Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves: 167-186). Also available on the Internet.

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 / Master / Master of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Local neuronal activity in the brain results in increased blood flow and is called neurovascular coupling. Such blood flow changes result in the blood-oxygen level dependent (BOLD) fluctuations detectable by functional magnetic resonance imaging (fMRI). The hemodynamic response is also an essential component of brain health and is impaired in various models of cognitive dysfunction. However, we still do not understand why functional hyperemia in the brain is important. To understand this question, various groups have studied brain metabolic activity as well as the mechanisms underlying neurovascular coupling. Over the years, several cell types have been proposed to contribute to functional hyperemia in the brain, including neurons, astrocytes and pericytes. However, the picture remains incomplete – controversies abound regarding the exact role of astrocytes, and pericytes in neurovascular coupling. Our lab has studies the mechanisms of neurovascular coupling from a mesoscopic perspective, as vasodilation in the rodent cortex involves capillaries and diving arterioles in the brain parenchyma as well as surface vasculature in the brain. We proposed that the vascular endothelium itself might provide a continuous conduit for transmitting vasodilatory signals initiated at the capillary level due to local neuronal activity. Given that systemic endothelial dysfunction could contribute to decreased neurovascular function, this hypothesis raised important concerns regarding endothelial vulnerabilities in common diseases like hypertension and diabetes and its role in diminished cognitive function and neurodegeneration. Based on findings from vascular research in other organ systems, we hypothesized that two distinct mechanisms of endothelium-derived vasodilation significantly contribute to neurovascular coupling the brain. These two mechanisms were expected to consist of fast long-range endothelium-derived hyperpolarization (EDH) dependent vasodilation (conducted vasodilation) and slower, more localized endothelium calcium-wave dependent vasodilation (propagated vasodilation). Together, we expected these mechanisms to shape the spatio-temporal evolution of hemodynamic responses in the brain. This dual mechanism of endothelial control of the hyperemic response in the brain might explain the complex spatiotemporal properties and non-linearities of the fMRI blood oxygen level dependent (BOLD) signal. My initial experiments were conducted in anesthetized rats, where I pharmacologically inhibited endothelial dependent vasodilation during functional hyperemia in the somatosensory cortex under a hind-paw electrical stimulus paradigm. While the results gleaned from these experiments were very revealing, it was important to consider the effect of the pharmacological manipulations on neuronal activity in the brain. In addition, neurovascular coupling and overall brain blood flow in anesthetized animals is dramatically altered when compared to awake animals. In order to accomplish these goals, I built a wide-field optical imaging system that could simultaneously measure fluorescence-based neuronal activity and reflectance-based hemodynamic activity in awake head-restrained mice. I then used non-blood brain barrier permeable pharmacology to study endothelial mechanisms of neurovascular coupling in awake Thy1-GCaMP6f mice, which express the calcium fluorophore in a subset of excitatory neurons in the cortex. I found that using this pharmacology I could dissect out the hypothesized two spatiotemporally distinct components of whisker-stimulus evoked neurovascular coupling in awake mice. With simultaneous recording of the neuronal activity driving this blood flow, I was able to build a mathematical model for neurovascular coupling that accounted for these two mechanisms by allowing for the superposition of a time-invariant, constant hemodynamic response with a hemodynamic response obtained by convolving the underlying neuronal response with a hemodynamic response function (HRF). I was able to linearize these apparent non-linearities in the hemodynamic response by studying the properties of deconvolved HRFs for stimuli of different durations before and after pharmacological manipulation of endothelial activity. Two important considerations remain. Firstly, our wide-field, mesoscopic view of the brain prevents observations of endothelial function (hyperpolarization and calcium activity) and the propagation dynamics of dilation best observed at the microscopic level. To accomplish this task, ongoing experiments currently use our high-speed volumetric imaging technology (SCAPE – Swept Confocally Aligned Planar Excitation microscopy) to study stimulus-evoked vascular dynamics in mouse lines expressing GFP and GCaMP8 in endothelial cells. Secondly, our longitudinal imaging of these animals is ideal for studying the acute and long-term effects of endothelial dysfunction on cognitive function. This requires adequate study of changes in mouse behavior during manipulations of endothelial function longitudinally in awake mice. Future experiments should involve the development and implementation of appropriate task-based behavior experiments, and analysis methods for more carefully exploring changes in neuronal activity in the mouse brain during stimulus and non-stimulus dependent activity.

