Atherosclerosis is the leading cause of death in the Western world. Although mitochondrial DNA (mtDNA) damage has been implicated in atherosclerosis, it is unclear whether the damage is sufficient to impair mitochondrial respiration, and mitochondrial dysfunction has not been demonstrated. Treatment of vascular smooth muscle cells (VSMCs) with an atherogenic lipid, oxidised low-density lipoprotein (OxLDL), dose dependently decreased basal and maximal respiration and fat-feeding of apolipoprotein E deficient (ApoE-/-) mice reduced mitochondrial DNA copy number relative to nuclear DNA in aortas. Mitochondrial respiration of ApoE-/- mouse aortas, assessed through a 24-well Seahorse extracellular flux analyser, was not affected prior to the development of atherosclerotic plaques. Developed human carotid atherosclerotic plaques were dissected into defined regions including healthy media, shoulder region, fibrous cap and core and their respiration was investigated. The respiratory reserve capacity (RRC) of the shoulder region was similar to the media. However, the cap RRC was significantly reduced compared to healthy media. In contrast, the extracellular acidification rates (ECAR) of the media, shoulder, cap and core regions were similar. In addition, mtDNA copy number was significantly reduced in tissues derived from human plaques compared to healthy arteries and expression of complexes I and II of the electron transfer chain (ETC) were significantly reduced in plaque VSMCs. OxLDL induced mitophagy in human VSMCs and plaque VSMCs demonstrated increased levels of mitophagy without compensatory upregulation of proteins involved in mitochondrial biogenesis. Understanding the role of mitochondrial metabolism and signalling is important for our understanding of disease progression and may lead to future therapeutic targets.

O Metotrexato (MTX) é um fármaco utilizado como anti-inflamatório no tratamento da artrite reumatóide (AR). O risco de doença cardiovascular em pacientes com AR é menor quando tratados com MTX. Apesar dessa evidência, há poucos relatos da utilização de MTX para o tratamento da aterosclerose. Foi desenvolvida em nosso laboratório uma nanopartícula rica em colesterol (LDE), a qual é reconhecida pelos receptores da lipoproteína de baixa densidade (LDLr) após injeção na corrente sangüínea. A LDE concentra-se em células com hiperexpressão de LDLr, em processos proliferativos como a aterosclerose. Dessa maneira, a LDE pode ser utilizada como veículo para o direcionamento de fármacos contra essas células. A molécula de MTX foi latenciada e a modificação do fármaco aumentou a sua incorporação à LDE. A proposta desse estudo foi avaliar a eficácia de MTX associado à LDE (LDE-MTX) no tratamento da aterosclerose em coelhos além de investigar o efeito desse complexo na expressão de genes inflamatórios que participam do processo aterogênico. Para realização do estudo foram utilizados quatro grupos de 10 coelhos (raça New Zealand) cada, sendo que todos foram submetidos a uma dieta rica em colesterol por 8 semanas. Após as primeiras 4 semanas de dieta, os grupos foram tratados com LDE-MTX (grupo LDE-MTX), MTX comercial (grupo MTX comercial), LDE (grupo controle LDE) ou solução salina (grupo controle Salina), via endovenosa por 4 semanas. O grupo LDE-MTX não apresentou toxicidade ao longo do tratamento de acordo com os parâmetros utilizados, enquanto que o grupo MTX comercial apresentou uma queda acentuada de eritrócitos ao final do tratamento (p<0,001). A análise morfométrica macroscópica mostrou que os grupos LDE-MTX e MTX comercial reduziram as lesões ateroscleróticas quando comparados ao grupo controle Salina (66 e 76%, respectivamente) (p<0,001). Por microscopia, a camada íntima do arco aórtico e torácico foi reduzida nos grupos LDE-MTX (67% e 75%) e MTX comercial (81% e 92%, respectivamente) quando comparados com os mesmos fragmentos do grupo controle Salina (p<0,05). A presença de macrófagos na camada íntima dos grupos LDE-MTX e MTX comercial foi reduzida em 59% e 57% (p<0,001) no arco aórtico e 37% e 38% na região da aorta torácica (p=0,016) em relação ao grupo controle Salina, respectivamente. A porcentagem de MMP-9 no arco aórtico foi reduzida em 48% em ambos os grupos tratados (p=0,0003), enquanto que na aorta torácica LDE-MTX e MTX comercial diminuíram 54% e 66% (p<0,0001) respectivamente, em relação ao grupo controle