Long noncoding RNAs (lncRNAs) are emerging as novel regulators of macrophage biology and related inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human-specific lncRNAs that are not conserved in rodents, are limited. Through deep RNA-seq of human monocyte-derived macrophages, we identified SIMALR (suppressor of inflammatory macrophage apoptosis lincRNA), a human macrophage-specific long intergenic noncoding RNA (lincRNA), to be highly induced in the nucleus of LPS/IFNγ-stimulated macrophages. Treatment of LPS/IFNγ-stimulated THP1 human macrophages with SIMALR antisense oligonucleotides induced apoptosis of inflammatory macrophages, as shown by increased Annexin V+ macrophages, and increased protein expression of cleaved PARP, caspase 9, and caspase 3. Differential expression analysis of RNA-seq data from SIMALR knockdown versus control in human macrophages revealed Netrin 1 (NTN1), a known regulator of macrophage apoptosis, to be one of the top downregulated genes. NTN1 knockdown in LPS/IFNγ-stimulated THP1 macrophages induced apoptosis. This apoptotic phenotype was attenuated by treating LPS/IFNγ-stimulated macrophages with recombinant human NTN1 after SIMALR knockdown. Furthermore, NTN1 promoter-luciferase reporter activity was increased in HEK293T cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α and RNA immunoprecipitation (RIP) showed that SIMALR binds HIF1α, suggesting that SIMALR may modulate HIF1α binding at the NTN1 promoter to regulate apoptosis of macrophages. In human translational studies, SIMALR was found to be upregulated in macrophages in unstable human atherosclerotic plaques, suggesting a possible mechanistic link between inflammation and cardiovascular diseases. In addition to SIMALR, through deep RNA-seq of human monocyte-derived macrophages, we identified RP11-184M15.1, a human macrophage-specific lincRNA, to be highly induced in the cytoplasm of IL-4-stimulated macrophage. Preliminary data showed that treatment of IL-4-stimulated THP1 human macrophages with RP11-184M15.1 small interfering RNA (siRNA) repressed apoptosis of resolving macrophages, as shown by decreased Annexin V+ macrophages, and reduced protein expression of cleaved PARP. Biotinylated RP11-184M15.1 pulldown coupled with mass spectrometry indicated an interaction between RP11-184M15.1 and zinc finger RNA-binding protein (ZFR). RIP corroborated the proposed interaction between RP11-184M15.1 and ZFR. RNAInter revealed mRNAs predicted to interact with ZFR, and some of those genes (e.g., ALYREF, CCNYL1) were also differentially expressed in RNA-seq data of control versus RP11-184M15.1 knockdown in IL-4-stimulated THP1 macrophages. qPCR validated that ALYREF and CCNYL1 expression are reduced with RP11-184M15.1 knockdown. In contrast, with ZFR siRNA, ALYREF and CCNYL1 mRNA expressions were elevated. Thus, a hypothesis to be further tested is that RP11-184M15.1 interacts with ZFR to regulate mRNA stability in IL-4-stimulated macrophages. Nuclear RNA export factor 1 (NXF1) was also validated by RIP to interact with RP11-184M15.1. NXF1 is a known interacting partner of ALYREF in the transcription-export (TREX) complex. With RP11-184M15.1 knockdown, the protein level of ALYREF decreased, and Ingenuity Pathway Analysis (IPA) of RNA-seq data of control versus RP11-184M15.1 knockdown revealed that THO complex subunit 5 homolog (THOC5), another component of the TREX complex, may be an upstream regulator. In addition, past studies have revealed that ALYREF and NXF1 are involved in nuclear export of inflammatory mRNAs and proinflammatory macrophage phenotype, respectively. With RP11-184M15.1 knockdown, there was decreased expression of inflammatory macrophage-associated genes. It may be possible that RP11-184M15.1 functions in mRNA export, along with NXF1 and ALYREF. In human translational studies, RP11-184M15.1 was found to be upregulated in macrophages in unstable human atherosclerotic plaques. Further work is needed to better understand the functions and molecular mechanism of RP11-184M15.1. In summary, we found that SIMALR may interact with HIF1α to regulate macrophage apoptosis via NTN1. Our preliminary work also revealed that RP11-184M15.1 may regulate apoptosis, mRNA stability and mRNA export in anti-inflammatory macrophages. Both lincRNAs may be upregulated in unstable human atherosclerotic plaques. By studying SIMALR and RP11-184M15.1, we were able to illustrate the importance of interrogating the functions of human-specific lincRNAs despite the lack of rodent models, and demonstrated roles in macrophage inflammation that may be relevant to human cardiovascular disease.

