The purpose of this project was to develop a practice guideline for screening patients at risk for cardiovascular disease, educate the staff at the site about the guideline, and implement the guideline at a primary care clinic. The intention was to identify and treat patients at risk for cardiovascular disease to prevent occurrence of heart disease. Cardiovascular disease includes hypertension, coronary heart disease, heart failure, and stroke. Coronary heart disease is one of the leading causes of death in the Western world. The local practice problem and focus of this project was underprescribed statin therapy for patients at risk for developing heart disease at a clinic in the southern United States. The practice-focused question that guided this project explored whether an evidence-based clinical guideline that might impact the prescription of statins for the prevention of cardiovascular disease would be approved for implementation in a primary care clinic serving adult and geriatric patients. The appraisal of guidelines for research and evaluation and the Fineout-Overholt model were used to guide this project. Sources of evidence to meet the purpose of this project were obtained from the literature and scholarly articles. The results of the presentation to the expert panel indicated that this clinical practice guideline would be implemented at the project site and would be used by nurse practitioners and physicians. The implications of this project for positive social change might include improved management of patients who are at risk for heart disease and a decrease in premature deaths related to cardiovascular disease.

A series of investigations were undertaken to determine whether physical or emotional stress, catecholamines or naloxone (B-endorphon blocker) would increase serum total cholesterol and LDL and HDL levels. Physical stress given daily over a period of time caused a steady increase in serum total cholesterol and LDL without a significantly altering high density lipoproteins (HDL) or serum triglycerides. Daily injections of epinerphrine in oil caused an increase in both serum total cholesterol and LDL levels while daily injections of norepinephrine did not. Reversal of the treatments caused a reversed response in both groups of rats. Similar increases in both total cholesterol and LDL levels occurred in graduate students during preparation for their comprehensive written or oral thesis/dissertation defense. Injection of eigher dichloroisoproterenol (M.W. = 248) or naloxone (M.W. = 346) in rats prior to stress inhibited the increase in total cholesterol and LDL levels, although naloxone at the dosage given was more effective, possibly due to its larger molecular weight. When naloxone plus epinephrine was injected into non-stressed rats, there was a significant increase in total cholesterol and LDL levels, but the increase was not as great as that of groups injected with epinephrine only. Stressed, adrenalectomized rats exhibited higher cholesterol and LDL levels than the normal reported range for rats of their age and weight, but their levels did not differ from those of stressed, sham-operated rats indicating that the adrenals per se are not needed for stress-induced elevation of blood LDL levels.

