Dravet syndrome (DS) is a severe form of pediatric epilepsy with characterizations of pharmacoresistant seizures and developmental delay. A rarer variant of the DS model is caused by heterozygous loss-of-function mutations in SCN1B, which is essential in regulating sodium channel gating, expression, localization, and the firing of action potentials. Mutations in SCN1B result in severe seizures as well as a higher risk of Sudden Unexpected Death in EPilepsy (SUDEP). Factors underlying SUDEP are poorly understood, although cardiac arrythmias have been implicated. Acebutolol (ACE) is a common beta-blocker used in the treatment of arrhythmias and hypertension. We hypothesized that treating mice with ACE will decrease cardiac arrhythmias and the incidence of SUDEP, prolonging lifespan of Scn1b null mice. Wild-type (WT) and null (KO) mice were given daily injections of 10 mg/kg ACE or saline starting at postnatal day 15 (after typical seizure onset) either during the day (09:00) or at night (21:00). ECG was recorded daily including a baseline and a 20-minute post injection measurement to analyze the long-term and acute effects of treatment. 20 minutes following ACE injection, KO mice displayed a significant reduction in heart rate compared to WT (38% vs. 11%). Interestingly, HR the day prior to death consistently dropped ~50% (average 414 bpm to 193 bpm) in our saline group; this was prevented in KO animals treated with ACE (421 bpm). A modest, but significant, increase in survival curves in our KO animals was observed compared to saline treated mice for those given injections during the day (a 2 day increase in median survival). In addition, in this group, the onset of animal death was delayed. Surprisingly, in the mice injected during the night hours, there was a trend towards a decrease in lifespan. From these findings there is a notable hypersensitivity to ACE in this DS model. Leading up to death, we believe it is possible ACE assisted in decreased cardiovascular deficits that could lead to SUDEP and contributed to the modest increased lifespan. While we are still seeing death in the ACE treated group because of unnoticed, prolonged seizures, or other mechanisms of SUDEP. In addition, our results demonstrate the importance of timing in delivery of drugs targeted at SUDEP. Further work includes testing this hypothesis by adding 24 hour drug delivery or an anti-epileptic drug to see if lifespan is further affected is warranted.

Indiana University-Purdue University Indianapolis (IUPUI) / While insomnia/sleep disturbance has been identified as an independent predictor of cardiovascular disease in the general population, no studies have examined whether insomnia contributes to the elevated cardiovascular disease (CVD) risk in people with human immunodeficiency virus (HIV). Thus, the current study examined whether insomnia symptoms predict incident atherosclerotic CVD in the Veterans Aging Cohort Study 9 (VACS9), a prospective cohort of HIV-infected (n = 3,138) and uninfected (n = 3,010) Veterans utilizing self-report measures and administrative data. In partial support of my hypotheses, I found that HIV-infected Veterans bothered a lot by difficulty falling or staying asleep have greater CVD risk than HIV-infected Veterans without these symptoms. This study failed to replicate previous findings that insomnia symptoms are predictive of incident CVD in uninfected adults, which may be due to issues related to the validity of the insomnia symptoms assessment. A number of methodological issues are identified and considered in the interpretation of the current study results. Given the novelty of examining insomnia as a predictor of incident CVD in HIV-infected adults and the limitations of the present study, future research is needed to better elucidate the association between insomnia and future CVD in this population.