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

Cardiovascular disease (CVD) affects millions of people and is a leading cause of death worldwide. CVD consists of a broad set of conditions including structural heart disease, coronary artery disease and stroke. Risk for each of these conditions accumulates over long periods of time depending on several risk factors. In order to reduce morbidity and mortality due to CVD, preventative treatments administered prior to first CVD event are critical. According to clinical guidelines, such treatments should be guided by an individual’s total risk within a window of time. A related objective is secondary prevention, or early detection, wherein the aim is to identify and mitigate the impact of a disease that has already taken effect. With the widespread adoption of electronic health records (EHRs), there is tremendous opportunity to build better methods for risk assessment and early detection. However, existing methods which use EHRs are limited in several ways: (1) they do not leverage the full longitudinal history of patients, (2) they use a limited feature set or specific data modalities, and (3) they are rarely validated in broader populations and across different institutions. In this dissertation, I address each of these limitations. In Aim 1, I explore the challenge of handling longitudinal, irregularly sampled clinical data, proposing discriminative and generative approaches to model this data. In Aim 2, I develop a multimodal approach for the early detection of structural heart disease. Finally, in Aim 3, I study how different feature inclusion choices affect the transportability of deep risk assessment models of coronary artery disease across institutions. Collectively, this dissertation contributes important insights towards building better approaches for risk assessment and early detection of CVD using EHR data and systematically assessing their transportability across institutions and populations.

published_or_final_version / Community Medicine / Master / Master of Public Health

No description available.

