Statins are one of the most widely prescribed drug classes because they lower circulating low density lipoprotein-cholesterol (LDL) and reduce the risk of cardiovascular events. Statin-mediated inhibition of HMG-CoA reductase also lowers substrates required for protein prenylation. This cholesterol-independent effect of statins can alter immune function. Lower protein prenylation can increase IL-1β. This pro-inflammatory cytokine can promote insulin resistance, which may be a factor in the recent evidence linking statins to increased incidence of diabetes. IL-1β is unique compared to most cytokines because it can be regulated by the NLRP3 inflammasome. In this thesis, we discovered that statins promote NLRP3-dependent insulin resistance in adipose tissue. We next hypothesized that statin-induced lowering of protein prenylation activated the NLRP3/Caspase-1 inflammasome, which would cause IL-1β-dependent insulin resistance in adipose tissue. We showed that atorvastatin impaired insulin signalling in adipose tissue from WT, but not IL-1β-/- mice. Treatment with a caspase-1 inhibitor prevented atorvastatin-inhibition of insulin signalling. The isoprenoid, Geranylgeranyl pyrophosphate (GGPP) also prevented atorvastatin-induced defects in insulin signalling. Interestingly, atorvastatin- inhibition of insulin action was associated with decreased insulin-stimulated lipogenesis in both white adipose tissue and 3T3-L1 adipocytes. The findings in this thesis suggest a statin-induced reduction in isoprenoids, required for protein prenylation production, impairs insulin action via IL-1β derived from activation of NLRP3/Caspase-1 inflammasome in adipose tissue. / Thesis / Doctor of Philosophy (PhD) / Statins are one of the most widely prescribed drugs and used in the treatment of cardiovascular disease. Statins lower LDL often called bad cholesterol. However, some patients experience side effects such as higher blood glucose and increased risk of type 2 diabetes. Type 2 diabetes is generally preceded by insulin resistance and this period of “prediabetes” is reversible. Inflammation is one factor involved in insulin resistance and in the development of type 2 diabetes. In this thesis, we discover that statins activate an inflammatory response known as the NLRP3 inflammasome, which leads to insulin resistance, particularly in fat tissue. Activation of this inflammasome by a statin, did not require lower cholesterol, but increased the inflammatory cytokine IL-1b, which was the key factor leading to insulin resistance in fat tissue. Inhibition of the NLRP3 inflammasome or IL-1b may prevent insulin resistance and risk of type 2 diabetes in people taking statins.

Statins are the mainstay of cholesterol-lowering therapy and are taken by millions of people worldwide. These drugs are generally well-tolerated but can cause myopathy ranging from mild muscle pain to fatal rhabdomyolysis. The mechanism of statin-induced myopathy (SIM) is not fully understood and there is currently no convenient and reliable marker of SIM, but mitochondrial dysfunction has been implicated. We sought to investigate the effect of statins on mitochondrial DNA (mtDNA) levels in order to gain information on the mechanism of SIM and to explore the possibility of utilizing changing mtDNA levels as a marker of SIM. Several approaches were used. First, mtDNA levels were quantified in skeletal muscle biopsies collected from a previously published 8-week clinical trial of high-dose simvastatin or atorvastatin versus placebo. Forty-eight hypercholesterolemic subjects were randomly assigned to receive placebo (N=16), high dose atorvastatin 40mg/day (N=16), or high dose simvastatin 80mg/day (N=16) for 8 weeks. Muscle mtDNA content was assessed by real-time PCR atbaseline and after 8-weeks on statin treatment and found to be significantly reduced in the groupreceiving simvastatin (P=0.005) but not the other two. In addition, a significant positive correlation was observed between mtDNA and muscle ubiquinone in all groups (R=0.63, P<0.01), with the strongest association found in the simvastatin-treated subjects (R=0.75, P=0.002). Next, in an attempt to determine whether statin-induced muscle pain may be associated with muscle mtDNA depletion, archived muscle biopsies collected from statin users with muscle complaints were sought through a review of a muscle biopsy database and possible study samples were identified; however, this was put on hold as too much information was missing from the pathology reports. Third, a series of cell culture experiments were carried out in which human skeletal muscle myotubes were exposed to various concentrations of simvastatin or atorvastatin, in order to determine an appropriate dose range for subsequent mitochondrial toxicity experiments. Lastly, mtDNA content and expression was quantified in skeletal muscle biopsies collected from 10 patients with statin-induced rhabdomyolysis (SIR) and compared to 8 healthy controls to investigate whether muscle mtDNA is altered in rhabdomyolysis. No differences in mtDNA content or expression were observed between the two study groups, but this may have been be due to the SIR subjects' marked heterogeneity. Statin therapy can be associated with considerable alterations in mtDNA content, which may play a role in the aetiology of SIM. MtDNA levels alterations with statin exposure should be investigated further to explore the involvement of mitochondrial alterations in the mechanism of SIM, and determine whether these may represent a useful clinical tool for assessing statin-induced muscle toxicity.

