Dissertations / Theses on the topic 'Clinical atherosclerosi'

Proprotein Convertase Subtilisin Kexin type 9 (PCSK9), together with LDLR and APOB genes, had been identified as the third gene associated with Familial Hypercholesterolemia (FH). Secreted PCSK9 in fact, targets the hepatic LDL receptor (LDLR) for degradation thus preventing its recycling on the cell surface. The decreased expression of LDLR determines an increase in the circulating LDL particles, leading to increased cholesterol levels. Secreted PCSK9 is mainly derived from the liver, but it is also expressed in other tissues such as the brain, the kidney, the pancreas as well as the cells that composed the arterial wall. It is therefore possible that it could exert multiple paracrine effects. Our research group found that PCSK9 is expressed and secreted by smooth muscles cells (SMCs) which constitute the atherosclerotic plaque. The aim of my project was to determine the paracrine role of PCSK9 on the neointima formation through preclinical and clinical approaches. The immunohistochemical analysis of Pcsk9-/- and Pcsk9+/+ mice revealed that, after vascular manipulation, the PCSK9-null mice were protected from the formation of neointima with lower intima area (28100 ± 4901 µm2 and 14350 ± 2990 µm2 for Pcsk9+/+ and Pcsk9-/- mice respectively, p<0.05), associated with decreased intima/media ratio of 1.48 ± 0.34 and 0.60 ± 0.18 for Pcsk9+/+ and Pcsk9-/- mice respectively, (p<0.05). The in vitro studies on isolated SMCs from Pcsk9-/- and Pcsk9+/+ mice showed that the absence of PCSK9 induced a more contractile phenotype, associated with a reduced proliferation rate (doubling time were 57.3 ± 2.1h and 106.3 ± 4.5h, respectively [p<0.001]). The response to the chemotactic agent PDGF-BB (Platelet-derived growth factor), measured with Boyden’s chamber assay, was also impaired in the absence of PCSK9. These were rescued after the reconstitution of PCSK9 in the Pcsk9-/- cell line, which led to a more synthetic phenotype associated with a doubling time of 32.2± 3.1h and 41.2 ± 1.9h [p< 0.001], for Pcsk9-/- and Pcsk9-/-REC SMCs, respectively. The difference in proliferation between Pcsk9-/- and Pcsk9-/-REC SMCs was maintained also after the incubation with 40µM of simvastatin, suggesting that PCSK9 could improve SMCs proliferation through mechanism independently from cholesterol levels. The cell cycle analyses of the Pcsk9-/- and Pcsk9-/-REC SMCs showed a decreased activation of p21 and p27, associated with an increased expression of cyclin E and cyclin D1; presumably due to a different activation of the PDGF receptor pathway mediated by LRP1. Finally, the observational study, carried out in collaboration with the Brisighella Heart Study research group, demonstrated that serum levels of PCSK9, together with aging, is positively correlated to the pulse wave velocity. This is an indirect parameter used to evaluate the arterial stiffness and hence the presence of atherosclerotic plaques. Taken together, these results demonstrated that aside from its function on in regulating cholesterol homeostasis, PCSK9 plays a direct pro-atherogenic role in the arterial wall by sustaining SMC synthetic phenotype, proliferation, and migration.

Contesto e scopo: La proproteina convertasi subtilisina/kexina di tipo 9 (PCSK9), uno dei principali regolatori del metabolismo del recettore delle LDL, è stata associata allo sviluppo di aterosclerosi. Diversi studi hanno confermato tale associazione attraverso vie lipidiche e non lipidiche. Tuttavia, le relazioni dirette tra PCSK9 circolante e marcatori di aterosclerosi subclinica e clinica sono ancora da chiarire. Pertanto, abbiamo valutato le relazioni tra i livelli plasmatici di PCSK9 ed alcuni indici di aterosclerosi subclinica (marcatori di imaging) e clinica (eventi vascolari; EV). Un altro obiettivo è stato l'identificazione dei determinanti indipendenti di PCSK9, con particolare attenzione ai lipidi e ai biomarcatori infiammatori. Infine, abbiamo anche valutato la relazione tra alcuni marcatori di imaging e quattro SNPs del gene PCSK9, noti per essere associati alla presenza di bassi livelli di colesterolo LDL. Per validare i risultati ottenuti in quest’ultima parte, le analisi genetiche sono state replicate in una coorte indipendente reclutata nel Regno Unito (UK). Metodi: Lo studio è stato realizzato sfruttando le banche dati, biobanche e la banca di immagini dello studio IMPROVE. 3,703 soggetti europei (54-79 anni; 48% uomini), privi di EV al basale e definiti ad alto rischio per la presenza di almeno tre fattori di rischio vascolare, sono stati reclutati e seguiti per 36 mesi. PCSK9 è stata misurata tramite ELISA e trasformata in logaritmo prima delle analisi. I marcatori di imaging convenzionali [spessore medio-intimale carotideo (cIMT, dall’inglese intima-media thickness) e dimensione della placca carotidea] ed emergenti [cambiamento di cIMT nel tempo, ecolucenza dello spessore del complesso medio intimale della carotide comune misurato in zone libere da placca (PF CC-IMTmean), ecolucenza della placca più grande rilevata in tutto l'albero carotideo e punteggio di calcio carotideo (cCS, dall’inglese carotid calcium score)] sono stati misurati su scansioni ultrasonografiche conservate nella banca di immagini. In particolare, l'ecolucenza è stata misurata in termini di mediana della scala dei grigi (GSM, dall’inglese grey scale median) della distribuzione dei pixel di una specifica regione d’interesse, mentre il cCS è stato calcolato come somma delle lunghezze dei coni d’ombra acustici generati dal calcio all'interno delle placche carotidee. I lipidi sono stati misurati con metodi enzimatici (ad eccezione del colesterolo LDL che è stato calcolato con la formula di Friedewald). Tra i marcatori infiammatori, la proteina C reattiva ad alta sensibilità (hs-PCR) è stata misurata con la turbidimetria, mentre il conteggio dei globuli bianchi (WBC, dall’inglese white blood cells) e la formula leucocitaria sono stati misurati localmente. Tutti i soggetti dello studio IMPROVE e della coorte UK (n=22,179; 48 % uomini) sono stati genotipizzati. Risultati: Nell'analisi univariata, PCSK9 correlava positivamente con colesterolo totale, LDL e HDL e con trigliceridi e basofili (tutte le p <0.0001), mentre correlava negativamente con neutrofili ed eosinofili (entrambe le p=0.04). Le correlazioni positive osservate con hs-PCR e con il conteggio dei WBC erano solo vicine alla significatività statistica (p=0.060 e 0.064, rispettivamente). Le terapie con fibrati o statine (positivamente; entrambe le p <0.0001), così come sesso maschile e storia familiare di diabete (negativamente; entrambe le p <0.05) erano i predittori indipendenti più forti dei livelli plasmatici di PCSK9. Nell'analisi non aggiustata, si osservava una correlazione negativa tra PCSK9 e variabili basali di cIMT (IMTmean, IMTmax, IMTmean-max, e PF CC-IMTmean), una correlazione negativa tra PCSK9 e la variazione di cIMT nel tempo (Fastest-IMTmax-progr) e cCS (tutte le p ≤0.01), mentre si osservava un trend positivo tra PCSK9 e GSM sia del PF CC-IMTmean che della placca carotidea (entrambe le p ≤0.0001). Il cCS (positivamente) e il GSM del PF CC-IMTmean (positivamente) erano associati significativamente (o vicini alla significatività) a PCSK9 in diversi modelli multivariati (tutte le p ≤0.064). Tutte le correlazioni osservate all’analisi univariata tra PCSK9 e le variabili basali di cIMT, Fastest-IMTmax-progr e GSM della placca carotidea perdevano la significatività statistica dopo aggiustamento delle stesse per età, sesso, latitudine ed altri potenziali confondenti. Durante il follow-up [mediana (intervallo interquartile): 3.01 (2.98; 3.12) anni], sono stati registrati 215 EV: 125 coronarici, 73 cerebrali e 17 EV periferici. Tra questi, 37 erano eventi hard (infarto miocardico, morte improvvisa ed ictus). Nell'analisi non aggiustata, PCSK9 era associata positivamente ad eventi combinati e coronarici (entrambe le p <0.01), ma non ad eventi cerebrovascolari. Anche in questo caso, tuttavia, tutte le associazioni osservate perdevano la significatività statistica dopo aggiustamento delle analisi per età, sesso e stratificazione per latitudine. La mancanza di associazione con EV era confermata anche nel modello aggiustato per tutti i fattori confondenti considerati e nelle analisi focalizzate sugli eventi hard. Per quanto riguarda il ruolo delle varianti genetiche, nessuno dei quattro SNPs considerati correlava con cIMT (IMTmean, IMTmax, IMTmean-max) quando l'analisi era effettuata nei soggetti reclutati nello studio IMPROVE. La variante rs11591147, invece, correlava negativamente con l’IMTmax misurato nella popolazione UK (p=0.002). Combinando le quattro varianti genetiche in uno score, la relazione con cIMT era non significativa nello studio IMPROVE, mentre era negativa e significativa nella popolazione UK (tutte le p <0.01). Conclusioni: I livelli plasmatici di PCSK9 non sono associati a EV. Per quanto riguarda i marcatori dell'aterosclerosi subclinica, i livelli plasmatici di PCSK9 non sono associati né alla dimensione della lesione, né all'ecolucenza della placca carotidea, ma sono associati all'ecolucenza dello spessore della parete carotidea e al carotid calcium score. Ulteriori studi sono pertanto necessari per comprendere meglio il ruolo di tale proproteina nell'ecolucenza dello spessore della parete carotidea e nel carotid calcium score. La terapia con fibrati o statine, così come il sesso maschile e la storia familiare di diabete sono i predittori indipendenti più forti di PCSK9 circolante. È stata inoltre confermata l'associazione, precedentemente osservata, tra PCSK9 circolante e alcuni marcatori lipidici ed infiammatori. La relazione tra i livelli plasmatici di PCSK9 ed altri marcatori infiammatori (neutrofili, basofili ed eosinofili) merita ulteriori indagini, così come merita ulteriori indagini l’associazione tra le quattro varianti genetiche di PCSK9 selezionate e il cIMT nella coorte britannica, in quanto lascia intravvedere un possibile ruolo di SNPs o polimorfismi genici di PCSK9 nell’aterosclerosi e nelle strategie della sua prevenzione.
Background and purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the main regulators of LDL receptor metabolism, has been associated with atherosclerosis development. Several studies have confirmed such association through both lipid and non-lipid pathways. However, the direct relationships between circulating PCSK9 and markers of subclinical and clinical atherosclerosis are still matter of debate. Therefore, we investigated the relationships between plasma PCSK9 levels and some indexes of subclinical (imaging markers) and clinical (vascular events; VEs) atherosclerosis. Another objective was the identification of the independent determinants of PCSK9, with particular attention to lipids and inflammatory biomarkers. Finally, we also assessed the relationship between some imaging markers and four SNPs of the PCSK9 gene, known to be associated with the presence of low levels of LDL-cholesterol. In order to validate the results obtained in this last part, the genetic analyses were replicated in an independent cohort recruited in the United Kingdom (UK). Methods: The study was carried out taking advantage of databases, biobanks and imaging-bank of the IMPROVE study. 3,703 European subjects (54-79 years; 48% men), free of VEs at baseline and defined at high risk for the presence of at least three vascular risk factors, were recruited and followed-up for 36 months. PCSK9 was measured by ELISA and log-transformed prior to analyses. Conventional imaging markers [carotid intima-media thickness (cIMT) and carotid plaque-size], and emerging imaging markers [cIMT change over time, echolucency of the intima-media thickess of common carotid measured in plaque free areas (PF CC-IMTmean), echolucency of the biggest plaque detected in the whole carotid tree, and carotid calcium score (cCS)] were measured on ultrasonographic scans stored in the imaging-bank. In particular, echolucency was measured in terms of grey scale median (GSM) of pixels distribution of a specific region of interest, whereas cCS was calculated as sum of lengths of acoustic shadow cones generated by calcium within carotid plaques. Lipids were measured with enzymatic methods (except for LDL-cholesterol, which was calculated by Friedewald's formula). Among inflammatory markers, high-sensitivity C-reactive protein (hs-CRP) was measured by turbidimetry, whereas white blood cells (WBC) count and the leukocyte formula had already been measured locally. All the IMPROVE study and UK (n=22,179; 48% men) subjects have been genotyped. Results: In the univariate analysis, PCSK9 was positively correlated with total, LDL-, and HDL-cholesterol, and with triglycerides and basophils (all p <0.0001), whereas was negatively correlated with neutrophils and eosinophils (both p=0.04). The positive correlations observed with hs-CRP and WBC count were just close to the statistical significance (p=0.060 and 0.064, respectively). Fibrates or statins therapies (positively; both p <0.0001), as well as male sex and family history of diabetes (negatively; both p <0.05) were the strongest independent predictors of plasma PCSK9 levels. In the unadjusted analysis, a negative correlation was observed between PCSK9 levels and basal cIMT variables (i.e. carotid IMTmean, IMTmax, IMTmean-max, and PF CC-IMTmean), a negative correlation between PCSK9 and cIMT change over time (Fastest-IMTmax-progr) and cCS (all p ≤0.01), whereas a positive trend was observed between PCSK9 and GSM of both PF CC-IMTmean and carotid plaque (both p ≤0.0001). The cCS (positively) and the GSM of PF CC-IMTmean (positively) were significantly (or almost significantly) associated with PCSK9 in several multivariate models (all p ≤0.064). All correlations observed in the univariate analysis between PCSK9 and basal cIMT variables, Fastest-IMTmax-progr and GSM of carotid plaque lost the statistical significance after adjustment for age, sex, latitude, and other potential confounders. During the follow-up [median (interquartile range): 3.01 (2.98; 3.12) years], 215 VEs were recorded: 125 coronary, 73 cerebral and 17 peripheral VEs. Among these, 37 were hard events (i.e. myocardial infarction, sudden death and stroke). In the unadjusted analysis, PCSK9 was positively associated with combined and coronary events (both p <0.01), but not with cerebrovascular events. Also in this case, however, all the associations observed lost the statistical significance after adjustment of the analyses for age, sex, and stratification for latitude. The lack of association with VEs was confirmed also in the model adjusted for all confounding factors considered, and in the analyses focused on hard events. With regard to the role of genetic variants, none of the four SNPs considered was correlated with cIMT (i.e. IMTmean, IMTmax, IMTmean-max) when the analysis was performed in the subjects recruited in the IMPROVE study. The rs11591147 variant, by contrast, was negatively correlated with IMTmax measured in the UK population (p=0.002). By combining the four genetic variants in a score, the relationship with cIMT was not significant in the IMPROVE study, whereas was negative and significant in the UK population (all p <0.01). Conclusions: Plasma PCSK9 levels are not associated with VEs. Regarding markers of subclinical atherosclerosis, PCSK9 levels are associated neither with lesion size, nor with carotid plaque echolucency, but are associated with echolucency of carotid wall thickness and with carotid calcium score. Therefore, further studies are needed to better understand the role of such circulating proprotein in carotid wall thickness echolucency and in carotid calcium score. Fibrates or statins therapies, as well as male sex and family history of diabetes are the strongest independent predictors of PCSK9 levels. The associations, previously observed, between circulating PCSK9 and some lipid and inflammatory markers have been confirmed. The relationship between plasma levels of PCSK9 and other inflammatory markers (neutrophils, basophils and eosinophils) deserves further investigation, as does the association between the four selected PCSK9 variants and cIMT in the UK cohort, as it suggests a possible role of PCSK9 SNPs or gene polymorphisms in atherosclerosis and in its preventive strategies.