Indiana University-Purdue University Indianapolis (IUPUI) / Depressed adults are at increased risk of developing atherosclerotic cardiovascular disease (CVD). However, heterogeneity in the depressed population engenders a key question: Are there subgroups of depressed adults at greater risk of developing CVD? Because other affective traits – i.e., anxiety, hostility/anger, and low trait positive affect – have also been associated with increased CVD risk, depressed adults with higher levels of these co-occurring affective traits may have an elevated risk of developing CVD. Consequently, the present study’s first aim was to examine, in depressed adults, which affective traits (depression, anxiety, hostility/anger, or low positive affect) are associated with endothelial function, a marker of cumulative CVD risk. In addition, because the other affective traits overlap with depressive symptom severity, this study’s second aim was to investigate which components of pairs of affective traits (shared versus unique) are related to endothelial function. Finally, given that the mechanisms underlying affective trait-endothelial function relationships in depressed adults are unknown, this study’s third aim was to explore traditional CVD risk status as a candidate mediator of observed relationships. To achieve these aims, I combined pre-treatment, cross-sectional data from three randomized controlled trials involving 138 depressed primary care patients with no history of clinical CVD. Assessments included validated self-report questionnaires for affective traits, brachial artery flow-mediated dilation (FMD) for endothelial function, and 10-year Framingham risk score for traditional CVD risk status. I conducted structural equation modeling (SEM) with confirmatory factor analysis to examine the relationships of interest after adjusting for age, sex, race/ethnicity, education, and baseline arterial diameter. Although the shared variance between each affective trait pair could not be modeled due to poor fit, adequate fitting models revealed that hostility/anger and the unique components of hostility/anger were associated with poorer endothelial function (standardized coefficients = -.18 and -.22, respectively). All of the other affective traits and their components (depression, anxiety, positive affect, unique depression, unique anxiety, and unique positive affect) were not related to endothelial function (all ps > .08). Traditional CVD risk status did not partially explain the relationship between the unique components of hostility/anger and endothelial function (standardized coefficient for the indirect effect = .00; p = .89). If my results are supported by future findings, it would suggest that depressed adults with hostility/anger (a) may be a subgroup of the depressed population at greater risk of developing CVD and (b) may be in need of earlier, more intense, and/or different CVD primary prevention efforts. Future studies are needed to confirm this relationship and identify underlying mechanisms.

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Paraoxonase-1 (PON1) has an anti-oxidative function in preventing the formation of oxidized lipoproteins (LDL and HDL) and hydrolyzing the active metabolites of some organophosphate insecticides (e.g., paraoxon and diazoxon) and other non-physiological substrates. PON1Q192R affects PON1 hydrolytic activity and its protective role against oxidative stress, thereby influencing susceptibility to cardiovascular disease among individuals. The objectives of this study were to determine the effect of race, gender, and age on PON1 activities and PON1192 genotypes in Caucasian and African American Southerners. Serum samples from 200 individuals (equally distributed race and gender classes, ages 25-55) were assayed spectrophotometrically for paraoxon and diazoxon hydrolysis. Data indicate a positive correspondence between PON1192 genotypes and race and PON1 activity and race. Data do not indicate an influence of gender and age on PON1 activities or PON1192 genotypes. These results are useful in explaining the increased risk of cardiovascular disease in African Americans compared to Caucasians

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Background: Diet is an important modifiable risk factor for cardiovascular disease. Numerous studies have examined the association between dietary intake and cardiovascular disease in North America and Europe, but little information is available on potential associations in many geographic regions including South Asia, South America, Africa, China, and the Middle East. Presently, it is unclear whether the findings from Western countries are applicable to these regions where population characteristics, background diet, and disease risk differ. This thesis aims to investigate the relationship between diet and cardiovascular disease and mortality in a heterogeneous, international population. Methods: Baseline data from the Prospective Urban Rural Epidemiology (PURE) study was used to investigate the availability, affordability, and consumption of fruits and vegetables. Additionally, PURE baseline and follow-up was used to examine the association between foods (fruits, vegetables and legumes) and macronutrients (total and fat subtypes, carbohydrate and protein) and cardiovascular outcomes and mortality. PURE is a prospective cohort study in individuals aged 35 to 70 years in 18 high-income middle-income and low-income countries on five continents. Availability and affordability of fruits and vegetables was collected from centrally located grocery stores and market places in each PURE community. Diet was measured using country and region-specific food frequency questionnaires at baseline. Case-report forms, death certificates, medical records and verbal autopsies were used to capture data about major cardiovascular events, and death during follow-up. The cost and diversity of fruits and vegetables was documented and mean fruit and vegetable intake by their relative cost was assessed. Associations between fruit, vegetable and legume consumption with risk of cardiovascular outcomes and mortality were examined. We investigated the association between macronutrients and risk of mortality and modeled nutrient replacement using energy-adjustment and joint effect models. Results: Results from the PURE study indicate that consumption of fruits and vegetables is low worldwide, particularly in low-income countries, and this is associated with low affordability. Higher fruit, vegetable and legume consumption was associated with a lower risk of non-cardiovascular, and total mortality and benefits appear to be maximal at three to four servings per day. This finding indicates that health benefits can be achieved at intake lower than most dietary recommendations, an approach that is likely to be more affordable in poor countries. Higher carbohydrate intake was positively associated with an increased risk of mortality, while total and fat subtypes, and protein was inversely associated with death. For the nutrient replacement analysis, the joint effect method demonstrated higher agreement with the single nutrient results compared to the conventional energy-adjustment method. This result suggests that traditional nutrient replacement modeling is not appropriate for international populations with diverse nutrient intake. Conclusions: Dietary intake varies across geographic regions and interventions to improve diet and nutrition recommendations should be tailored to the geographic setting. / Thesis / Doctor of Philosophy (PhD)