Statins are the mainstay of cholesterol-lowering therapy and are taken by millions of people worldwide. These drugs are generally well-tolerated but can cause myopathy ranging from mild muscle pain to fatal rhabdomyolysis. The mechanism of statin-induced myopathy (SIM) is not fully understood and there is currently no convenient and reliable marker of SIM, but mitochondrial dysfunction has been implicated. We sought to investigate the effect of statins on mitochondrial DNA (mtDNA) levels in order to gain information on the mechanism of SIM and to explore the possibility of utilizing changing mtDNA levels as a marker of SIM. Several approaches were used. First, mtDNA levels were quantified in skeletal muscle biopsies collected from a previously published 8-week clinical trial of high-dose simvastatin or atorvastatin versus placebo. Forty-eight hypercholesterolemic subjects were randomly assigned to receive placebo (N=16), high dose atorvastatin 40mg/day (N=16), or high dose simvastatin 80mg/day (N=16) for 8 weeks. Muscle mtDNA content was assessed by real-time PCR atbaseline and after 8-weeks on statin treatment and found to be significantly reduced in the groupreceiving simvastatin (P=0.005) but not the other two. In addition, a significant positive correlation was observed between mtDNA and muscle ubiquinone in all groups (R=0.63, P<0.01), with the strongest association found in the simvastatin-treated subjects (R=0.75, P=0.002). Next, in an attempt to determine whether statin-induced muscle pain may be associated with muscle mtDNA depletion, archived muscle biopsies collected from statin users with muscle complaints were sought through a review of a muscle biopsy database and possible study samples were identified; however, this was put on hold as too much information was missing from the pathology reports. Third, a series of cell culture experiments were carried out in which human skeletal muscle myotubes were exposed to various concentrations of simvastatin or atorvastatin, in order to determine an appropriate dose range for subsequent mitochondrial toxicity experiments. Lastly, mtDNA content and expression was quantified in skeletal muscle biopsies collected from 10 patients with statin-induced rhabdomyolysis (SIR) and compared to 8 healthy controls to investigate whether muscle mtDNA is altered in rhabdomyolysis. No differences in mtDNA content or expression were observed between the two study groups, but this may have been be due to the SIR subjects' marked heterogeneity. Statin therapy can be associated with considerable alterations in mtDNA content, which may play a role in the aetiology of SIM. MtDNA levels alterations with statin exposure should be investigated further to explore the involvement of mitochondrial alterations in the mechanism of SIM, and determine whether these may represent a useful clinical tool for assessing statin-induced muscle toxicity.