Thesis (DScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Vascular endothelium forms the first line of defence against harmful stimuli in the circulation. Endothelial dysfunction is a valuable predictor of cardiovascular disease and therapies aimed at improving endothelial function are therefore needed. The anti-dyslipidaemic agents, simvastatin and fenofibrate, are known for their beneficial effects on lipid parameters, however additional pleiotropic effects have been shown for both. These include improved endothelial function due to increased levels of nitric oxide (NO), as well as anti-oxidant and anti-inflammatory actions. NO is produced by the enzyme, nitric oxide synthase (NOS), which exists in the endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) isoforms. Most studies investigating the endothelial effects of simvastatin and fenofibrate are performed on macrovascular-derived endothelial cells, and there is a lack of data on endothelial cells (ECs) from the microcirculation, particularly the cardiac microvessels. This dissertation aimed to investigate and elucidate mechanisms underlying the pleiotropic effects of simvastatin and fenofibrate on ECs and vascular tissue using in vitro, ex vivo and in vivo experimental models. In vitro investigations included flow cytometry-based intracellular measurements of NO, as well as different types of reactive oxygen species (ROS) and cell viability parameters. Signalling pathways involved with these changes were measured by western blot analyses of the expression and phosphorylation of critical proteins involved in vascular function. Results on cardiac microvascular ECs (CMECs) demonstrated that fenofibrate (50 μM) exerted a potent, increasing effect on NO production after short periods (1 and 4 hour treatments), but after 24 hours the effects were less robust. Exhaustive investigations suggested that the NOincreasing effects of fenofibrate in baseline CMECs were NOS-independent, a novel finding as far as we are aware. Fenofibrate’s ability to protect ECs against injury was demonstrated when CMECs incubated with the pro-inflammatory cytokine, TNF-α, were pre-treated with fenofibrate, resulting in increased NO and improved cell viability parameters. Simvastatin (1 μM) increased NO to a lesser extent in baseline CMECs, and resulted in increased apoptosis and necrosis. Following the cell studies, their effects on vascular reactivity was measured by aortic ring isometric tension studies. The effects of acutely administered fenofibrate to pre-contracted aortic rings were investigated, and results showed a modest, but significant NOS-dependent vasodilatory response. Next, an in vivo model of Wistar rats treated with simvastatin (0.5 mg/kg/day) and fenofibrate (100 mg/kg/day) for 6 weeks was established. Data showed that neither drug was able to improve aortic ring contraction and dilation above baseline values. Both drug treatments increased iNOS expression, which is usually associated with harmful actions. However, in our hands, increased iNOS expression was associated with a beneficial anticontractile response in the simvastatin-treated animals. Fenofibrate treatment increased NO bioavailability in the blood of these animals. In conclusion, fenofibrate showed endothelio-protective pleiotropic effects with regards to NO production after short treatment periods in CMECs. These effects were mediated via a NOSindependent mechanism, a novel finding. Fenofibrate pre-treatment was also protective against the harmful effects of TNF-α. Simvastatin did not show pronounced pleiotropic effects in vitro or in vivo on endothelial function. / AFRIKAANSE OPSOMMING: Die vaskulêre endoteellaag is die eerste linie van verdediging teen skadelike stimuli in die bloedsirkulasie. Endoteeldisfunksie is ‘n waardevolle voorspeller van kardiovaskulêre siektes en enige terapeutiese behandeling wat kan bydra tot verbeterde endoteelfunksie is belangrik. Simvastatien en fenofibraat word as anti-dislipidemiese middels voorgeskryf en hoewel hulle primêr gebruik word om cholesterolvlakke te verbeter, toon hulle ook pleiotropiese (cholesterolonafhanklike) eienskappe. Dit sluit in bevordering van endoteelfunksie (via verhoogde stikstofoksied (NO) produksie), asook anti-oksidant en anti-inflammatoriese effekte. NO word vervaardig deur die ensiem, stikstofoksiedsintase (NOS) wat voorkom in drie isovorme: endoteelafgeleide NOS (eNOS), induseerbare NOS (iNOS) en neuronale NOS (nNOS). Die meerderheid studies wat pleiotropiese effekte van simvastatien en fenofibraat ondersoek, gebruik endoteelselle van makrovaskulêre bloedvate, wat beteken daar is ‘n tekort aan data aangaande endoteelselle vanaf mikrovaskulêre vate, veral kardiale mikrovaskulêre vate (CMECs). Hierdie proefskrif het dit ten doel gehad om meganismes betrokke by die pleiotropiese effekte van simvastatien en fenofibraat te ondersoek deur van in vitro, ex vivo en in vivo modelle gebruik te maak. Die in vitro ondersoeke het gefokus op vloeisitometrie-gebaseerde metings van intrasellulêre NO, reaktiewe suurstof-radikale (ROS) en sellewensvatbaarheid. Seintransduksie paaie betrokke by hierdie veranderinge was bepaal deur proteienuitdrukking en -fosforilasie vlakke te meet van belangrike proteïene, met behulp van die Western-blot tegniek. Resultate van die CMEC eksperimente het getoon dat fenofibraat (50 μM) ‘n kragtige en verhogende effek op NO produksie uitgeoefen het na kort behandelingstye (1 en 4 ure), maar na 24 uur was hierdie effek minder uitgesproke. Uitvoerige ondersoeke het getoon dat fenofibraat se basislyn effekte op CMECs deur NOS-onafhanklike meganismes teweeggebring is, en sover ons kennis strek, is dit ‘n nuwe bevinding. Fenofibraat se endoteel-beskermende effekte kon ook aangetoon word deur CMECs vir een uur te behandel voor byvoeging van die pro-inflammatories sitokien, tumor nekrose faktor alpha (TNF-α), wat gelei het tot verhoogde NO vlakke en verbeterde seloorlewing. Simvastatien (1 μM) het tot ‘n mindere mate NO produksie verhoog in CMECs, tesame met pro-apoptotiese en -nekrotiese effekte. Vervolgens was die effekte op vaskulêre reaktiwiteit geëvalueer d.m.v. isometriese spanningsondersoeke. Akute effekte van fenofibraat is gemeet deur byvoeging daarvan tot ‘n vooraf saamgetrekte aorta-ring, wat tot matige, maar beduidende NOS-afhanklike verslapping gelei het. Hierna is ‘n in vivo model opgestel deur Wistar rotte vir ses weke met 0.5 mg/kg/dag simvastatien of 100 mg/kg/dag fenofibraat te behandel. Resultate toon dat geen van die behandelings basislyn kontraksie of verslapping van aorta ringe kon verbeter nie. Beide behandelings het tot verhoogde iNOS uitdrukking gelei, wat gewoonlik met nadelige effekte geassosieer word, maar in ons studies was dit met voordelige, anti-kontraktiele effekte in aortaringe van simvastatien-behandelde rotte geassosieer. Fenofibraat behandeling het die NObiobeskikbaarheid in die rotte se bloed verhoog. Ten slotte, fenofibraat het met endoteel-beskermende, pleiotropiese effekte op endoteelselle gepaard gegaan, veral t.o.v. NO-produksie na akute middeltoediening in die CMECs. Die meganisme was ‘n NOS-onafkanklike proses, wat ‘n nuwe bevinding is. Fenofibraat prebehandeling het teen die skadelike effekte van TNF-α beskerm. Geen uitgesproke pleiotropiese effekte is in vitro of in vivo gevind met simvastatien behandeling nie.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Community Medicine / Master / Master of Public Health

The purpose of this research was to describe the relationships among patient characteristics, care processes, and care outcomes for patients in a coronary care unit (CCU). The sample consisted of 179 CCU patients. Data collectors reviewed charts and retrieved the chart information needed to measure the operational variables of APACHE II score (Acute Physiology and Chronic Health Evaluation II), years of age, CCU length of stay, nurse to patient ratio, and mortality. Descriptive statistics were used to analyze the demographic data of the patient characteristics. Correlational statistics were used to analyze the five operational variables in the "CCU Patient Outcomes Model." Pearson correlations revealed significant positive relationships between APACHE II score and age and nurse to patient ratio. Point Biserial correlations revealed significant positive relationships between mortality and APACHE II score and nurse to patient ratio. Patient characteristics were related to care processes. Patient characteristics and care processes were related to patient outcomes.