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.

Background: South Asians demonstrate high coronary heart disease mortality, largely unexplained by conventional risk factors and unidentified by risk stratification tools. Developments in technology allow us to visualize coronary atherosclerosis non-invasively, thus providing the potential to identify presence of coronary atherosclerosis before it manifests clinically. Coronary artery calcification is closely correlated with total plaque burden and provides an assessment of coronary plaque burden. Myocardial perfusion scintigraphy provides an estimate of myocardial blood flow and thus, severity of coronary artery disease. Increased coronary artery calcification and silent myocardial ischemia predict future risk of coronary heart disease mortality, independent of conventional factors. Inflammation is a key factor in initiation and progression of atherosclerosis. High sensitivity C-reactive protein (CRP) is an important marker of active inflammation and is considered an independent predictor of future cardiovascular events. Thus, markers of subclinical atherosclerosis and inflammation could provide us with a tool for early identification of South Asians at risk of coronary events, unidentified by traditional means. However, majority of the data for such markers is from North American and European populations, with no data evaluating the role of coronary artery calcification, myocardial perfusion scintigraphy and CRP in assessing the coronary heart disease risk in South Asians. Methods and Results: I carried out assessments including coronary artery calcium, myocardial perfusion imaging and assessment of high sensitivity C-reactive protein for a cohort of asymptomatic South Asians and Europeans men and women, aged 35 to 75 years, who were part of the London Life Sciences Population (LOLIPOP) study. I found that: 1) Coronary artery calcification scores were closely associated with age, male gender, cigarette smoking, hypertension, systolic blood pressure, diabetes and total cholesterol. 2) There were no differences in either coronary artery calcification prevalence or mean levels of coronary artery calcification between South Asians and Europeans, after adjustment for the measured cardiovascular risk factors. 3) Presence of diabetes and increasing coronary artery calcification were independent predictors for silent myocardial ischemia. 4) South Asian ethnicity did not influence the prevalence or the extent of silent myocardial ischemia, after adjustment for conventional risk factors. 5) C-reactive protein levels did not correlate with measures of plaque burden. 5) South Asian ethnicity was an independent predictor of inflammation as seen by levels of high sensitivity C-reactive protein. This effect was independent of, and remained significant after adjusting for conventional cardiovascular risk factors and novel factors linked to inflammation such as diabetes and indices of abdominal obesity. Conclusions: While traditional risk factor correlate well with markers of atherosclerosis, the higher coronary heart disease risk and mortality observed in South Asians is not identified by markers of atherosclerotic burden such as coronary artery calcification and myocardial perfusion scintigraphy. South Asians have elevated levels of inflammation as seen by high sensitivity C-reactive protein levels. C-reactive protein levels are not correlated with coronary artery calcium or myocardial ischemia measured by myocardial perfusion scintigraphy. These findings suggest a role of factors such as systemic and plaque inflammation, unrelated to and unmeasured by plaque burden assessment in the higher coronary heart disease mortality observed among South Asians. The study therefore suggests a role of potential risk stratification tools reflecting the multisystem nature of CHD. These could be a combination of clinical risk factors contributing towards CHD, imaging of atherosclerotic plaque and assessment of plaque or systemic inflammation.

A large number of studies have established that raised cholesterol levels increase the probability of the development of atherosclerotic vascular disease, and that reducing serum cholesterol will result in fewer cardiac events in the treated population, both in those with and without evidence of pre-existing coronary disease. More direct evidence that this is due to alteration of the progression of the atheromatous plaques has resulted from angiographic studies demonstrating the halting of progression or even regression of the stenotic lesions. Some workers have found a relationship between the extent of lowering of the serum lipoproteins and the likelihood of regression, although it has not been clear whether this continues to hold true at the lower extremes, nor whether there may be a threshold level which requires to be achieved before regression may take place. The principal purpose of these studies was to investigate the effects of applying very intensive lipid-lowering therapy, including LDL-apheresis, in a group of patients with coronary artery disease and moderately severe hypercholesterolaemia to achieve subnormal lipoprotein levels, and comparing the effects of such treatment with those achieved in another group of subjects treated with drug therapy to the currently recommended therapeutic targets for such patients. The studies involved the measurement of lipids and lipoproteins before and after apheresis and at regular intervals throughout the two-year study period. ApoB metabolism was assessed at baseline and following completion of the treatment period, and the data analysed using a multicompartmental mathematical model. The patients were assessed non-invasively by exercise electrocardiography at regular intervals, and by thallium scintigraphy at baseline and at annual intervals. The principal end-point was the proportion of arterial segments undergoing regression or progression in each group assessed by computer-assisted analysis of coronary angiograms performed at baseline and on completion of the intervention. The results from these studies demonstrated radical differences in lipoprotein concentration and composition during treatment. There was increased catabolism of LDL precursors with diminished flux of apoB which may reflect up-regulation of the LDL-receptor, but a rapid return to pre-treatment lipid levels indicated the effects on lipoprotein metabolism were transient. There was a reduction in the progression of coronary disease in the majority of lesions, with a small number in each group undergoing definite regression. There were significant differences in the changes in exercise tolerance with treatment, and the likely mechanisms for this are discussed. The thallium scans demonstrated no difference between the groups in the number of segments with improved perfusion, but were shown to have some value in the non-invasive assessment of predicting angiographic changes in the proximal segments, particularly in the right coronary artery. The findings are put into the context of the recent publications on cholesterol reduction in coronary disease; implications for clinical management are drawn, and areas of potential future research are highlighted.

Atherosclerotic renovascular disease (ARVD) is a significant cause of chronic kidney disease (CKD) and is associated with an increased risk for cardiovascular morbidity and mortality. Randomised controlled trials, representing over 2100 patients, have failed to demonstrate any prognostic benefit of percutaneous renal revascularisation when utilised in addition to standard medical therapy. This negative finding has been interpreted in three ways. Firstly, that ARVD may be an association of CKD and not a specific disease process. Secondly, that published studies have recruited low-risk patients who are least likely to benefit from revascularisation. Thirdly, that the focus of treatment for patients with ARVD should be optimal medical therapy, not renal revascularisation. This research project had a series of linked aims. These were investigated in two large patient cohorts that had been accumulated at this centre over the last decade. These cohorts comprised > 900 patients with ARVD, the Salford Renovascular Database (SRVD), and > 2500 patients with all-cause CKD, the Chronic Renal Insufficiency Standards Implementation Study (CRISIS). The first aim was to consider whether ARVD should be considered as a specific cause of CKD. Here risks for death and progression to renal replacement therapy were compared between patients having ARVD as their primary cause of renal failure and patients with other coded causes of CKD. In this analysis, patients with ARVD had a greater risk for death and a lesser risk for RRT than patients with other forms of CKD.The second aim of this thesis was to consider if specific patient sub-groups of ARVD could be identified. Patients in the SRVD with currently accepted high- risk clinical presentations were selected and outcomes compared to patients without a high-risk presentation. In this analysis, presentation with flash pulmonary oedema (but with not refractory hypertension or rapidly declining renal function) was associated with an increased risk for death and cardiovascular event. When the effects of revascularisation were considered in patients with high-risk presentations, a mortality benefit was observed in patients with flash pulmonary oedema and in patients presenting with rapidly declining renal function and refractory hypertension in combination. A separate analysis was performed in the SRVD to consider if a high-risk sub-group of ARVD patients could be identified using laboratory measurements. Here, a classification tree methodology was employed to identify ARVD patients with the greatest risk for progression to end stage kidney disease. The results of this analysis were converted into a practically applicable clinical scoring system incorporating renal function, proteinuria, medications, smoking history and renal artery occlusion. The final aim of this thesis was to describe how the majority of ARVD patients should be treated. In this analysis of the SRVD effects of treatment with anti- platelet and beta-blocker therapy were considered, and shown to be associated with reduced risks for cardiovascular events and death.

Stroke is the third leading cause of death and the most common cause of disability in the world. To relieve the heavy burden of stroke, we need to understand the mechanisms that will form the basis of improved prevention and treatment. Epidemiological studies have found evidence for a genetic influence on the common form of stroke. However the genetic of stroke is still in its infancy. Subclinical intracranial atherosclerosis is sometime a predisposing factor for ischemic stroke (IS). This study was carried out to elucidate genetic factors influencing the complex phenotype of IS and subclinical intracranial atherosclerosis.

In the Belgium Stroke Study (BSS), we collected 237 middle-aged (45-60 yrs) patients with small vessel occlusion (SVO) or large vessel atherosclerosis (LVA) IS, according to the Acute Stroke Treatment (TOAST) criteria, 326 ethnicity and gender matched subjects were used as controls. We tested variants in cholesterol-related candidate genes (sterol regulatory element binding protein, SREBP, SREBP-cleavage activating protein, SCAP, Apolipoprotein E, APOE, and Proprotein convertase subtilisin/kexin type 9, PCSKA) for association with IS. Significant gene-IS associations were further tested in a Finnish autopsy collection of 1004 cases with a quantitative assessment of atherosclerosis in the circle of Willis.

While we could not detect any significant association between polymorphisms in the SREBP and SCAP genes and IS, we found evidence for association at the APOE and PCSK9 loci. The APOE &949;4+ genotype was related to a more severe intracranial atherosclerosis score in men, and within the most common APOE &949;3/&949;3 genotype group a higher risk of IS was associated with the G-allele at the -219G/T promoter polymorphisms. At PCSK9, the minor allele (G) of the tagging E670G polymorphism appeared as a significant predictor of LVA (OR = 3.52, 95% CI 1.25-9.85; p = 0.017). Accordingly, in the Finnish autopsy series, G-allele carriers tended to have more severe allele copy number-dependent (p=0.095) atherosclerosis in the circle of Willis and in its branches.

Our findings in this unique combination of clinical and autopsy data suggest a multifaceted role of apoE on the risk of cerebrovascular diseases. The APOE &949;4+ genotype did not predict the risk of IS, but was associated with severity of subclinical intracranial atherosclerosis in men. In contrast, the promoter variants affecting apoE expression were significant predictors of IS, suggesting that quantitative rather than qualitative variation of apoE is related to IS independently of subclinical intracranial atherosclerosis. Furthermore, we demonstrated that PCSK9 associates with the risk of LVA stroke subtype, and suggest that the risk is related to the severity of the underlying intracranial atherosclerosis.

Atherogenesis is considered as an active, inflammatory process, interleukin (IL)-18 a proinflammatory cytokine, is thought to play a central role in the development of atherosclerosis and more specifically in plaque rupture. We genotyped four haplotype tagging polymorphisms at the IL18 gene in the BSS and the Finnish autopsy series. The minor alleles of the IL18 -607 and +127 polymorphisms, as well as the haplotype carrying both minor alleles, associated with IS after adjustment for all cardiovascular risk factors. No association was seen with the development of subclinical intracranial atherosclerosis. Our findings suggest that variation in the IL18 gene influences the acute atherosclerotic IS event, but not the previous development of subclinical intracranial atherosclerosis, suggesting a causal role of IL18 in the vulnerability of cerebral arterial atherosclerotic plaques to acute rupture and subsequent thrombosis.

Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Public Health Epidemiology." Discipline: Epidemiology; Department/School: Public Health.

Atherosclerosis is a progressive inflammatory disease that is characterized by the accumulation of lipids, infiltrated cells and fibrous elements in large arteries.

This thesis focuses on the molecular mechanisms behind foam cell formation and inflammation, two central processes in the development of atherosclerosis. More specific, we studied the effects of proinflammatory cytokines on CXCL16 expression and its role as scavenger receptor on macrophages and smooth muscle cells in atherogenesis. CXCL16 is defined as a chemokine and a scavenger receptor, regulating adhesion and chemoattraction of CXCR6 expressing cells and uptake of oxLDL. We show that the expression of CXCL16 and its receptor CXCR6 are more pronounced in human atherosclerotic lesions compared with non-atherosclerotic vessels. Increased expression of CXCL16 was also seen in atherosclerotic aortas of apoE-/- mice compared with aortas of non-atherosclerotic, age-matched C57BL/6 mice. In vitro, IFN gamma induced CXCL16 expression in primary human monocytes and smooth muscle cells which resulted in an increased uptake of oxLDL. Treatment of mice with IFN gamma also induced CXCL16 expression in atherosclerotic lesions. Thus, we have demonstrated a role for IFN gamma in foam cell formation through upregulation of CXCL16. The expression of CXCR6 was defined to the same regions as for CXCL16 in the lesion, indicating the presence of cells able to respond to CXCL16. Consequently, CXCL16 could serve as a molecular link between lipid metabolism and immune activity in atherosclerotic lesion.

CD137 belongs to the TNF family and mediates several important processes in inflammation. CD137 is involved in the activation of T cells, NK cells, B cells and monocytes and regulate cytokine production, proliferation and apoptosis in these cells. A limited number of studies have demonstrated CD137 expression on smooth muscle cells and endothelial cells. Our results show that CD137 mRNA is higher expressed in human atherosclerotic lesions compared with unaffected vessels. We found that endothelial cells express CD137 in atherosclerotic lesions and that cultured endothelial cells and smooth muscle cells express CD137 and CD137 ligand in vitro. CD137 was regulated differentially by proinflammatory cytokines (i.e. IFN gamma, TNF alpha, IL-1 beta) and bacterial lipopolysaccharide depending on cell type. Furthermore, we investigated the effects of CD137 signalling, demonstrating that binding of the CD137 ligand to its receptor increases proliferation and migration of smooth muscle cells.

In summary, this thesis has focused on the expression, regulation and role of CXCL16 and CD137, two genes that have not been described earlier in the concept of atherosclerosis. The findings demonstrate some of the molecular mechanisms involved in vascular inflammation and may increase our knowledge about the development of atherosclerosis.

Prevention programs targeting Standard Modifiable cardiovascular Risk Factors (SMuRFs: hypertension, diabetes, hypercholesterolemia, smoking) are critical, yet ST elevation myocardial infarction (STEMI) in the absence of SMuRFs is not infrequent. Clinical outcomes of SMuRF-less STEMI patients have not been adequately assessed. Primary prevention risk assessments using SMuRFs do not adequately capture individual host response to risk factors. Novel markers reflecting disease activity and susceptibility are needed. Aims • Assess proportion of SMuRF-less STEMI patients • Examine outcomes of SMuRF-less STEMI patients • Describe rationale and methodology of the BioHEART cohort study • Assess coronary artery disease (CAD) phenotypes in BioHEART-CT • Assess for novel CAD biomarkers in BioHEART a. plasma metabolites b. IgE-sensitisation to the mammalian oligosaccharide galactose-α-1,3-galactose (α-Gal) Methods & Results The proportion and clinical outcomes of SMuRF-less STEMI patients were assessed in 3 retrospective cohort studies. 15%-25% of STEMI patients without prior CAD have no SMuRFs. SMuRF-less STEMI patients have higher early mortality (11% vs 8%, P<0.0001, n=62,048), with the highest rate in SMuRF-less women (18%). The BioHEART-CT cohort study was established to identify novel biomarkers, mechanistic pathways, and improve risk assessment. Associations between SMuRFs and CAD phenotypes were assessed, identifying marked differences between the sexes. Novel metabolite associations with CAD phenotypes were demonstrated, as were strong associations between α-gal sensitization secondary to tick bites and clinically relevant CAD phenotypes including non-calcified plaque, obstructive CAD and STEMI. Conclusion 15-25% of STEMI patients have no SMuRFs and SMuRF-less STEMI patients have a higher 30-day mortality rate. The BioHEART-CT cohort study utilising precise imaging and molecular phenotyping is well placed for largescale a priori and unbiased biomarker discovery studies.