Cryopreservation of germplasm is widely used in agriculture, biotechnology, conservation of threatened species and human reproductive medicine. There is a need however to improve the reproductive efficiency of breeding with cryopreserved semen, which may involve increasing the post-thaw quality of sperm through improvements in cryopreservation extenders. Extenders including egg yolk from chickens are successfully used worldwide for cryopreservation of bovine semen, whereas the protective agent in the egg yolk is believed to be the low-density lipoprotein (LDL) fraction. Egg yolks of different avian species vary in their cholesterol, phospholipid and polyunsaturated fatty acid content which have been shown to have important effects on sperm’s freezing capability. The purpose of this study was to determine the cryoprotective effect of clarified egg yolk and LDLs extracted from different egg yolk sources (chicken, chicken omega-3, pigeon, quail and turkey) on bovine sperm. Semen from six bulls was collected four times each by electroejaculation, split and diluted with the 10 following extenders: chicken clarified (Ccl), chicken omega-3 clarified (O3cl), pigeon clarified (Pcl), quail clarified (Qcl), turkey clarified (Tcl), chicken LDL (CLDL), chicken omega-3 LDL (O3LDL), pigeon LDL (PLDL), quail LDL (QLDL) and turkey LDL (TLDL). The extended semen was evaluated, cryopreserved and examined directly after thawing (0h) and after two hours at 37 ˚C (2h). Computer assisted sperm analysis (CASA) was used to determine total sperm motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). Intact plasma membrane (IPM) and intact acrosomes (IA) were measured by flow cytometry. The percentage change (loss; Δ%) of each sperm characteristic was calculated and used to compare the effect of the extenders. From extending to 0h post-thaw, the pigeon LDL extender lead to greater losses in sperm total and progressive motility, as well as of intact acrosomes, than the other nine extenders tested (P < 0.05). During 0h to 2h post-thaw, the sperm in PLDL extender experienced greater losses in total and progressive motility (P < 0.0001), as well as in curvilinear velocity (P < 0.05), than in all the other nine extenders. Sperm in turkey clarified extender had a greater loss in the velocity parameters (VSL, VAP, VCL) than sperm in several of the other extenders such as O3cl, CLDL, O3LDL, QLDL and TLDL from 0h to 2h (P < 0.05). Concomitantly, sperm in the Tcl extender had a greater loss in the velocity parameters and of intact acrosomes compared to sperm in its counterpart, the turkey LDL extender, from 0h to 2h post-thaw (P < 0.05). The differences produced in post-thaw quality of cryopreserved bovine sperm in the pigeon LDL and turkey clarified extenders were attributed to methodological differences in these egg yolk preparations compared with the other eight extenders. Importantly, the results demonstrate that with most egg yolk preparations derived from a variety of species, there are equivalent cryoprotective effects afforded by the use of omega-3 chicken, pigeon, quail, or conventional chicken egg yolk in a clarified form in freezing extenders for bovine semen. We further proved that the freezing capabilities of bovine semen extenders containing the low-density lipoprotein fraction of omega-3 chicken, quail, turkey and conventional chicken egg yolk were similar.

Si certains cancers sont maintenant traités avec succès, la lutte à finir avec plusieurs autres tels les cancers cérébraux, passe impérativement par une meilleure connaissance des cellules constituant ces tumeurs et de ce qui les entoure. Les tumeurs cérébrales sont isolées du reste du corps par la barrière hémato-encéphalique, ce qui rend le traitement de ces dernières un défi de taille. Cibler le microenvironnement dans lequel évoluent ces tumeurs est un nouveau moyen d'augmenter peut être l'efficacité des thérapies anticancéreuses. En effet, les cellules endothéliales tumorales (CET) sont connues pour avoir un phénotype distinct des CE normales. Ces CE peuvent donc être une cible potentielle supplémentaire pour l'inhibition sélective de la croissance tumorale. En même temps, les tumeurs semblent être dirigées et initiées par une sous-population de cellules au sein de la tumeur, les cellules souches cancéreuses (CSC). Le concept des CSC a pris de l'envergure ces trois dernières années et la recherche de caractéristiques intrinsèques particulières des CSC semblerait un bon début pour l'avancement de l'oncologie. Une des signatures phénotypiques de plusieurs CSC est le marqueur CD133 ou Prominine-1. Comme les cellules cancéreuses ne sont pas la seule cible thérapeutique envisageable, les cellules endothéliales, composantes du milieu tumoral, ont servi de modèle pour l'étude de récepteurs aux facteurs plaquettaires. À l'aide de la lignée HBMEC (human brain microvascular endothelial cell), nous avons mis en évidence une migration préférentielle marquée des HBMEC en réponse à l'acide lysophosphatidique (LPA) comparativement à la sphingosine 1- phosphate (S1P), le LPA et la S1P étant deux facteurs plaquettaires. La voie des MAP (Mitogen-activated protein) kinases semble être une des voies activée chez les HBMEC stimulées au LPA. Nous avons démontré sous condition hypoxique, que cette migration était fortement inhibée (>50%) suite à l'invalidation de l'un ou l'autre des récepteurs au LPA, LPAR-1 ou LPAR-3, deux récepteurs qui s'exprimaient à la hausse suite à l'étude du profil génique des LPAR chez les HBMEC. Dans le but d'investiguer la réponse au LPA et à la S1P dans un modèle de résistance tumorale, une sous-population de cellules CD133(+) a été triée à partir de la lignée cellulaire de médulloblastome DAOY. Il a été observé que les cellules CD133(+) sont plus sensibles au LPA que les CD133(-) , fait qui a été constaté via une réponse précoce et plus intense de l'activation de la voie des MAP kinases chez les cellules CD133(+) ainsi que par une surexpression des récepteurs au LPA, en particulier, LPAR-2 et LPAR-4 chez cette même sous-population. En plus, la résistance des cellules CD133(+) a été reliée à un phénotype d'expression différentielle des récepteurs lipoprotéiques de basse densité (LRP). En effet, la déprivation en nutriments régule à la hausse l'expression des récepteurs LRP-1, LRP-1b et LRP-5 ainsi que celle du marqueur CD133. En somme, nous proposons que les LPAR constituent une cible propice pour combattre le cancer, que ce soit au niveau du compartiment tumoral cérébral ou vasculaire cérébral. Nos résultats suggèrent aussi que les CSC s'adaptent entre autre par l'intermédiaire de récepteurs LRP, qui constituent une autre cible thérapeutique du compartiment tumoral cérébral. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : cellule endothéliale, cellule souche cancéreuse, CD133, acide lysophosphatidique, récepteur au LPA, médulloblastome, low-density lipoprotein receptor-related protein.

