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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

L’arthrose (OA) est la maladie articulaire la plus répandue dans le monde faisant l’objet de nombreux travaux de recherche en raison de son lourd impact socioéconomique. Plusieurs travaux dans ce domaine ont pour objectif de déterminer les mécanismes moléculaires impliqués dans sa physiopathologie. Plusieurs travaux ont appuyés l’implication de la prostaglandine (E2) PGE2 dans sa physiopathologie, contrairement à la prostaglandine (D2) (PGD2) dont le rôle reste à déterminer. C’est pourquoi, nous nous sommes penchés dans cette thèse à l’étude de cette dernière molécule. Dans la première partie de nos travaux, nous avons montré que la PGD2 diminue au niveau du cartilage articulaire et au niveau niveau des explants de cartilage humains, la production des métalloprotéases-1(MMP-1) et MMP-13 induites par (Interleukine-1β) l’IL-1β. Cette diminution de la production protéique est accompagnée d’une diminution de l’expression au niveau de l’ARNm, et d’une diminution de l’activité du promoteur de MMP-1 et MMP-13. Cet effet est exercé via le récepteur D prostanoïde (DP1), bien que le Chemoattractant receptor expressed on Th2 cells (CRTH2) soit également exprimé chez les chondrocytes humains, mais ne semble pas être impliqué dans l’effet observé. Cette action inhibitrice se fait via la voie DP1/AMPc/protéine kinase A (AMPc/PKA). Dans la suite de nos travaux, nous avons montré pour la première fois l’expression des prostaglandines D-synthases responsables de la biosynthèse de la PGD2 au niveau des chondrocytes humains par immunohistochimie, avec des niveaux d’expression de l’ARNm plus élevés de la L-PGDS au niveau du cartilage OA comparativement au cartilage normal. L’IL-1β pourrait être responsable de cette augmentation via l’activation de la voie JNK et p38 MAPK, ainsi que par la voie NF-κB. L’ensemble de ces données indiquent que la modulation des niveaux de la PGD2 au niveau de l’articulation pourrait être pourvue d’un important potentiel thérapeutique. La L-PGDS pour sa part semble avoir un rôle important dans la physiopathologie de l’OA. / Osteoarthritis (OA) is the most common joint disease world wide, because of its higher socioeconomic impact it is one of the most studied joint diseases. The aims of these studies was to determine the molecular mechanisms involved in the pathophysiology of osteaarthritis. Previous studies have mainly focused on the involvement of prostaglandin (E2) PGE2 in contrast to PGD2 in the pathogenesis osteoarthritis as such the role of PGD2 remains unclear. In this thesis we examined the involvment of PGD2 in the pathogenesis of OA. In the first part of our work, we showed that in a dose dependent manner PGD2 decreased the interleukin-1β (IL-1β)–induced mettalloproteases (MMP-1) and MMP-13 expression both at protein and mRNA levels by supression of their promoter activity. The inhibitory effect was exerted via the D prostanoid receptor (DP1) and mediated through the cAMP/protein kinase A (PKA) signalling pathway. Although human chondrocytes do express the Chemoattractant Receptor Expressed on Th2 cells (CRTH2) the latter were not implicated in the inhibiton of MMP-1 and MMP-13. In the second part of our work, we showed the expression of prostaglandin D synthases (PGDS) responsible for the biosynthesis of PGD2 in human chondrocytes, with higher levels of mRNA expression of lipocaline type prostaglandin D-synthase (L-PGDS) in OA cartilage compared to normal cartilage. IL-1β may be responsible for this increase via the activation of Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK), as well as the nuclear factor-κB (NF-κB). Together, these data indicate that modulation of the levels of PGD2 at the joint may be provided with an important therapeutic potential. L-PGDS in turn seems to have an important role in the pathogenesis of OA.

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Dietary fat quality influences the risk of type 2 diabetes and cardiovascular disease. A low-grade inflammation is suggested to contribute to the disease development, often accompanied by obesity. Whereas n-3 polyunsaturated fatty acids (PUFA) have been considered anti-inflammatory, n-6 PUFA have been proposed to act pro-inflammatory. Saturated fatty acids (SFA) act pro-inflammatory in vitro. This thesis aimed to investigate effects of different fatty acids on low-grade inflammation in observational and interventional studies. In Paper I and II, fatty acid composition in serum cholesterol esters was used as objective marker of dietary fat quality and related to serum C-reactive protein (CRP) and other circulating inflammatory markers in two population-based cohorts, conducted in middle-aged men and elderly men and women, respectively. In Paper III and IV, the impact of diets differing in fat quality on inflammation and oxidative stress was investigated in randomised controlled studies, in subjects with metabolic syndrome and abdominal obesity. In Paper I and II, a low proportion of linoleic acid (18:2 n-6) in serum was associated with higher CRP concentrations, indicating that a low intake of vegetable fats may be related to low-grade inflammation. High CRP concentrations were also associated with high proportions of palmitoleic (16:1) and oleic (18:1) acids and high stearoyl coenzymeA desaturase index, possibly reflecting altered fat metabolism and/or high SFA intake in this population. When comparing two high-fat diets rich in either saturated or monounsaturated fat, and two low-fat diets with or without long-chain n-3 PUFA supplementation during 12 weeks (Paper III), no differences in inflammation or oxidative stress markers were observed. Moreover, a 10-week intervention (Paper IV) with high linoleic acid intake showed no adverse effects on inflammation or oxidative stress. Instead, interleukin-1 receptor antagonist and tumor necrosis factor receptor-2 decreased after linoleic acid intake compared with a diet high in SFA. The results in this thesis indicate that dietary n-6 PUFA found in vegetable fats is associated with lower inflammation marker levels, and to some extent reduces systemic inflammation when compared with SFA. Supplementation of n-3 PUFA did not exert any systemic anti-inflammatory effects, maybe due to a relatively low dose.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of hematological disorders. However, to be effective, transplanted HSCs must efficiently “home” to supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution, or in some cases, bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation efficiency. We have previously shown that ex vivo treatment of mouse or human HSCs with 16-16 dimethyl PGE2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting in part from upregulation of surface CXCR4 expression. We now show that pulse-treatment of mouse or human HSPCs with dmPGE2 stabilizes HIF1α in HSPCs, and that similar treatment with the hypoxia mimetic DMOG produces analogous effects to dmPGE2 on HSPC CXCR4 expression and homing. This suggests that HIF1α is responsible for PGE2’s enhancing effects on HSPCs. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP), confirms the requirement of HIF1α for dmPGE2-enhanced migration and CXCR4 upregulation. Additionally, we confirm the requirement for HIF1α in dmPGE2-enhanced in vivo homing using a conditional knockout mouse model of HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after PGE2 treatment. Interestingly, we also observe an increase in the small GTPase Rac1 after dmPGE2 treatment, as well as a defect in PGE2-enhanced migration and CXCR4 expression in Rac1 knockout HSPCs. Using state-of-the-art imaging technology we, confirm an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment that likely explains enhanced sensitivity of PGE2-treated HSPCs to SDF-1. Taken together, these results define a precise mechanism through which ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing, and describe a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammatory disorders are characterized by an increase in excitability of small diameter sensory neurons located in dorsal root ganglia (DRGs). This sensitization of neurons is a mechanism for chronic inflammatory pain and available therapies have poor efficacy and severe adverse effects when used chronically. Prostaglandin E₂ (PGE₂) is an inflammatory mediator that plays an important role in sensitization by activating G-protein coupled receptors (GPCRs) known as E-series prostaglandin receptors (EPs) coupled to the protein kinase A (PKA) pathway. EPs are known to downregulate upon prolonged exposure to PGE₂ or in chronic inflammation, however, sensitization persists and the mechanism for this is unknown. I hypothesized that persistence of PGE₂-induced hypersensitivity is associated with a switch in signaling caused by prolonged exposure to PGE₂ or the neurotrophin nerve growth factor (NGF), also a crucial inflammatory mediator. DRG cultures grown in the presence or absence of either PGE₂ or NGF were used to study whether re-exposure to the eicosanoid is able to cause sensitization and activate PKA. When cultures were grown in the presence of NGF, PGE₂-induced sensitization was not attenuated by inhibitors of PKA. Activation of PKA by PGE₂ was similar in DRG cultures grown in the presence or absence of NGF when phosphatase inhibitors were added to the lysis and assay buffers, but significantly less in cultures grown in the presence of NGF when phosphatase inhibitors were not added. In DRG cultures exposed to PGE₂ for 12 hours-5 days, sensitization after re-exposure to PGE₂ is maintained and resistant to PKA inhibition. Prolonged exposure to the eicosanoid caused complete loss of PKA activation after PGE₂ re-exposure. This desensitization was homologous, time dependent, reversible, and insurmountable by a higher concentration of PGE₂. Desensitization was attenuated by reduction of expression of G-protein receptor kinase 2 and was not mediated by PKA or protein kinase C. The presented work provides evidence for persistence of sensitization by PGE₂ as well as switch from the signaling pathway mediating this sensitization after long-term exposure to NFG or PGE₂.