L’aterosclerosi è una patologia degenerativa e progressiva delle arterie di grosso e medio calibro, caratterizzata da disfunzione endoteliale, accumulo di lipidi, infiltrazione di linfociti, migrazione e proliferazione di cellule muscolari lisce e deposizione di matrice extracellulare nella parete vascolare, con conseguente formazione di una placca ateromasica. Il processo aterosclerotico è alla base della patogenesi della malattia cardiovascolare nelle sue diverse manifestazioni cliniche, quali la malattia coronarica (CAD), l’infarto del miocardio (MI), l’arteriopatia ostruttiva periferica e l’ictus. L’aterosclerosi e’ responsabile delle prime tre cause di mortalità e morbilità nel mondo. L’aterosclerosi è una patologia complessa, alla quale concorrono molteplici fattori genetici ed ambientali. Lo scopo di questa tesi è stato l’identificazione di nuove componenti genetiche e molecolari della patologia aterosclerotica. Lo studio si e’ sviluppato a livello di DNA, RNA e proteina, applicando diverse metodologie e integrando dati ottenuti mediante: i) analisi di polimorfismi a singolo nucleotide (SNPs) in soggetti con e senza CAD, ii) analisi dei livelli di espressione genica in cellule muscolari lisce vascolari (VSMCs) e in placche aterosclerotiche umane, tramite microarray e Real time PCR, iii) analisi dei livelli di proteina mediante immunoistochimica di specimens di parete arteriosa carotidea. Partendo dai dati relativi a 91 SNPs analizzati in 510 pazienti con CAD e MI e in 388 soggetti di controllo senza patologia coronarica nella fase di replicazione dello studio di associazione “genome-wide” del Consorzio MiGen, sono stati selezionati 15 SNPs associati nominalmente al CAD (P<0.1). Riassunto Riassunto 2 Per studiare la potenziale associazione di questi SNPs con il processo aterosclerotico, i livelli di espressione dei 71 geni prossimali ai 15 SNPs sono stati analizzati sia in VSMCs, che rivestono un ruolo primario nel processo aterosclerotico, sia in porzioni di parete arteriosa derivanti da interventi di endoarteriectomia carotidea. A tal fine sono state condotte due consecutive ed indipendenti analisi del trascrittoma mediante microarray, la prima in culture primarie di VSMCs isolate da placche aterosclerotiche e dalla porzione prossimale virtualmente sana, la seconda in specimens di placche carotidee e delle corrispondenti porzioni “sane”. L’analisi dei profili di espressione ha permesso di identificare tre geni differenzialmente modulati, BCL3, PVRL2 e ABCA1, quest’ultimo già ampiamente studiato in relazione alla patologia aterosclerotica. Ponendo l’attenzione ai due geni adiacenti sullo stesso cromosoma (19q13) sono stati analizzati 4 SNPS intragenici, due per BCL3 e due per PVRL2 ed uno intergenico BCL3-PVRL2 in 393 soggetti di controllo e 442 pazienti con CAD senza pregresso MI. Quest’ultima coorte è stata selezionata per poter investigare in modo preferenziale l’eventuale associazione con l’aterosclerosi piuttosto che con la sua complicanza trombotica acuta. L’analisi dei genotipi ha mostrato che i portatori dell’allele G del polimorfismo BCL3 rs2965169 erano più rappresentati tra la popolazione con CAD e l’associazione con la patologia rimaneva significativa anche dopo correzione per i tradizionali fattori di rischio cardiovascolare. L’allele A del polimorfismo BCL3rs8100239 correlava con l’indice di massa corporea, l’ipertensione e il profilo lipidico. L’ analisi della distribuzione dei genotipi non ha identificato associazioni significative con la patologia coronarica o con variabili metaboliche sia per entrambi gli SNPs di PVRL2 che per la variante intergenica BCL3-PVRL2. L’analisi immunoistochimica di placche aterosclerotiche (n=10) e di porzioni adiacenti virtualmente sane (n=5) ha evidenziato espressione della proteina BCL3 solamente nella porzione aterosclerotica, associata a VSMCs e foam cells. Nella parete vascolare aterosclerotica l’espressione del mRNA per BCL3 sembra pertanto correlare con l’espressione della proteina. Complessivamente l’approccio integrato utilizzato nello sviluppo di questa tesi supporta il coinvolgimento della proteina BCL3 nel processo aterosclerotico.
Atherosclerosis is the common ground of several clinical manifestations of cardiovascular diseases (CVD), including coronary artery disease (CAD), myocardial infarction (MI), peripheral artery occlusive disease and stroke. CVD is still one of the major causes of mortality and morbidity in the worldwide. Atherosclerosis is a complex multifactorial disease of the wall of medium-sized and large arteries, characterized by endothelial cell dysfunction, smooth muscle cell proliferation (VSMCs) and migration, inflammation, lipid and matrix accumulation. Susceptibility to atherosclerosis is in turn influenced by interplay of genetic and environmental factors. The aim of this thesis was to unravel new potential genetic and molecular signatures of the atherosclerosis, by using an integrated approach which joins information from i) single nucleotide gene polymorphisms (SNPs) analysis in a case-control study of subjects with or without CAD, ii) microarray-based gene expression analysis on human cultured VSMCs and on carotid artery specimens, and iii) immunohistochemical analysis in carotid artery specimens. Firstly, 15 SNPs nominally associated with CAD (P< 0.1) were selected from 91 SNPs, investigated within replication of a genome-wide association study –MiGen- (510 patients with CAD and MI and 388 subjects with normal coronary arteries CAD-free). The expression levels of 71 genes proximal to the 15 tag-SNPs were evaluated by two subsequent steps of microarray-based RNA profiling, the former in VSMC populations isolated from grossly non-atherosclerotic (NP) and atherosclerotic (DP) human carotid portions, and the latter in whole carotid specimens. BCL3 and PVRL2, located on chromosome 19, and ABCA1, extensively investigated before, were found to be differentially expressed. Focusing the attention on BCL3 and PVRL2, the only couple of contiguous genes differentially expressed in the transcriptomic analysis, a total of 5 SNPs, two within BCL3 gene, Abstract Abstract 4 two within PVRL2 gene and one BCL3-PVRL2 intergenic, were genotyped within CAD-free subjects (n=393) and CAD patients without MI history (n=442). This cohort enabled to preferentially investigate the atherosclerosis pathways, rather than its acute thrombotic complication. The carriership of the BCL3 rs2965169 G allele was more represented among CAD patients and remained independently associated with CAD after adjustment for all the traditional cardiovascular risk factors, while the BCL3 rs8100239 A allele correlated with metabolic abnormalities. No significant associations were found for either PVRL2 SNPs or BCL3-PVRL2 intergenic variant. A BCL3 positive immunostaining was detected in the intima-media of atherosclerotic specimens, but not within non-atherosclerotic ones, thus indicating a correlation between BCL3 mRNA and protein levels. This thesis, which integrates GWAS data with the downstream changes in the RNA and protein levels in human arterial wall specimens, supports a role for BCL3 in atherosclerosis.

Previous studies demonstrated that coplanar PCBs promote inflammation by release of pro-inflammatory cytokines like TNF, MCP-1, and VCAM-1 from endothelial cells as well as adipocytes. Also these PCBs at small doses may contribute to the development of obesity by inducing adipocyte differentiation. Obesity is a known risk factor that promotes cardiovascular disorders like atherosclerosis and AAAs. Evidence shows Ang II, a component of the RAS, leads to the formation of atherosclerosis and AAAs in both normal as well as hyperlipidemic mice. Earlier studies in our laboratory have also shown that coplanar PCB-77 promotes atherosclerotic lesion formation in ApoE-/- mice. The purpose of this study was to define the effects of PCB77 on Ang II induced vascular diseases like atherosclerosis and AAAs. Two different hyperlipidemic mouse models, which require different diets to get atherosclerosis, the ApoE deficient mice (ApoE-/-) requiring the normal mouse diet (Chow diet) and the Low Density Lipoprotein Receptor deficient mice (LDLr-/-) requiring the Western diet, were used for this study as both are susceptible to Ang II induced vascular disorders. The timing of PCB administration was also studied in LDLr-/- mice to see the profound effects of PCB77 on atherosclerosis and AAAs.

This study examined racial/ethnic differences in pre-clinical disease, social support, and tested whether social support was a moderator of racial/ethnic differences in subclinical atherosclerosis. Participants were NHWs, NHBs, and Latinos (n = 283) from the baseline and cross-sectional sample of the North Texas Heart Study. Results from unadjusted models showed no significant racial/ethnic differences for common or bifurcation intima-media thickness (cIMT). However, unadjusted models for cIMT showed a main effect for race/ethnicity F(2, 229) = 3.12, p = .046, partial η2 = .027, with Latinos demonstrating significantly greater internal cIMT compared to NHB but not NHWs. In minimally adjusted models, there was a main effect for race/ethnicity, F(2, 227) = 3.10, p = .047, partial η2 = .027, with significantly greater internal cIMT in Latinos compared to NHBs but not NHWs. In fully adjusted models, racial/ethnic differences in cIMT were attenuated. Contrary to study hypotheses, no racial/ethnic differences in social support were found and social support was not a moderator of racial/ethnic differences in subclinical disease. In the North Texas Heart Study, few racial/ethnic differences emerged, with fully adjusted risk factor models accounting for these differences.

Il a été bien établi chez l’Homme qu'il existe une forte association entre les faibles concentrations plasmatiques de cholestérol associé aux lipoprotéines de haute densité (HDL-C) et le risque accru de maladie cardiovasculaire (MCV). Augmenter le taux de HDL-C a donc été proposé comme stratégie thérapeutique visant à réduire le risque de MCV. En effet, les HDL présentent de multiples fonctions athéroprotectrices, notamment la capacité d’efflux du cholestérol, ainsi que des activités antioxydantes, anti-inflammatoires, vasodilatatrices, cytoprotectrices, anti-infectieuses et anti- thrombotiques. Cependant, des essais cliniques à grande échelle ont révélé que l'augmentation du HDL-C induite par un médicament ne réduisait pas nécessairement le risque CV. En outre, des études de randomisation mendélienne ont montré que des concentrations faibles de HDL-C déterminées génétiquement ne se traduisaient pas toujours par un risque accru de MCV. Récemment, plusieurs études épidémiologiques à grande échelle ont mis en évidence une dépendance en forme de U entre la maladie cardiovasculaire et les taux de HDL-C, établissant un lien entre un taux de HDL-C extrêmement élevé et un risque CV élevé. Afin de surmonter les limites du HDL-C en tant que facteur de risque CV, le concept de fonctionnalité des lipoprotéines HDL a été étudié, ce qui a permis de développer un test permettant la mesure de la capacité des HDL à faire de l’efflux de cholestérol comme approche de prédiction du risque. Cependant, ce concept révèle plusieurs faiblesses, telles que la préservation de l'efflux de cholestérol tissulaire chez les patients ayant un taux de HDL-C bas d'origine génétique. Dans la circulation, le métabolisme des HDL est intimement lié à celui des triglycérides (TG) par divers facteurs, notamment des enzymes, telles que la lipoprotéine lipase (LPL), et des protéines de transfert lipidique, telles que la protéine de transfert d'esters de cholestérol (CETP). La contribution des niveaux circulants de TG au risque élevé de MCV a été établie dans des modèles multivariés. On pense que les lipoprotéines riches en triglycérides (TGRL) contribuent à l'athérosclérose via leurs particules résiduelles produites lors de la lipolyse des TGRL par la LPL. Des études antérieures ont montré que les HDL sont capables d'empêcher l'accumulation de ces particules residuelles de TGRL dans la paroi artérielle. Il a donc été proposé que le faible taux de HDL-C représente un biomarqueur de niveaux élevés de résidus de TGRL générés par la lipolyse. On ignore actuellement si cette association peut expliquer la relation en forme de U entre le risque CV et le taux de HDL-C. En outre, les mécanismes sous-jacents à l'association entre le HDL-C, les résidus de TG et les MCV restent obscurs. Dans la présente étude, nous proposons une hypothèse selon laquelle les HDL circulants peuvent se charger en lipides, tels que le cholestérol libre (FC) et le phospholipide (PL), et des protéines provenants de la surface des résidus des TGRL générés au cours de la lipolyse médiée par la LPL, puis les transporter vers le foie dans un processus appelé le transport inverse des résidus (RRT). Nous suggérons en outre que les modifications de la RRT sous-tendent les relations entre HDL-C et MCV. Pour évaluer cette hypothèse, nous avons conçu un nouveau test in vitro évaluant les transferts lipidiques des TGRL aux HDL au cours de la lipolyse et l'avons appliqué à plusieurs populations de sujets présentant des taux plasmatiques de HDL-C différents. Les mécanismes de transfert de lipides de surface vers les HDL ont également été étudiés. Nous avons observé que les HDL, isolés par ultracentrifugation ou par déplétion plasmatique de l'apolipoprotéine B, acquéraient des lipides de surface, y compris FC et PL, des TGRL lors d'une lipolyse induite par le LPL à 37 ° C, en fonction du temps, comme l'a révélé la photométrie [...]
It has been well established that there is a strong association between low concentrations of high-density lipoprotein-cholesterol (HDL-C) in human plasma and the risk of cardiovascular disease (CVD). Raising HDL-C level was therefore proposed as a therapeutic strategy to decrease CV risk. Indeed, HDL displays multiple atheroprotective functions, including cholesterol efflux capacity as well as antioxidative, anti-inflammatory, vasodilatory, cytoprotective, anti-infectious and anti-thrombotic activities. However, large-scale clinical trials revealed that drug-induced HDL-C raising did not necessarily reduce CV risk. Furthermore, Mendelian randomization studies reported that genetically determined low HDL-C concentrations did not always translate to increased risk of CVD. Recently, a U-shape dependence between CV disease and HDL-C levels was observed in several large-scale epidemiological studies, linking extremely high HDL-C to elevated CV risk. To overcome the limitations of HDL-C as a CV risk factor, a concept of HDL functionality was developed which resulted in the development of the measurement of cholesterol efflux capacity of HDL as a risk-predicting approach. However, this concept reveals several weaknesses, such as, preservation of tissue cholesterol efflux in patients with genetically low HDL-C. In the circulation, HDL metabolism is intimately linked to that of triglyceride (TG) by various factors, including enzymes, such as lipoprotein lipase (LPL), and lipid transfer proteins, such as cholesteryl ester transfer protein (CETP). The contribution of circulating TG levels to the elevated risk of CVD was established in multivariate models. Triglyceride-rich lipoproteins (TGRLs) are thought to contribute to atherosclerosis via their remnant particles produced during lipolysis of TGRLs by LPL. Earlier studies showed that HDL is capable of preventing TGRL remnants from accumulation in the arterial wall. Low HDL-C was therefore proposed to represent a biomarker of elevated levels of TGRL remnants generated by the lipolysis. It is presently unknown whether this association can account for the U-shape relationship between CV risk and HDL-C. In addition, mechanisms underlying the association between HDL-C, TG remnants and CVD remain obscure. In the present study, we propose a hypothesis that in the circulation HDL can acquire lipids, such as free cholesterol (FC) and phospholipid (PL), and proteins from TGRL surface remnants generated during LPL-mediated lipolysis, and subsequently transport them to the liver in a process termed reverse remnant transport (RRT). We further suggest that RRT alterations underlie the relationships between HDL-C and CVD. To assess this hypothesis, we designed a novel in vitro assay evaluating lipid transfers from TGRL to HDL during lipolysis and applied it to several populations of subjects greatly differing in plasma HDL-C levels; mechanisms of surface lipid transfer to HDL were also studied. We observed that HDL, isolated by ultracentrifugation or by apolipoprotein B depletion of plasma, acquired surface lipids, including FC and PL, from TGRL upon LPL-induced lipolysis at 37°C in a time-dependent fashion as revealed by photometry [...]

Background: Small non coding RNAs (sncRNAs) are endogenous short non coding molecules that regulate gene expression at post-translational level and are involved in several physiopathological processes. Circulating sncRNAs could be found free in biological fluids or loaded into extracellular vesicles, such as microparticles (MPs) in order to reach other tissues and amplify their signal. Next-generation sequencing (NGS) has become the main platform for biological research and biomarker discovery in the profiling of sncRNAs. Aim: The aim of this study was: 1) to set up a protocol using NGS technology for the identification and quantification of circulating sncRNAs involved in atherosclerotic plaque composition in type 1 diabetic patients (T1DM); 2) to characterize the phenotypes of circulating MPs derived from T1DM, associated with the plaque composition to evaluate the impact of these extracellular vesicles as carrier of specific small non coding RNAs, involved in these pathways. Material and Methods: Total RNA of 61 T1DM patients with fibrous (CFP; n 30) or calcified (CCP; n 31) carotid plaques was extracted from plasma samples, using a kit for biological fluids. For NGS sequencing, 25 CFP and 26 CCP were evaluated. The preparation of libraries was assessed using the Qiagen system. The sncRNA libraries pool was sequenced through the NGS sequencer MiSeq (Illumina), and the analysis performed by two bioinformatics tools (Partek Flow and CLC Genomics Workbench software). MPs derived from plasma of 40 T1DM patients with fibrous (CFP; n 20) or calcified (CCP; n 20) carotid plaques was assessed by centrifugation (40min x 14,000 rpm a 4°C) and characterized using flow cytometry (CytoFLEX, Beckman Coulter). Results: An unbiased and accurate sncRNome-wide quantification was obtained, detecting already known circulating sncRNAs (miRNAs, n 2632; piRNAs, n 3286; and tsRNAs, n 640). The bioinformatic analysis using two software on the already known 2632 miRNAs showed a different profile in T1DM with CCP compared to T1DM with CFP. Circulating level of several miRNA implicated in vascular remodeling and glucose metabolism were upregulated in patients with CCP, compared to CFP (miR-503-5p, miR-93-5p, miR-106b-5p and let-7d-5p) and downregulated (miR-451a, miR-10a-5p and miR-29b-3p) in patients with CCP, compared to CFP. We found that MPs released from endothelial cells and Platelets are enhanced in T1DM with vascular calcification (CCP) compare with T1DM with fibrous plaque (CFP); interestingly, a population of MPs derived from a niche of cells positive for CD34 and α-smooth muscle actin (αSMA) is also increased in CCP. Furthermore, the subgroup of MPs positive for calcification marker was significantly enhanced in patients with CCP in comparison to CFP patients with the main contribution given by CD34+ cells, suggesting a key role of these cells in the development of this vascular complication. Conclusions: In conclusion, our results demonstrate the power of NGS technology to identify a huge amount of circulating sncRNAs and to discover RNA molecules present in human plasma. The identification of new molecular biomarkers with this ultra-high throughput and sensitive technique (NGS) will help to go further insight specific pathophysiological processes, such as atherosclerotic plaque composition in diabetes, allowing a potentially more targeted therapeutic approach. Furthermore, we demonstrate that microparticles exhibit differential markers in the presence of vascular calcification suggesting a potential role as carrier of small molecules to amplify their signal.

A aterosclerose e suas complicações são a principal causa de morbidade e mortalidade no mundo ocidental. O aumento da espessura da camada médio-intimal da carótida está associado com risco para doenças cardiovasculares, pois representa um marcador de aterosclerose subclínica, podendo ser detectada precocemente em indivíduos assintomáticos. O objetivo desse estudo foi identificar variáveis clínicas e nutricionais associadas com a aterosclerose subclínica em mulheres hipertensas. Estudo transversal envolvendo uma amostra de conveniência composta por 116 mulheres hipertensas entre 40 e 65 anos. Dados clínicos, como pressão arterial (PA) sistólica e diastólica, história de tabagismo, atividade física, uso de medicamentos foram coletados; foi feita a análise do perfil lipídico, glicemia e proteína C reativa (PCR); a avaliação dietética obtida pelo Recordatório de 24 horas e pelo Registro de três dias. A espessura médio-intimal (EMI) de carótidas foi realizada pelo aparelho de ultrassonografia. As pacientes foram divididas em dois grupos, de acordo com os valores da espessura médio-intimal de carótidas: EMI 0,9mm ou EMI > 0,9mm. Houve diferença significativa entre os grupos em relação à idade (50,846,62 vs 53,547,13; p=0,044), PA sistólica (134,5216,54 vs 142,9821,47; p=0,020), pressão de pulso (PP) (49,3611,03 vs 60,15 17,77; p<0,001), HDL (48,988,54 vs 44,057,45; p=0,004) e PCR (2,311,21 vs 3,051,34; p=0,016). Não houve diferença significativa em relação aos parâmetros antropométricos, exceto em relação à reactância (65,199,69 vs 61,447,88; p=0,036), avaliada pela bioimpedância elétrica (BIA). Quanto ao padrão de consumo alimentar, somente o consumo de gordura monoinsaturada foi diferente entre os grupos, sendo o maior consumo no grupo com menor valor de EMI (7,882,09 vs 7,022,06; p=0,031). Não houve diferença em relação à frequência de tabagismo e atividade física. Quando foi feita a análise de correlação da amostra, foi encontrada uma correlação entre a EMI de carótidas e idade (r=0,25; p=0,0067), PAS (r=0,19; p=0,0086); PP (r=0,30; p=0,0009), LDL (r=0,19; p=0,0434), assim como com gordura monoinsaturada (r= -0,25; p=0,0087), PCR (r=0,31; p=0,007) e HDL (r=-0,33; p=0,0004), porém apenas as variáveis HDL, PCRus e pressão de pulso mostraram ser preditoras independentes da EMI de carótida após feita uma análise de regressão linear multivariada. A proteína C reativa, HDL colesterol e pressão de pulso são importantes preditores independentes de aterosclerose subclínica.
Atherosclerosis and its complications are the main cause of morbidity and mortality in the Western world. Increased carotid intima-media thickness is associated with cardiovascular risk,and it represents a marker of subclinical atherosclerosis, which can be detected early in asymptomatic individuals. The aim of this study was to identify clinical and nutritional variables associated with subclinical atherosclerosis in hypertensive women. Cross-sectional study involving a convenience sample composed by 116 hypertensive women aged between 40 and 65. Clinical data such systolic and diastolic blood pressure (BP), smoking history, physical activity, medication use were collected, a lipid profile, blood glucose and C-reactive protein (CRP)analysis was performed, the dietary assessment was obtained by dietary recall 24 hours and three days food record. Carotid intima-media thickness was performed by the high resolution ultrasound. Patients were divided into two groups according to the values of carotid IMT: IMT 0.9 mm or IMT > 0.9 mm. There was significant difference between the groups regarding age (50.846.62 vs 53.547.13; p=0.044), systolic BP(134.5216.54 vs 142.9821.47; p=0.020), pulse pressure (PP) (49.3611.03 vs 60.15 17.77; p<0.001), HDL-cholesterol (49.3611.03 vs 60.1517.77; p<0.001)and CRP(2.311.21 vs 3.051.34; p=0.016). There was no significant difference regarding to anthropometric parameters, except for the reactance (65.199.69 vs 61.447.88; p=0.036), measured by bioelectrical impedance analysis (BIA). Regarding the dietary pattern, only the monounsaturated fat intake was different between the groups 65.199.69 vs 61.447.88; p=0.036),. There was no difference in smoking and physical activity. In the correlation analysis, we have found a correlation between carotid IMT and age (r = 0.25, p = 0.0067), SBP (r= 0.19, p = 0.0086); PP (r = 0.30, p = 0.0009), LDL (r = 0.19, p = 0.0434), and monounsaturated fat (r = -0.25, p = 0.0087), CRP (r = 0.31, p = 0.007) and HDL (r =- 0.33, p = 0.0004), but only HDL-cholesterol, hsCRP and pulse pressure were shown to be independent predictors of carotid IMT after made a multivariate analysis. Conclude that C reactive protein, HDL-cholesterol and pulse pressure are important predictors for subclinical atherosclerosis.

Patients with chronic inflammatory conditions such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) experience premature cardiovascular mortality and morbidity compared with the general population. The increased risk of cardiovascular disease (CVD) may in part, result from an interaction between traditional and non-traditional risk factors, modulated by chronic inflammation. The aim of this project was to look at lipid associated biomarkers in patients with SLE/RA and the association between these markers and cardiovascular disease outcomes. We also aimed to study the effect of inflammation reduction on vascular biomarkers. In the first study we examined 168 SLE patients median (IQR) age was 53 (46-61) years and median disease duration 13 (7, 23) years and 56 healthy controls median age 50 (39-60) years. We demonstrated elevated level of oxidised-LDLin SLE patients compared with healthy controls (76 (57, 99) U/l vs 56 (42, 88)U/l P= 0.02). We further explored the association between oxidant stress and premature atherosclerosis as measured by carotid intima media thickness (cIMT) and plaque. In addition to age and systolic blood pressure, oxidised-LDL and urinary 8-isoprostane were significantly and independently associated with cIMTin SLE patients _ coefficient 95%CI [0.00007 (5.29−6, 0.0001) and 0.003 (0.0008,0.004)], respectively. In healthy controls, age was the only independent variable. In the Norfolk Arthritis Register, 1266 patients with early inflammatory polyarthritis (IP) were studied. A linear regression analysis revealed a significant negative association between CRP and lipid profile namely TC, LDL, TG and ApoA-1. During a median (IQR) follow up = 5.5 (3.7-7.7) years 100 (7%) patients died (all causes) of which 33% (33) deaths were attributed to CVD. Forward stepwise regression analysis demonstrated that a low total cholesterol was independently associated with all cause mortality HR (95%CI) 0.75 (0.61, 0.91) and CVD mortality HR (95%CI) 0.49 (0.29, 0.85). In a small cohort 27 SLE patients and 15 healthy controls. We measured endothelial function using flow mediated dilatation of the brachial artery. At baseline we found a significant increase in TG level [1.36 (0.9, 1.87) mmol/l vs0.88 (0.64, 1) mmol/l P= 0.009] and a significant impaired endothelial function in SLE patients compared to the healthy controls [2.86 (0.6, 5.3) vs 6.81 (3.46,8.57), P= 0.03]. After treatment, there was a trend towards reduced TG level and improved endothelial function. Oxidised-LDL did not change significantly. In conclusion, oxidant stress is increased in SLE patients and relates to some measures of subclinical atherosclerosis. Control of inflammation may not be sufficient to completely control this in routine practice. In early RA, active inflammationmay mask any tendency to hyperlipidemia in this population. Low total cholesterol may be the best biomarker of the overall metabolic and inflammatory status of the patients as well as indicating a group with increased risk of future mortality.

Em estudos anteriores, mostramos que uma nanoemulsão artificial (NEm) de composição semelhante à da lipoproteína de baixa densidade se liga a receptores de lipoproteínas de baixa densidade após sua injeção na corrente sanguínea. Como tais receptores estão superexpressos em células com altas taxas de proliferação, como ocorre no cancer e na aterosclerose, a NEm pode ser utilizada como veículo para direcionar drogas a estas células, diminuindo sua toxicidade e aumentando sua ação farmacológica. Recentemente, reportamos que a associação de um derivado do agente antiproliferativo paclitaxel, o oleato de paclitaxel (OPTX) à NEm reduziu em 60% a área lesionada de aortas de coelhos submetidos à dieta rica em colesterol. Neste estudo, testamos o efeito sinérgico da terapia combinada do OPTX-NEm com um derivado do metotrexato, o di-dodecil metotrexato (DMTX), também associado à NEm. O MTX, além de sua ação antiproliferativa, também possui propriedades imunossupressoras. Coelhos machos da raça Nova Zelândia foram submetidos à dieta enriquecida com 1% de colesterol durante 8 semanas. A partir da quinta semana de consumo da dieta, 8 animais foram injetados semanalmente com solução salina por via endovenosa (grupo controle) e 8 receberam o tratamento combinado de OPTX-NEm (4mg/Kg) com DMTX-NEm (4mg/Kg), por 4 semanas. Ao final das 8 semanas, os animais foram sacrificados. As aortas dos animais foram retiradas, abertas longitudinalmente, fixadas em formalina tamponada a 10% e coradas com Escarlate R para a análise macroscópica da lesão. Os arcos aórticos foram seccionados em fragmentos de 5mm, embebidos em parafina e os cortes realizados foram corados com hematoxilina-eosina, para a determinação da área das camadas íntima e média. O tratamento combinado de OPTX-NEm com DMTX-NEm reduziu a área das lesões em 82%, em comparação ao grupo controle, e a razão da área da lesão/área total diminuiu de 0,82±0,08 para 0,08±0,06 (p<0,01). Por meio das avaliações da variação do consumo de ração, peso corporal e contagem de leucócitos totais (p>0,05), pode-se afirmar que os tratamentos não apresentaram toxicidade significativa, exceto pela queda na contagem de eritrócitos (p<0,05). Como conclusão, a quimioterapia combinada de OPTX e DMTX associados à NEm como veículo mostrou-se eficaz na redução da área de lesão aterosclerótica em coelhos e a toxicidade relacionada aos fármacos foi nitidamente reduzida.
In previous studies we have shown that an artificial nanoemulsion (NEm) that resemble LDL composition are taken-up by LDL receptors after injection into the bloodstream. As those receptors are upregulated in cells with higher proliferation rates, as occurs in cancer and atherosclerosis, NEm can be used as vehicle to direct drugs against those cells, diminishing toxicity and increasing pharmacological action. Recently, we reported that association of antiproliferative agent paclitaxel derivative, paclitaxel oleate (OPTX) to NEm reduced by 60% the lesion area of cholesterol-fed rabbits. In this study, the combined chemotherapy of OPTX-NEm with a methotrexate derivative, di-dodecil methotrexate (DMTX), also associated with NEm, was tested for synergistic effects. MTX, besides antiproliferative action, has also immunosuppressant properties. Male New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30, 8 animals were treated with 4 weekly I.V. saline solution injections (control group) and 8 with combined OPTX-NEm (4 mg/kg) plus DMTX-NEm (4 mg/kg) for additional 30 days. On day 60, the animals were sacrificed for analysis. Aorta was excised, open longitudinally, placed in 10% buffered formalin and stained in Scarlet R for lesion macroscopic analysis. Segments of 5mm of the aortic arch were embedded in paraffin and sections were taken and stained in hematoxylin-eosin for intima and media area measurement. In comparison with controls, treatment with combined OPTX-NEm plus DMTX-NEm reduced the lesion area by 82% and the lesion/total area ratio was decreased from 0,82±0,08 to 0,08±0,06 (p<0.01). Except for decrease in erythrocyte count (p<0.05), treatments were devoid of significant toxicity, as evaluated by food intake, body weight and leucocyte count (p>0.05). In conclusion, this novel approach consisting in combined chemotherapy of OPTX and DMTX using NEm as a drug-targeting vehicle showed effective lesion area regression in rabbits and marked toxicity reduction.

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Sociedade de Cardiologia do Estado de São Paulo
Introduction: About half of the cases of atherosclerotic coronary disease (a mean of 15% of women deaths and 25% of men) cannot be explained by most of the known risk factors. Coronary vasa vasorum are associated with coronary artery disease; however, their anatomy and physiopathology are not well clear. Objective: The aim of this study was to carry out a post mortem stereological study of adventitial vasa vasorum in different histopathological degrees of coronary atherosclerosis intending to correlate vasa vasorum, myocardial infarction physiopathology and histopathological degrees of atherosclerosis. Method: Ten consecutive autopsies of adults (5 men, 5 women, from 35 to 83 years-old, frozen at 4o C) were performed. Six proximal, medium and distal biopsies of the anterior and posterior interventricular coronary branches (at intervals of 1.5 cm) were performed per autopsy (a total of 60 coronary biopsy fragments). Fragments were processed by histological routine technique and cut in 4 fragments of 4 mm thickness. The first two consecutive histological fragments were stained by hematoxylin-eosin, and the two remaining by Masson´s trichrome. The fragments were histopathologically analysed according to Stary´s coronary atherosclerosis classification and examined by Zeiss Jenaâ, a light microscope with a bright chamber attached a Zeissâ micrometer scale, to outline adventitial vasa vasorum as well as to measure the coronary intraluminal diameter and the medial thickness. Intersection points of vasa vasorum with Merzâ´s grille were manually counted. For all types of vasa vasorum, points on Merzâ´s grille were counted to obtain the following stereological parameters of vasa vasorum: diameter, wall thickness, volumetric and superficial density, and adventitial connective tissue density. Parametric data were analysed by Pearson s linear correlation and principal component analysis. Agreement in determining coronary atherosclerosis degree in laminas stained by hematoxylin-eosin or Masson´s trichrome was assessed by kappa statistics. Differences among variables at each atherosclerosis degree was assessed by analysis of variance or Kruskal-Wallis test. Results: Coronary intraluminal diameter correlated negatively with coronary medial thickness and number of adventitial vasa vasorum (r>0.50; P-value<0.05). These correlations may be explained by sex, age and coronary atherosclerosis degree (r>0.50; P-value<0.05). All stereological parameters of vasa vasorum correlated negatively with coronary intraluminal diameter and positively with medial thickness, both explained by sex and atherosclerosis degree (r>0.50; P-value<0.05). The size of all types of vasa vasorum augmented proportionally to atherosclerosis histopathological degree aggravation. Kappa statistics for hematoxylin-eosin and Masson´s trichrome presented agreements varying from substantial or good to almost perfect or fine . All variables presented significant differences since the degree II of atherosclerosis. Conclusions: Coronary medial thickness and number of vasa vasorum correlated negatively with coronary intraluminal diameter. These correlations may be explained by sex and coronary atherosclerosis degree. Stereological parameters of vasa vasorum (except coronary adventitial connective tissue density) correlated positively with number of adventitial vasa vasorum as well as medial thickness, and negatively with coronary intraluminal diameter. Both correlations were determined by degree of atherosclerosis. Venular rupture in vulnerable atherosclerotic plaques may be associated with myocardial infarction arising since the degree II of atherosclerosis.
Introdução: Cerca de 50% dos casos de doença arterial coronária aterosclerótica (15% das mortes masculinas e 25% femininas) não são explicados pelos clássicos fatores de risco. Vasa vasorum coronários podem associar-se à aterosclerose coronariana; contudo, sua anatomia e fisiopatologia não estão completamente elucidadas. Objetivos: Realizar estudo estereológico dos vasa vasorum da túnica externa de artérias coronárias autopsiadas buscando correlação entre vasa vasorum, fisiopatologia do infarto do miocárdio e graus histopatológicos de aterosclerose. Material e Método: Em dez autópsias consecutivas de adultos (5 homens, 5 mulheres, 35 a 83 anos, congelados a 4º C) efetuaram-se biópsias proximal, média e distal dos ramos interventriculares anterior e posterior em cada autópsia, a cada 1,5 cm, totalizando 6 biópsias por autópsia. Cada fragmento foi processado histologicamente com cortes sucessivos de 4 cm de espessura, totalizando 4 fragmentos (24 fragmentos por autópsia). Os dois primeiros fragmentos foram corados em hematoxilina-eosina e os dois últimos em tricrômico de Masson. Lâminas histológicas foram diagnosticadas histopatologicamente quanto ao grau de aterosclerose coronariana pela classificação de Stary e examinadas em microscópio de luz Zeiss Jena com câmara clara e escala micrométrica Zeissâ para delineamento dos vasa vasorum da túnica externa coronária e mensuração do diâmetro do lúmen coronário e da espessura da túnica média coronária. Pontos dos delineamentos dos vasa vasorum sobre a grade estereológica foram manualmente contados obtendo-se diâmetro do lúmen, espessura da parede e densidades volumétrica e superficial dos vasa vasorum, e densidade do tecido conectivo da túnica externa. Dados paramétricos foram analisados por correlação linear de Pearson e análise de componentes principais, concordância no diagnóstico do grau aterosclerótico pela estatística kappa, e diferenças entre valores das variáveis a cada grau aterosclerótico por análise de variância ou teste de Kruskal-Wallis Resultados: Diâmetro do lúmen coronário correlacionou-se negativamente com espessura da túnica média e número de vasa vasorum da túnica externa (r>0,50; valor-p<0,05), com correlações explicadas pelo sexo e grau de aterosclerose (r>0,50; valor-p<0,05). Parâmetros estereológicos dos vasa vasorum correlacionaram-se negativamente com diâmetro do lúmen coronário e positivamente com espessura da túnica média, com correlações explicadas pelo grau de aterosclerose (r>0,50; valor-p<0,05). Tamanho de todos os tipos de vasa vasorum aumentou proporcionalmente ao agravamento das lesões ateroscleróticas. Estatística kappa para lâminas histológicas coradas em hematoxilina-eosina ou em tricrômico de Masson apresentou concordâncias variando de substancial ou boa a quase perfeita ou ótima . Todas as variáveis envolvidas apresentaram diferenças significativas a partir do grau II de aterosclerose coronariana. Conclusões: Espessura da túnica média e número de vasa vasorum da túnica externa correlacionaram-se negativamente com diâmetro do lúmen coronário, com correlações explicadas pelo sexo e grau de aterosclerose. Parâmetros estereológicos (exceto densidade do tecido conectivo da túnica externa) variaram proporcionalmente com espessura da túnica média e com número de vasa vasorum, e inversamente com diâmetro do lúmen coronário, com correlações explicadas pelo grau histopatológico de aterosclerose. Tamanho de cada tipo de vasa vasorum aumentou proporcionalmente à progressão dos graus ateroscleróticos. Ruptura venular precoce em placas ateroscleróticas vulneráveis poderia propiciar infarto do miocárdio desde o grau II ou III de aterosclerose.

INTRODUÇÃO: A estenose carotídea de origem aterosclerótica é um importante marcador de aterosclerose sistêmica avançada. A oclusão (obstrução completa da artéria) é rara e corresponde ao evento morfológico final da progressão da placa de ateroma na bifurcação carotídea. Muitos pacientes são sintomáticos no momento do diagnóstico e apresentam novos sintomas neurológicos na evolução apesar de tratamento clínico adequado. A literatura médica é escassa em determinar os principais fatores que podem levar a oclusão carotídea. A participação e intensidade das comorbidades e/ou fatores de risco, associados a dados demográficos peculiares foram pouco explorados. OBJETIVOS: Caracterizar o paciente com oclusão carotídea (OC) quanto a aspectos demográficos, doenças associadas e fatores de risco; detectar novos eventos neurológicos, cardiovasculares e óbitos no seguimento clínico destes pacientes. MÉTODO: Informações demográficas, clinicas e evolutivas de pacientes com oclusão carotídea e estenose carotídea não significativa foram recuperadas de um banco de dados compÍetado prospectivamente e complementadas com prontuário hospitalar e novos dados obtidos via convocação e/ou entrevista telefônica. RESULTADOS: No período de janeiro de 2005 a janeiro de 2013 foram analisados 213 pacientes portadores de OC e 172 portadores de estenose hemodinamicamente não significativa (ENS), ou abaixo de 50%. Foram analisados 4 dados demográficos e 9 fatores de risco, bem como sintomas neurológicos na apresentação e na evolução. No grupo OC predominaram indivíduos do sexo masculino, hipertensos, tabagistas, portadores de doença arterial obstrutiva periférica (DAOP), insuficiência renal crônica (IRC) com significância estatística em relação ao grupo ENS (p < 0,05).\\.Entre os pacientes com OC, 76,1% apresentaram sintomas neurológicos inicialmente contra 35,5% do grupo ENS (p = 0,000001). Quanto à evolução, os pacientes com OC apresentaram progressão significativa da estenose carotídea contralateral, quando comparada com a progressão da estenose nas carótidas do grupo ENS. (15,0% e 2,3%, p = 0,00011). O aparecimento de novos sintomas foi determinado pelo estado clínico de apresentação dos pacientes: 10,8% de novos sintomas nos inicialmente sintomáticos e 4,3% nos assintomáticos (p = 0,0218). Constatou-se maior número de óbitos na amostra OC (14,1%) do que na ENS (6,4%) com diferença significativa (p = 0,0150). CONCLUSÕES: OS pacientes portadores de OC apresentam maior prevalência de fatores de risco e comorbidades e maior mortalidade que o grupo ENS. No seguimento, os pacientes que se apresentavam sintomas neurológicos no momento do diagnóstico foram aqueles que mais desenvolveram novos eventos neurológicos. Este estudo representa um esforço em identificar uma amostra da população com estenose carotídea que pode necessitar de diagnóstico precoce e intervenção clínica vigorosa na prevenção de novos eventos e/ou óbitos
INTRODUCTION: Carotid stenosis is an important marker of severe systemic atherosclerosis. Internal Carotid occlusion (ICO) is rare and represents the final event when it comes to atherosclerotic plaque progression at the carotid bifurcation. Many patients are symptomatic when diagnosed with this condition and some of them will present more neurologic symptoms despite proper clinical management. So far only few studies have investigated if more comorbidities andjor risk factors, associated to demographic characteristics can lead to ICO. OBJETIVES: To identify the patient with ICO as regard to his demographic data, associated diseases and risk factors. Primary end-points were new neurologic events, cardiovascular symptoms and deaths during follow-up. METHOD: A prospective database was completed with demographic data and clinical information from patients with ICO and from a control group of patients with a non-significant stenosis (NSS), ar below 50%. Information was collected retrospectively from clínical records and missing data were completed with a medical appointment or teJephone interview. RESULTS: From [anuary 2005 to [anuary 2013, 213 patients with ICO and 172 patients with NSS were studied. Demographic data, risk factors for atherosclerosis and neurological symptoms at diagnosis and during follow-up were verified. Among patients with [CO there were more men and those with history of smoking, and more patients presenting with peripheral arterial disease (PAD) and chronic renal failure (CRF) than those in the NSS group (p < 0,05). At the time of diagnosis 76.1% of patients with ICO were symptomatic, while 35.5% in the NSS group (p=0.000001). Patients in the ICO group presented significant progression of the contralateral stenosis when compared to progression on any side in the control grouP\'\"(15.0% versus 2.3%, p = 0.00011). New symptoms were determined by the patient\'s clinical status. As regard to new neurological symptoms during follow-up, 10.8% of those initially symptomatic (both groups combined), presented new symptoms, opposed to 4.3% of those initially asymptomatic (p=0.0218). Number of deaths was significantly higher among patients in the ICO group (14.1% versus 6.4%, p=0.0150). CONCLUSIONS: Patients presenting with ICO have more risk factors and higher mortality by any cause. Those initially symptomatic will likely present more neurological symptoms during follow up. This study aims to identify those who are more at risk before the occlusion and could benefit of earJy diagnosis and vigorous c1inical intervention before new neurological events andjor death

A aterosclerose é um processo complexo, multifatorial que ainda não está totalmente esclarecido. Foi proposto que a resposta imune mediada por processos infecciosos e/ou inflamatórios influencia na patogênese de lesões ateroscleróticas. Os receptores TolI-likes (TLRs) estão envolvidos na resposta inata e em outros eventos fisiológicos através da interação com seus ligantes endógenos e exógenos e talvez envolvidos no processo aterogênico. Tem por objetivo analisar a expressão dos receptores Toll-like 2 e 4 (TLR2 e TLR4) associando o processo de sinalização com a presença de agentes infecciosos tais como a Chlamydia pneumoniae (CP) e Mycoplasma pneumoniae (MP), em pacientes com infarto do miocárdio (MI) e em aneurismas aórticos. Foram obtidos fragmentos de aortas ascendentes de pacientes submetidos à cirurgia de revascularização do miocárdio (G1, n=13) e de fragmentos de pacientes submetidos à cirurgia de correção de aneurisma aórtico (G2, n=14). Amostras congeladas e parafinadas foram analisadas por Imunohistoquímica (lHO) e Hibridização in situ (HIS) para detecção e localização da presença dos patógenos e TLRs. Realizou-se uma semiquantificação em microscópio (O, ausente; 1, discreto e focal; 2, moderado e focal e 3, intenso e difuso). Observou-se o grau de inflamação e de acúmulo de gordura. Outrossim, realizou-se PCR em tempo real (SYBR Green) para pesquisa de DNA de CP e MP, como também análise da expressão de mRNA de TLR2 e de TLR4. Na lHQ, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), maior quantidade de MP (p=0,012) e de TLR4 (p=0,017) no G2. Houve correlação de CP com MP (r=0,810 e p=0,003) e de TLR2 com TLR4 (r=0,569 e p=0,034). Na HIS, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), não houve diferenças significativas comparando-se os grupos (G1 x G2), porém houve correlação, no G1, de CP com TLR4 (r=0,730 e p=0,040) e de infiltrado inflamatório com células adiposas (r=0,700 e p=0,036). No G2, houve várias correlações: MP com CP (r=0,620 e p=0,016), MP com TLR4 (r=0,662 e p=0,010), CP com TLR2 (r=O,733 e p=0,003), CP com TLR4 (r=0,589 e p=0,027) e de TLR2 com TLR4 (r=0,714 e p=0,004). A PCR em tempo real mostrou presença de CP, pela segunda extração de DNA realizada (G2). Não houve diferença de expressão dos TLRs entre os grupos. A expressão de TLR2 foi maior do que de TLR4 no G1 (p=0,006). O grau de inflamação e o acúmulo de gordura foram maiores no G2 do que no G1(p=0,001). Estes dados sugerem uma relação da co-infecção CP e MP, na gravidade do processo inflamatório presente em placas ateroscleróticas e em pacientes com infarto do miocádio, como também, participação dos receptores Toll-like 2 e 4.
The atherosclerosis is a complex and multifactorial process that is not still completely elucidated. It has been proposed that immune-mediate response to inflammatory and/or infectious processes is implicated in the pathogenesis of the atherosclerotic lesions. Toll-like receptors (TLRs) are involved in the innate response and other physiological events through binding to endogenous and exogenous ligands and it may be involved in the atherogenic process To investigate the Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) expression in atheroma plaques and its association with the presence of infectious agents such as Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) in patients with myocardial infarction (MI) and aortic aneurysms. Fragments of ascending aorta were obtained from MI patients submitted to surgeries of revascularization of the myocardium (G1, n=13) and correction of aortic aneurism (G2, n=14). Frozen and paraffined samples slices were analyzed by Immunohistochemistry (lHQ) and in situ Hybridization for detection and localization of TLR2 and TLR4 expression and CP and MP antigens. There was semiquantification in microscope (0, absent; 1, discreet and focal; 2, moderate and focal; and 3, intense and diffuse). Histopathology was also carried out to investigate the inflammation degree and fat accumulation in these tissues. Real time PCR using SYBR Green System detection was used to stydy DNA CP and MP, also to analyze expression of mRNA TLR2 and TLR4. Using lHQ, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), larger amount of MP (p=0.012) and TLR4 (p=0.017) in G2. In G1 group, MP was positively correlated with CP (r=0.810, p=0.003), in G2, TLR2 with TLR4 (r=0.569, p=0.034). Using HIS, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), there were not significant differences between groups (G1 x G2), however, It was shown correlation between in G1, CP with TLR4 (r=0.730, p=0.040) and also inflammation with fat accumulation (r=0.700, p=0.036). In G2, there were several correlations: presence of MP with CP (r=0.620, p=0.016), MP with TLR4 (r=0.662, p=0.010), CP with TLR2 (r=0.733 p=0,003), CP with TLR4 (r=0.589, p=0.027) and TLR2 with TLR4 (r=0.714, p=0.004). Real time PCR showed presence of CP DNA using second purification accomplished (G2). There was not difference of expression TLRs among the groups. The expression of TLR2 was higher than TLR4 in G1 (p=0.006). Increased degree of inflammation and fat accumulation was also find in G2 than in G1 (p=0.001). These results are suggesting that the gravity of the inflammatory process in atherosclerotic plaques strongly are related to the presence of MP and CP co infection and expression of TLR2 and TLR4, as well in MI patients under myocardial revascularization.

Esta tese foi motivada por uma pesquisa anterior (COSTA, G.B. Relevância da obesidade para o incremento do risco cardiovascular global na criança e no adolescente. Aracaju, 2002. 109p. Dissertação (Mestrado) - UFS, na qual se caracterizou o perfil pró-aterosclerótico. Na pesquisa atual realizou-se um ensaio clínico controlado em 52 indivíduos, entre 10 e 18 anos, todos com o percentil do IMC > 85%, divididos em dois grupos: Grupo IR, que recebeu intervenção não-farmacológica multidisciplinar por 16 semanas consecutivas e Grupo IU que recebeu uma única intervenção não-farmacológica. Não houve diferenças estatísticas em relação a: idade, raça e gênero. Na análise multivariada verificou-se interação significativa para grupo x tempo (p<0,05) para as seguintes variáveis: peso (p<0,0001), altura (p=0,0083), índice de massa corpórea (p=0,0053), %GC (p=0,0022), circunferência da cintura (p=0,0359), pressão arterial sistólica (p=0,0021), pressão arterial diastólica (p=0,0004), triglicérides (p=0,029), Apo A-1 (p=0,001), glicemia em jejum (p=0,018), IGFBP-3 (p=0,005), TSH (p=0,045) e testosterona total (p=0,030). Não se verificou interação significativa para grupo x tempo (p<0,05) para as seguintes variáveis: freqüência cardíaca(bpm) (p=0,6809), colesterol total(mg/dL) (p=0,445), HDL-C(mg/dL) (p=0,726), LDL-C(mg/dL) (p=0,926), Apo B(mg/dL) (p=0,069), insulina(uU/mL) (p=0,866), HOMA-ir (p=0,088), IGF-1(nanog/mL) (p=0,424), cortisol sérico(mcg/mL) (p=0,175), PCR-us(mg/mL) (p=0,594) e estradiol(picog/mL) (p=0,507). Observou-se uma baixa incidência das variáveis depressão (23,0%), ansiedade (15,4%) e compulsão alimentar (13,5%) na população estudada. Não se obteve dados estatísticos em relação à nutrição nem ao condicionamento físico, uma vez que os mesmos tiveram papel educativo no presente estudo.
This hypothesis was developed by reason of another research (COSTA G.B. Importance of the obesity increasing cardiovascular risk factors in children and adolescents. Aracaju, 2002. 109p.), in which pro-atherosclerotic profile was determined. At this current survey a controlled clinical trial was performed in fifty-two subjects, aged 10 to 18 years, all of them with IMC percentile > 85% and divided in two groups: Group IR, which received non-pharmacological multidisciplinary interventions for sixteen weeks and Group IU, which received only one intervention. There were no statistical differences in relation to: age, race and gender. There was significant interaction group x time (p<0,05) for: weight(Kg) (p<0,0001), height(m) (p=0,0083), body mass index(Kg/m2) (p=0,0053), percentage of body fat(%) (p=0,0022), waist circumference(cm) (p=0,0359), systolic blood pressure(mm Hg) (p=0,0021), diastolic blood pressure( mm Hg) (p=0,0004), TG(mg/dL) (p=0,029), Apo A-1(mg/dL) (p=0,001), fasting glucose(mg/dL) (p=0,018), IGFBP-3(mcg/mL) (p=0,005), TSH(uUi/mL) (p=0,045) and total testosterone(ng/mL) (p=0,030). However, there was not significant interaction group x time (P<0,05) for: heart rate (bpm) (p=0,6809), total cholesterol(mg/dL) (p=0,445), HDL-C(mg/dL) (p=0,726), LDL-C(mg/dL) (p=0,926), Apo B(mg/dL) (p=0,069), insuline(uU/mL) (p=0,866), HOMA-ir (p=0,088), IGF-1(nanog/mL) (p=0,424), cortisol(mcg/mL) (p=0,175), PCR-us(mg/mL) and estradiol(picog/mL) (p=0,507). There was low incidence of depression (23,0%), anxiety(15,4%) and alimentary compulsion(13,5%) on whole studied population. In regarding to nutrition and exercise, there are no statistic data because the aim was the education.

Introdução: O estudo MASS III não mostrou diferença significativa entre a cirurgia de revascularização miocárdica (CRM) com e sem circulação extracorpórea (CEC) em relação ao desfecho composto primário de morte por todas as causas, infarto agudo do miocárdio, acidente vascular cerebral ou revascularização adicional em pacientes com doença coronariana multiarterial. No entanto, a custo-efetividade dessas estratégias permanece desconhecida. Métodos: Pacientes com doença coronariana multiarterial estável e função ventricular esquerda preservada foram randomizados para CRM com CEC (n=153) ou sem CEC (n=155). Os dois grupos eram bem semelhantes quanto às características basais. A análise dos custos foi realizada a partir da perspectiva do sistema público de saúde brasileiro, e as utilities foram avaliadas pelo questionário SF-6D. Um modelo de Markov, com base nos dados de 5 anos de seguimento, foi utilizado para extrapolar os custos e os anos de vida ajustados pela qualidade (QALY) para doença coronariana crônica. Resultados: A qualidade de vida de ambos os grupos melhorou significativamente após a cirurgia durante o seguimento, em comparação com os dados pré-cirurgia, embora os ganhos de vida adquiridos (LYG) e QALYs tenham sido semelhantes entre os grupos durante o seguimento de 5 anos. Os custos para o período total do estudo não diferiram entre os grupos sem e com CEC (R$ 19.180,65 e R$ 19.909,18, respectivamente, p=0,409). Ao longo de um horizonte de tempo ajustado para a expectativa de vida da população do estudo, a razão de custo-efetividade incremental da CRM com versus sem CEC foi R$ 45.274 por QALY ganho, que foi robusto nas simulações de Monte Carlo e nas análises de sensibilidade. Para um limiar de custo-efetividade de R$ 34.212 por QALY ganho, a CRM sem CEC tem 65% de probabilidade de ser custo-efetiva quando comparada com CRM com CEC. Conclusão: Cirurgia de revascularização miocárdica sem CEC é clinicamente tão segura e efetiva quanto a cirurgia com CEC e parece ser uma estratégia economicamente atraente em comparação com a CRM com CEC em pacientes com doença arterial coronariana estável
Background: The MASS III trial revealed that in patients with multivessel coronary disease, no significant difference was observed between on-pump and off-pump coronary artery bypass surgery (CABG) in the primary composite outcome. However, long-term cost-effectiveness of these strategies is unknown. Methods: Patients with stable multivessel coronary artery disease and preserved left ventricular function were randomized to onpump (n=153) or off-pump CABG (n=155). The 2 groups were well matched for baseline characteristics. Costs analysis was conducted from a Brazilian public healthcare system perspective, and health state utilities were assessed using the SF-6D questionnaire. A Markov\'s model based on the 5- year in-trial data was used to extrapolate costs and quality-adjusted life-years (QALY) for chronic coronary disease. Results: Both groups\' quality of life improved significantly after surgery during follow-up compared with baseline, and life-years gained (LYG) and QALY gains were similar between on-pump and off-pump CABG over the 5-year time frame of the trial. The costs for the overall period of the trial - the mean cost in U.S. dollars per patient - did not differ significantly between the off-pump group and the on-pump group ($5674.75 and $5890.29 respectively, p=0.409). Over a lifetime horizon, the incremental cost-effectiveness ratio of on-pump vs. off-pump CABG was $12,576 per QALY gained, which was robust in Monte Carlo replications and in sensitivity analyses. Using a cost-effectiveness threshold of $10,122 per QALY gained, off-pump has 65% probability of being cost-effective versus on-pump CABG. Conclusions: Off-pump CABG was clinically as safe and effective as on-pump CABG and appears to be an economically attractive strategy compared with on-pump CABG among patients with stable coronary artery disease

Chlamydophila pneumoniae è un batterio Gram-negative ed intracellulare obbligato, con un ciclo di sviluppo bifasico, in quanto assume due diverse forme: la forma extracellulare infettiva chiamata corpo elementare e la forma intracellulare replicativa chiamata corpo reticolare. C. pneumoniae è l’agente eziologico delle infezioni respiratorie e sembra giocare un ruolo immuno-patogenico nell’aterosclerosi contribuendo all’infiammazione ed instabilità della placca. Le Fosfolipasi D (PLDs) sono enzimi coinvolti nel metabolismo lipidico ed in altri eventi che direttamente o indirettamente agiscono sulla virulenza e la risposta infiammatoria. Per capire meglio il ruolo della PLD di C. pneumoniae (CpPLD) nella biologia cellulare e durante l’infezione, il gene Cppld è stato clonato ed espresso in Escherichia coli e la proteina ricombinante rCpPLD è stata purificata. Questa proteina si è dimostrata molto immunogenica e capace di individuare anticorpi anti-CpPLD di una popolazione generale di soggetti esposti a C. pneumoniae. Esperimenti in vitro di trascrizione genica ed espressione in cellule Hep-2 infettate, ha dimostrato che il gene Cppld è molto espresso all’inizio e alla fine del ciclo di sviluppo della Chlamydia e che la proteina CpPLD è localizzata alla periferia del corpo d’inclusione a 72 ore dall’infezione. Della proteina CpPLD è stata studiata anche l’attività enzimatica. rCpPLD è stata in grado di sintetizzare cardiolipina da 2 molecole di fosfatidil glicerolo, dimostrando di essere un enzima cardiolipina sintetasi. Inoltre, lo scopo di questo studio è stato anche quello di valutare la risposta sierologica alla rCpPLD in pazienti con sindrome coronarica acuta (ACS) ed in sangue di donatori sani. Tutti i sieri esaminati sono stati analizzati per microimmunofluorescenza (MIF). I campioni positivi sono stati categorizzati come soggetti con presuntiva infezione da C. pneumoniae o passata esposizione (solo specifiche IgG) ed infezione cronica (presenza di specifiche IgG e IgA). Nessun siero MIF-negativo ha mostrato anticorpi contro rCpPLD. In soggetti MIF-positivi, anticorpi contro rCpPLD sono stati consistentemente trovati in sieri di pazienti affetti da ACS con un’infezione cronica. In più è stato individuato un epitopo immunodominante P5 (aa 233-252) della proteina CpPLD, il quale reagisce fortemente con sieri di soggetti con ACS. La CpPLD ed il peptide P5 potrebbero essere utilizzati come antigeni per la diagnosi di C. pneumoniae in pazienti ACS con infezione cronica. Questi dati suggeriscono che rCpPLD potrebbe essere utile per futuri studi in merito al ruolo che quest’enzima gioca nella patologia e nella risposta immunitaria all’infezione da C. pneumoniae.
Chlamydophila pneumoniae is a Gram-negative obligate intracellular eubacteria, with a biphasic developmental cycle and two distinct morphological forms: the extracellular infectious elementary body and the intracellular replicating reticulate body. C. pneumoniae is an aetiological agent of respiratory infection also suspected to play an immuno-pathogenetic role in atherosclerosis by contributing to inflammation and plaque instability. Phospholipases D (PLDs) are enzyme involved in lipid metabolism and others events which can direct or indirect impact on virulence and inflammatory response. To better understand the role of C. pneumoniae PLD (CpPLD) in cell biology and during the infection, the Cppld gene was cloned and expressed in Escherichia coli and the recombinant protein rCpPLD was purified. This generated protein was highly immunogenic in mice and capable to elicit anti-CpPLD antibodies in the general population exposed to C. pneumoniae. In vitro experiments of gene transcription and expression in Hep-2 infected cells, showed that Cppld gene was expressed highly to early and late chlamydial development, and the CpPLD protein was localized at the periphery of inclusions at 72 h post infection. Enzymatic activity was also investigated. The rCpPLD was able to synthesize cardiolipin from 2 molecules of phosphatidyl-glycerol, demonstrating that the CpPLD was a cardiolipin sinthase enzyme. Furthermore, the purpose of this study was to evaluate the serological response to the rCpPLD in patients with acute coronary syndromes (ACS) and in healthy blood donors. All serum samples were screened by microimmunefluorescence (MIF). the positive samples were categorized as subjects with presumptive C. pneumoniae infection or past exposure (only specific IgG) and chronic infection (presence of specific IgG and IgA). MIF-negative sera showed antibodies against rCpPLD. In MIF-positive subjects antibodies against to rCpPLD were consistently found in sera of ACS patients with chronic infection. Additionally, it was recognized an immunodominant epitope in position 233-252 aa (P5) of the CpPLD protein which strongly reacted with ACS sera. The CpPLD protein and its P5 peptide could be plausible antigens for the diagnosis of C. pneumoniae chronic infections in ACS patients. These data suggest that rCpPLD may be a useful tool for future studies concerning the role that this enzyme plays in the pathology and immune response to C. pneumoniae infection.

Les édulcorants ont été progressivement introduits dans l'alimentation humaine dans le but, de réduire l'apport calorique mais aussi de limiter les répercussions sur les niveaux de glycémie, tout en préservant le gout sucré, élément essentiel du plaisir gustatif. Plusieurs travaux de recherche ont néanmoins suggéré, contrairement aux intentions premières, que cette consommation d’édulcorants quotidienne pouvait participer à augmenter le risque de devenir obèse ou diabétique mais aussi le risque de mortalité cardiovasculaire. Dans ce contexte, l’objectif de ce travail de thèse a été d’identifier les effets d’une consommation chronique de boissons contenant des édulcorants de synthèse et non caloriques, seuls ou en synergie, sur le contrôle glycémique et la physiologie vasculaire, afin de comprendre le risque accru d’évènements cardiovasculaires observé chez les consommateurs fréquents d’édulcorants. Ainsi, nous avons pu mettre en évidence chez le rongeur la sensibilité du tissu vasculaire à l’exposition de l’acésulfame de potassium (Ace-K) et du sucralose, sensibilité qui semble indépendante de l’activation des récepteurs du gout sucré. De manière intéressante, les effets vasomoteurs de ces édulcorants sont apparus antagonistes l’un envers l’autre. Les études actuelles suggèrent également l’implication des édulcorants dans la survenue de facteurs de risque cardiovasculaire tel que l’obésité. Toutefois dans notre modèle, la consommation de sucralose dans le cadre d’un régime gras a permis au contraire de limiter les altérations de ce régime sur l’accumulation de tissu adipeux et sur le contrôle de la glycémie, mais également de potentialiser la réponse vasodilatatrice artérielle chez les souris. Ainsi, nos résultats démontrent l’importance d’identifier les conséquences propres directes (sur les cellules du tissu vasculaire) et indirectes (modulation des facteurs de risque cardiovasculaires) de chaque molécule sur la physiologie vasculaire. Néanmoins, des études supplémentaires sont nécessaires afin d’évaluer les effets spécifiques de chaque molécule sur cette balance vasomotrice
Artificial sweeteners have been extensively introduced into human diets with the objective of decreasing caloric intake and normalize the levels of blood glucose, while preserving an essential element of taste pleasure, the sweet taste. Nevertheless, several studies have suggested that a daily and sustained consumption of sweeteners could contribute to the increased risk of becoming obese or diabetic but also to the increased cardiovascular mortality risk. In this context, the objective of this work was to identify the effects of chronic consumption of beverages containing synthetic and non-caloric sweeteners on glycemic control and vascular physiology. Thus, we were able to demonstrate in rodents the sensitivity of vascular tissue to acesulfame potassium (Ace-K) and sucralose exposure that appears to be independent of sweet taste receptor activation. Interestingly, the vasomotor effects of these artificial sweeteners appeared to be antagonistic to each other. Current studies also suggest the involvement of artificial sweeteners in the occurrence of cardiometabolic disease such as obesity. Surprisingly, in our model, the consumption of sucralose appears to limit the deleterious effects of a high fat diet on body composition and glycemic control. The sucralose consumption even potentiated the vasodilatory arterial response in our mice model. Thus, according to the effects observed on glucose homeostasis, our results demonstrate the importance of identifying the specific direct (on vascular tissue cells) and indirect (modulation of cardiovascular risk factors) consequences of each molecule on vascular physiology. Nevertheless, further studies are needed to evaluate the specific effects of each molecule on this vasomotor balance

博士
國立臺灣大學
臨床醫學研究所
97
Background: Rupture of atherosclerotic plaques and subsequent formation of thrombi are currently recognized as the major cause of morbidity and mortality in ischemic stroke and cardiovascular diseases, including acute coronary syndrome. Several imaging modalities have shown promises as potential surrogate markers for atherosclerosis. They may help clinicians and investigators to directly visualize atherosclerotic plaque burden, refine cardiovascular risk assessments, and allow serial monitoring of disease activity once interventions have been initiated. The proposed major criteria for defining vulnerable plaques, based on the autopsy studies, include active inflammation (monocyte/macrophage and sometimes T-cell infiltration), thin cap with large lipid core, endothelial denudation with superficial platelet aggregation, fissured plaque and severe luminal stenosis. Conventional imaging of atherosclerosis is based on the degree of luminal stenosis and morphologic characteristics of atheromas, including X-ray contrast angiography, computed tomography (CT) and magnetic resonance (MR) imaging. Nuclear imaging also has the ability to non-invasively image pathophysiological process of atherosclerosis. Recent studies have shown that 18F-fluorodeoxyglucose (18F-FDG) accumulates in macrophage-rich plaques and the inflammatory activity of individual plaques could be detected and measured by using positron emission tomography (PET). In addition, hybrid PET/CT scanners could detect calcification and provide better localization of systemic atherosclerotic plaques, thus may allow to screen, diagnose or monitor treatment response in patients with atherosclerosis. Atherosclerosis is a complex and multifactorial disorder, and a specific profile of biomarkers may help identify subjects at risk for progressive atherosclerosis or plaque rupture. Inflammation plays a significant role in the pathogenesis and progression of atherosclerosis. Numerous systemic serological markers have been reported to provide additional information about the risk of developing cardiovascular disease and reflect the overall burden of atherosclerosis. However, many of these are not specific to systemic atherosclerosis or plaque vulnerability, and the clinical significance remains to be elucidated. Levels of cellular adhesion molecules (such as E-selectin, ICAM-1, VCAM-1) may reflect extent of expression and/or shedding of molecules on endothelial surface that are influenced by pro-inflammatory cytokines. Recent data have suggested that macrophages and vascular smooth muscle cells are important component of vulnerable plaques, and are the sources of matrix metalloproteinases (MMPs) production. Several immunochemical findings have showed that MMP-1 is abundant in the macrophage-rich shoulder regions of atherosclerotic plaques and is robustly induced by cytokines and growth factor. However, scant studies have reported that MMP-1 is associated with acute coronary syndrome and the presence of complex coronary lesions. The significance of circulating levels of MMP-1 remains to be evaluated in larger-scale clinical studies. Macrophages are important for intracellular lipid accumulation and foam cell formation in the process of atherosclerosis. MMPs secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability. Diabetes is a major risk factor for atherosclerosis, thus we investigate the regulatory mechanism of MMP-1 in THP-1 differentiated macrophages under high glucose media. Purposes: The aims of this study are to evaluate the feasibility of 18F-FDG PET/CT as a non-invasive imaging modality in the detection of systemic atherosclerosis, and correlation between characteristics of atherosclerotic plaques by using 18F-FDG PET/CT and circulating biomarkers among subjects with or without carotid stenosis. We further evaluate the role of cellular adhesion molecules in patients with transplant vasculopathy and MMP-1 in patients with significant atherosclerosis. We establish an in-vitro system of THP-1 monoblastic leukemic cell-line to explore MMP-1 and hypoxia-inducible factor-1α (HIF-1α) expression and regulation signal pathways including nuclear factor-kB (NF-kB) or c-Jun N-terminal kinase (JNK) pathways, especially in high glucose conditions. We also evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) and MMP-1. Research Designs and Results: 1. We analyzed ICAM-1, VCAM-1 and E-selectin levels from the coronary sinus of 25 cardiac allograft recipients, correlated with the degree of acute rejection detected in endomyocardial biopsy specimens and the presence of transplant vasculopathy assessed with coronary angiography. We found that VCAM-1 significantly increased in patients with transplant vasculopathy compared with those without transplant vasculopathy, whereas E-selectin and ICAM-1 did not. 2.We examined 25 patients with significant carotid stenosis (≥ 50%) and 22 controls using 18F-FDG PET/CT. 18F-FDG arterial uptake, as well as calcifications, was significantly higher in extensive distributions in patients with established carotid stenosis; but their distribution was not consistently overlapping. The values of circulating MMP-1 and leukocyte counts were significantly higher in patients with carotid stenosis (all P < 0.05). In addition, subjects with higher 18F-FDG uptake (SUVmax > 2.0) on target lesions had higher baseline and post-stenting MMP-1 levels (all P < 0.05). We further measured hs-CRP and MMP-1 in 37 patients with significant carotid stenosis and 84 controls. We also confirmed that patients with carotid stenosis exhibited significant higher MMP-1 as compared with controls, but no difference in hs-CRP. Moreover, MMP-1 elevated immediately after stenting (n = 30). In multivariate analyses, MMP-1 was negatively correlated with statin and angiotensin converting enzyme inhibitor/angiotensin-II receptor blocker use in controls. 3. MMP-1, hs-CRP and adiponectin were measured in 217 subjects with angiographically documented multivessel coronary artery disease (CAD, two or three-vessel disease by luminal stenosis ≥ 50%) and 81 controls. MMP-1 and hs-CRP were notably higher in patients with CAD; while adiponectin was not significantly different between two groups. Levels of hs-CRP positively correlated with body mass index and left ventricular dysfunction (R2 = 0.16, P < 0.0001); while adiponectin was significantly associated with age, gender, and levels of cholesterol and triglyceride (R2 = 0.09, P < 0.0001). On the contrary, MMP-1 was not associated with any clinical cardiovascular risk factors, and still an independent predictor (OR = 1.49, P < 0.0001) of multivessel CAD after the adjustment of clinical risk factors and hs-CRP. 4. We have established an in-vitro THP-1 macrophage cell model. THP-1 treated with PMA may mimic inflammatory stimulation. High glucose concentration could augment PMA-stimulated MMP-1 expression in THP-1. MMP-1 mRNA expression is through cytokines/inflammatory process, via NF-κB and JNK pathways, especially NF-κB. Of glimepiride, metformin and BRL-49653 (rosiglitazone, PPAR γ agonist), BRL-49653 notably attenuates PMA-stimulated MMP-1 expression in THP-1 in high glucose concentration. We have demonstrated that PMA could upregulate HIF-1α which is suppressed by NF-κB inhibitor. HIF-1α inducers could upregulate MMP-1 while HIF-1α inhibitor could attenuate MMP-1, suggesting MMP-1 could be regulated by HIF-1α. Conclusions: This study has demonstrated that an increased coronary sinus level of sVCAM-1 is a reliable marker in assessing cardiac transplant vasculopathy. Our study has also shown that 18F-FDG PET/CT imaging could be used as an adjunct to the clinical management of high-risk atherosclerosis and an in vivo tool to study plaque biology. Elevated MMP-1 could predict the presence of advanced atherosclerosis. Higher levels and rapid surge after stenting in patients with carotid stenosis support MMP-1 is an important composition of plaques. Our investigation provides a link between 18F FDG uptake and MMP-1. In THP-1 cell model, increased transcription of macrophage MMP-1 under high glucose conditions provides a mechanism for accelerated atherosclerosis in diabetes. MMP-1 expression is regulated via NF-κB and JNK pathways, as well as HIF-1α., thus providing a molecular basis for regulation of MMP-1 in differentiated THP-1 cells.

在亞洲人群包括中國人群中，顱內動脈粥樣硬化（ICAS）發病率很高，是缺血性卒中和短暫性腦缺血發作（TIA）的首要病因。然而，目前ICAS 並未被深入研究。因此我們在一系列研究中通過運用傳統及創新的神經影像學方法，來研究ICAS 的臨床及血流動力學特徵，以期促進其全面評價及危險分層。
既往研究發現亞洲人群和西方人群在頭頸部動脈粥樣硬化的發生和發展上存在種族差異。爲了進一步驗證這些種族差異，我們開展了一項社區研究，以探索無癥狀性顱內外動脈粥樣硬化在中國社區成年居民中的發病情況，以及二者之間的相互關係。在該研究中，我們分別採用經顱多普勒（TCD）和頸部血管超聲（CD）來評價顱內和顱外動脈的粥樣硬化。在研究納入的537 例研究對象中，我們發現顱內動脈粥樣硬化的發展優先於頸動脈粥樣硬化，而且不同階段的頸動脈粥樣硬化與顱內動脈粥樣硬化並無獨立相關關係。該結果提示，在中國人群的全身系統性粥樣硬化的過程中，顱內動脈粥樣硬化可能是一個比較早期而且相對獨立的過程，這與西方人群的情況不同。本研究結果進一步支持東西方人群在顱內外動脈粥樣硬化進程上存在的種族差異。
根據既往研究結果，病因為癥狀性ICAS的缺血性卒中或TIA患者卒中復發的風險很高。目前，癥狀性ICAS患者的危險分層大多基於其動脈管腔的狹窄程度。然而，管腔的解剖學狹窄程度並不一定與其血流動力學的嚴重程度成比例，而後者也可能影響相關患者的卒中復發風險。因此，我們進行了以下的一系列研究， 以評價癥狀性ICAS的血流動力學特徵，同時初步探索其在相關患者危險分層中的應用價值。
我們首先進行了三項研究，採用一種基於磁共振血管成像（MRA）的創新方法來評價癥狀性ICAS的血流動力學嚴重程度。基於時間飛躍法（TOF）MRA的信號對比機制，我們提出了一項名為信號強度比值（SIR）的參數來定量地評價癥狀性ICAS 的血流動力學效應；該參數代表TOF MRA 影像上經過背景信號強度校正后的ICAS 病變后和病變前的信號強度比值。在一項初步研究中，我們確定了該參數的評價和計算方法。在26例癥狀性ICAS病變中，我們發現該參數的計算操作簡便，在臨床上可行，且在同一評價者的兩次評價中具有很高的可重複性。在隨後的一項研究中，我們在102例癥狀性ICAS病變中發現該參數在兩位評價者之間具有顯著的可重複性。在第三項研究中，我們在36例具有癥狀性ICAS的缺血性卒中患者中發現SIR與患者的急性梗死灶體積顯著相關，但我們並未發現該參數與患者1年的卒中復發風險相關，可能由於該研究的樣本量過小。以上三項研究證實，SIR作為一種基於TOF MRA的評價癥狀性ICAS血流動力學嚴重程度的方法，具有可重複性及臨床可行性；而其對於相關患者危險分層的價值需要在前瞻性的較大型研究中進一步驗證。
在如下的另外兩項研究中，我們採用計算機流體動力學（CFD）技術對癥狀性ICAS患者的計算機斷層掃描血管成像（CTA）影像進行重建，從而評價其ICAS 病變的血流動力學特徵。首先，在一項初步研究中，我們探索了採用臨床常規CTA影像進行CFD模型重建的可行性。在10例癥狀性ICAS病變中，9例病變的CTA原始圖像成功重建為CFD模型。重建的CFD模型可以定量地反映ICAS病變的各種血流動力學特徵。該初步研究證實了基於臨床常規CTA進行CFD建模從而評價ICAS血流動力學特徵的可行性。在隨後的一項研究中，我們探索了CFD 模型反映的癥狀性ICAS 的血流動力學特徵對於相關患者卒中復發的預測價值。在32例具有70-99%管腔狹窄的癥狀性ICAS病例中，我們發現病變前後血流動力學參數的變化（包括壓力，剪切應變率及血流速度）可能預測患者的卒中復發風險。以上兩項研究證實，基於臨床常規CTA進行CFD模型重建從而定量評價癥狀性ICAS的血流動力學特徵具可行性，同時，這些血流動力學特徵可能對相關患者的卒中復發風險具有預測價值。
綜上所述，通過以上研究，我們進一步證實了亞洲人群和西方人群在顱內外動脈粥樣硬化的進程上存在的種族差異。更重要的是，我們的研究證實評價癥狀性ICAS病變的血流動力學特徵具有臨床意義。對於相關患者，採用以上研究中的兩種方法評價癥狀性ICAS的血流動力學特徵，可能對患者的危險分層具有潛在的指導意義。在未來的前瞻性大樣本研究中，上述方法對癥狀性ICAS患者卒中復發風險的預測價值需要進一步證實，以期促進相關的臨床決策，從而在長遠目標上降低相關患者的卒中復發風險。
Intracranial atherosclerosis (ICAS) is of high prevalence in Asia, which is the leading cause for ischemic stroke and transient ischemic attack (TIA) in Asians, including the Chinese. However, it has not been fully appreciated or adequately investigated in relevant studies. In this thesis, we tried to delineate the hemodynamics and clinical implications of ICAS, by using several traditional and novel neuroimaging methods.
Previous studies had suggested differences in atherogenesis of intra- and extracranial arteries between Asians and Caucasians. To find further evidence, we performed a study to investigate asymptomatic ICAS and carotid atherosclerosis and their correlations in community-dwelling Chinese adults, by using transcranial Doppler and carotid duplex ultrasonography, respectively. For the 537 subjects studied, we found more advanced asymptomatic ICAS than carotid atherosclerosis, and there were no independent correlations between different stages of carotid atherosclerosis and presence of ICAS. The results suggested that atherogenesis of intracranial arteries might be a relatively independent course in systemic atherosclerosis in the Chinese population, which is unlike the case in Caucasians. By combing with previous findings, results of this study further supported the existence of racial differences in cervicocerebral atherogenesis between Asians and Caucasians.
According to previous studies, stroke patients with symptomatic ICAS are at high risk of recurrence. Currently, risk stratification of symptomatic ICAS are usually based on the percentage of luminal stenosis. However, the anatomic severity does not always proportionate to its hemodynamic significance, which may also impact on the risk of stroke recurrence in symptomatic ICAS. Therefore, we performed a series of studies as follows to evaluate the hemodynamics of symptomatic ICAS, and to assess its value in risk stratification of those with such lesions.
We first performed three studies based on time-of-flight (TOF) magnetic resonance angiography (MRA), to gauge the hemodynamic significance of symptomatic ICAS. Based on the signal contrast mechanism of TOF MRA, we developed a novel index, signal intensity ratio (SIR), representing changes of signal intensities (SI) across an ICAS on maximum intensity projections of TOF MRA, to quantify its hemodynamic significance: SIR = (mean post-stenotic SI - mean background SI) / (mean pre-stenotic SI - mean background SI). In a pilot study to establish the methodology of this index, we found it easy to perform, and highly reproducible between repetitive measurements by a same observer in 26 symptomatic ICASs. In a subsequent study, we also found this index to be substantially reproducible between measurements from two observers in 102 symptomatic ICAS lesions. In a third study, we tried to investigate the relationships between SIR of ICAS, other imaging features, and 1 year outcomes of patients with symptomatic ICAS. In the 36 patients enrolled, SIR was found to be significantly, linearly and negatively correlated to acute infarct volume on diffusion-weighted MR images. However, we did not establish a definite correlation between SIR and recurrent ischemic stroke, probably due to the small sample size. These studies suggested that SIR as evaluated on MRA was a feasible and reproducible method to gauge the hemodynamic and functional significance of ICAS. The role of this index in predicting further recurrent risks in those with symptomatic ICAS needs to be verified in future studies.
In another two studies, we applied the computational fluid dynamics (CFD) modeling technique in processing computed tomography angiography (CTA) images, to evaluate the hemodynamic characteristics of ICAS. In a pilot study, we tested the feasibility of CFD modeling of ICAS based on CTA images. Among 10 cases of symptomatic ICAS identified on CTA, the CTA source images of 9 were successfully processed to CFD models, which were able to quantitatively delineate the hemodynamic environment across the lesions. This pilot study demonstrated the feasibility of constructing CFD models of ICAS out of routinely obtained CTA source images. Then in a second study, we preliminarily explored the values of hemodynamics of ICAS revealed by such CFD models, in predicting recurrent risks in patients with symptomatic ICAS of 70-99% luminal stenosis. In the 32 cases evaluated, we found that changes of hemodynamic features across an ICAS lesion, including the changes of pressure, shear strain rate, and blood flow velocity, may be able to predict the recurrent risk in this patient subset. Therefore, it was feasible to model hemodynamics of symptomatic ICAS based on CTA images, and future prospective studies with larger sample sizes are warranted to further validate the role of CFD modeling in risk stratification of affected patients.
In conclusion, in this thesis we found further evidence to support the existence of racial differences in atherogenesis of cervicocerebral arteries between Caucasians and Asians. More importantly, we demonstrated that hemodynamics of symptomatic ICAS could be of clinical significance in characterization of such lesions. In patients with symptomatic ICAS, the two methods to evaluate hemodynamic features of ICAS as used in this thesis, may yield potential values in predicting the recurrent risk of these patients. In the near future, prospective studies enrolling more patients are warranted to further validate findings in this thesis, to embrace more reasonable and comprehensive evaluation of symptomatic ICAS, so as to facilitate decision making in clinical scenarios and patient selection in clinical studies, which in the long run may help reduce the risk of stroke recurrence in affected patients.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Leng, Xinyi.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 131-146).
Abstracts also in Chinese.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2016
The relationship between osteoporosis (OP) and atherosclerosis is still unclear. Despite the epidemiological association and the existence of common risk factors such as the influence of age in both pathologies, there are clinical, genetic and pathogenic data, suggesting the possibility of a common pathway. Vascular calcification has been an important target of investigation. There are several studies that demonstrate increased vascular calcifications in patients with OP. The present study aimed to histologically evaluate the calcium deposits in atherosclerotic plaques of patients with and without osteoporosis, through a semi-quantitative analysis of calcium deposits. In this study were included 32 patients undergoing elective carotid endarterectomy, from which were obtained blood samples and the atheroma plaque. A dual-energy X-ray absorptiometry (DXA) was performed and a structured clinical protocol was applied. Samples were stained with Alizarin Red S and scanned with NanoZoomer®, in order to observe the calcium deposits within the atherosclerotic plaques. The semiquantitative analysis by ImageJ® showed numerically higher median values of relative calcium area in the atheroma plaques from osteoporotic patients (median: 45.46%) comparing to patients with normal bone mineral density (median: 15.73%), although this difference was not statistically significant (p-value = 0.175). In multivariate analysis, we do not found an independent relationship, between the relative area of calcium in atherosclerotic plaques with serum HDL levels and statins therapy, in the presence of osteoporosis, adjusted to gender and age.
A relação entre osteoporose e aterosclerose é ainda pouco clara. Contudo, além da associação epidemiológica e de factores de risco, nomeadamente, a influência da idade em ambas as patologias, existem outros dados de cariz clínico, genético e patogénico que sugerem a possibilidade de uma via comum de desenvolvimento. A calcificação vascular tem sido alvo de estudo, existindo várias investigações que demonstram um aumento de depósitos de cálcio em placas de ateroma nos doentes com osteoporose. O presente estudo consistiu na avaliação histológica dos depósitos de cálcio nas placas de ateroma de doentes com e sem osteoporose, por meio de uma análise semi-quantitativa dos depósitos de cálcio. Neste estudo foram incluídos 32 doentes submetidos a endarterectomia carotídea dos quais se obtiveram amostras de sangue e placa de ateroma. Os doentes realizaram também uma densitometria óssea e foi aplicado um protocolo clínico estruturado. As amostras foram coradas com Alizarin Red S e as lâminas fotografadas com o NanoZoomer®, por forma a conseguimos observar nas placas de ateroma os depósitos de cálcio. A partir da análise semiquantitativa realizada pelo Image J® constatámos que os doentes com osteoporose apresentam áreas relativas de depositos de cálcio numericamente superiores (mediana: 45.46%) aos indivíduos sem osteoporose (mediana: 15.73%) mas esta diferença não é estatisticamente significativa (p-value = 0.175). A análise multivariada não revelou uma relação independente entre a área relativa de cálcio nas placas de ateroma com os níveis séricos de HDL e terapêutica com estatinas, na presença de osteoporose, tendo em conta a idade e o sexo.

Study 1:

The success of regenerative cardiac therapy requires reestablishing a capable blood supply via vasculature. The objective of this study was to develop an optimal scaffold formulation for de novo collateral vessel growth of aortic grafts using modified fibrin clots. This ex vivo vascular outgrowth model can be used to interrogate the complex cell or tissue interactions on the angiogenic front as vessels are formed. Based on formulation constraints, the methods used here may provide a clinically applicable option for guided collateral formation. Once understood, the methods and procedures can be tested and modified as necessary for in vivo, in situ regenerative therapy. Aortic segments from wild-type (C57BL/6J) and apolipoprotein-E deficient (ApoE) atherosclerosis-prone mice were cultured in a 3D environment created by various formulations of PEGylated fibrin. Aortic outgrowth was assessed and the optimal formulation was chosen to test the formation of de novo vascular circuits -- the first step necessary for collateral artery formation. The cultures were examined by conventional and confocal microscopy as well as by optical coherence tomography. Experiments testing the relationship between fibrin PEGylation and aortic vascular outgrowth showed that PEGylating fibrinogen prior to clot formation increased outgrowth over non-PEG control (n=6, p<.05) at lower fibrin concentrations. Lowering fibrin concentration to 10, 5, or 2.5mg/ml resulted in significantly higher outgrowth that was 1.92, 2.04, or 2.20 times that of 20mg/ml PEGylated fibrin gels. When multiple aortic segments are cultured in proximity, microvascular outgrowths visually anastamose suggesting that aorta-aorta conduits can be formed in fibrin based hydrogels. Anastomosing circuits appeared between wild-type aortic segments as well as between wild-type and atherosclerotic prone ApoE knockout segments. Fibrin gels, with or without PEGylation, form scaffolds suitable for regenerative vascular outgrowth ex vivo in normal and atherogenic environments. PEGylating fibrin prior to thrombin-initiated polymerization will allow the incorporation of growth factors or other bioactive components, making this a customizable therapy for guided collateral formation. Additionally, the incorporation of PEG itself does not limit and may actually increase the outgrowth from aortic segments in lower density gels. Finally, PEGylated fibrin gels offer an environment that will promote vascular extensions that visually anastamose, making this a viable model for ex vivo collateral formation.
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La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.
Mass spectrometry is the measurement of the mass over charge ratio of ions. It is broadly applicable and capable of analyzing complex mixtures. Imaging mass spectrometry (IMS) is a branch of mass spectrometry that analyses ions across a surface while conserving their spatial organization on said surface. At this juncture, the most studied IMS samples are thin tissue sections from plants and animals. Among the molecules routinely imaged by IMS, lipids have generated significant interest. Lipids are important in disease and normal cell function as they form cell membranes and act as signaling molecules for cellular events among many other roles. Considering the potential of lipids in biological and clinical applications and the capability of MALDI to ionize lipids, we developed analytical strategies for the handling of samples and analysis of large lipid MALDI IMS datasets. Lipid degradation is massively important in the food industry with oxidized products producing a bad smell and taste. Similarly, lipids in thin tissue sections cut from whole tissues are subject to degradation, and their degradation products can introduce IMS artifacts and the loss of normally occurring species to degradation can skew accuracy in IMS measures of abundance. Oxidized lipids are also known to be important mediators in the progression of several diseases and their accurate preservation is critical. As IMS studies become multi-institutional and collaborations lead to sample exchange, the need for validated protocols and measures of degradation are necessary. We observed the products of lipid degradation in tissue sections from multiple mouse organs and reported on the conditions promoting and inhibiting their presence as well as the timeline of degradation. Our key findings were the increase in oxidized phospholipids and lysophospholipids from degradation at ambient conditions, the decrease in the presence of lipids containing unsaturations on their fatty acyl chains, and the inhibition of degradation by matrix coating and cold storage of sections under N2 atmosphere. At ambient atmospheric and temperature, lipids degraded into oxidized phospholipids on the time-scale of a normal IMS experiment sample preparation (within 30 min). Lipids then degraded into lysophospholipids’ on a time scale on the order of several days. Validation of sample handling is especially important when a greater number of samples are to be analyzed either through a cohort of samples, or analysis of multiple sections from a single tissue as in serial 3D IMS. Atherosclerosis is disease caused by accumulation of cellular material at the arterial wall. The accumulation implanted in the cell wall grows and eventually occludes the blood vessel, or causes a stroke. Atherosclerosis is a 3D phenomenon and serial 3D IMS is useful for its ability to localize molecules throughout the length of a plaque and help to define the molecular mechanisms of plaque development and rupture. Serial 3D IMS has many challenges, many of which are simply a matter of producing 3D reconstructions and interpreting them in a timely fashion. In this aim and using analysis of lipids from atherosclerotic plaques from a human carotid and mouse aortic sinuses, we described 3D reconstruction methods using open-source software. Our methodology provides means to obtain high quality visualizations and demonstrates strategies for rapid interpretation of 3D IMS datasets through multivariate segmentation. Mouse aorta from model animals provided a springboard for developing the methods on lower risk samples with less variation with interesting molecular results. 3D MALDI IMS showed localized phospholipid accumulation in the mouse aortic sinuses with correlation between separate positive and negative ionization datasets. Silver-assisted LDI imaging presented differential localization of free fatty acids, cholesterol / cholesterol esters, and triglycerides. The human carotid’s 3D segmentation shows molecular histologies (spatial groupings of imaging pixels with similar spectral fingerprints) correlating to the degree of arterial stenosis. Our results outline the potential for 3D IMS in atherosclerotic research. Molecular histologies and their 3D spatial organization, obtained from the IMS techniques used herein, may predict high-risk features, and particularly identify areas of plaque that have higher-risk of rupture. These investigations would help further unravel the biological complexities of atherosclerosis, and predict clinical outcomes. Colorectal cancer liver metastasis (CRCLM) is the metastatic disease of primary colorectal cancer, one of the most common cancers worldwide. CRC is a cancer of the endothelial lining of the colon or rectum. CRC itself is often cured with surgery, while CRCLM is more deadly and treated with chemotherapy with more limited efficacy. Prognosticating and assessment of tumors is performed using classical histopathology with a margin of error. We have used lipid IMS to identify the histological compartments and extract their signatures. Using these IMS signatures we obtained a quantitative and objective histopathological score that correlates with prognosis. Additionally, by dissecting out the lipid signatures we have identified single lipid moieties that are unique to different histologies that could potentially be used as new biomarkers for assessing response to therapy. Particularly, we found a series of plasmalogen and sphingolipid species that differentiate infarct-like and usual necrosis, typical of chemotherapeutic response and normal tumor function, respectively.