAE-mice. Lastly, using in vitro cell models which mimic the intestinal epithelium, we verified that OA protected against intestinal barrier dysfunction induced by injurious agents produced in both EAE and MS. CONCLUSION: These findings reveal that OA ameliorates the gut dysfunction found in EAE mice. OA normalizes the levels of gut mucosal dysfunction markers, as well as the pro- and anti-inflammatory immune bias during EAE, thus reinforcing the idea that OA is a beneficial compound for treating EAE and suggesting that OA may be an interesting candidate to be explored for the treatment of human MS.

Secreted Phospholipase A2-IIA Modulates Transdifferentiation of Cardiac Fibroblast through EGFR Transactivation: An Inflammation-Fibrosis Link.

Secreted phospholipase A2-IIA (sPLA2-IIA) is a pro-inflammatory protein associated with cardiovascular disorders, whose functions and underlying mechanisms in cardiac remodelling are still under investigation. We herein study the role of sPLA2-IIA in cardiac fibroblast (CFs)-to-myofibroblast differentiation and fibrosis, two major features involved in cardiac remodelling, and also explore potential mechanisms involved. In a mice model of dilated cardiomyopathy (DCM) after autoimmune myocarditis, serum and cardiac sPLA2-IIA protein expression were found to be increased, together with elevated cardiac levels of the cross-linking enzyme lysyl oxidase (LOX) and reactive oxygen species (ROS) accumulation. Exogenous sPLA2-IIA treatment induced proliferation and differentiation of adult rat CFs. Molecular studies demonstrated that sPLA2-IIA promoted Src phosphorylation, shedding of the membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) ectodomain and EGFR phosphorylation, which triggered phosphorylation of ERK, P70S6K and rS6. This was also accompanied by an up-regulated expression of the bone morphogenic protein (BMP)-1, LOX and collagen I. ROS accumulation were also found to be increased in sPLA2-IIA-treated CFs. The presence of inhibitors of the Src/ADAMs-dependent HB-EGF shedding/EGFR pathway abolished the CF phenotype induced by sPLA2-IIA. In conclusion, sPLA2-IIA may promote myofibroblast differentiation through its ability to modulate EGFR transactivation and signalling as key mechanisms that underlie its biological and pro-fibrotic effects.

A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells.

Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications.

Oleanolic acid controls allergic and inflammatory responses in experimental allergic conjunctivitis.

Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivitis, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A2 type-IIA (sPLA2-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA2-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases.

Secreted PLA2 induces proliferation in astrocytoma through the EGF receptor: another inflammation-cancer link.

We have investigated mechanisms that contribute to reinforce the relationship between inflammation and cancer. Secreted phospholipase A(2) group IIA (sPLA(2)-IIA) is a molecule relevant in inflammatory events and has been proposed as a marker for some of these. Previously, we reported the mitogenic properties of this sPLA(2) in the human astrocytoma cell line 1321N1. Here, we go deeper into the mechanisms that link this inflammatory protein with proliferation in one of the most aggressive types of tumors. We found that phosphorylation of the extracellular regulated kinase (ERK) was preceded by the activation of the small GTPase Ras, and both failed to be activated by inhibiting protein kinase C (PKC). Fractionation and immunofluorescence studies revealed translocation of PKC alpha, delta, and epsilon to the membrane fraction upon stimulation with sPLA(2)-IIA. Immunoprecipitation analysis showed that sPLA(2)-IIA induces phosphorylation of the epidermal growth factor receptor (EGFR) through a PKC-dependent pathway. We found that phosphorylation of this receptor contributed to Ras and ERK activation and that inhibition of ERK, PKC, and EGFR blocked the mitogenic response induced by sPLA(2)-IIA. This study showed that sPLA(2)-IIA is able to bring into play EGFR to trigger its signaling and that PKC leads the distribution of resources. Interestingly, we found that this is not a cell-specific response, because sPLA(2)-IIA was also able to transactivate EGFR in MCF7 human breast cancer cells. Therefore, this mechanism could contribute to worsen the prognosis of a tumor in an inflammatory microenvironment. We also present more links of the tumor chain possibly susceptible to targeting.

Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease for which there exist no therapies without undesired side effects. Thus, the establishment of less toxic treatments is an ongoing challenge. Nowadays, research on medicinal plants has been attracting much attention, since screening of its active principles could prove useful in identification of safe and innovative pharmaceutical molecules. In this study we investigated the therapeutic effect of oleanolic acid (OA) a plant-derived triterpene with potent anti-inflammatory and immunomodulatory activities, whose actions on CNS diseases remain far from completely characterized. We focussed on the potential therapeutic effect of oleanolic acid (OA) on an accepted experimental model of MS, the experimental autoimmune encephalomyelitis (EAE). We have found that OA treatment, before or at the early onset of EAE, ameliorates neurological signs of EAE-mice. These beneficial effects of OA seem to be associated with a reduction of blood-brain barrier leakage and lower infiltration of inflammatory cells within the CNS, as well as with its modulatory role in Th1/Th2 polarization: inhibition of proinflammatory cytokines and chemokines, and stimulation of anti-inflammatory ones. Moreover, EAE-animals that were treated with OA had lower levels of anti-MOG antibodies than untreated EAE-mice. Our findings show that the administration of the natural triterpenoid OA reduces and limits the severity and development of EAE. Therefore, OA therapy might be of clinical interest for human MS and other Th1 cell-mediated inflammatory diseases.

Inflammatory protein sPLA(2)-IIA abrogates TNFalpha-induced apoptosis in human astroglioma cells: Crucial role of ERK.

Brain injury induces the expression of well-known cytokines, such as tumor necrosis factor-alpha (TNFalpha), and other, which functions are less understood, as secreted phospholipase A(2) group IIA (sPLA(2)-IIA). Since in pathological processes, cytokines function coordinately in networks, to further explore the actions of sPLA(2)-IIA in tumorigenesis, we investigated the effect of sPLA(2)-IIA in the presence of TNFalpha in human 1321N1 astrocytoma cells. In these cells, TNFalpha activates the apoptotic programme that is accompanied of cytoskeleton changes; however, simultaneous treatment with sPLA(2)-IIA prevents TNFalpha-mediated apoptosis and reverses the modification of the markers associated to this response. In fact, the mitogenic activity elicited by the phospholipase alone is preserved. This inhibitory effect is not found in other TNFalpha-mediated responses, even a functional cooperation is observed on COX-2 protein induction. The cross-talk between TNFalpha and sPLA(2)-IIA is associated with ERK activity since its pharmacological inhibition attenuates both synergistic and inhibitory responses. We have also observed that upon sPLA(2)-IIA stimulation, endogenous ERK has the capacity to bind and phosphorylate sequences present within the cytoplasmic domain of TNFR1/CD120a. These findings thus indicate that sPLA(2)-IIA and TNFalpha transduction pathways interact to modulate inflammatory responses and provide additional insights about the capacity of sPLA(2)-IIA to promote apoptosis resistance in astrocytoma cells.

Secreted phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells.

Human group IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA(2)-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA(2)-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA(2)-IIA-induced cell proliferation. We found that sPLA(2)-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA(2)-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA(2)-IIA treatment, effect that was abrogated by N-acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N-acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA(2)-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.

Natural triterpenic diols promote apoptosis in astrocytoma cells through ROS-mediated mitochondrial depolarization and JNK activation.