Yeung Lam. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENT --- p.V / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.2 --- Vascular endothelial growth factors (VEGFs) --- p.2 / Chapter 1.2.1 --- VEGF-A --- p.2 / Chapter 1.2.2 --- P1GF --- p.4 / Chapter 1.2.3 --- VEGF-B --- p.5 / Chapter 1.2.4 --- VEGF-C and VEGF-D --- p.6 / Chapter 1.3 --- VEGF receptors (VEGFRs) --- p.9 / Chapter 1.3.1 --- VEGFR-1 (or flt-1) --- p.9 / Chapter 1.3.2 --- VEGFR-2 ( or flk-1) --- p.10 / Chapter 1.3.3 --- VEGFR-3 ( or flt-4) --- p.11 / Chapter 1.4 --- Hormonal regulation of VEGFs by LH/hCG --- p.14 / Chapter 1.5 --- General review of the testis --- p.17 / Chapter 1.5.1 --- Structure and function of the testis --- p.17 / Chapter 1.5.2 --- Testicular vasculature --- p.18 / Chapter 1.5.3 --- Testicular angiogenesis --- p.19 / Chapter 1.6 --- Localization of VEGF and VEGF receptors in the testis --- p.20 / Chapter 1.7 --- Aims of the present study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Animals --- p.23 / Chapter 2.1.1 --- Depletion of Leydig cell --- p.23 / Chapter 2.1.2 --- Suppression of Leydig cell and stimulation by hCG --- p.24 / Chapter 2.1.3 --- Collection of tissue --- p.25 / Chapter 2.2 --- Preparation of primary cells from rat testes --- p.27 / Chapter 2.2.1 --- Sertoli cell preparation --- p.27 / Chapter 2.2.2 --- Germ cell preparation --- p.29 / Chapter 2.2.3 --- Interstitial cell and Leydig cell preparation --- p.30 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Reagents and cell lines --- p.32 / Chapter 2.3.2 --- "Mouse Leydig cell line, TM3 and Sertoli cell line, TM4" --- p.33 / Chapter 2.3.3 --- "Mouse tumor Leydig cell line, MLTC-1" --- p.34 / Chapter 2.3.4 --- "Rat tumor Leydig cell line, R2C" --- p.34 / Chapter 2.3.5 --- "Rat tumor Leydig cell line, LC540" --- p.35 / Chapter 2.4 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.35 / Chapter 2.4.1 --- Extraction of total RNA --- p.35 / Chapter 2.4.2 --- Quantitation of total RNA --- p.37 / Chapter 2.4.3 --- RT-PCR --- p.37 / Chapter 2.4.4 --- Purification and authentication of PCR products --- p.47 / Chapter 2.5 --- Immunohistochemical staining --- p.48 / Chapter 2.5.1 --- Perfusion and processing of testes for histological sections --- p.48 / Chapter 2.5.2 --- Immunohistochemical staining of tissue sections --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.52 / Chapter 2.6.1 --- Extraction and quantitation of total protein --- p.52 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Immunoblotting --- p.55 / Chapter 2.7 --- Statistical analyses --- p.57 / Chapter 3. --- Results --- p.58 / Chapter 3.1 --- Expression and localization of VEGFs in the rat testis --- p.58 / Chapter 3.1.1 --- VEGF-A --- p.58 / Chapter 3.1.2 --- VEGF-B --- p.64 / Chapter 3.1.3 --- VEGF-C --- p.69 / Chapter 3.1.4 --- VEGF-D --- p.73 / Chapter 3.1.5 --- P1GF --- p.77 / Chapter 3.2 --- Effect of Leydig cell depletion on VEGFs expression in the rat testis --- p.81 / Chapter 3.2.1 --- Effect on VEGF-A --- p.81 / Chapter 3.2.2 --- Effect on VEGF-B --- p.82 / Chapter 3.2.3 --- Effect on VEGF-C --- p.88 / Chapter 3.2.4 --- Effect on VEGF-D --- p.91 / Chapter 3.2.5 --- Effect on P1GF --- p.94 / Chapter 3.3 --- Effect of Leydig cell suppression and hCG stimulation on VEGFs expression in the rat testis --- p.97 / Chapter 3.3.1 --- Effect on VEGF-A --- p.97 / Chapter 3.3.2 --- Effect on VEGF-B --- p.107 / Chapter 3.3.3 --- Effect on VEGF-C --- p.113 / Chapter 3.3.4 --- Effect on VEGF-D --- p.119 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- References --- p.134