Salina. Na região da aorta abdominal, a redução de MMP-9 também foi observada nos grupos LDE-MTX (68%) e MTX comercial (70%) (p=0,016). Quando se comparou ao grupo controle LDE, os dois grupos tratados tiveram porcentagens semelhantes de redução em todas as análises morfométricas. Nos estudos de expressão gênica in vivo, 5 genes inflamatórios se mostraram hipoexpressos (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 e MMP-12) e um gene (IL-10) apresentou aumento de expressão no arco aórtico de coelhos tratados com LDE-MTX em relação ao grupo controle. No experimento in vitro, 5 dos 10 genes (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) avaliados tiveram redução da expressão e 1 (TGF-&#946;1) se mostrou hiperexpresso na linhagem de endotélio humana (HUVEC) tratada com as duas formulações de MTX. Os resultados indicam que tanto a LDE-MTX quanto MTX comercial reduzem acentuadamente as lesões ateroscleróticas em coelhos e parecem minimizar a resposta inflamatória na doença aterosclerótica. Contudo, LDE-MTX apresentou uma tolerabilidade maior ao tratamento, pois não apresentou toxicidade hematológica em comparação com MTX comercial. / Methotrexate (MTX) is the most frequently used drug for rheumatoid arthritis treatment. The incidence of vascular disease in these patients is lower when treated with MTX. However, few studies have been done using MTX for atherosclerosis treatment. In previous studies, we showed that, after injection into blood stream, a cholesterol-rich nanoparticle (LDE) binds to low density lipoprotein receptors (LDLr) and concentrates in tissues with intense cell proliferation such as atherosclerosis. LDE may thus carry drugs directed against those tissues reducing the toxicity of chemotherapeutic agents. For stable association with LDE, a lipophylic methotrexate derivative was used. The purpose of this study was to test MTX associated to LDE (LDE-MTX) in rabbits with atherosclerosis and investigate their anti-inflammatory effects on inflammatory mediators. Atherosclerosis was induced in rabbits by cholesterol rich diet during eight weeks. After 4 weeks from the introduction of the atherogenic diet, 4 groups of 10 animals were treated with LDE-MTX (LDE-MTX group), commercial MTX (commercial MTX Group), LDE (LDE control group) and saline solulion (Saline control group). MTX dose in both preparations was 4mg/kg/week during 4 additional weeks. LDE-MTX group showed superior tolerability with pronouncedly lesser hematologic toxicity in comparison to commercial MTX (p< 0.001). By morphometric analysis, both LDE-MTX and commercial MTX treatment groups showed a pronounced reduction of lesion area compared with Saline control group (66-76% respectively) (p<0001). By microscopy, intimal width at aortic arch and thoracic segments was reduced by 67% and 75% in LDE-MTX group compared to Saline control group, respectively (p<0.05). Commercial MTX group showed a reduction of 81% and 92% at aortic arch and thoracic segments compared to Saline control group, respectively (p<0.05). Presence of macrophages in intima layer at aortic arch was reduced by 59% and 57% (p<0.001) in LDE-MTX and commercial MTX groups while at thoracic segments was diminished by 37% and 38% (p=0.016) compared to Saline control group, respectively. MMP-9 percentage was diminished by 48% in both treated groups at aortic arch (p=0.0003) while at thoracic segment, LDE-MTX and commercial MTX reduced by 54% and 66% (p<0.0001) compared to Saline control group, respectively. Furthermore, MMP-9 percentage also diminished at abdominal segment in LDE-MTX group (68%) and commercial MTX (70%) when compared to Saline control group (p=0,016). When compared to LDE control group, both treated groups had similar percentage reduction in all morphometric analysis. In vivo studies, the expression of 5 inflammatory genes was downregulated (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 and MMP-12) and 1 was upregulated (IL-10) when rabbits with induced atherosclerosis were treated with LDE-MTX. Besides, 5 inflammatory genes were downregulated (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) and 1 was upregulated (TGF-&#946;1) when human endothelial cell line (HUVEC) was treated with both MTX preparations. Therefore, MTX can be an effective drug for atherosclerosis treatment and associated to LDE, side effects of this chemotherapeutic agent can be minimized.

Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. Since it is believed that OxC blocks the removal of normal cholesterol from cells in the artery wall, it is possible that selective removal of OxC in the vessel wall in-vivo may prevent or reverse atherosclerosis.As a prelude to major studies, this research project was designed to answer two critical questions; 1/. What is the best route for delivery of CD. 2/. How do animals (apoE-/- mice) tolerate it. Pilot studies were established and results noted. These studies have provided valuable information in the apoE-/- mouse for subsequent studies to prevent or reverse atherosclerosis in this animal model. / Master of Science (Hons)

People with diabetes suffer from an increased incidence of atherosclerosis, possibly due to the hyperglycaemia associated with this disease. Glucose may covalently modify proteins via glycation and glycoxidation reactions. Reactive aldehydes (e.g. methylglyoxal and glycolaldehyde) generated from these glycation and glycoxidation reactions, lipid peroxidation and other metabolic pathways may also modify proteins in glycation and glycoxidation reactions. These reactions can result in the formation of advanced glycation end-products, which are increased in diabetes and associated complications such as atherosclerosis. Low-density lipoproteins (LDLs) are the main source of lipid in atherosclerotic plaques, and the lipid-laden foam cells contained within. Modification of the single protein in LDL, apolipoprotein B-100 (apo B) by glucose and aldehydes may result in recognition of these altered LDL particles by macrophage scavenger receptors and cellular accumulation of cholesteryl esters; such accumulation is characteristic of atherosclerotic foam cells. The extent and nature of the modifications of LDLs that give rise to this behaviour have been poorly characterised, especially in regards to modification/oxidation of protein versus lipid components induced by glucose and low-molecular-mass aldehydes. Therefore the aims of this project were to: 1) characterise LDL modification by glucose, methylglyoxal and glycolaldehyde; 2) examine the effect of these modified LDLs on arterial cells by monitoring cellular viability, proliferation and cholesterol and cholesteryl ester levels; and 3) examine macrophage handling of apo B from these modified LDLs. Glycolaldehyde induced more rapid and more extensive changes to LDL than methylglyoxal, which was significantly more modified than LDL exposed to glucose, in the presence or absence of Cu2+. LDL was modified by glycolaldehyde and methylglyoxal in a time- and concentration-dependent manner. These aldehyde-modified LDLs were significantly more negatively charged relative (determined by changes in relative electrophoretic mobility), more aggregated (by SDS-PAGE) and lost more Arg, Lys and Trp residues (assessed by fluorescence-based assays) than glucose-modified and control LDLs. Glucose-modified LDL had more modest increases in net negative charge, aggregation and only significantly lost Arg residues. Under the conditions examined none of the modified LDLs contained significant levels of the protein oxidation products DOPA and o-tyrosine, the lipid oxidation products 7-ketocholesterol and cholesteryl ester hydro(pero)oxides, nor marked depletion of the major antioxidant &alpha;-tocopherol or significant radical formation (EPR spectroscopy). Therefore these LDLs were glycated, but not (glyc)oxidised, and so allowed the cellular uptake of glycated LDL, rather than glycoxidised LDL, to be examined. These glycated LDLs had no effect on the cellular viability (assessed by LDH release), cell protein (BCA assay), and cholesterol and cholesteryl ester levels (quantified by reverse-phase HPLC) of endothelial and smooth muscle cells. The glycated LDLs also had no effects on human and mouse macrophage viability, protein and free cholesterol levels. However, exposure of macrophages to some of the glycated LDLs resulted in significant accumulation of cholesteryl esters and apo B. The greatest cellular accumulation of cholesteryl esters was in cells exposed to glycolaldehyde-modified LDL, which occurred in a time- and concentration-dependent manner. Less cholesteryl ester accumulation was observed in cells exposed to methylglyoxal-modified LDL, but some conditions resulted in significantly more cellular cholesteryl esters as compared to control LDLs, unlike glucose-modified LDL. Macrophages endocytosed significantly more apo B from glycolaldehyde-modified LDL labelled with 125I on the apo B, than methylglyoxal-modified 125I-LDL. Apo B from methylglyoxal-modified 125I-LDL was also endocytosed and degraded in greater amounts than control 125I-LDLs, unlike glucose-modified 125I-LDLs. The glycation of LDL by some low-molecular-mass aldehydes have been shown to result in model foam cell formation as characterised by cholesteryl ester and apo B accumulation. This accumulation correlated with increases in net negative charge, aggregation and loss of Lys and Trp residues of the apo B in glycated LDL particles. However, the differences in cellular uptake of glycolaldehyde- versus methylglyoxal-modified LDL were not completely resolved and it is postulated that this may arise from the extent or type of products formed on key amino acid residues, resulting in differential uptake by macrophage scavenger receptors, rather than loss of particular amino acids per se. Therefore these studies provide a potential mechanism to explain the increased atherosclerosis in people with diabetes, and a suitable model to examine the potential inhibition of the effects of glycated LDLs. This could provide potential therapeutic interventions to reduce diabetes-induced atherosclerosis.