The proprotein convertase subtilisin/kexin type 9 (PCSK9) was first characterized in 2003 by Seidah and colleagues and marked the beginning of what is now considered by many as the greatest advancement in the field of cardiovascular disease (CVD) research since the discovery of the LDLR nearly half of a century ago. Since its discovery, PCSK9 was shown to enhance the degradation of cell-surface low-density lipoprotein (LDL) receptor (LDLR) and gain-of-function (GOF) mutations were shown to correlate with CVD risk. In contrast, patients carrying loss-of-function (LOF) mutations in PCSK9 highlighted a novel therapeutic approach for LDL lowering as they exhibit a life-long state of hypocholesterolemia and reduced CVD risk. A decade after the cloning of the PCSK9 gene, pharmaceutical companies have now developed a variety of PCSK9 inhibitors, ranging from monoclonal antibodies (mAbs) to small interfering RNA (siRNA) and vaccines, which have been shown to markedly reduce LDL cholesterol levels in pre-clinical models, as well as in patients at high risk of CVD. Despite these advances, there remained several unanswered questions regarding the mechanisms by which PCSK9 expression and secretion is regulated in the liver; the tissue from which the circulating pool of PCSK9 almost exclusively originates. The thought that further development of our understanding of PCSK9 biology may lead to the discovery of a signaling cascade that could be targeted by small molecules, the only class of inhibitor that has not yet been developed, has now merited additional research attention. The focal point of my doctoral studies represents the axis between a cellular process known as endoplasmic reticulum (ER) stress and PCSK9 expression/biosynthesis. ER stress is a deleterious cellular process that is known to occur in secretory cell types, such as liver hepatocytes, and is a well-established causative driver of an array of human diseases ranging from CVD to neurodegenerative diseases. ER stress is prevalent in the livers of patients with metabolic disease and is also known to activate the transcription factor capable of regulating PCSK9 levels, the sterol regulatory element-binding protein 2 (SREBP2). Based on this information, the first aim during the course of my PhD studies was to determine whether ER stress affected the expression and secretory status of PCSK9. In the past several years, I demonstrated that ER stress caused by ER Ca2+ depletion led to a marked increase in PCSK9 protein expression, but blocked its secretion as a result of its retention in the ER. Such a result was also associated with heightened hepatic LDLR expression and reduced LDL cholesterol levels in mice. Additional studies also characterized a variety of agents, including caffeine, as potent inhibitors of PCSK9 expression via increasing ER Ca2+ levels, which antagonized SREBP2 activity. As our initial studies revealed ER PCSK9 retention as a viable strategy for PCSK9 inhibition and LDL lowering, follow-up studies were also carried out to determine the outcome of such a strategy on liver function and injury. Given that heritable mutations in proteins that transit the ER can accumulate in this compartment and cause ER storage disease (ERSD), it was critical to further evaluate whether ER PCSK9 retention would lead to a similar outcome. In a series of experiments with rather surprising outcomes, we observed that the retention of the LOF Q152H PCSK9 mutant in the ER failed to cause ER stress; even in mice overexpressing the protein. Interestingly, tissue culture and mouse models demonstrated that the retention of PCSK9 in this cellular compartment increased the cellular abundance of ER stress response chaperones, such as the glucose-regulated proteins of 78- and 94-kDa (GRP78 and GRP94, respectively), but did not activate transducers of the ER stress signaling cascade. Strikingly, mice expressing the ER-retained PCSK9 Q152H mutant were protected against ER stress, suggesting a novel co-chaperone-like role of intracellular PCSK9. Collectively, the ER environment including secondary messengers like Ca2+ as well as its chaperones, plays a critical regulatory role on PCSK9 expression and secretion. Agents that increase ER Ca2+ levels can be utilized to block PCSK9 expression at the mRNA level to increase hepatic LDL clearance, and ER PCSK9 retention may also represent a safe avenue with a similar LDL lowering outcome. Beyond LDL lowering, hepatic ER PCSK9 retention may also serve as a novel strategy to enhance ER function and protect against ER stress-driven diseases of the liver. / Thesis / Doctor of Philosophy (Medical Science)