Statins are the mainstay of cholesterol-lowering therapy and are taken by millions of people worldwide. These drugs are generally well-tolerated but can cause myopathy ranging from mild muscle pain to fatal rhabdomyolysis. The mechanism of statin-induced myopathy (SIM) is not fully understood and there is currently no convenient and reliable marker of SIM, but mitochondrial dysfunction has been implicated. We sought to investigate the effect of statins on mitochondrial DNA (mtDNA) levels in order to gain information on the mechanism of SIM and to explore the possibility of utilizing changing mtDNA levels as a marker of SIM. Several approaches were used. First, mtDNA levels were quantified in skeletal muscle biopsies collected from a previously published 8-week clinical trial of high-dose simvastatin or atorvastatin versus placebo. Forty-eight hypercholesterolemic subjects were randomly assigned to receive placebo (N=16), high dose atorvastatin 40mg/day (N=16), or high dose simvastatin 80mg/day (N=16) for 8 weeks. Muscle mtDNA content was assessed by real-time PCR atbaseline and after 8-weeks on statin treatment and found to be significantly reduced in the groupreceiving simvastatin (P=0.005) but not the other two. In addition, a significant positive correlation was observed between mtDNA and muscle ubiquinone in all groups (R=0.63, P<0.01), with the strongest association found in the simvastatin-treated subjects (R=0.75, P=0.002). Next, in an attempt to determine whether statin-induced muscle pain may be associated with muscle mtDNA depletion, archived muscle biopsies collected from statin users with muscle complaints were sought through a review of a muscle biopsy database and possible study samples were identified; however, this was put on hold as too much information was missing from the pathology reports. Third, a series of cell culture experiments were carried out in which human skeletal muscle myotubes were exposed to various concentrations of simvastatin or atorvastatin, in order to determine an appropriate dose range for subsequent mitochondrial toxicity experiments. Lastly, mtDNA content and expression was quantified in skeletal muscle biopsies collected from 10 patients with statin-induced rhabdomyolysis (SIR) and compared to 8 healthy controls to investigate whether muscle mtDNA is altered in rhabdomyolysis. No differences in mtDNA content or expression were observed between the two study groups, but this may have been be due to the SIR subjects' marked heterogeneity. Statin therapy can be associated with considerable alterations in mtDNA content, which may play a role in the aetiology of SIM. MtDNA levels alterations with statin exposure should be investigated further to explore the involvement of mitochondrial alterations in the mechanism of SIM, and determine whether these may represent a useful clinical tool for assessing statin-induced muscle toxicity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Class of 2016 Abstract / Objectives: The goals of this study were: to assess patients’ knowledge of grapefruit interactions when taking statin class (dyslipidemia) medications, to identify any pertinent demographic characteristics that may influence knowledge of grapefruit statin interactions, and to identify patient preferred sources of health information. Methods: Questionnaires were administered at community health fairs during the academic school year 2014 through 2015. The survey addressed grapefruit consumption, frequency and amount, for both whole fruit and juice; examined knowledge of the potential for harmful interactions of grapefruit juice with statin medications; and how or where the participant learned this information. Results: A total of 74 participants completed surveys, of which, 72 submitted fully completed surveys, mean age was 64 (SD=+/- 15.6), 71.2% were female (N=52), and 78.1% were white. Of those surveyed, 63.5% (N= 47) reported consuming grapefruit in the past 12 months, and 36.1% (N=26) reported taking a statin. Those taking statins, 50% (N=13) reported consuming grapefruit as well. The majority of people, 61.3% (N=45), reported obtaining health related information from healthcare sources. Those with a college education were more likely to have consumed grapefruit in the last 12 months (X2=4.88, p=0.027) and to have ever consumed grapefruit (X2 =4.40, p=0.036). Conclusions: The majority of the health fair attendees surveyed were highly educated, reported having health insurance, had consumed grapefruit in the past year, and had heard about grapefruit-drug interactions.