BACKGROUND: Triterpene alcohols and acids are multifunctional compounds widely distributed throughout the plant kingdom that exhibit a variety of beneficial health properties, being synthetic analogs of oleanolic acid under clinical evaluation as anti-tumoral therapeutic agents. However, the antineoplastic activity of two natural occurring triterpenoid alcohols extracted from olive oil, erythrodiol (an intermediate from oleanolic acid), and its isomer, uvaol, has barely been reported, particularly on brain cancer cells. Astrocytomas are among the most common and aggressive type of primary malignant tumors in the neurological system lacking effective treatments, and in this study, we addressed the effect of these two triterpenic diols on the human 1321N1 astrocytoma cell line. PRINCIPAL FINDINGS: Erythrodiol and uvaol effectively affected cell proliferation, as well as cell cycle phases and induced 1321N1 cell death. Both triterpenes successfully modulated the apoptotic response, promoting nuclear condensation and fragmentation. They caused retraction and rounding of cultured cells, which lost adherence from their supports, while F-actin and vimentin filaments disappeared as an organized cytoplasmic network. At molecular level, changes in the expression of surface proteins associated with adhesion or death processes were also observed. Moreover, triterpene exposure resulted in the production of reactive oxygen species (ROS) with loss of mitochondrial transmembrane potential, and correlated with the activation of c-Jun N-terminal kinases (JNK). The presence of catalase reversed the triterpenic diols-induced mitochondrial depolarization, JNK activation, and apoptotic death, indicating the critical role of ROS in the action of these compounds. CONCLUSIONS: Overall, we provide a significant insight into the anticarcinogenic action of erythrodiol and uvaol that may have a potential in prevention and treatment of brain tumors and other cancers.

Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis.

AIMS: Human atherosclerotic plaques express markers of macrophage/dendritic cells as well as high levels of inflammatory proteins such as secreted phospholipase A(2) type IIA (sPLA(2)-IIA). To understand the cellular changes associated with the progress of atherosclerosis, we evaluated the role of sPLA(2)-IIA in mediating monocyte recruitment and differentiation into antigen-presenting cells. METHODS AND RESULTS: The effect of sPLA(2)-IIA on monocyte differentiation was evaluated in human THP-1 cells, a cellular line widely used as a model for monocyte-macrophage differentiation. Changes in functional processes, morphology and expression of antigens, characteristic of differentiated cells, were monitored over a 1-3 day period. sPLA(2)-IIA inhibited CD14 expression in a time- and concentration-dependent manner and upregulated dendritic cell-specific ICAM-3 grabbing non-integrin levels at the cell surface, findings that were the same for human monocytes. In addition, sPLA(2)-IIA-differentiated cells showed a dendritic cell phenotype characterized by the generation of fine dendritic protrusions and an increase in surface markers such as CD40, CD83, CD54, CD61, and CD62L. Furthermore, cell adhesion, migration, endocytic activity, and allogeneic T cell proliferation capacity were markedly increased after sPLA(2)-IIA treatment. CONCLUSION: sPLA(2)-IIA induces the differentiation of mononuclear cells and increases their adhesive and migratory capabilities, which suggests a novel function for sPLA(2)-IIA as a mediator connecting innate and adaptive immunity. These findings may provide insight into the immuno-inflammatory processes occurring in atherosclerosis, helping us to understand the cellular changes associated with the development of atherosclerosis.

Acidic triterpenes compromise growth and survival of astrocytoma cell lines by regulating reactive oxygen species accumulation.

Several studies have shown how pentacyclic triterpenes can inhibit proliferation and induce apoptosis of some tumor cell lines; however, its effect on astrocytic tumors, one of the most malignant forms of cancer, has rarely been reported. The aim of this study was to examine how the pentacyclic triterpenes, oleanolic acid and maslinic acid, isolated from olive juice, affected astrocytoma cell morphology and survival. Cell proliferation was inhibited in 1321N1 astrocytoma cells by using 1 to 50 micromol/L of either oleanolic acid or maslinic acid, with an average IC(50) of 25 micromol/L. Growth inhibition led to morphologic and cytoskeletal alterations associated with the loss of stellate morphology and characterized by a retraction of the cytoplasm and collapse of actin stress fibers. Using 4',6-diamidino-2-phenylindole and Annexin V, we showed that astrocytoma cell death induced by oleanolic acid or maslinic acid were mainly due to apoptotic events. Furthermore, we showed that caspase-3 is activated as a consequence of triterpene treatment. Finally, we found that exposure of the cells to oleanolic acid or maslinic acid resulted in a significant increase of intracellular reactive oxygen species, followed by loss of mitochondrial membrane integrity. Importantly, enzymatic scavengers, such as catalase, or phenolic antioxidants, such as butylated hydroxytoluene, rescued cells from the triterpene-mediated apoptosis, suggesting that the potential therapeutic effect of these acidic triterpenes is dependent on oxidative stress. Our data show that acidic triterpenes play a major role in 1321N1 astrocytoma morphology and viability and support the conclusion that oleanolic acid and maslinic acid may thus be promising new agents in the management of astrocytomas.

