Dissertations / Theses on the topic 'Atherosclerosis – Pathogenesis'

C-Myb is a DNA-binding transcription factor that functions in apoptosis, proliferation and differentiation. The role of c-Myb in vascular injury has been investigated in vitro and in vivo. Knock-down of c-Myb leads to a reduction in proliferation and an increase in apoptosis in vascular smooth muscle cells (VSMCs). Reduction of c-Myb activity has also been shown to reduce the incidence of neointimal formation in vivo by reducing VSMC proliferation. In contrast, over-expression of c-Myb in vivo leads to increased survival rates in certain cell types. Despite these observations, c-Myb expression has not yet been investigated in atherosclerosis. The balance between apoptosis and proliferation is pivotal in the pathogenesis of the disease. The aim of this thesis w as to investigate c-Myb expression in atherosclerotic lesions in addition to elucidating possible mechanisms by which c-Myb functions in atherosclerosis.

This thesis describes studies into the pathogenesis of Cholesterol—related early vascular injury. In addition, this thesis examines the potential reversibility of this injury with 2 agents: a) simvastatin, an HMGCoA reductase inhibitor, in a model independent of any reduction in lipids and 2) high dose aspirin. Hypercholesterolaemia led to a decrease in NO bioavailability, in association with a decrease in the enzyme, endothelial nitric oxide synthase and increased oxidative stress. In addition, there was an increase in the pro—inflammatory transcription factor, NF—KB, in the intima of the epicardial coronary arteries. Moreover, activated NF—KB was present in macrophages, foam cells and vascular smooth muscle cells in coronary atheromatous plaque and its expression increased in unstable coronary syndromes. These data support a role for NF—KB in the pathogenesis of early atherosclerosis and the development of unstable coronary syndromes. In addition, this thesis demonstrated for the first time that simvastatin, an HMGCoA reductase inhibitor, preserves endothelium—dependent vasorelaxation in both large and small coronary vessels in porcine experimental HC, despite no reduction in plasma lipids. This effect was associated with normalisation of eNOS protein levels. Furthermore, in vivo plasma markers of oxidative stress were attenuated by treatment with simvastatin. However, there was no attenuation in the activation of the proinflammatory transcription factor, NF—KB. These studies suggest a role for the HMGCoA reductase inhibitors in reducing cardiac morbidity and mortality, beyond their effect on cholesterol levels. The current studies also demonstrated that high dose aspirin therapy preserved endothelial function in large coronary vessels. This alteration in the generation of prostanoids in favour of vasodilatation may be an important component of the therapeutic benefit of aspirin in HC-induced atherosclerosis. In summary, results of studies described in this thesis provide insights into the molecular mechanisms that may be responsible for early vascular injury in hypercholesterolaemia and its reversibility with the therapeutic agents, simvastatin and high dose aspirin.

We comparatively analyzed the natural killer (NK) cell response against HCMV-infected pro-inflammatory (M1) and anti-inflammatory (M2) M[Fi] derived from autologous monocytes. M1 M[Fi] were more resistant to infection, secreting TNF-[alfa], IL-6, IL-12 and type I IFN. By contrast, in HCMV-infected M2 M[Fi] the production of proinflammatory cytokines, type I IFN and IL-10 was limited, and IL-12 undetectable. NK cell degranulation was triggered by interaction with HCMV-infected M1 and M2 M[Fi] and was partially inhibited by specific anti-NKp46, anti-DNAM-1 and anti-2B4 mAbs, thus supporting a dominant role of these activating receptors. By contrast, only HCMV-infected M1 M[Fi] efficiently promoted NK cell-mediated IFN-[gamma] secretion, an effect partially related to IL-12 production. These observations reveal differences in the NK cell response triggered by distinct HCMV-infected monocyte-derived cell types, which may be relevant in the pathogenesis of this viral infection. HCMV infection has been proposed to contribute to the development of atherosclerosis, a chronic inflammatory process in which M[Fi] play a key role. The contribution of HCMV to vascular disease may depend on features of the immune response not reflected by the detection of specific antibodies. Persistent HCMV infection in healthy blood donors has been associated with changes in the distribution of NK cell receptors (NKR). The putative relationship among HCMV infection, NKR distribution, subclinical atherosclerosis and coronary heart disease was assessed. An association of overt and subclinical atherosclerotic disease with LILRB1+ NK and T cells was observed, likely reflecting a relationship between the immune challenge by infections and cardiovascular disease risk, without attributing a dominant role for HCMV.
