Rubén Martín

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.

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.

DIOL Triterpenes Block Profibrotic Effects of Angiotensin II and Protect from Cardiac Hypertrophy

Background The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. Methodology/Principal Findings The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. Conclusions/Significance Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ.

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 conjunctivits, 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 phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells

Human group IIA secreted phospholipase A2 (sPLA2‐IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA2‐IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA2‐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 sPLA2‐IIA‐induced cell proliferation. We found that sPLA2‐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, sPLA2‐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 sPLA2‐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 sPLA2‐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.

Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding

BackgroundActivation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells.MethodsPrimary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events.ResultsThe direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA.ConclusionThese results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.

Inflammatory protein sPLA2-IIA abrogates TNFα-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.