Cooperation between secretory phospholipase A2 and TNF-receptor superfamily signaling: implications for the inflammatory response in atherogenesis.

Atherogenesis is the consequence of a variety of effector mechanisms rather than the result of a single functional molecule. In this connection, type IIA secretory phospholipase A2 (sPLA2) is an acute-phase reactant, which accumulates in atherosclerotic arterial walls, elicits several effects on monocytes, and has been related to the development of atherosclerosis. CD40/CD40 ligand pair is also a strong proatherogenic system. sPLA2 produced an increase of the surface expression of CD40 in THP-1 monocytes and enhanced the effect of CD40 ligation on the expression of both Fas and FasL, thus indicating the existence of a positive cooperation between sPLA2 and different elements of the TNF-receptor superfamily. Activation of the CD40/CD40L dyad with anti-CD40 antibody produced a small release of arachidonic acid and lacked any significant effect on the induction of cyclooxygenase-2, whereas the secretion of the chemokine MCP-1 and the surface display of CD11b, the alpha chain of the integrin Mac-1, were upregulated. Engagement of CD40 did not influence the survival of THP-1 monocytes, but coincubation of THP-1 monocytes pretreated with anti-CD40 antibody and Jurkat cells induced a significant increase of the number of Jurkat cells showing binding of annexin-V, and nuclear condensation and fragmentation, thus indicating that this treatment might trigger a juxtacrine/paracrine mechanism of apoptotic death in sensitive cell types. This data indicates the existence of overlapping routes for the response to CD40, TNF-alpha, and sPLA2, thus allowing the development of distinct patterns of response in monocytic cells.

Biological effects of group IIA secreted phosholipase A(2).

Group IIA secreted phospholipase A(2) (sPLA(2)-IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A(2), which shows properties different from those displayed by the cytosolic phospholipase A(2) involved in the release of arachidonic acid. sPLA(2)-IIA behaves as a ligand for a group of receptors inside the C-type multilectin mannose receptor family and also interacts with heparan sulfate proteoglycans such as glypican, the dermatan/chondroitin sulfate-rich decorin, and the chondroitin sulfate-rich versican, thus being able to internalize to specific compartments within the cell and producing biological responses. This review provides a short summary of the biological actions of sPLA(2)-IIA on intracellular signaling pathways.

Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis.

Type IIA secretory phospholipase A(2) (sPLA(2)) is an acute-phase reactant that plays a role in atherogenesis and is expressed in atherosclerotic arterial walls displaying inflammatory features. This generates a relevant question addressing the biological effects of this enzyme on monocytic cells, in view of the role of these cells in the inflammatory process associated with atherosclerosis. sPLA(2) produced a mild activation of the p42 mitogen-activated protein module of the mitogen-activated protein kinase (MAPK) cascade and a prominent activation of c-Jun N-terminal kinase in THP-1 monocytes. This activation showed both an early and a late peak, different from that elicited by tumor necrosis factor-alpha (TNF-alpha), which only showed the first peak. This was accompanied by activation of arachidonate metabolism, as judged from both the activation of the cytosolic phospholipase A(2) (cPLA(2)) and the induction of cyclooxygenase-2 (COX-2) expression. sPLA(2) also elicited the production of monocyte chemoattractant protein-1 (MCP-1) and showed a synergistic effect with TNF-alpha on both COX-2 induction and MCP-1 production. sPLA(2) upregulated the expression of Fas ligand at the cell surface, but it did not influence Fas expression nor cell survival of monocytes. In summary, these data indicate that some of the atherogenic effects of sPLA(2) can be exerted by engagement of an sPLA(2)-binding structure on monocytic cells, most probably the M-type receptor for sPLA(2), which produces the activation of the MAPK cascade, induces a proinflammatory phenotype, and upregulates the cell surface expression of Fas ligand.