Effects of copper sulfate-oxidized or myeloperoxidase-modified LDL on lipid loading and programmed cell death in macrophages under hypoxia

Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia

Effects of copper sulfate-oxidized or myeloperoxidase- modified LDL on lipid loading and programmed cell death in macrophages under hypoxia

Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia

PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis

Aims Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε. Methods and results Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1(Ser133) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. Conclusions PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury

Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1(-/-) mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.National Heart and Lung Institute Foundation UK charity studentship: (Charity no. 1048073); National Institute for Health Research (NIHR); Biomedical Research Centre; Imperial College Healthcare NHS; Trust and Imperial College London

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis

Étude comparative des effets des LDL oxydées avec du sulfate de cuivre ou de la myéloperoxydase sur des macrophages et des cellules endothéliales en culture – Importance de la voie de signalisation Nrf2

Atherosclerosis is an arterial inflammatory and degenerative disease characterized by a subendothelial accumulation of lipids, leading to the formation of an atheroma. The rupture of the atheroma is associated with clinical outcomes such as myocardial infarction and thrombosis. Atherosclerotic lesions develop preferentially in arterial regions subjected to disturbed or low shear stress, which represents the tangential frictional force of the blood flow at the surface of endothelial cells. These local hemodynamic conditions promote the intimal accumulation of low-density lipoproteins (LDL), especially in case of hypercholesterolemia. In the intima, LDL are modified, notably through their oxidation, and initiate an inflammatory response, promoting the recruitment of circulating monocytes by endothelial cells. The differentiation of monocytes into macrophages and the excessive and uncontrolled internalization of modified LDL lead to the formation of lipid-laden macrophages, also termed foam cells. The apoptotic death of foam cells contributes to the formation of the necrotic core and to the development of the atherosclerotic plaque. The physiological oxidizing agents implicated in LDL oxidation in vivo are still unambiguously identified. In vitro, LDL are commonly oxidized using copper sulfate, leading to the formation of LDL modified both at the lipid and protein moieties. However, the physiological relevance of copper in LDL oxidation is more and more called in question. In this way, alternative and more physiological methods of LDL oxidation have emerged, such as myeloperoxidase. In this study, we evaluated the effects of native, copper-oxidized LDL (OxLDL) and myeloperoxidase-oxidized LDL (MoxLDL) on the activation of the anti-oxidant and anti-inflammatory Nrf2-dependent signalling pathway in macrophages and endothelial cells: two cell types largely involved in the development of atherosclerotic lesions. Moreover, the MoxLDL used in this study are exclusively modified on the protein moiety. Our results showed that both types of oxidized LDL activate the Nrf2 transcription factor in macrophages, contrary to native LDL. Moreover, the activation of the Nrf2 pathway triggered by the incubation with MoxLDL is more important than the activation triggered by the incubation with OxLDL. This differential activation can be explained, at least partly, by a more important production of reactive oxygen species (ROS) in cells exposed to MoxLDL through a specific pathway implicating cytosolic PLA2. Moreover, we showed that the non-oxidized lipids of MoxLDL are certainly involved in this ROS production. On the other hand, MoxLDL are not able to induce the activation of the Nrf2 pathway in endothelial cells, in spite of the induction of an important intracellular oxidative stress. On the contrary, the incubation of endothelial cells with OxLDL induces the activation of the Nrf2 pathway, but through a ROS-independent pathway, involving PKCs. In vivo, endothelial cells are constantly exposed to local hemodynamic forces induced by blood flow. In this study, we evaluated the effects of a high or low laminar shear stress, representative of the hemodynamic conditions in arterial regions respectively protected and susceptible to the development of atherosclerotic lesion, on the expression of genes in cultured endothelial cells. Using micro-fluidic cards in Real-time PCR, we showed that a low laminar shear stress globally induces the expression of pro-atherogenic genes in endothelial cells, whereas cells exposed to a high laminar shear stress exhibit an anti-atherogenic expression profile. Moreover, a high laminar shear stress induces the nuclear translocation of the Nrf2 transcription factor. Interestingly, the addition of oxidized LDL disturbs the endothelial response to a high shear stress. These preliminary results suggest that the study of the effects of risk factors on endothelial cells should be performed in relevant hemodynamic conditions, in order to better understand their pro-atherogenic role in vivo. In conclusion, the results of this thesis notably underline that macrophages and endothelial cells are able to discriminate copper-oxidized LDL, modified both at the lipid and protein moieties, of myeloperoxidase-oxidized LDL, more physiologically relevant and only modified on the protein fraction, and respond very differently to these two types of oxidized LDL. This reality should be kept in mind in