L’angiogenèse ou la mise en place de nouveaux vaisseaux sanguins à partir de vaisseaux préexistants, est indispensable à la croissance tumorale et constitue donc une cible intéressante en thérapie anticancéreuse. Depuis 2004, plusieurs thérapies anti-angiogéniques ciblant les cancers du colon, poumon, sein et rein, ont été développées et sont appliquées en clinique. Le gène VE-statine/egfl7 a été découvert au sein du laboratoire en 2003 et il est majoritairement exprimé par les cellules endothéliales au cours du développement embryonnaire et du remodelage vasculaire. Plusieurs publications ont montré l’implication de la VE-statine dans la formation des vaisseaux. L’expression de la VE-statine par des tumeurs humaines a récemment été décrite, et une forte expression est associée avec un haut grade tumoral, une forte incidence métastatique et un mauvais pronostic dans les hépatocarcinomes, les gliomes et les tumeurs colorectales. L’objectif de ma thèse était de mettre au point les approches et d’étudier les effets de la VE-statine dans la croissance et l’angiogenèse tumorale. Cette analyse permet de mieux comprendre les mécanismes modulant la progression tumorale. / Angiogenesis, which corresponds to the growth of new blood vessels from pre-existing vessels; is necessary for tumor growth and is an attractive target for anticancer therapy. Since 2004, several antiangiogenic therapies against colon, lung, breast, kidney cancers have been developed and used in clinic.VE-statin/egfl7 was discovered by our team in 2003. It is specifically expressed by endothelial cells during embryonic development and vascular remodelling. Several publications have described the role of VE-statin in vessel formation. VE-statin expression by human tumors has been recently associated with a higher grade and metastatic score and with a poorer prognosis in hepatocellular carcinoma, glioma and colorectal cancer.The aim of my work was to develop tools for the study of the functions of VE-statin in tumor growth and angiogenesis. This analysis allows a better understanding of the mechanisms which modulate tumor progression.

[Truncated abstract] Dyslipidaemia in obesity and the metabolic syndrome is typically characterized by elevated plasma concentrations of apolipoprotein (apo) B and chylomicron remnants, and low apoA-I levels. This may account for the increased risk of cardiovascularrelated diseases. Although the precise mechanisms whereby visceral obesity confers the onset of dyslipidaemia have not been fully established, it may relate chiefly to insulin resistance. Insulin resistance leads to increased hepatic secretion of very low density lipoprotein (VLDL) apoB, as well as impaired catabolism of VLDL, intermediate density lipoprotein (IDL), low-density lipoprotein (LDL) and chylomicron remnants, and high density lipoprotein (HDL) apoA-I. This thesis tests the unifying hypothesis that lipoprotein metabolism, in particular apoB, chylomicron remnants and apoA-I, is abnormal in the metabolic syndrome, and that medical intervention can correct for these abnormalities. The primary objectives were to examine firstly, the kinetics of apoB and apoA-I by stable isotope technology and secondly, chylomicron remnant kinetics by using an indirect assessment of a new breath test. Six observational statements and related hypotheses were constructed and derived from the unifying hypothesis that examine the kinetics of lipoprotein metabolism, adipose tissue mass compartments and liver fat accumulation, as well as the impact of plasma adipocytokines in subjects with visceral adiposity and features of the metabolic syndrome. The first four observational statements related to cross-sectional studies of lipoprotein kinetics, adipose tissue mass distribution and liver fat accumulation as well as plasma adipocytokines in both obese and non-obese men. The latter two observational statements related to the effect of statin therapy and dietary weight loss on the improvement of lipoprotein kinetics in obesity. The findings from these studies collectively support the unifying hypothesis. The kinetics of apoB in VLDL, IDL and LDL, and apoA-I in HDL were assessed by gas-chromatography mass spectrometry following either a primed-constant infusion of 13C-leucine or an intravenous bolus injection of d3-leucine. ... This is the first study to examine the effects of dietary weight loss on LDL and HDL metabolism and the relationships with adipocytokines in men with the metabolic syndrome. The data support the unifying hypothesis that medical intervention with dietary weight loss could correct the kinetic abnormalities in VLDL, LDL and HDL. The aforementioned studies showed that plasma lipid and lipoprotein abnormalities in visceral obesity are chiefly regulated by the combination of hepatic over-secretion of VLDL particles, and catabolic defects in apoB and chylomicron remnants as well as apoA-I-containing lipoprotein particles. These kinetic defects may also relate to low and high plasma adiponectin and RBP-4 levels, respectively. The data arising from the thesis are consistent with the unifying hypothesis and support the role of dietary intervention and pharmacotherapy as a recommended treatment in correcting the abnormalities in lipoprotein metabolism within the metabolic syndrome.