Thesis (M.Sc.), Memorial University of Newfoundland, 2000. / Restricted until June 2003. Bibliography: leaves 98-117.

No description available.

Macrophage death is likely to contribute to the transformation of fatty streaks into advanced atherosclerotic lesions. Previous work in the laboratory showed that OxLDL promotes cell death in human macrophages by a mechanism involving intracellular peroxide formation. Here we show that glutathione depletion induced by OxLDL occurs independent of peroxyl radical formation. Our data suggest that the depletion of glutathione is the fundamental defect that renders macrophages susceptible to OxLDL-induced cell injury, but alone is not sufficient to kill macrophages. We indicate that increased protein-Sglutathionylation is involved in OxLDL-induced macrophage death. A potentiation of OxLDL toxicity was observed in macrophages transfected with siRNA directed against either glutathione reductase or glutaredoxin. Our data suggests that OxLDL-induced cell injury in human macrophage is mediated by the depletion of GSH, a decreased in the GSH/GSSG ratio and peroxyl radical formation. All three signals are required for OxLDL-induced macrophage death. Our results also show that the glutathione reductase/glutaredoxin system protects macrophages from OxLDL-induced cell death.

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

The proprotein convertase subtilisin/kexin type 9, PCSK9, belongs to the proprotein convertase (PC) family. Human mutations in the gene encoding PCSK9 lead to either familial hyper- or hypocholesterolemia, resulting from a gain or loss of function, respectively. Mice lacking PCSK9 are viable and show a 42% decrease in plasma cholesterol levels. The enzyme triggers the degradation of the low density lipoprotein receptor (LDLR) through a partially unknown mechanism. / PCSK9 is very abundant in the liver and intestine during development and adulthood. Hepatocytes have a capacity to reproduce themselves and, upon injury, can repopulate the liver. For a better understanding of the role of PCSK9 in the liver, partial hepatectomy was performed on Pcsk9 +/+, Pcsk9+/- and Pcsk9-/- mice. The absence of PCSK9 resulted in defective liver regeneration, while wild type (WT) and heterozygous mice had no phenotype. Regeneration defects could be prevented by a high cholesterol diet. PCSK9 deficiency, by contributing to maintaining low circulating cholesterol levels may thus hamper liver regeneration. This knowledge is critical for the analysis of future PCSK9 inhibitors expected to be developed in the near future. / Key words. Proprotein convertase subtilisin/kexin 9 (PCSK9), a familial hyper- or hypocholesterolemia, low density lipoprotein receptor, knockout mouse model, partial hepatectomy.