Hem analitzat la resposta de la cèl•lula NK als macròfags proinflamatoris (M1) i antiinflamatoris (M2) derivats de monòcits autòlegs infectats pel citomegalovirus humà (HCMV). Els macròfags M1 son més reistents a la infecció i secreten TNF-[alfa], IL-6, IL-12 i IFN de tipus I. Per altra banda, en els macròfags M2 infectats per HCMV la producció de citoquines proinflamatories, IFN de tipus I i IL-10 es limitada i la IL-12 indetectable. La cèl•lula NK degranula al interaccionar amb els macròfags M1 i M2 infectats. Aquesta degranulació s’inhibeix parcialment al bloquejar amb anticossos específics anti-NKp46, anti-DNAM-1 i anti-2B4, això indica que aquests receptors tenen un rol important en el procés. En canvi, només els macròfags M1 infectats amb HCMV promouen de manera eficient la producció d’IFN-[gamma] per part de la cèl•lula NK, degut parcialment a la producció de IL-12. Aquestes observacions posen de manifest diferències en la resposta de la cèl•lula NK a diferents tipus de macròfags infectats per HCMV que pot ser relevant en la patogènesis d’aquesta infecció viral. S’ha proposat que la infecció per HCMV contribueix al desenvolupament de l’aterosclerosis, un procés inflamatori crònic en el que els macròfags tenen un paper clau. La contribució del HCMV a la malaltia cardiovascular pot dependre de la resposta immune. La infecció per HCMV en donants de sang sans s’ha associat a canvis en la distribució dels receptors de les cèl•lules NK. S’ha evaluat la possible relació entre la infecció per HCMV, la distribució dels receptors de les cèl•lules NK i l’infart agut de miocardi. S’ha observat una associació de l’infart agut de miocardi i l’aterosclerosi subclínica tant amb les cèl•lules NK LILRB1+ com amb les cèl•lules T LILRB1+. Això possiblement reflexa la relació entre la pressió que les infeccions exerceixen en el sistema immunitari i el risc cardiovascular sense atribuir un paper principal al HCMV.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on May 16, 2008 ). Research advisor: Gerald I. Byrne, Ph.D. Document formatted into pages (xi, 114 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 65-107).

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Atherosclerosis is a common degenerative disease in which the clinical manifestations are often through stroke or myocardial infarction. Some of the established risk factors for atherosclerosis include elevated plasma low-density lipoprotein (LDL)-cholesterol levels, obesity, diabetes mellitus (DM) and cigarette smoking. Of the risk factors, an elevation in plasma LDL is one of the most established and the most researched. This is partly a consequence of the deposition of cholesterol within arterial intima being a crucial step in the progression of atherosclerosis, combined with the finding that LDL particles are a major transporter of cholesterol in circulation. Recently there is increasing evidence showing a role of the other major transporter of cholesterol in circulation, chylomicron remnants, in the progression of atherosclerosis. The notion of atherosclerosis as a postprandial phenomenon has been further substantiated by the emergence of evidence showing a direct role of chylomicron remnants in arterial cholesterol deposition. Based on evidence that chylomicron remnants are proatherogenic, the suggestion arises that accumulation of postprandial lipoproteins in plasma may add another dimension of risk to the development of coronary artery disease (CAD). This thesis tests the general hypothesis that individuals with or at high risk of CAD have postprandial dyslipidaemia and that this metabolic abnormality is correctable with a class of lipid-lowering drugs called statins. To test the hypothesis, clinical studies were conducted in normolipidaemic CAD patients, heterozygous familial hypercholesterolaemia (FH) and postmenopausal women with type 2 DM. Determination of postprandial dyslipidaemia by comparison with control populations were conducted initially in each patient group (Studies 1, 3 and 5), followed by intervention studies investigating possible modulation of the dyslipidaemia with a statin (Studies 2, 4 and 6). Six observation statements based on case-control comparisons of postprandial lipaemia in patients with or at risk of CAD and the effects of statins on postprandial dyslipidaemia in the patient groups were derived from the general hypothesis. The observation statements were examined in the individual studies described below. Postprandial lipoprotein metabolism was assessed using a number of methods. For comparison of postprandial lipaemia in Studies 1 and 2, a classic oral fat challenge was utilised. As markers of chylomicrons and chylomicron remnants, retinyl