Statins can act as preconditioning agents against ischemia reperfusion (IR)-injury through a mechanism involving cyclooxygenase (COX)-2 and the upregulation of prostaglandin synthesis. The following study investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against IR-induced endothelial dysfunction in the human forearm vasculature. Healthy volunteers were randomized to drugs with different COX-inhibiting properties: 81mg aspirin (OD), 325mg aspirin (OD), 400mg ibuprofen (QID), 200mg celecoxib (BID) or placebo. A single dose of 40mg rosuvastatin was also administered 24-hours prior to IR. Endothelial function before and after IR was assessed by measuring flow-mediated dilation of the radial artery. Our results show that 81mg and 325mg aspirin (more COX-1 selective), 400mg ibuprofen (similar selectivity for COX-1/2) and 200mg celecoxib (COX-2 selective) all effectively abolished statin-mediated protection against IR-induced endothelial dysfunction in the forearm (2-way ANOVA, p<0.05). These findings indicate that even partial COX-2 inhibition is sufficient to attenuate statin-induced preconditioning.

Statins can act as preconditioning agents against ischemia reperfusion (IR)-injury through a mechanism involving cyclooxygenase (COX)-2 and the upregulation of prostaglandin synthesis. The following study investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against IR-induced endothelial dysfunction in the human forearm vasculature. Healthy volunteers were randomized to drugs with different COX-inhibiting properties: 81mg aspirin (OD), 325mg aspirin (OD), 400mg ibuprofen (QID), 200mg celecoxib (BID) or placebo. A single dose of 40mg rosuvastatin was also administered 24-hours prior to IR. Endothelial function before and after IR was assessed by measuring flow-mediated dilation of the radial artery. Our results show that 81mg and 325mg aspirin (more COX-1 selective), 400mg ibuprofen (similar selectivity for COX-1/2) and 200mg celecoxib (COX-2 selective) all effectively abolished statin-mediated protection against IR-induced endothelial dysfunction in the forearm (2-way ANOVA, p<0.05). These findings indicate that even partial COX-2 inhibition is sufficient to attenuate statin-induced preconditioning.

Venous thromboembolism (VTE) which includes pulmonary embolism (PE), or deep venous thrombosis (DVT) poses an important disease burden, however, much remains unknown about the risk factors that cause it. In recent years, more attention has focused on medications that play a role in the development of drug-induced venous thrombosis. The three studies in this dissertation explore the risk of VTE in relation to the use of specific pharmacologic agents; glucocorticoids, 5ARIs and statins in three distinct patient populations with; asthma, benign prostatic hyperplasia (BPH) and hypercholesterolemia, using different approaches to address the inevitable confounding present in etiologic pharmacoepidemiologic research. Using a nested case-control design for the first two studies and cohort design for the last, we assessed the risk of VTE in relation to timing of drug exposure, duration of use, number, and dose of prescriptions. The base populations comprised subjects who received at least one prescription for any of the pharmacologic agents of interest (during 1995–2015 for study 1 and 2 and 1995–2018 for study 3) in the UK-based Clinical Practice Research Datalink. We used descriptive analyses as well as conditional logistic regression and Poisson regression models to evaluate the relationship between these drugs and the risk of VTE. Study 1 examined the relationship between glucocorticoid use and venous thromboembolism among asthma patients age 20–59. We found that current and systemic glucocorticoid use was associated with an increased risk of VTE, with a dose-response relationship. Study 2 explored the relationship between VTE and 5ARI use compared to alpha blocker use among patients, age 40–79, with BPH. We observed that cumulatively high doses of 5ARI+/-AB increases the risk of incident VTE. In study 3, we examined the relationship between VTE and statin use compared to fibrate use among 40–79-year-old patients with hypercholesterolemia. We observed that statin use decreases the risk of incident VTE. These three studies in a large population-based database of high quality, efficiently evaluate the safety/unintended benefit of glucocorticoids, 5ARIS and statins, with the hope to guide the development of guidelines for their use in prolonged care of asthma, BPH and hypercholesterolemia, respectively. / 2023-08-25T00:00:00Z