Marita Hernández

Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1

The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such asp-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptorp-aminophenyl-α-d-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation.

Selection of the optimal stress test for the diagnosis of coronary artery disease

Objective To compare the value and limitations of exercise testing, dipyridamole echocardiography, dobutamine-atropine echocardiography, and MIBI-SPECT (technetium-99m methoxyisobutyl nitrile single photon emission computed tomography) during dobutamine infusion in the diagnosis of coronary artery disease. Design The performance of these four tests was assessed in random order on a consecutive cohort of patients. The presence or absence of coronary artery disease was confirmed by coronary angiography. Setting Two tertiary care and university centres. Patients 102 consecutive patients with chest pain and no previous history of coronary artery disease. Ten patients with left bundle branch block were excluded for further analysis of exercise testing and scintigraphy results. Results MIBI-SPECT was the most sensitive (87%) but the least specific test (70%). Exercise stress testing had a sensitivity of 66%, which increased to 80% when patients with inconclusive results were excluded. Dipyridamole and dobutamine echocardiography had similar sensitivity (81%, 78%) and specificity (94%, 88%). All four tests had similar accuracy and positive and negative predictive values. Agreement between the echocardiographic techniques was excellent (detection of coronary artery disease 87%, κ = 0.72; regional analysis 93%, κ = 0.72; diagnosis of the “culprit” vessel 95%, κ = 0.92), and it was good between echocardiographic techniques and MIBI-SPECT (diagnosis of the culprit vessel 90%, κ = 0.84 with dobutamine and 92%, κ = 0.85 with dipyridamole). Conclusions Exercise stress testing has a sensitivity comparable to other tests in patients capable of exercising and with no basal electrical abnormalities. The greatest sensitivity is offered by MIBI-SPECT and the greatest specificity is obtained with stress echocardiography. Redundant information is obtained with dipyridamole echocardiography, dobutamine echocardiography, and MIBI-SPECT.

Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by Tumor necrosis factor-α : Phosphorylation of cytosolic phospholipase A2 and transactivation of cyclooxygenase-2

Abstract : Tumor necrosis factor‐α (TNF‐α) is a cytokine that elicits cell responses by activating the mitogen‐activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor‐kB (NF‐kB). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase‐2 (COX‐2), the effect of TNF‐α on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF‐α produced a phosphorylation of cPLA2, which was preceded by an activation of both c‐Jun N‐terminal kinase (JNK) and p38‐MAP kinase, and this was associated with the release of [3H]AA. In contrast, TNF‐α did not activate the extracellular signal‐regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic response. Analysis of [3H]AA metabolites by reverse‐phase HPLC showed that all of the [3H]AA released during the first hour after TNF‐α addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF‐α, 25% of the [3H]AA had been converted into COX products as compared with only 9% in control cells. In keeping with these findings, TNF‐α produced an increase of COX‐2 expression, as judged from both RT‐PCR studies and immunoblot of COX‐2 protein, and a long‐lasting activation of NF‐kB. These data show that TNF‐α produces in astrocytoma cells an early activation of both p38‐MAP kinase and JNK, which is followed by the phosphorylation of cPLA2 and the release of AA. On the other hand, the activation of NF‐kB may explain the induction of the expression of COX‐2 and the delayed generation of prostanoids.

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.

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 occuring 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.

Biological effects of group IIA secreted phosholipase A2

Group IIA secreted phospholipase A2 (sPLA2‐IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A2, which shows properties different from those displayed by the cytosolic phospholipase A2 involved in the release of arachidonic acid. sPLA2‐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 sPLA2‐IIA on intracellular signaling pathways.

Cooperation Between Secretory Phospholipase A2 and TNF-Receptor Superfamily Signaling Implications for the Inflammatory Response in Atherogenesis

Abstract— 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 &agr; 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-&agr;, and sPLA2, thus allowing the development of distinct patterns of response in monocytic cells.

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.

Natural triterpenes modulate immune-inflammatory markers of experimental autoimmune encephalomyelitis: therapeutic implications for multiple sclerosis

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases that develop as a result of deregulated immune responses causing glial activation and destruction of CNS tissues. Oleanolic acid and erythrodiol are natural triterpenes that display strong anti‐inflammatory and immunomodulatory activities. Oleanolic acid beneficially influences the course of established EAE. We now extend our previous observations to erythrodiol and address the efficacy of both compounds to protect against EAE, given under different regimens.

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.