Atherosclerosis, hardening and narrowing of the arteries, is the principal underlying cause of heart attacks, strokes, and peripheral vascular disease, which kills more than 600,000 Americans each year. High plasma levels of low-density lipoproteins (LDL) are linked to the formation of atherosclerotic plaques in arteries. LDL is the last metabolic product of very low-density lipoprotein (VLDL), which is secreted from the liver along with one molecule of apolipoprotein B (apoB). Current therapies to control levels of LDL include: cholesterol synthesis inhibitors or statins, low-fat diets and antisense oligonucleotides to reduce cholesterol levels. Recent studies recommend lower clinical levels of plasma LDL to maintain an individual’s health, especially of those who have already developed atheroscle- rotic plaques. However, existing therapies are often unable to achieve these aggressive limits. Furthermore, patients have shown various levels of intolerance to these treatments. In order to develop new, targeted drugs, that can control LDL levels with minimal side effects, it is imperative to understand, in detail, the process of apoB-containing lipoprotein formation. ApoB is one of the largest human proteins known (4563 residues) and previous attempts to solve the structure have been unsuccessful, mainly due to analyzing the protein as a whole or by large sections. To advance the field we will go by a different approach. I present here a construct that represents roughly 8% of the whole protein, apoB9-17 (residues 430 to 782). This section of the protein is believed to play a pivotal role in the assembly process of LDL. My hypothesis is that this construct will be well-behaved and suitable for structural and functional analysis. The study shows that apoB9-17 can be produced in considerable quantities from bacterial cells and can be purified by means of a 6-histidine tag with a good yield. Furthermore, circular dichroism analysis shows the construct contains the expected secondary structure at room temperature and is stable at a wide temperature range (50 to 70 ◦C) at low concentrations. The construct here described will be useful to test the effect of mutations such as the one found in patients with Familial hypobetalipoproteinemia (FHBL). Furthermore, this construct contains two regions believed to be of vital importance for LDL particle formation: the alpha-helical region (residues 430 to 570) is believed to associate with MTP at the initial stages of LDL formation and the c-sheet (residues 614 to 782), which may form part of the lipid recruiting process. Both essential aspects to ultimately develop therapies that can modulate VLDL particle formation.

Increased triglyceride (TG) concentration has been generally accepted as a risk factor for ischemic heart disease and, therefore, lowering TG is therapeutic target that should reduce cardiovascular disease risk. Traditionally, concentration of TG is measured in the fasting state (8-12 hours after an overnight fasting) mainly because the rise in TG levels after meal leads to the high variation in TG values. However, human beings spend larger portion of the day in a postprandial state and postprandial hypertriglyceridemia may then play a substantial role in determination of cardiovascular disease risk. The increased and prolonged postprandial lipemia has been found in patients with coronary heart disease. Moreover, recent data from Copenhagen Heart Study point out that the non-fasting TG concentration is associated with cardiovascular disease risk more tightly than the fasting TG concentration. Importantly, concentration of non-fasting TG is substantially affected by individual behavioural habits such as diet composition and physical activity. It remains to be determined whether it would be appropriate to identify individuals at higher risk of cardiovascular disease due to increased postprandial TG using tolerance test analogous to glucose tolerance test. The protocol of standardized fat tolerance...

BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

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Die Doppelfiltrationsapherese stellt eine Therapieform zur extrakorporalen Entfernung von atherogenen Lipoproteinen bei Patienten mit schweren Lipidstoffwechselstörungen und konsekutiven kardiovaskulären Erkrankungen dar. Das Ziel der vorliegenden Studie bestand darin, optimale Behandlungsbedingungen für eine neuere synthetische Fraktionierungsmembran (FractioPESTM 200; 3M Deutschland GmbH, Neuss) für die Doppelfiltrations-Lipoproteinapherese im Rahmen eines In-vivo-Modells am Schaf zu definieren. In einer prospektiven und randomisierten "Crossover–Studie" an vier Schafen wurde hierzu die Permselektivität der Fraktionierungsmembran unter unterschiedlichen Plasmaflussraten (PF 30, 36 und 42 ml/min), umgekehrter Plasmaflussrichtung (Outside- In-Filtration) und erhöhter Plasmatemperatur untersucht. Nach definierten behandelten Plasmavolumina wurde dafür die In-vivo-Performance der Fraktionierungsmembran anhand von Reduktionsrationes und Siebkoeffizienten für die relevanten Moleküle LDL, HDL, Fibrinogen, Albumin und IgG gemessen. Entsprechend des Therapieziels war die Fraktionierungsmembran für LDL-Cholesterin während aller Behandlungseinstellungen nahezu undurchlässig, was sich an niedrigen SK und statistisch sich nicht unterscheidenden Reduktionsrationes (49,0 ± 8,9 (Outside-In) bis 60,6 ± 9,7 % (PF 36)) zeigte. Lediglich bei 600 ml behandeltem Plasmavolumen wurde unter PF 42 und Outside-In ein signifikant höherer LDL-SK (0,165 ± 0,022 bzw. 0,194 ± 0,068) im Vergleich zu PF 30 und 36 (p < 0,05) bestimmt. Eine gewünschte geringe Membrandurchlässigkeit fand sich ebenfalls für Fibrinogen, wobei signifikant höhere und damit ungünstigere SK für Outside-In nach 600 ml (0,229 ± 0,03 (p < 0,05)) gegenüber allen anderen Behandlungsmethoden und nach 900 ml (SK 0,369 ± 0,12 (p < 0,05)) im Vergleich zu PF 30 und PF 42 gemessen wurden. Bezüglich der unerwünscht entfernten Substanzen waren zwischen den Behandlungsmethoden keine Unterschiede bei HDL-Cholesterin und Albumin nachweisbar. Lediglich für IgG lag nach 900 ml ein höherer SK (1,047 ± 0,070 (p = 0,049)) bei PF 42 im Vergleich zu PF 30 (0,573 ± 0,321) vor.Grundsätzlich stiegen bei allen Behandlungsarten die SK für alle Substanzen mit zunehmendem Plasmavolumen teilweise signifikant an. Eine Ausnahme stellte der Outside-In-Modus dar, bei dem es nach 600 ml zu einem Abfall der SK kam. Bei PF 42 war die günstigste HDL/LDL-Ratio der Reduktionsrationes nachweisbar, d.h. die höchste Retention atherogenen LDL bei geringster Entfernung des vasoprotektiven HDL. Die Anwendung verschiedener Behandlungsbedingungen bei Verwendung der FractioPESTM 200-Membran führte nur zu geringen Unterschieden bei der Entfernung der Zielsubstanzen. Als günstigste Einstellung erwies sich die höchste Plasmaflussrate, PF 42 ml/min, in standardmäßiger Flussrichtung, während sich die Outside-In-Filtration nachteilig auswirkte. Der Grund dafür dürfte im asymmetrischen Wandaufbau der Fraktionierungsmembran mit den kleinsten Poren, d.h. der Separationsschicht, innen liegen, der zu Unterschieden beim Verstopfen („Clogging“) der Membranporen führt. Das Schafsmodell erwies sich erneut als zuverlässiges und auf die klinische Doppelfiltrations-Lipoproteinapherese übertragbares In-vivo-Experiment. / Double filtration apheresis is a form of therapy for the extracorporeal removal of atherogenic lipoproteins in patients with severe lipid metabolism disorders and consecutive cardiovascular diseases. The aim of this study was to define optimal treatment conditions for a newer synthetic fractionation membrane (FractioPESTM 200; 3M Germany GmbH, Neuss) for double filtration plasmapheresis within a setting of an in-vivo model on a sheep. In a prospective and randomized "crossover study" on four sheeps, the permselectivity of the fractionation membrane was investigated during different plasma flow rates (PF 30, 36 and 42 ml/min), reverse plasma flow direction (outside-in filtration) and increased plasma temperature. After defined treated plasma volumes, the in-vivo performance of the fractionation membrane was measured based on reduction rations and sieving coefficients for relevant molecules LDL, HDL, fibrinogen, albumin and IgG. Accordingly the therapy goal, the fractionation membrane was nearly impermeable to LDL-cholesterol during all treatment settings, evidenced by low SK and statistically indistinguishable reduction rations (49.0 ± 8.9 (outside-in) to 60.6 ± 9.7% (PF 36)). Only at 600 ml treated plasma a significantly higher LDL-SK (0.165 ± 0.022 and 0.194 ± 0.068) during PF 42 and outside-in compared to PF 30 and 36 (p < 0.05) was determined. A desired low membrane permeability was also found for fibrinogen, shown by the significantly higher and thus less favorable SK for outside-in after 600 ml (0.229 ± 0.03 (p < 0.05)) compared to all other treatment methods and after 900 ml (SK 0.369 ± 0.12 (p<0.05)) compared to PF 30 and PF 42. Regarding the undesirably removed substances, no differences in HDL-cholesterol and albumin could be detected between the treatment methods. Only for IgG there was a higher SK (1.047 ± 0.070 (p = 0.049)) at PF 42 compared to PF 30 (0.573 ± 0.321) after 900 ml. Basically, with all types of treatment, the SK for all substances increased, partially significant, with increasing plasma volume. The outside-in mode was an exception, in which there was a decrease in the SK after 600 ml. At PF 42, the most favorable HDL/LDL ratio of the reduction ration was detectable, i.e. the highest retention of atherogenic LDL with the lowest removal of the vasoprotective HDL. The application of different treatment conditions when using the FractioPESTM 200 membrane resulted in only minor differences in the removal of the target substances. The best setting proved to be the highest plasma flow rate, PF 42 mL/min, in the standard flow direction, while outside-in filtration had a disadvantageous effect. The reason for this may be the asymmetrical wall structure of the fractionation membrane and the small pores, i.e. a separation layer lying inside, which leads to differences in clogging of the membrane pores. The sheep model again proved to be a reliable in-vivo experiment transferable to clinical double filtration lipoprotein apheresis.

