Abdominal aortic aneurysm (AAA) is a disease of the aorta characterized by pathological remodeling and progressive weakening of the vessel resulting in the increased risk of rupture and sudden death. In a mouse model of the disease induced by chronic Angiotensin II (AngII) infusion, progression of AAAs is associated with reduced differentiation of smooth muscle cells (SMCs) at the site of lesion development. In the mouse model, the effectiveness of cyclooxygenase-2 (COX-2) inhibition for attenuating AAA progression is associated with maintenance of a differentiated SMC phenotype. However, the safety of COX-2 inhibitors is currently in question due to the increased risk of adverse cardiovascular events. Thus, it is crucial to identify mediators downstream of COX-2 that may provide new targets for treatment of this disease. Recent studies in humans and mouse models have suggested that the microsomal prostaglandin E synthase (mPGES-1) enzyme, which acts downstream of COX-2, may also be involved in the pathogenesis of the disease. We hypothesized that increased prostaglandin E2 (PGE2) synthesis resulting from the induction of both COX-2 and mPGES-1 may result in reduced differentiation of SMCs, and that disruption of this pathway would preserve the differentiated phenotype. To test this hypothesis, human aortic smooth muscle cells (hASMCs) were utilized to examine the effects of a variety of agents involved in AAA development and the COX-2 pathway. My findings suggest that one of the effects of exposing hASMCs to AngII involves a specific induction of mPGES-1 expression. Furthermore, although different COX-2-derived products may have opposing effects, mPGES-1-derived PGE2 may be the primary prostanoid synthesized by SMCs which functions to attenuate differentiation. Therefore, mPGES-1 inhibition may provide inhibition of PGE2 that is more specific than COX-2 inhibitor treatment and may serve as a therapeutic target for attenuating AAA progression by maintaining a differentiated SMC phenotype.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

Human skin has distinct lipid metabolism and production of bioactive lipid mediators that can be modulated by nutritional supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFA), of which eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids exert anti-inflammatory effects. The aims of this project were to gain better understanding of their individual mechanisms in human epidermis and dermis. HaCaT keratinocytes, 46BR.1N fibroblasts, primary human epidermal keratinocytes and dermal fibroblasts were treated with EPA or DHA for 72h and then sham-irradiated or exposed to 15 mJ/cm2 ultraviolet radiation (UVR). Viability was measured by the MTT assay. The expression of cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) proteins was explored by western blotting. Human skin explants (n=4 donors) were cultured for 3 or 6 days and supplemented with EPA, DHA or vehicle. Culture media were collected to evaluate tissue damage and PUFA cytotoxicity (lactate dehydrogenase assay). Epidermal and dermal lipid profiles were assessed by gas chromatography and liquid chromatography coupled to tandem mass spectrometry. Primary keratinocytes were treated with fatty acids and various lipid mediators for 48h. Their effect was determined by the scratch assay and transepithelial electrical resistance. UVR upregulated COX-2 in HaCaT and primary epidermal keratinocytes, but did not affect mPGES-1 and 15-PGDH protein expression. UVR upregulated COX-2 and mPGES-1 in 46BR.1N fibroblasts but had no effect on 15-PGDH expression. The same UVR dose did not alter the expression of COX-2, mPGES-1 and 15-PGDH in primary dermal fibroblasts. Only EPA attenuated COX-2 expression in HaCaT and primary keratinocytes and either EPA or DHA had any effect in 46BR.1N and primary fibroblasts. Skin explants showed initial post-biopsy tissue damage. EPA and DHA supplementation augmented cellular levels of the corresponding fatty acids in both epidermis and dermis to a different extent. Increased uptake of DHA in the dermis was accompanied by reduced arachidonic acid levels. EPA treatment stimulated the production of PGE3 and various HEPE in epidermis, while DHA treatment caused high levels of HDHA species in dermis. N-3 PUFA and their derivatives delayed wound healing, cell migration and epidermal barrier permeability, while n-6 PUFA lipids showed the opposite effect. Overall, these findings suggest that EPA and DHA differently affect skin cells and skin, with EPA preference in epidermis and DHA in the dermis. These results highlight the importance of differential skin responses that could be important in skin health and disease.