Diferents estudis assenyalen que el consum de peix, a través dels àcids grassos poliinsaturats omega-3 (AGPI omega-3), pot prevenir malalties vasculars lligades a l'aterosclerosi. Els AGPI omega -3 són altament insaturats, la qual cosa els fa especialment susceptibles a l'atac oxidatiu. Com que la lipoperoxidació de les lipoproteïnes de baixa densitat (LDL) és un dels esdeveniments clau en les etapes inicials de la formació de la placa d'ateroma, això faria d'una suplementació amb oli de peix de la dieta un factor potencialment pro-aterogènic. Així doncs, hi ha d'haver altres mecanismes que contrarestin l'efecte pro-oxidant dels AGPI omega-3 i que expliquin les seves propietats antiaterogèniques. A partir dels AGPI omega-6 i omega-3, se sintetitzen dues sèries diferents d'eicosanoids. Generalment, els primers presenten una activitat biològica més gran que els segons. Les dues sèries d'eicosanoids constitueixen un element clau en les accions diferenciades dels AGPI omega-3 respecte dels AGPI omega -6. Entre les accions del AGPI omega -3, i pel que fa a l'aterogènesi, hipotetitzem la modulació de la producció de radicals lliures com l'òxid nítric (·NO), sintetitzat per l'enzim eNOS en l'endoteli vascular, i d'efecte relaxador, i l'anió superòxid, d'efecte vasoconstrictor.Per tal d'estudiar els efectes d'una suplementació amb AGPI omega-6 i amb AGPI omega -3 en vasos i en lipoproteïnes, s'alimentaren rates Sprague-Dawley, amb una dieta rica o bé en oli de blat de moro (5%, ric en AGPI omega -6), o bé en oli de peix (5%, ric en AGPI omega-3) durant 8 setmanes. Part dels animals, a més, reberen una suplementació addicional de L-arginina lliure, substrat de l'eNOS. Després del període d'alimentació, els animals eren sacrificats i se n'extreia l'aorta (per fer estudis vasculars) i la sang (per obtenir-ne les lipoproteïnes de molt baixa densitat (VLDL) i les LDL).Els estudis en bany d'òrgans van permetre d'observar un augment en la relaxació induïda per acetilcolina i mediada per ·NO en les rates suplementades amb oli de peix. L'augment d'aquestes relaxacions no era degut ni a una menor producció de superòxid (O2·-, principal agent limitador de la biodisponibilitat vascular de ·NO), ni a una major resposta del vas al ·NO (no hi havia diferències en les relaxacions induïdes per nitroprussiat sòdic, un donador de ·NO).D'altra banda, també augmentava, en les rates suplementades amb oli de peix, la producció basal de ·NO (avaluada per ressonància de spin electrònic), fenomen vinculat, a més, a l'estimulació de l'expressió del ARNm i de la proteïna d'eNOS. Això s'acompanyava d'una davallada del contingut de L-arginina lliure del teixit i d'un augment del GMPc. Quant a la suplementació de L-arginina en les dietes, aquesta produïa també un augment, en tots dos tipus d'olis, de la producció vascular de ·NO.Pel que fa a la fracció de VLDL+LDL, no s'hi observaven canvis deguts a la dieta en la mobilitat electroforètica, que és un indicador del grau d'oxidació nadiu d'aquestes. En canvi, la suplementació amb oli de peix davallava les reserves d'alfa-tocoferol de les lipoproteïnes. En consonància amb això, davant un atac oxidatiu ex vivo induït per Cu2+, les lipoproteïnes de les rates suplementades amb oli de peix, iniciaven l'oxidació abans, però alhora s'hi propagava menys ràpidament i el nivell màxim de diens conjugats era més baix.L'augment de la producció vascular de ·NO generat per la suplementació amb oli de peix, és un mecanisme potencialment antiaterogènic, a través del manteniment del to vascular mitjançant l'activitat vasorelaxadora del ·NO. A banda d'això, el ·NO, d'acord amb diversos estudis in vitro, actua com a antioxidant en lipoproteïnes que podria interferir en la propagació de l'oxidació in vivo i explicar la paradoxa de l'oli de peix en la prevenció de l'aterosclerosi.