Ph.D. / Biomedical Engineering / The cardiovascular disease atherosclerosis is directly linked to the functions of the endothelium, the monolayer of endothelial cells (ECs) that line the lumen of all blood vessels. EC functions are affected by fluid shear stress (FSS), the tangential force exerted by flowing blood. In vivo FSS is determined by vascular geometry with relatively straight vessels producing high, unidirectional FSS and vessel branch points and curvatures producing low, oscillatory FSS. While these distinct FSS conditions differentially regulate EC functions, they also dramatically affect EC shape and cytoskeletal structure. High and unidirectional FSS induces EC elongation and cytoskeletal alignment, while concurrently promoting EC functions that are atheroprotective. In contrast, low and oscillatory FSS induces cobblestone-shaped ECs with randomly oriented cytoskeletal features, while simultaneously promoting EC functions that create an athero-prone vascular environment. Whether these distinct EC shapes and cytoskeletal structures influence EC functions, independent of FSS, is largely unknown. The overall hypothesis of this study is that cell shape and cytoskeletal structure regulate EC functions through mechanisms that are independent of FSS. Due to advances in surface engineering in the field of micropatterning, EC shape can be controlled independent of external forces by creating spatially localized surface cues. In this research, lanes of protein were micropatterned on glass surfaces to induce EC elongated shape in the absence of FSS. In Aim 1, micropattern-elongated EC (MPEC) shape and cytoskeletal structure were fully characterized and determined to be comparable to FSS-elongated ECs. Thus, inducing EC elongation on micropatterned lanes provides a platform for studying the functional consequences of EC shape, independent of FSS. Using this model, the following important markers of EC functions related to atherosclerosis were evaluated to determine the influence of EC shape and cytoskeletal alignment: extracellular matrix deposition (Aim 2), inflammatory function(Aim 3), and thrombotic potential (Aim 4). The results indicate that EC-elongated shape and cytoskeletal alignment participate in promoting selected EC functions that are protective against atherosclerosis, independent of FSS. Since EC shape is governed by the cytoskeleton, this data suggests that the cytoskeleton plays an active role in the regulation of EC functions that promote cardiovascular health.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 209-255).

Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-activated protein present on endothelial cells (ECs). Our laboratory has provided evidence that DNA methylation, a mark associated with gene silencing, is fundamental for regulating VCAM-1 expression. First, we showed that RNA polymerase II, preferentially associates with VCAM-1 hypomethylated alleles. This finding was confirmed using fluorescence-activated cell sorting (FACS) to sort populations of cytokine-activated ECs with high vs. low cell surface VCAM-1 expression. We found that ECs with high VCAM-1 expression were hypomethylated at the promoter. We then went on to show that populations of cells generated from single ECs exhibit differential VCAM-1 methylation from one another, and from the original founder population. Intriguingly, our data shows that VCAM-1 mRNA levels differ between the clones, and correlate with the observed differences in DNA methylation. Taken together, this data provides exciting evidence that DNA methylation is important in the regulation of VCAM-1 gene expression.