palmitate and triglyceride were measured postprandially as well as apolipoprotein (apo) B48 concentrations, a specific marker of intestinal lipoproteins. ApoB48 was also measured in the fasting state and found to predict the postprandial responses of retinyl palmitate, triglyceride and apoB48. This suggested that fasting measurement of apoB48 could be used as a simple indicator of postprandial dyslipidaemia. Consequently for Studies 3 - 6, fasting apoB48 measurements were used as primary markers of postprandial dyslipidaemia. Other markers for chylomicrons and their remnants utilised were fasting plasma concentrations of remnant-like particle-cholesterol (RLP-C) and apoC-III. As well as these static markers, chylomicron remnant catabolism was measured using a stable isotope breath test. The breath test involves the intravenous injection of a chylomicron remnant-like emulsion labelled with ¹³C-oleate and measurement of enriched ¹³CO2 in expired breath by isotope ratio mass spectrometry. The fractional catabolic rate (FCR) of the injected emulsion was subsequently calculated using multi-compartmental modeling (SAAM II). The studies are presented in this thesis as published and unpublished works. In Study 1, postprandial lipoprotein metabolism was compared between 18 normolipidaemic CAD patients (cholesterol 4.54 ± 0.12 mmol/L, triglyceride 1.09 ± 0.16) with 13 asymptomatic healthy controls using an oral fat challenge. Normolipidaemic CAD patients had higher postprandial area-under-curve (AUC) for triglyceride (+34%, p=0.019), retinyl palmitate (+74%, p=0.032) and apoB48 (+36%, p<0.001). Fasting apoB48 was also higher (+41%, p=0.001) and found to correlate significantly with AUC of triglyceride (p=0.017), retinyl palmitate (p=0.001) and apoB48 (p<0.001). The data suggest that normolipidaemic CAD patients have increased concentrations of intestinal lipoproteins in the fasting and postprandial state. In addition to these findings, significant correlations of fasting apoB48 with postprandial markers (p<0.02) suggests the fasting marker to be a simpler surrogate marker for the degree of total postprandial lipaemia. Study 2 investigated the effect of atorvastatin treatment on postprandial dyslipidaemia found in the 18 near-normolipidaemic CAD patients from Study 1. The trial was conducted in a randomised, placebo-controlled design, using oral fat challenges before and after 12-weeks atorvastatin/placebo treatment. Compared with the placebo group, atorvastatin decreased the total postprandial AUC for iii triglyceride (-22%, p=0.05) and apoB48 (-34%, p=0.013). Fasting markers of apoB48 (-35%, p=0.019) and RLP-C (-36%, p=0.032) also decreased significantly. Atorvastatin was also found to increase LDL-receptor activity by +218% (p<0.001) as reflected in binding studies. The data suggest atorvastatin reduces the fasting levels of intestinal lipoproteins as well as total postprandial lipaemia, but without acute dynamic changes in postprandial lipaemia. The reduction in fasting and total postprandial lipoprotein levels could be partly attributed to an increase in LDL-receptor mediated removal from circulation. In Study 3, postprandial lipaemia was compared in 15 heterozygous FH patients with 15 healthy controls. FH patients had higher fasting concentrations of apoB48 (+56%, p<0.001) and RLP-C (+48%, p=0.003). The elevation in these fasting markers of chylomicrons and their remnants suggests FH patients have postprandial dyslipidaemia due to an accumulation of these particles in plasma. Study 4 examined the effects of long- (> 6 months) and short-term (4 weeks) simvastatin treatment on modulating postprandial dyslipidaemia found in the 15 FH patients from Study 3. Short- and long-term simvastatin treatment decreased the fasting concentrations of apoB48 (-29% and 15% respectively, p<0.05) and RLP-C (both -38%, p<0.001), but did not significantly alter the FCR of the injected chylomicron remnant-like emulsion. The data suggest that in heterozygous FH both long- and short-term simvastatin treatments decrease the fasting markers of postprandial lipoproteins by mechanisms that may not be mediated via processes differentiated by the 13CO2 breath test. This implies that the effect on postprandial lipaemia may be from a decrease in production and/or a possible increase in catabolism of triglyceride-rich lipoproteins (TRLs). In Study 5, postprandial lipaemia was compared in 24 postmenopausal women age and body mass index matched with 14 postmenopausal women with type 2 DM. Postmenopausal diabetic women were found to have higher fasting concentrations of apoB48 (+21%, p=0.021) and apoC-III (+16%, p=0.042) as well as lower FCR of the chylomicron