The vascular system transports components essential to the survival of the individual and acts as a barrier to substances that may injure the organism. Atherosclerosis is a dynamic, lesion producing disease of the arterial system that compromises the functioning of the organ by occlusive and thrombogenic processes. This investigation was undertaken to elucidate some of the normal biochemical processes related to the development of atherosclerosis. A significant part of the investigation was directed toward developing and combining methods and protocols to obtain the data in a concerted manner. A postmitochondnal supernatant of bovine aorta, using mevalonate-2-�����C as the substrate, was employed in the investigation. Methods included paper, thin layer, and silica gel chromatography; gel filtration, high performance liquid chromatography (HPLC), and mass spectrometry. This current research demonstrated direct incorporation of mevalonate-2- �����C into the trans-methyiglutaconic shunt intermediates. The aorta also contains alcohol dehydrogenase activity, which converts dimethylallyl alcohol and isopentenol to dimethylacrylic acid, a constituent of the trans-methylgiutaconate Small, radioactive peptides, named Nketewa as a group, were biosynthesized using mevalonate-2-�����C as the substrate. They were shown to pass through a 1000 D membrane. Acid hydrolysis and dabsyl-HPLC analysis defined the composition of the Nketewa peptides. One such peptide, Nketewa 1, had a molecular weight of 1038 and a sequence of his-gly-val-cys-phe-ala-ser-met (HGVCFASM), with a farnesyl group linked via thioether linkage to the cysteine residue. Methods were developed for the concerted investigation of the trans-methylglutaconate shunt, the isolation of mevalonate-2-�����C labeled peptides, and characteristics of neutral and acidic metabolites of mevalonate. The question as to whether or not mevalonate was the direct precursor was answered in the affirmative. These results contribute to the understanding of the biochemistry of the vessel wall and the associated atherogenic processes. Mevalonate-derived volatile and acidic compounds may represent an alternate metabolic pathway. The prenylated Nicetewa peptide may be, as are other prenylated peptides, participants in the intracellular signaling process, release of cytokines, expansion of extracellular matrix, and calcium release. / Graduation date: 2001

Introduction: Subjects with insulin resistance (IR) and diabetes are at increased risk of cardiovascular disease (CVD) than those without diabetes, however the mechanistic basis remains elusive. Despite LDL-cholesterol lowering by statin therapy, two-thirds of all CVD events remain, constituting a significant 'residual risk' for CVD. This ‘residual risk’ has been found to be greater for patients with diabetes than those without diabetes. This suggests the role for alternative sources of lipoprotein-derived cholesterol in CVD during diabetes. Both type-1 diabetic as well as IR subjects have been found to have increased plasma concentrations of fasting intestinal derived apoB48 containing remnants (CM-r). However it is not known if the diabetic metabolic milieu indeed increases the susceptibility of the arteries to CM-r and if these indeed bind to arterial proteoglycans (PGs). Objectives: To determine arterial retention of CM-r in type-1 diabetes and IR using ex vivo perfusion methodology in a streptozotocin rat model of type 1 diabetes and JCR-LA-cp rat model of IR. To determine the direct binding affinity and capacity of CM-r to biglycan using an in vitro approach. Methods and Results: We observed increased arterial CM-R retention in type 1 diabetic vessels as well as in IR vessels when compared to control vessels. The retained CM-r colocalized with arterial biglycan in type 1 diabetic vessels and a direct correlation was observed between the CM-r and the presence of glycated proteins in type I diabetic arteries. The increased arterial CM-r retention in the IR rats was associated with increased arterial biglycan protein content. We have conclusively demonstrated for the first time that CM-r indeed bind to human biglycan. Conclusion: Tight glycemic control in patients with type 1 diabetes can alleviate CVD by reducing hyperglycemia and subsequent retention of CM-r. A significant increase in biglycan protein core content during IR is suggestive of early vascular remodeling and may help to explain how CM-r accumulate more readily during diabetes induced CVD. Based on the results from this study, individuals with IR may be at increased risk for atherogenesis due to increased atherogenicity of the post-prandial CM-r when compared to normal population. / Nutrition and Metabolism

Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes. The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems. Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis. A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner. Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression. <b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types.

