Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction.

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...

Objective: We proposed that women who develop preeclampsia have a low ratio of 'protective' toxicity preventing activity (TxPA) to 'toxic' very low density lipoproteins (VLDL) late in pregnancy. Having confirmed this hypothesis, we then tested whether this low ratio would manifest itself early in the pregnancy of women who develop preeclampsia. Methods: Serially collected plasma from women who developed preeclampsia and from matched controls was assayed blind for TxPA, triglycerides, cholesterol, high-density lipoproteins, albumin, and nonesterified fatty acids (NEFA). Main Outcome Measures: Plasma concentrations of lipids, NEFA, and proteins which bind NEFA (TxPA and albumin) were measured in normal and preeclamptic women. These parameters were formulated prior to data collection because of the low albumin/triglyceride' ratios and the elevated NEFA levels reported to occur in preeclampsia. Results: In late pregnancy, TxPA was lower (1.82 ± 0.63 vs. 2.30 ± 0.40 g/dL, P = 0.008) and VLDL higher (292 ± 130 vs. 206 ± 60 mg/dL, P = 0.013) in preeclamptics than in controls. Discrimination analysis (TxPA and triglyceride), correctly classified 95% of the preeclamptics and 79% of the controls in late pregnancy. The ratio of TxPA to non-TxPA and triglyceride correctly classified 92% of the preeclamptics and 85% of the controls in early pregnancy. Conclusions: The ratio of TxPA to VLDL accurately distinguishes preeclamptic from normal pregnant women, suggesting that both these factors are involved in the development of preeclampsia.

7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA 2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [ 3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of CPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388B1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA 2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.

Multiple GWAS have identified SNPs in the 8q24 locus near the TRIB1 gene that significantly associate with plasma lipids and coronary artery disease. While subsequent studies have uncovered roles for hepatic and myeloid Trib1 in contributing to plasma lipids or atherosclerosis, eQTLs linking TRIB1 expression to the 8q24 GWAS SNPs have not been identified in any metabolic tissue, making the causal tissue for these GWAS associations unclear. The same 8q24 SNPs significantly associate with plasma adiponectin levels in humans as well, suggesting a role for TRIB1 in adipose tissue. To investigate the role of adipocyte-specific TRIB1 in plasma adiponectin and lipid regulation, we generated adipocyte-specific Trib1 knockout mice (Trib1_ASKO) and show that they have increased plasma adiponectin levels and decreased plasma cholesterol and triglycerides. We demonstrate that loss of Trib1 increases adipocyte production and secretion of adiponectin independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice suggests that alterations in adipocyte function underlie the plasma lipid changes observed in these mice. Secretomics and RNA-seq analysis reveal that Trib1_ASKO mice have increased production of LPL and decreased production of ANGPTL4 in adipose tissue, and fluorescent substrate assays confirm an increase in adipose tissue LPL activity, which likely underlies the observed triglyceride phenotype. To better understand the role of the 8q24 SNPs in regulation of TRIB1 expression, we also present a short study of putative enhancer regions identified through DNA accessibility and ChIP-seq enhancer marks in Huh-7 cells. CRISPRi and CRISPRa perturbation of these regions do not affect TRIB1 expression, but instead affect the expression of a lncRNA that overlaps with the GWAS signal. In summary, we demonstrate here a novel role for adipocyte TRIB1 in regulating plasma adiponectin, total cholesterol, and triglycerides in mice, confirming previous genetic associations observed in humans and providing a novel avenue through which TRIB1 regulates plasma lipids and coronary artery disease.

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Atherosclerosis is a major cause of cardiovascular disease, which is among the leading causes of death globally. Elevated plasma concentration of low density lipoprotein (LDL) cholesterol is a risk factor for atherosclerosis, while a high plasma level of high density lipoprotein (HDL) cholesterol is considered protective. Uptake of HDL cholesterol by hepatocytes during reverse cholesterol transport, and athero-protective signaling induced by HDL in other cells are mediated by the scavenger receptor class B, type 1 (SR-B1). SR-B1 deficiency in mice that are susceptible to atherosclerosis results in exacerbation of atherosclerosis, and in mice with mutations in apolipoprotein E (apoE), renders mice uniquely susceptible to occlusive coronary artery (CA) atherosclerosis and myocardial infarction. In this thesis, the impact of a lack of SR-B1 on the development of atherosclerosis is characterized in otherwise wild type mice, and in mice that also lack the LDL receptor (LDLR). We demonstrate that after prolonged feeding of a high fat, high cholesterol cholate-containing diet, SR-B1 knockout (KO) mice develop similar levels of diet-induced atherosclerosis to LDLR KO mice and apoE KO mice in traditionally susceptible arteries, and significantly more atherosclerosis in arteries that are typically resistant to plaque development, such as the CAs. SR-B1/LDLR double KO mice develop extensive occlusive CA atherosclerosis accompanied by myocardial infarction, and exhibit reduced iv survival compared to LDLR KO control mice when fed a variety of atherogenic diets. In both SR-B1 single KO and SR-BI/LDLR dKO mice, CA atherosclerosis is accompanied by splenomegaly, elevated numbers of circulating leukocytes and increased expression of VCAM-1 in CA endothelium. Interestingly, removal of the spleen has no effect on circulating leukocyte numbers or atherosclerosis in SR-B1/LDLR dKO mice, suggesting the enlarged spleens in SR-B1 deficient mice do not influence atherosclerosis in these animals. We conclude that SR-B1 is important for the protection against atherosclerosis in mice, particularly in CAs. This is likely through roles in multiple cell types including hepatocytes, endothelial cells and bone marrow-derived cells. Future studies should focus on evaluating the impact of cell-specific SR-B1 activity in different cell types on murine atherosclerotic CA disease. / Thesis / Doctor of Philosophy (PhD)

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