remnant-like emulsion (-50%, p<0.001). The data suggest that postmenopausal diabetic women have postprandial dyslipidaemia, and that this is due to delayed catabolism of chylomicron remnants. Study 6 was an hypothesis-generating exercise examining the effects of 4-weeks pravastatin treatment on postprandial dyslipidaemia found in 7 postmenopausal women with type 2 DM from Study 5. Although plasma LDL-cholesterol was reduced (-19%, p=0.028), there were no significant effects found on fasting apoB48 concentrations (-12%, p=0.116) or the FCR of the chylomicron remnant-like emulsion (+38%, p=0.345). A larger sample size of patients and/or treatment with a more potent statin at a dosage known to affect chylomicron remnant metabolism would be required to demonstrate a significant reduction in postprandial dyslipidaemia in postmenopausal women with type 2 DM. The results of the above mentioned studies combined support the general hypothesis that postprandial dyslipidaemia is a feature of patients with or at risk of CAD. This defect may be demonstrated using fasting apoB48 as an indicator of the degree of postprandial lipaemia. Postprandial dyslipidaemia may reflect a reduction in catabolism, as suggested with the breath test in type 2 DM, and/or an over overproduction of chylomicrons. Both these mechanisms would also increase competition for lipolysis and clearance pathways between hepatically and intestinally-derived lipoproteins. The exact mechanisms by which postprandial dyslipidaemia occurs are yet to be determined. Statins appear to improve defective postprandial lipaemia in patients with or at risk of CAD, which is in agreement with the general hypothesis. The effectiveness of a statin is dependant on their potency in inhibiting cholesterol biosynthesis and increasing receptor mediated clearance of LDL and chylomicron remnants. The studies conducted in this thesis show that postprandial dyslipidaemia can be reduced by statins but not to the extent demonstrated in controls. However, the demonstrated reduction in fasting and total postprandial lipaemia translates to a lowering in overall arterial exposure to circulating proatherogenic particles. The elevation in fasting and postprandial levels of proatherogenic chylomicron remnants found in the patient groups described in this thesis indicates another dimension to their risk of coronary disease. The reductions in the overall levels of proatherogenic particles in patients with or at high CAD risk, infers a possible reduction in the risk of coronary disease in these patients.

Human Immunodeficiency Virus Type I, or HIV, is one of the most well-known and well-researched viruses in the world. The current standard of care for HIV infected individuals is an antiretroviral drug therapy regiment, or ART, started immediately after diagnosis. While this treatment is generally quite effective at keeping the viral load low and stopping the progression from HIV infection to AIDS, patients receiving ART therapy still have a lower life expectancy than uninfected individuals. Many times, the cause of death in these patients is not the common opportunistic pathogens and cancers linked to HIV and AIDS, but chronic health conditions that develop. One of these conditions that is seen in many of the HIV infected patients undergoing the antiretroviral therapy is cardiovascular disease, such as atherosclerosis and myocardial infarction. Research shows that one of the key players in developing these conditions in HIV patients is the chronic inflammation caused by the immune system attempts to control the level of the virus. By studying the links between HIV, inflammation, and cardiovascular disease, we may be able to find solutions to the development of chronic disease in HIV patients on antiretroviral therapy.

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Die Pathogenese der Hypertonie und der Arteriosklerose sind multifaktorieller Prozesse, welche die Interaktion von genetischen und Umweltfaktoren involviert. Die molekularen Mechanismen an den Gefäßen sind ähnlich, jedoch nicht im Detail verstanden. Bei beiden Gefäßerkrankungen ist das Endothel der zentrale Angriffspunkt, der zu einer chronischen Entzündungsreaktion führt, ist. Der Transkriptionsfaktor NF-kappaB ist der Hauptschalter bei der Aktivierung der Gene, die zu der Aktivierung von Leukozyten und zu der Entwicklung der Entzündungsreaktion führen. Die hier aufgezeigten Untersuchungen befassen sich mit dieser Problematik und sind in 4 Teilprojekte gegliedert. Teilprojekt 1 befasst sich mit der physiologischen Funktion von Bcl-3, einem Mitglied der IkappaBalpha Familie. Da über die Funktion von Bcl-3 wenig bekannt ist, sollte mit Hilfe des Yeast-Two-Hybrid Systems neue Interaktionspartner identifiziert werden. Insgesamt würden fünf Proteine identifiziert, die alle binäre Komplexe in vitro (Co-Immunopräzipitation und GST-puldown) und im Yeast-Two-Hybrid bilden. Wir konnten zeigen, dass Bcl-3 mit den Ankyrin Repeats zur gleichen Zeit mit (mindestens) zwei Proteinen interagieren kann. Die endogene Aktivität von p50/Bcl-3 Komplexe wird durch die Transfektion der Interaktionspartner gesteigert. Den größten Effekt hat dabei Jab1. In transienten Transfektionen modulieren Jab1, Tip60, Bard1 und Pirin modulieren die Transkriptionsaktivität von Bcl-3 in verschiedenen Zellinien und mit verschiedenen Reporterkonstrukten. Den stärksten aktivierenden Effekt auf p50/Bcl-3 übt Tip60 aus. Tip60, Jab1 und Pirin können ebenfalls in vivo mit Bcl-3 co-immunoprezipitiert werden, wenn beide Proteine transient transfiziert werden. Auf Grund dieser Ergebnisse spekulieren wir, dass Bcl-3 eine Adapterfunktion ausübt, die NF-kappaB p50/p52 mit anderen Transkriptionsfaktoren (NF-1, c-jun, thyroid rezeptor, HIV-Tat) bzw. basalen Transkriptionsfaktoren (Histonacetylasen, RNA Polymerasen) verbindet. Teilprojekt 2 hat sich mit neuen Risikofaktoren für die koronare Herzerkrankung beschafft. Es gibt Hinweise, dass ein infektiöses Agens neben etablierten Risikofaktoren ursächlich an der Pathogenese und Progredienz der Arteriosklerose beteiligt ist. Unter den möglichen Erregern haben Untersuchungen der letzten Jahre gezeigt, dass Chlamydia pneumoniae das infektiöse Agens sein könnte. Infektion mit C. pneumoniae führte zu einer Aktivierung von pro-koagulatorischen Faktoren (Tissue Faktor und PAI-1) und Zytokinen (Interleukin 6). vermehrt expremiert werden. Wir konnten zudem eine vermehrte membranständige Expression von Rho-A und Rac-1 nachweisen, welche durch Statine gehemmt wurde. Zudem zeigte sich eine vermehrte Sauerstoffradikalfreisetzung nach Infektion mit C.pneumoniae. Die untersuchten Faktoren werden durch den Transkriptionsfaktor NF-kappaB reguliert, der ebenfalls nach Infektion induziert wurde. IkappaBalpha wurde degradiert. Alle Prozesse wurden durch Zugabe von Statinen reduziert. Wir zeigen einen möglichen molekularen Mechanismus auf, wie durch eine C. pneumoniae Infektion das lokale Milieu in der arteriosklerotischen Plaque derart verändert wird, dass Prozesse gefördert werden, die zu einer instabilen Koronarsituation führen können. Ziel des Teilprojekts 3 war es, neue molekulare Mechanismen bei der Pathogenese Angiotensin II-vermittelter Endorganschäden herauszufinden. Wir haben dazu ein transgenes Tiermodell (dTGR) untersucht, welches das humane Renin- und Angiotensinogen-Gen überexprimiert und 3 bis 5-fach erhöhte Ang II-Spiegel aufweist. Ang II stimulierte die Transkriptionsfaktoren NF-kappaB und AP-1, sowie von ihnen regulierte pro-entzündliche Proteine. Wir sind der Hypothese nachgegangen, ob Entzündungsreaktionen eine entscheidende Rolle bei der Pathogenese spielen könnten. Sowohl anti-entzündliche Inhibitoren, wie Aspirin in hoher Dosierung, die die Aktivierung von NF-kappaB verhindern, verringerten die Organschäden. Pleiotrope Eigenschaften des HMG-CoA-Reduktase-Hemmers Cerivastatin verminderten, cholesterinunabhängig, die Organschädigung. Auch hier ist der zu Grunde liegende Mechanismus eine Hemmung der Ang II induzeirten Entzündungsreaktion. Teilprojekt IV befasst sich mit der Präeklampsie, einer schwangerschaftsspezifischen Erkrankung, die durch das erstmalige Auftreten von arterieller Hypertonie und Proteinurie nach der 20. Schwangerschaftswoche charakterisiert ist. Die Präeklampsie ist unverändert eine der häufigsten Ursachen für mütterliche und kindliche Morbidität und Mortalität. Wir haben festgestellt, dass Schwangere mit Präeklampsie spezifische, agonistische Antikörper gegen den Angiontensin-II-AT1-Rezeptor (AT1-AA) entwickeln. Dieser Autoantikörper ist gegen die zweite extrazelluläre Schleife des AT1-Rezeptors gerichtet. Wir konnten zeigen, dass AT1-AA eine Aktivierung von NF-kappaB und AP-1 bewirken. Zudem konnten wir nachweisen, das vermehrt reaktive Sauerstoffmetabolite (ROS) induziert werden und dass dieser Prozess über die NADPH-Oxidase vermittelt ist. Auch konnten wir zeigen, dass die Expression der NADPH oxidase durch den AT1-AA induziert wird. Präeklamptische Plazenten weisen eine stärkere Produktion von ROS und eine vermehrte Expression der NADPH oxidase auf als gesunde Kontrollplazenten. Die Autoantikörper könnten einen wesentlichen endothelialen Schädigungsmechanismus bei Präeklampsie darstellen. Die konsekutive Aktivierung von NF-kappaB und AP-1 eine wichtige, mögliche Ursache, dass eine chronische vaskuläre Entzündungsreaktion iniitiert und unterhalten wird.
Atheroscleosis and hypertension are considered to be a complex process involving the interaction of genetic and environmental factors and the interplay between several cell types. Besides established risk factors for atherosclerosis and hypertension induced vascular pathology, chronic inflammation is regarded as a key event in the pathogenesis. The transcription factor family NF-kappaB is a key regulator of abroad rage of genes involved in inflammatory processes, proliferation and apoptosis. Aim of the investigations is to identify molecular mechanisms in vascular biology in order to understand pathophysiological vascular processes. The investigations are divided into 4 projects In project 1 we tried to identify the physiological role of the proto-oncoprotein Bcl-3, which is a member of the IkappaB family,. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase. Project 2 concentrated on investigating novel mechanism in arteriosclerosis. Recent reports link C. pneumoniae infection of arteriosclerotic lesions to the precipitation of acute coronary syndromes, which also feature tissue factor and plasminogen activator inhibitor 1 (PAI-1) overexpression. We investigated whether or not C. pneumoniae can induce thrombogenicity by upregulation of procoagulant proteins. Human vascular endothelial and smooth muscle cells were infected with a strain of C. pneumoniae isolated from an arteriosclerotic coronary artery. Tissue factor, PAI-1, and interleukin-6 expression was increased in infected cells. Western blotting We could show, that C.pneumoniae infection resulted in increased cell membrane-associated RhoA and Rac1, implying increased prenylation of these proteins. This effect was blocked by statin but circumvented by mevalonate. Cytochrome C assays showed that infected VSMCs produced increased reactive oxygen species that was blocked by statin. Infection increased nuclear transcription factor-kappaB expression in VSMCs that was dose-dependently suppressed by statin. Infected VSMCs produced and released RANTES and MCP-1. Statin dose-dependently blocked this production both at the mRNA and protein levels. These data provide evidence that C. pneumoniae infection can induce procoagulant protein and proinflammatory cytokine expression. This cellular response is accompanied by activation of NF-kappaB. Our results demonstrate how C. pneumoniae infection may initiate acute coronary syndromes. In project 3 we investigated molecular mechanism in Angiotensin II induced endorgan cardiovascular endorgan damage. Therefore we established a double transgenic rat (dTGR) model, in which rats transgenic for the humanangiotensinogen and renin genes are crossed. We show evidence that Ang II stimulates NF-kappaB and AP-1, which are involved with initiating chemokine and cytokine expression, leading to the above cascade in this model. Various pharmacological, such as the endothlin-1 receptor blocker bosentan, the immunosuppressant cyclosporin, the NF-kappaB inhibitor PDTC, the aldosteron antagonist spironolactone all ameliorated the ANG II-induced end-organ damage. However, all inhibitors were able to reduce NF-kappaB activation and inflammation. Stain also reduced cholesterol-independent the cardiovascular endorgan damage in dTGR. The pleiotrophic effects of statins reduced NF-kappaB and AP-1 activity, indicating that the local Angiotensin II induced inflammatory reaction is the key event in initiating the endorgan damage. Project 4 is focused on preeclampsia. Preeclampsia occurs after the 20th week of gestation and features hypertension and an increased peripheral vascular resistance; the mechanisms are unknown. The is a major cause of maternal and fetal morbidity We recently described autoantibodies (AT1-AA) directed at the AT1 receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). We proved AT1-AA specificity by coimmunoprecipitating the AT1 receptor with AT1-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT1-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT1-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect. AT1-AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. AT1-AA activated NF-kappaB. IkappaBalpha expression was reduced in response to AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-kappaB was activated in placentas from preeclamptic women. We conclude that AT1-AA and Ang II both stimulate the AT1 receptor and initiate a signaling cascade resulting in TF expression. NADPH oxidase is potentially an important source of ROS that may upregulate NF-kappaB in preeclampsia These results show an action of AT1-AA on human cells that could contribute to the pathogenesis of preeclampsia. We suggest that AT1-AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia.

Ziel dieser Dissertationsschrift war es, die Effekte des oxidativen Stresses in Form von oxLDL auf die Expression der atherogenen Scavenger-Rezeptoren CD36, SR-BI, des Transkriptionsfaktors PPARγ und pro-inflammatorischer Zytokine zu untersuchen. Die durchgeführten Untersuchungen beruhen auf der Annahme, dass modifizierte LDL durch Induktion der genannten Scavenger-Rezeptoren und nachfolgende unregulierte Aufnahme in Makrophagen mit Bildung von Schaumzellen entscheidend zur Entwicklung einer Atherosklerose beitragen. Dabei scheint vor allem die im Rahmen des oxidativen Stresses auftretende oxidative Modifizierung der LDL eine Rolle zu spielen. Oxidativem Stress wird in der Pathogenese vieler Krankheiten eine Bedeutung zugesprochen, deren pathobiochemische Grundlagen jedoch noch nicht vollständig geklärt sind und zu deren Erforschung diese Dissertation beitragen sollte. Im ersten Versuchsteil wurden deshalb in vitro – Versuche zu Expressionsraten oben genannter Rezeptoren durchgeführt. Dabei wurden Hypochlorit-oxidierte LDL genutzt, denen im Gegensatz zu den in vielen Versuchen verwendeten Kupfer-oxidierten LDL auch in vivo eine pathophysiologische Relevanz zukommt. Desweiteren ist bekannt, dass Diabetiker neben einem hohen Risiko an Atherosklerose zu erkranken, auch laborchemisch erhöhte Marker des oxidativen Stresses zeigen, inklusive einer gesteigerten Menge an zirkulierenden oxLDL. Im zweiten Versuchsteil wurde deshalb ex vivo untersucht, inwiefern LDL, gewonnen von Patienten im prädiabetischen Stadium der gestörten Glukosetoleranz, ähnliche Auswirkungen auf die Expression von Scavenger-Rezeptoren zeigen, wie in den mit Hypochlorit-oxidierten LDL durchgeführten in vitro - Untersuchungen beobachtet werden konnte. Es zeigte sich, dass CD36, der für die Aufnahme von oxidativ modifizierten LDL als der bedeutendste Rezeptor gilt, durch Hypochlorit-oxidierte LDL stark induziert wird. Zusammengenommen mit der ebenfalls beobachteten Heraufregulation des Transkriptionsfaktors PPARγ, sowie der signifikanten Korrelation beider Gene kann damit für Hypochlorit-oxidierte LDL eine atherogene Wirkung nachgewiesen werden. Die Ergebnisse bestätigen zudem die von Nagy und Tontonoz zuerst beschriebene Feed-Forward-Schleife, bei der oxidative LDL über Induktion und Aktivierung von PPARγ die Expression von CD36 erhöhen, mit nachfolgender unregulierter Aufnahme von Cholesterolestern in Makrophagen und subsequenter Bildung von Schaumzellen. Die Versuche zeigen zudem, dass der Scavenger-Rezeptor SR-BI, obwohl strukturell stark mit CD36 verwandt, eine differentielle Regulation aufweist und durch oxidativ modifizierte LDL signifikant supprimiert wird. Eine Induktion durch PPARγ, wie sie eindrucksvoll bei CD36 beobachtet werden kann, ist für SR-BI in den in vitro - Versuchen nicht vorhanden. Mit der in vielen Studien SR-BI, jedoch nicht CD36 zugesprochenen Funktion als Vermittler des HDL-Membranstransports gehen damit auch unterschiedliche Regulationsmechanismen einher. Die Ausschüttung pro-inflammatorischer Zytokine in murinen Makrophagen wurde durch Hypochlorit-oxidierte LDL im Vergleich mit nativen LDL signifikant vermindert, eine Assoziation mit dem Transkriptionsfaktor PPARγ konnte nicht gefunden werden. Die für die ex vivo - Versuche untersuchten Gruppen der insgesamt 40 Probanden mit gestörter und normaler Glukosetoleranz wiesen, mit Ausnahme eines pathologischen Glukosetoleranztests für IGT-Patienten, in der klinischen Untersuchung, beim Vergleich der Laborparameter, ebenso wie in der Zusammensetzung der LDL keine signifikanten Unterschiede auf. Dennoch fand sich eine signifikante Erhöhung der CD36-Expression in Makrophagen schon nach einstündiger Inkubation mit LDL aus IGT-Patienten im Vergleich zu LDL von Probanden mit normaler Glukosetoleranz. Für die Expressionsraten von SR-BI, PPARγ und die Zytokinausschüttung konnten nur marginale Unterschiede zwischen den Probandengruppen erfasst werden. Auch in den ex vivo – Untersuchungen wiesen eine insignifikante PPARγ - Erhöhung und eine positive Korrelation zwischen PPARγ und CD36 auf die Existenz und Aktivierung der beschriebenen atherogenen Feed-Forward-Schleife auch in IGT-Patienten hin. Zusammenfassend unterstreicht diese Dissertationsschrift die Bedeutung des oxidativen Stresses in der Entwicklung der Atherosklerose, indem erstmals auch für das pathophysiologisch relevante Hypochlorit-oxidierte LDL eine deutliche Expressionssteigerung des Scavenger-Rezeptors CD36 und des Transkriptionsfaktor PPARγ nachgewiesen werden konnte. Zudem zeigen die Experimente, dass bereits bei Patienten im Vorstadium des Diabetes mellitus Typ 2 funktionelle Veränderungen der LDL eingetreten sind, die zu einer verstärkten Atherosklerose führen können und die denen gleichen, die durch künstlich oxidierte LDL ausgelöst werden. Die Versuche liefern damit eine mögliche Erklärung für den bereits epidemiologisch nachgewiesenen Zusammenhang zwischen oxidativem Stress, Atherosklerose und Diabetes mellitus.