Prior studies of lipid and lipoprotein levels alterations associated with the administration of danazol, a testosterone derivative, in patients treated for endometriosis have been conflicting. We administered danazol to 7 normal menstruating women and measured plasma lipid and lipoprotein cholesterol levels prior to and 2 months after treatment. Small, non-significant decreases in total plasma cholesterol and triglyceride levels were seen, largely due to a dramatic decline in one woman with type IV hyperlipoproteinemia. No significant change in low density or very low density lipoprotein cholesterol levels was seen. However, a marked (40%) reduction of high density lipoprotein cholesterol level in the mean was found. These findings have implications for the atherogenic potential of danazol, the treatment of hyperlipidemia, and the relationship between gonadal hormones and lipoprotein levels.

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The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.

Atherosclerosis is a chronic inflammatory disease affecting large- and medium-sized arteries and is considered the major cause behind cardiovascular diseases (CVD). Elevated low-density lipoprotein (LDL)-cholesterol and low high-density lipoprotein (HDL)-cholesterol are considered major risk factors for the CVD. HDL mediates a variety of atheroprotective actions, many of them involving the interaction with the scavenger receptor class B, type 1 (SR-B1). Despite the efforts placed in raising HDL-cholesterol, no improvement has been achieved in reducing CVD risk, suggesting that other components of the HDL particles may be responsible for HDL-mediated atheroprotection. One of these may be sphingosine-1-phospate (S1P). In this thesis, the role of S1P receptors (S1PRs) in atherosclerosis is explored, with emphasis in macrophage apoptosis. In particular, the importance of the macrophage S1PR1 and its role in apoptosis and atherosclerosis was evaluated. We demonstrated that diabetes exacerbates atherosclerosis development and myocardial infarction in SR-B1 KO/apoE-hypomorphic mice and that treatment with FTY720, a S1PR agonist, protects against diabetes pro-atherogenic effects. We also show that S1PR1 agonists protected macrophages against apoptosis through phosphoinositide 3-kinase (PI3K)/AKT, and that HDL failed to protect S1PR1 deficient macrophages against apoptosis. In vivo, macrophage S1PR1 deficiency translated into increased atherosclerosis, necrotic core formation and numbers of apoptotic cells in the atherosclerotic plaque. BIM deficiency in BM cells was protective against atherosclerosis development and HDL treatment reduced BIM protein levels in cells exposed to ER stressors, suggesting that the pro-apoptotic protein may be an important target for HDL in macrophages. We conclude that signaling through the S1PRs, in particular S1PR1 is important in controlling macrophage apoptosis and atherosclerosis development. Our data suggests that S1PR1 signaling axis and the pro-apoptotic protein BIM play an important role in mediating HDL anti-apoptotic signaling, however further studies are required to clarify the interaction between all of these factors. / Thesis / Doctor of Philosophy (PhD)