order to better understand the physiological mechanisms implicated in atherogenesis, but also to suggest intervention studies in mice, and maybe new therapies in the future, complementary to the battery of therapeutics currently available.L’athérosclérose est une pathologie inflammatoire et dégénérative des artères caractérisée par une accumulation de lipides au niveau de l’intima conduisant à la formation d’une plaque d’athérome, dont la rupture est associée à des complications cliniques telles que l’infarctus du myocarde ou l’accident vasculaire cérébral. Les lésions athéroscléreuses se développent préférentiellement dans les régions artérielles où les forces de friction du flux sanguin au niveau de l’endothélium - « shear stress » - sont faibles ou perturbées. Ces conditions hémodynamiques locales favorisent l’accumulation subendothéliale des lipoprotéines de faible densité (LDL), particulièrement en cas d’hypercholestérolémie. Dans l’intima, les LDL sont modifiées, notamment par oxydation, et initient une réponse inflammatoire, permettant le recrutement des monocytes sanguins et leur différenciation en macrophages. L’internalisation non régulée des LDL modifiées par les macrophages, via les récepteurs « scavenger », conduit à la formation de cellules gorgées de lipides, appelées cellules spumeuses, dont la mort par apoptose contribue à la formation du cœur nécrotique et au développement de la plaque d’athérome. La nature des agents oxydants physiologiques responsables de l’oxydation des LDL est toujours sujette à controverse. In vitro, les LDL sont couramment oxydées par du sulfate de cuivre, générant des LDL modifiées au niveau protéique et lipidique. Cependant, la pertinence physiologique de cette méthode d’oxydation est de plus en plus controversée et des méthodes alternatives et plus physiologiques d’oxydation des LDL impliquant généralement des enzymes telles que la myéloperoxydase ont émergé. Au cours de ce travail, nous avons évalué les effets des LDL natives, des LDL oxydées avec du sulfate de cuivre (OxLDL) et des LDL oxydées avec de la myéloperoxydase (MoxLDL) sur l’activation de la voie anti-oxydante et anti-inflammatoire dépendante de Nrf2 dans deux types cellulaires jouant un rôle prépondérant dans le développement des lésions athéroscléreuses : les macrophages et les cellules endothéliales. Par ailleurs, les MoxLDL utilisées au cours de ce travail sont modifiées exclusivement au niveau protéique. Nos données ont montré que les LDL oxydées activent le facteur de transcription Nrf2 dans les macrophages, contrairement aux LDL natives. De plus, les MoxLDL activent la voie de signalisation Nrf2 de manière plus importante que les OxLDL. Cette activation différentielle est due à une production d’espèces réactives dérivées de l’oxygène (ROS) accrue par les MoxLDL via une voie spécifique impliquant les PLA2 cytosoliques et dans laquelle les lipides non-oxydés des LDL pourraient jouer un rôle important. D’autre part, nous avons montré que les MoxLDL sont incapables d’activer le facteur de transcription Nrf2 dans les cellules endothéliales, malgré l’induction d’un stress oxydatif important. Par contre, l’incubation des cellules endothéliales avec les OxLDL induit l’activation de la voie Nrf2, mais indépendamment des ROS et via un mécanisme impliquant les PKCs. Par ailleurs, afin de nous rapprocher des conditions de développement de la pathologie in vivo, les effets d’un « shear stress » laminaire élevé ou faible, respectivement représentatifs des conditions hémodynamiques des régions protégées ou propices au développement de l’athérosclérose, ont été évalués sur les cellules endothéliales en culture. De manière générale, nous avons montré qu’un « shear stress » faible favorise l’expression de gènes plutôt pro-athérogènes, alors qu’un « shear stress » laminaire élevé induit notamment la translocation nucléaire de Nrf2 et l’expression de ses gènes cibles, codant pour des protéines considérées comme anti-athérogènes. De manière intéressante, l’ajout de LDL oxydées altère la réponse des cellules endothéliales soumises à un « shear stress » laminaire élevé. Ces données préliminaires soulignent l’importance de l’étude des facteurs de risque de l’athérosclérose dans un environnement physiologique, et plus particulièrement de l’étude de la réponse des cellules endothéliales en conditions hémodynamiques. En conclusion, les résultats obtenus au cours de cette thèse auront notamment permis de mettre en évidence que les macrophages et les cellules endothéliales font la distinction entre une LDL modifiée au niveau protéique et lipidique, et une LDL modifiée uniquement sur la fraction protéique et probablement plus représentative d’un type de LDL oxydées in vivo, et réagissent de manière très différente à ces deux entités. C’est donc une réalité qu’il faudra à l’avenir mieux intégrer dans le processus patho-physiologique in vivo, pour continuer à mieux comprendre les mécanismes moléculaires de l’athérogenèse, mais aussi pour pouvoir proposer des études d’intervention en modèles murins et peut-être un jour de nouvelles approches thérapeutiques, complémentaires à l’arsenal de thérapies actuellement disponible.(DOCSC03) -- FUNDP, 201

Low-density lipoproteins are directly or indirectly carbamylated by myeloperoxidase

info:eu-repo/semantics/nonPublishe

Copper and myeloperoxidase-modified LDLs activate Nrf2 through different pathways of ROS production in macrophages.

Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROS-producing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggest that results obtained until now with copper oxidized-LDLs should be carefully reevaluated, taking into consideration physiologically more relevant oxidized LDLs.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe