Frankfurt (Main), Univ., Diss., 2000.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O ciclo vicioso de doenÃas entÃricas na infÃncia à um problema de saÃde pÃblica com consequÃncias graves e seus efeitos no desenvolvimento infantil nÃo estÃo totalmente elucidados. Orià e colaboradores, em 2005, demonstraram que crianÃas portadoras do gene APOE 4 com alta morbidade de diarreia apresentavam um melhor desempenho em testes cognitivos. O objetivo desse trabalho foi avaliar o papel protetor do gene APOE 4 em camundongos C57BL6J submetidos à desnutriÃÃo induzida por uma raÃÃo pobre em proteÃna (2%) e pela infecÃÃo intestinal induzida pelo Criptosporidium parvum. Utilizamos camundongos C57BL6J machos com peso mÃdio de 14 g, submetidos a um perÃodo de 14 dias de desnutriÃÃo e a 7 dias de infeÃÃo por C. parvum meio por meio da gavagem de 107 oocistos. Os animais foram separados segundo o genÃtipo: wildtype, APOE nocaute (ApoE Ko), APOE 3/3 (com gene APOE 3 humano) e APOE 4/4 (com gene APOE 4 humano). Os animais controles receberam PBS via gavagem. O peso dos animais foi monitorado diariamente. Os camundongos foram sacrificados em cÃmara de CO2 seguido de deslocamento cervical apÃs 14 dias do inÃcio do protocolo. Durante o perÃodo de pÃs-infecÃÃo foram coletadas as fezes dos animais infectados em dias alternados, para a realizaÃÃo do PCR quantitativo em tempo real (qPCR) para a anÃlise da quantidade de C. parvum liberada nas fezes. Amostras de Ãleo foram congeladas em nitrogÃnio lÃquido e armazenadas em freezer a -80ÂC para as anÃlises moleculares. Outras amostras foram fixadas em paraformaldeÃdo tamponado (4%) para processamento histolÃgico. Foram avaliados os parÃmetros morfomÃtricos de altura de vilo e profundidade de cripta nos segmentos ileais. Para a detecÃÃo de citocinas prÃinflamatÃrias de interesse (IL-1&#946;, IFN- &#947;, TNF-&#945; e IL-17), utilizou-se o ensaio multiplex (Luminex xMAP). Ainda por qPCR avaliou-se o transportador catiÃnico de aminoÃcidos (CAT-1), arginase 1, iNOS e TLR9. No peso encontramos uma maior adaptaÃÃo a perda de peso nos animais APOE 4 no 2o e 3o dias de desnutriÃÃo em comparaÃÃo a todos os grupos (p<0,05). No perÃodo de pÃs-infecÃÃo verificou-se diferenÃa significante no 2o dia (p<0,05) em comparaÃÃo a todos os grupos. Nas anÃlises morfomÃtricas, encontramos uma reduÃÃo na altura de vilos e profundidade de criptas nos animais APOE nocautes, jà nos animais APOE 4/4 ocorreu uma proteÃÃo contra esses danos em comparaÃÃo a todos os grupos (p<0,05). Os dados da anÃlise de liberaÃÃo de oocistos nas fezes evidenciaram um aumento do estado prÃ-inflamatÃrio e antiparasitÃrio nos animais APOE Ko e APOE 4/4, verificado por meio de uma reduÃÃo na quantidade de C. parvum liberado nas fezes de maneira significativa. Houve um aumento dos nÃveis intestinais das citocinas prÃ-inflamatÃrias IL-1&#946; (p<0,05) nos animais APOE Ko desnutridos e infectados em comparaÃÃo com APOE3/3 e APOE4/4, e altos nÃveis de IFN-&#947; (p<0,05) em comparaÃÃo com os controles selvagens e o grupo APOE Ko desnutrido controle. Os animais desnutridos controles APOE Ko tiveram aumento dos nÃveis intestinais de IL-17 (p<0,05) quando comparados aos animais APOE Ko desnutridos infectados. Dados de qPCR evidenciam que a presenÃa do genÃtipo APOE4 em camundongos aumenta os transcritos primÃrios de CAT-1 e arginase - 1 no Ãleo em relaÃÃo aos selvagens, APOE Ko e APOE3 (p<0,05) e que os animais nocautes aumentaram a expressÃo de iNOS em relaÃÃo aos outros grupos (p<0,05). Os animais APOE 4 desnutridos e infectados apresentaram uma expressÃo significativamente aumentada nos nÃveis de mRNA para TLR9 no Ãleo comparado com os APOE Ko igualmente desafiados (p<0,05). A partir dos nossos achados, podemos concluir que o animais com genÃtipo APOE 4 possuem uma aÃÃo prÃ-inflamatÃria controlada, o que favorece o combate ao C. parvum, visto que reduz a quantidade de DNA do parasita liberado nas fezes e melhora a taxa de crescimento de animais submetidos pela desnutriÃÃo/infecÃÃo, sugerindo que hospedeiros com genÃtipo APOE 4 possuem uma maior proteÃÃo contra as alteraÃÃes intestinais induzidas pela combinaÃÃo de C. parvum e desnutriÃÃo. / The vicious cycle of enteric infections and malnutrition during childhood is a major public health problem with devastating consequences and its effects are not fully elucidated. Oria and colleagues in 2005 showed that children with heavy diarrhea burdens when carrying the APOE 4 gene had a better cognitive performance. The aim of this study was to evaluate the protective role of APOE 4 gene in C57BL6J mice challenged by malnutrition induced by a 2% protein diet and intestinal infection caused by Cryptosporidium parvum. We used male C57BL6J mice weighing in average 14g, challenged by malnutrition for a period of 14 days compound with 7 days of C. parvum infection through a single dose of 107 oocysts given by gavage. Study animals were separated according to their genotype, as following: wild-type, APOE knock-out, APOE 3/3 (carriers of the human APOE 3 gene) and APOE 4/4 (carriers of human APOE 4 gene). Control animals received PBS by gavage. Body weight of the animals was monitored daily. Mice were sacrificed in CO2 chamber with posterior cervical dislocation after 14 days from the beginning of the protocol. During the post-infection period, stools samples were collected from the infected mice every other day for real time quantitative PCR (qPCR) assays in order to quantify C. parvum oocysts released in the stools. Ileal samples were immediately frozen in liquid nitrogen and then stored in a freezer at -80ÂC for molecular analyses. Other samples were fixed in buffered paraformaldehyde (4%) for histological processing. Morphometric parameters were evaluated for villus height and crypt depth in the ileal segments. For detection of a proinflammatory cytokine panel (IL- 1&#946;, IFN-&#947;, TNF-&#945;, and IL-17), we used the multiplex assay (Luminex xMAP). In addition by qPCR, the cationic amino acid transporter (CAT-1), arginase 1, iNOS, and TLR9 were assessed. Regarding weight, we found a greater adaptation to weight loss in APOE 4 animals in the 2nd and 3rd days of malnutrition (p<0.05) and in the postinfection time there was a significant difference on the 2nd day (p<0.05) compared to all groups. In the morphometric analyses, we found villus blunting and crypt disorganization in APOE knockout mice. We found APOE 4 protection against these alterations compared to all groups (p<0.05). The C. parvum oocyst shedding data indicate an increase in the pro-inflammatory state and anti-parasitic effects seen in the APOE Ko and APOE 4/4 mice, as confirmed by a significant reduction of the C. parvum released in the stools. In addition, we found increased levels of the intestinal pro-inflammatory cytokine (IL-1&#946;) (p<0.05) in the APOE Ko when compared with APOE3/3 and APOE4/4, higher levels of IFN-&#947; (p<0.05) when compared with wild-type and undernourished APOE Ko controls. The APOE Ko undernourished mice have increased intestinal levels of IL-17 compared with APOE Ko undernourished infected mice. qPCR data demonstrate that the presence of the APOE4 genotype in mice increased the primary transcripts of CAT-1 and arginase 1 in comparison to wild types, APOE Ko, and APOE 3/3 (p<0.05). Furtermore, APOE knockout mice had higher iNOS expression in comparison to all groups (p<0.05). The APOE 4 mice showed significant increase in the expression of TLR9 mRNA in the ileum when compared to APOE Ko mice (p<0.05). Altogether we concluded that the APOE 4 carriers have a balanced pro-inflammatory response, benefiting the C. parvum control, as seen by reduction of the parasite DNA released in the stools, and by improvements in the growth rates in the mice challenged malnutrition/infection, suggesting that the hosts carrying the APOE4 genotype have a better protection against the intestinal alterations induced by the compound challenge of C. parvum infection and malnutrition.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A periodontite crÃnica inflamatÃria (PC) caracteriza-se por um processo inflamatÃrio nos tecidos de suporte dos dentes. à causada inicialmente por bactÃrias, mas sua progressÃo està relacionada à resposta individual do hospedeiro. à uma doenÃa multifatorial e complexa, na qual fatores genÃticos e ambientais interagem, promovendo e modificando a expressÃo clÃnica da doenÃa. Polimorfismos de genes envolvidos no processo inflamatÃrio tÃm sido estudados no intuito de identificar possÃveis marcadores genÃticos e elucidar diferenÃas na expressÃo de citocinas mediadoras da inflamaÃÃo. ApolipoproteÃna E (apoE) à uma proteÃna de importÃncia no metabolismo lipÃdico e està envolvida em processos fisiopatolÃgicos. O objetivo deste estudo foi investigar se hà associaÃÃo do polimorfismo do gene da apoE com a susceptibilidade à PC em indivÃduos que procuraram o serviÃo odontolÃgico da clÃnica de Periodontia da Universidade Federal do Cearà e avaliar achados sociodemogrÃficos relacionados com essa doenÃa. Foram selecionados 109 indivÃduos entre 30 e 70 anos (mÃdia = 44,5  9,64) de ambos os gÃneros e agrupados da seguinte forma: grupo controle n=53 e grupo Periodontite CrÃnica n=56. Foi extraÃdo DNA a partir de um bochecho e esfregaÃo da mucosa oral e o polimorfismo da apoE foi identificado pelo mÃtodo de PCR-RFLP (reaÃÃo de polimerase em cadeia â polimorfismo com restriÃÃo de fragmentos) e submetidos à eletroforese em gel de agarose a 5%. As distribuiÃÃes da frequÃncia alÃlica e dos genÃtipos foram avaliadas pelo teste qui-quadrado (p&#706;0,05). O risco associado com alelos e genÃtipos foi calculado como odds ratio (OR) com intervalo de confianÃa (IC) de 95%. Para relacionar os achados sociodemogrÃficos com a PC em indivÃduos com alelos especÃficos foi utilizada a anÃlise de regressÃo logÃstica. Os resultados da anÃlise individual do polimorfismo da apoE nÃo evidenciaram associaÃÃo dos alelos e genÃtipos com a susceptibilidade à PC. Observou-se associaÃÃo entre a doenÃa e a renda familiar mensal, de maneira que a chance de adoecer aumenta 3 vezes quando a renda diminui de mais que 3 salÃrios-mÃnimos para a renda de 1 a 3 salÃrios-mÃnimos. Ainda, hà um aumento significativo na chance de desenvolver a doenÃa em 5,1% a cada ano de vida. IndivÃduos que reportaram hipertensÃo arterial tiveram uma chance quase 2,5 vezes maior de ter a PC do que o grupo controle apesar de nÃo ter dado significado estatÃstico. Da mesma forma, foi encontrada maior chance de desenvolver a doenÃa em indivÃduos com baixo nÃvel de escolaridade (OR=3,7). O polimorfismo da apoE nÃo està associado à PC na populaÃÃo estudada. / Chronic periodontitis (CP) is characterized by an inflammation in the supporting tissues of the teeth. It is primarily caused by bacteria, but progression is associated with individual host response. CP is a complex and multifactorial disease. Genetic and environmental factors interacting can modify the clinical cause of the disease. Genetic polymorphisms have been studied to identify possible genetic markers and explain differences in the inflammatory cytokines expression. Apolipoprotein E (apoE) is an important protein in lipid metabolism and is also envolved in pathophysiological processes. The aim of this study was to investigate whether there is an association of the APOE polymorphisms with the CPÂs susceptibility in subjects that sought periodontal treatment at Dental School of Federal University of Ceara and evaluate sociodemographic status related with the disease. A sample of 109 subjects between 30 and 70 years (mean age = 44,5  9,64) were grouped into: 53 controls and 56 subjects with CP. DNA was obtained through a mouthwash and oral mucosa scraping and genotyped by PCR-RFLP method (Polimerase Chain Reaction â Restrict Fragment Length Polymorphism). Differences in the allele and genotype frequencies were assessed by Chi-squared test (p&#706;0.05). The risk associated with alleles and genotypes was calculated as odds ratio (OR) with 95% confidence intervals (CI). Logistic regression was used to associate sociodemographic status with CP and genotypes. No differences were observed in the apoE allelic distribution regarding control and periodontitis groups. Age and socio-economic status increase the risk for having CP, since individuals with lower socio-economic status was about 3-fold more likely to develop periodontitis (OR=3.1) and increase in age enhances the risk in 5.1% to develop disease in each year of age. Hypertension was a factor of clinic importance in development of CP, since subjects who self-reported hypertensive have 2,5-fold more likely to develop disease than individuals who not self-reported hypertension, although no statistic difference was reached. Similarly, CP was also associated with lower education level (OR=3.7). The polymorphism in the APOE gene was not associated with the susceptibility to CP in the studied population.

Orientador: Joyce Maria Annichino-Bizzacchi, Valder Roberval Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T11:25:31Z (GMT). No. of bitstreams: 1 Fabri_DanielaRamos_D.pdf: 28613634 bytes, checksum: 506450d54c214e02520051f098953569 (MD5) Previous issue date: 2008 / Resumo: As complicacoes associadas a aterosclerose sao as causas mais comum de morte na populacao ocidental. O entendimento da aterosclerose como uma doenca inflamatoria e a associacao da coagulacao com fenomenos inflamatorios e complicacoes da doenca arterial oferecem novas oportunidades de prevencao e tratamento.Estudos populacionais demonstraram que o risco de infarto agudo do miocardio esta reduzido em 80% em homens hemofilicos A (HA) se comparados com controles pareados em idade e sexo. No entanto, lesoes ateroscleroticas iniciais foram identificadas em pequenos grupos de pacientes com coagulopatias por estudo ultrassonografico de arterias carotida e femural de forma semelhante aos controles pareados.O objetivo deste estudo e determinar o papel dos fatores VIII e IX dacoagulacao no desenvolvimento da doenca aterosclerotica em dois diferentesmodelos animais de dilipidemia. Comparamos grupos de animais deficientes deFVIII (HA) ou animais deficientes de FIX (HB) deficientes apoliproteina E (APOE-/-) ou deficientes de receptores de lipoproteina de baixa densidade (LDLR-/-) comanimais hemostaticamente normais deficientes apenas de apoE (APOE-/-) ou deLDLR (LDLR-/-). Todos animais da linhagem C57Bl/6, pareados em sexo e idade.Nossos resultados sugerem que o FVIII tem um papel protetor e tempodependente no desenvolvimento da aterosclerose nos animais deficientes de apoE, independente dos seus elevados niveis de colesterol. No entanto, essaprotecao nao foi evidente nos animais LDLR-/-.A deficiencia severa do FIX (<1%) nao protegeu contra o desenvolvimento da aterosclerose. Surpreendentemente, estes animais apresentaram significantemente mais lesoes nos periodos mais tardios nos dois modelos de doenca aterosclerotica utilizados. Os animais HB/APOE-/- apresentaram altos indices de mortalidade a partir de 20 semanas do periodo de dieta. Os elevados niveis de FIX nao influenciou o desenvolvimento da doenca arterial nos modelos apoE, mas mostrou-se como fator de risco para trombose venosa.Este estudo demonstra que o FVIII e o FIX tem papeis heterogeneos na evolução da aterosclerose, sugerindo participacao em mecanismos independentes ao da coagulacao no desenvolvimento da doenca arterial, e novos estudos devem ser realizados visando novas possibilidades terapeuticas tambem para os pacientes hemofilicos de idade mais avancada com risco de doenca cardiovasculares. / Abstract: Complications of atherosclerosis are the most common causes of death in Western societies. The knowledge that atherosclerosis is an inflammatory disease and coagulation affects the disease's complications offers new opportunities for prevention and treatment. Population studies demonstrated that the risk for myocardial infarction is reduced by 80% among hemophilia A (HA) men compared to age and gender-matched controls. However, early atherosclerotic lesions were readily identified in small cohorts of adults HA and hemophilia B (HB) by ultrasonography of carotid and femoral arteries in a similar fashion to an agecontrol male group. Here we sought to determine the role of FVIII and FIX on the development of atherosclerosis in two different mouse models. We compared a group of FVIII (HA) or FIX deficient mice (HB) lacking the low-density lipoprotein receptor (LDLR- /-) or apolipoprotein E (APOE-/-) with hemostatically normal littermate controls lacking either LDLR (LDLR-/-) or apoE (APOE-/-). All mice were on C57Bl/6 background and all groups were matched for gender and age. Our results suggest that FVIII has a protective and time dependent effect on development of atherosclerosis in apoE deficient mice, independently of higher cholesterol levels. Notably, FVIII deficiency did not influence the vascular disease of LDLR-/- mice. Severe FIX deficiency (<1% of normal) did not protect against the development of atherosclerosis. Unexpectedly, the rates of lesions were higher at late time points in the APOE-/- model. In addition, high levels of FIX was did not influence the vascular disease in APOE-/- whereas venous thrombosis was documented in 3/6 mice. Moreover, in the HB/APOE-/- model, the rates of atherosclerosis were higher at week 22 and beyond this age these mice presented high rates of mortality than controls. These data demonstrate that FVIII and FIX play a rather heterogeneous role in the development of atherosclerosis which suggest that there are coagulation independent mechanisms in the development of vascular diseases. Further studies in hemophilia B subjects and carriers are warranted to define the FIX effect on the onset and progression of occlusive vascular disease which may raises concerns on the onset cardiovascular risks on an aging hemophilia population. / Doutorado / Doutor em Clínica Médica

Neuroinflammation occurs in Alzheimer’s disease (AD) brain, and plays a role in neurodegeneration. The main aim of this study was to determine how treatments with exogenous apolipoprotein E (ApoE2, E3 and E4 isoforms), a genetic risk factor for AD, affects the amyloid-β (Aβ) induced inflammatory response in vitro in astrocytes. Recombinant, lipid-free ApoE4 was found not to affect Aβ-induced inflammation in rat astrocytes, while ApoE2 showed a protective effect. Mouse cells expressing human ApoE isoforms, which have similar lipidation and modification to native human ApoE, showed ApoE4 promoting inflammation, and no ApoE2 protective effect upon Aβ treatment. A Protein/DNA array was used to screen 345 transcription factors in rat astrocytes treated with Aβ and/or ApoE isoforms, in order to determine which contribute to the observed ApoE2 protection. Some candidates were validated by Western Blot or EMSA and/or by inhibition or activation. The findings suggest ApoE isoforms differentially regulate Aβ-induced inflammation, and multiple signalling pathways are involved in the process.

No description available.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Increasing evidence indicates that the sooner treatment begins for patients with Alzheimer's Disease (AD), the better the chance of delaying progression of the disease. As a result, studies have begun focusing on risk factors for AD with the goal of identifying individuals with AD at the earliest possible stage. Such studies have found that individuals with Mild Cognitive Impairment (MCI) are at increased risk for AD and other forms of dementia. This study examines the potential mediating effect of a set of prospective risk factors, smoking and ApolipoproteinE (ApoE) genotype, on the incidence of MCI and AD. Although results of this study provide some preliminary evidence of an interaction, the study models presented here fail to reach significance. Additional studies are needed to confirm the hypothesis of an ApoE mediated relationship of smoking on MCI and AD. / 2031-01-01

Yes / African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE. Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis. / This work, including the efforts of Stijn Deborggraeve, was funded by Research Foundation Flanders (1501413N). This work, including the efforts of Bart Cuypers, was funded by Research Foundation Flanders (11O1614N). This work, including the efforts of Jean-Claude Dujardin and Etienne Pays, was funded by Interuniversity Attraction Poles Program of Belgian Science Policy (P7/41). This work, including the efforts of Jean-Claude Dujardin, was funded by Flemish Ministry of Sciences (SOFI-B SINGLE). This work, including the efforts of Etienne Pays, was funded by EC | European Research Council (ERC) (APOLs 669007).

Endothelial dysfunction is a broad term which implies alteration of the overall functions of endothelial cells, including impairment of the barrier functions, vasodilation, and disturbances in proliferative and angiogenic capacities, migratory as well as tube formation, and deterrence of leukocyte transmigration. Such a dysfunction is triggered by pro-inflammatory stimuli and has been associated to several pathological conditions including atherosclerosis. Myeloperoxidase is a heme peroxidase secreted by activated neutrophils at site of inflammation near blood vessels and plays an important role in the initiation of atherosclerotic plaque by interfering with endothelial function. ApoLs represent a family of newly discovered apolipoproteins with yet unrevealed function, but predicted to be involved in inflammatory processes and cell death mechanisms. We aimed to study the expression of ApoLs as well as Myeloperoxidase in endothelial cells and their possible contribution to endothelial dysfunction. We performed RNA sequencing on MPO-treated endothelial cells and found that most of the induced genes are related to angiogenesis and blood vessel morphogenesis mechanisms. MPO treatment resulted in intracellular MPO localization and mimicked the effects of VEGF on several signal transduction pathways, such as Akt, Erk and Fak involved in angiogenesis. Accordingly MPO, independently of its enzymatic activity, stimulated cellular proliferation, migration and tubules formation by endothelial cells. RNA interference also pointed at a role of endogenous MPO in tubulogenesis and endothelium wound repair in vitro.On the other hand, ApoL3 among other family members was shown to be a downstream responsive gene to MPO, VEGF and FGF treatment. ApoL3 invalidation reduces tubules formation in MPO and VEGF-induced angiogenesis and wound repair in vitro. Accordingly, pro-angiogenic signaling pathways (Erk1/2 and FAK but not Akt) and some pro-angiogenic genes were partially inhibited in ApoL3 Knock out cells. These findings uncover for the first time an important and unsuspected role for ApoL3 and MPO as drivers of angiogenesis. / Le dysfonctionnement endothélial est un terme qui désigne un dérèglement général de la fonction endothéliale, caractérisé par des perturbations de l’intégrité membranaire, de la croissance endothéliale, du rôle anti-inflammatoire ;anti-coagulant, ainsi que leur propriété angiogenique principalement la migration endothéliale et la formation des structures tubulaires. Cette condition patho-physiologique pourrait être déclenchée par des stimuli pro-inflammatoire et elle est souvent associée à l’athérosclérose. La myéloperoxydase est une enzyme secrétee par les neutrophiles et contribue à la formation de la plaque d’athérome. Une nouvelle famille de protéines, les apolipoprotéines L, susceptibles d'intervenir dans le processus inflammatoire est bien exprimée dans les cellules endothéliales. Néanmoins, aucune fonction ne lui a été attribuée jusqu’à présent dans ce type cellulaire.dans le cadre de ce travail, Nous nous sommes intéressés à étudier l’implication des ApoLs ainsi que la Myeloperoxydase dans la dysfonction endothéliale. L’analyse du transcriptome des cellules traitées avec la MPO a montré que lamajorité des génes induits contrôlent le processus angiogenique. La myeloperoxidase stimule la proliferation,migration et la tubulogenese des cellules endotheliales. Cet effet est médié par l’activation des cascades (ERK1/2, Akt et FAK) et des genes pro-angiogeniques. Tandis que la suppression de l’expression de la MPO endogène entraine l’inhibition de la capacité des cellules à migrer et de former des tubes.D’autre part, l’invalidation de l’ApoL3 inhibe la migration cellulaire et la tubulogenése dépendente de la MPO et le VEGF. Sur le plan mécanistique, ces altérations phénotypiques sont les conséquences d’une part, une baisse de phosphorylation des kinases Erk1/2 et FAk (mais pas Akt) et d’autre part de la réduction du taux d’expression des gènes pro-angiogeniques dans les cellules ApoL3 Knock out stimulées par la MPO et le VEGF. ce résultat nous permet de définir l’ApoL3 et la MPO en tant que nouvels acteurs dans le processus angiogenique. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.