Capítulo I: Para evaluar el efecto sinérgico de IL-12/IL-18 y M-CSF/GM-CSF en la diferenciación de los monocitos y su impacto en el estallido respiratorio y el metabolismo del colesterol de los monocitos derivados en macrófagos, los monocitos fueron diferenciados durante 7 días en presencia de la combinación de la granulocyte-macrophage colony-stimulating factor (GM-CSF) y macrophage colonystimulating factor (M-CSF) o la interleucina 12 (IL-12) e IL-18 para producir respectivamente la M / GM-Ф y IL12/IL18- Ф. Como control, los monocitos fueron diferenciados sólo con M-CSF, GM-CSF, IL-12 e IL-18 para producir respectivamente la M-Ф, GM-Ф, IL12-Ф y IL18-Ф. El análisis de muestras de cuatro donantes de monocitos demostró una diferencia en el metabolismo del colesterol y el estallido respiratorio de las subpoblaciones de macrófagos. Los M/GM-Ф y IL12/IL18-Ф estimulados producieron alto nivel de H2O2 y mieloperoxidasa, y generaron gran cantidad de HOCl en respuesta al PMA. Por el contrario, mostraron bajos niveles de enzima antioxidante catalasa. Además intensificaron la oxidación de LDL y acumularon de forma espontánea gran cantidad de colesterol cuando incubado con la lipoproteína de baja densidad nativa. Los resultados sugieren que las vías M-CSF/GM-CSF o IL12/IL18 podría ser algunas señales sinérgica crítica experimentado por los monocitos / macrófagos durante su diferenciación in-vivo, en el que su potencial aterogénico o su capacidad de oxidar el LDL aumenta. Capítulo II: Estudios recientes muestran que el rebote de la carga del VIH-1 después de largos períodos de interrupciones del tratamiento (IT), resulta en un estallido de biomarcadores de la enfermedad arterial coronaria (CAD). Hemos investigado si las interrupciones cortas inducen un estallido de estos biomarcadores, si los niveles de estos biomarcadores vuelven a la basal durante la reintroducción del tratamiento y si los estallidos eran relacionados con el número de interrupciones. Los biomarcadores de CAD CRP, CXCL8, dímero-D, MMP-9 y los lípidos plasmáticos fueron medidos a partir de muestras de plasma almacenadas de 21 sujetos con infección crónica por el VIH-1 sometidos en un estudio de evaluación de seis ciclos de "2 semanas de interrupción" / "4 semanas reintroducción" de la terapia antirretrovirales. Los sujetos fueron agrupados en aquellos con un rebote de la carga viral después de interrumpir el tratamiento y los que no sufrieron del rebote. Los niveles de CRP, MMP-9, CXCL8, dímero-D y los triglicéridos aumentaron significativamente después de cada IT en los pacientes con el rebote de la carga viral. Los cambios de incremento medio en los sujetos sin rebote de la carga viral eran muy bajos en comparación con la basal y sin interés clínico como los valores se mantuvieron entre los rangos plasmáticos normales. Ningún efecto tiempo se observó durante la IT a la excepción de la CRP. Todos los biomarcadores volvieron a los niveles basales después de cada reinicio del tratamiento. Los resultados sugieren que las IT antirretroviral de tan sólo dos semanas son asociadas con un estallido de relevancia clínica de los biomarcadores de CAD aguda, que indica la importancia de la adherencia al tratamiento. / We organized the thesis into two chapters: Chapter I: To evaluate the synergic effect of IL-12/IL-18 and M-CSF/GM-CSF on monocytes differentiation and their impact on the respiratory burst and cholesterol metabolism of monocytes-derived macrophages, monocytes were differentiated for 7 days in the presence of both granulocyte-macrophage colonystimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) or Interleukin-12 (IL- 12) and IL-18 to produce respectively M/GM-Ф and IL12/IL18-Ф. As control, monocytes were differentiated only with M-CSF, GM-CSF, IL-12 and IL-18 to produce respectively M-Ф, GM-Ф, IL12- Ф and IL18-Ф. Samples analyses of four monocytes donors demonstrated a differential in the cholesterol metabolism and respiratory burst of macrophage subpopulations. Stimulated M/GM-Ф and IL12/IL18-Ф produce high level of H2O2 and myeloperoxidase; and generate significant amount of HOCl in response to PMA. In contrast, they show low levels of anti-oxidant enzyme catalase. Moreover they intensify LDL oxidation and spontaneously accumulate significant amount of cholesterol when incubated with unmodified low-density lipoprotein. The results suggest that M-CSF/GM-CSF or IL12/IL18 pathways might be some critical synergic signals experienced by monocytes/macrophages during their differentiation in-vivo; in which their atherogenic potential or their capacity to oxidize LDL increase. Chapter II: Recent studies show that HIV-1 load rebound after long periods of treatment interruptions (TI), results in a burst of coronary artery disease (CAD) biomarkers. We investigate whether short interruptions induce a burst of these biomarkers, whether their levels return to the baseline during treatment resumption and if the burst were related to the number of interruptions. CAD biomarkers CRP, CXCL8, D-dimer, MMP-9 and plasma lipids were measured from stored plasma samples of 21 chronically HIV-1 infected subjects enrolled in a study evaluating six cycles of “2 weeks off” / “4 weeks on” antiretroviral therapy. Subjects were clustered into those with a viral load rebound after stopping treatment and those without. The levels of CRP, MMP-9, CXCL8, D-dimer and triglycerides rose significantly after each TI in subjects with viral load rebound. Changes of means increment in subjects without viral load rebound were too low relative to the baseline and without clinical interest as values stayed between the normal plasma ranges. No times effect was observed during TI except for CRP. All biomarkers return to baseline levels after each treatment resumption. The results suggest that antiretroviral TI as short as two weeks are associated with a clinically relevant burst of acute CAD biomarkers, that indicating the importance of adhering to treatment.

Flow mediated dilation (FMD) is a non-invasive test that assesses endothelial health and nitric oxide bioavailability; it is commonly used to examine changes in vascular health due to disease or de-conditioning. Currently, a wide variety of protocols are being used to assess upper and lower extremity conduit artery health. The current project was embarked upon to gain a better understanding of the FMD protocols currently being used to asses conduit artery FMD and how these results impact our understanding of a participant’s vascular health. More specifically occlusion duration, cuff placement and artery location were examined in three commonly examined conduit arteries. The FMD responses in the brachial artery (BA), superficial femoral artery (SFA), and popliteal artery (PA) of ten healthy men, mean age of 27, after five and/or two-minutes of distal occlusion were examined. When the two-minute protocol was performed on the SFA and PA, low-resistance static calf exercise was added to augment the shear stimulus. It was hypothesized that percent FMD and shear stress responses of the SFA and PA would not be significantly different after five-minutes of occlusion, thereby allowing leg conduit artery FMD to be performed on either artery. It was further hypothesized that there would be no significant differences between the shear stress and percent FMD responses of the leg conduit arteries after five or two-minutes of occlusion; inferring that shorter occlusion durations when combines with ischemic muscle contractions can be used to assess SFA or PA FMD. With regards to comparisons between arm and leg conduit arteries, it was hypothesized that there would be significant between limb differences in baseline diameter, FMD and shear stress post five-minutes of distal occlusion. These differences will be used to better understand the effects of artery location and size on conduit artery FMD IV responses. Limitations with the traditional edge-detection method of determining arterial diameter prompted the creation of a new method of measuring artery diameter, the center-based method. It was hypothesized that there would be no significant differences in the percent FMD and time to FMD after five-minutes of BA occlusion (n=7). The results of the current study demonstrated that five-minutes of calf occlusion elicited a significant PA FMD but not a significant SFA FMD. FMD post two-minutes of PA occlusion with exercise was not significantly different than that produced by five-minutes of occlusion. Conversely, two-minutes of calf occlusion with exercise was unable to elicit a SFA FMD response. Significant differences in shear stress and FMD were reported between arm and leg conduit arteries, demonstrating different responses to five-minutes of distal occlusion due to artery size and location. Finally, no significant differences were noted between FMD and time to FMD when the center-based or edge-detection method was used. This study has demonstrated that the calf occlusion protocol was unable to elicit a FMD response in the SFA FMD; this occlusion location is only able to elicit a PA FMD response. Furthermore, two-minutes of occlusion with one-minute of exercise can be used in place of the five-minute protocol to examine PA FMD but not SFA FMD. Differences between arm and leg conduit arteries are noted and it has been suggested that this is likely due to leg conduit artery adaptations to gravity. Lastly, preliminary data suggest that the center-based method is an appropriate method of measuring conduit artery diameter.