Hemodynamics (the patterns of blood flow along blood vessels), such as laminar shear stress (LSS) and oscillatory shear stress (OSS), plays an important role in maintaining vascular homeostasis and also the pathogenesis of atherosclerosis. LSS, occurring mainly in the straight part of the vascular tree, is protective against the formation of atherosclerotic plagues, while OSS is the predominant hemodynamic pattern experienced by the branched regions and curvatures in the vascular system, in which most of atherosclerotic plaques are developed. Recent evidence indicates that the Hippo pathway is important in transducing mechanical stimulation. However, the exact role of Hippo signaling in hemodynamics and the development of atherosclerosis is unclear. Therefore, the present study was designed to investigate how the Hippo pathway responds to hemodynamic stimulation and the impact of this pathway in the development of atherosclerosis.
The central axis of the Hippo pathway consists of a chain of kinase cascade including serine/threonine-protein kinase 1/2 (LATS1/2) and downstream YAP/TAZ effectors. Activation of the Hippo pathway leads to phosphorylation, nuclear exportation and inhibition of YAP/TAZ transcription factors. The present study is probably for the first time to demonstrate that the Hippo pathway can be activated in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) subjected to LSS. Western blotting results showed that Hippo activation was most likely mediated by phosphorylation of Hippo kinase YAP. The results from the nuclear fractioning assay showed that the amount of the nuclear fraction of YAP/TAZ was reduced in HUVECs subjected to LSS. Repression of the established YAP/TAZ target genes CTGF and CYR61, and inhibition of Hippo reporter gene after LSS treatment, further supported the notion that activation of the Hippo pathway inhibits the YAP/TAZ downstream genes expression. In vivo experiments showed an increased YAP phosphorylation in the straight area of C57 mouse thoracic aortas as compared with aortic arch, which favors that LSS activates Hippo pathway under the physiological condition. To further understand whether different flow patterns have different impacts on the Hippo pathway, the effects of OSS were also studied. As expected, OSS inhibited the Hippo pathway through inducing the de-phosphorylation of YAP, and promoting the nuclear localization of YAP/TAZ. The induction of YAP/TAZ target genes (CTGF and CYR61) further confirms that OSS-induced inhibition of the Hippo pathway enhanced the YAP and TAZ transcription activity.
Since unfavorable hemodynamics is among the most important factors in the development of atherosclerotic plaques, I hypothesized that inhibition of the Hippo pathway is involved in the formation of atherosclerotic plaques. Adhesion molecules are a group of cell surface proteins that mediate cell-cell interaction. During the initiation phase of atherosclerosis, adhesion molecules are highly expressed on the surface of endothelial cells to enhance the attachment of monocytes to the vascular wall, leading to monocyte accumulation within the vascular wall. OSS up-regulates the expression of several adhesion molecules; however, the underlying mechanisms remain elusive. To identify the role of Hippo pathway in adhesion molecules expression, the gene expression of HUVECs with YAP/TAZ knockdown was investigated using real-time PCR. The expression of the four adhesion molecule, ICAM1, E-Selectin, MCP1 and CXCL1 were down-regulated by YAP and TAZ gene silencing. To further study the mechanism, the promoter region of the four target genes were amplified from HUVECs genomic DNA, and subcloned into PGL3 reporter plasmids. The promoter activity was tested by co-transfection of the reporter plasmids with different dosages of constructively active YAP and TAZ. The present results showed that activated YAP and TAZ dose-dependently increased the promoter activity of these four genes. CXCL1 was selected for further examination because it has been recently reported to play an important role in oxidized LDL-induced elevation of monocyte attachment to endothelial cells. Analysis of the CXCL1 promoter revealed that two TEAD1 binding sites located within the promoter region of CXCL1. The reporter gene assay results showed that TEAD1 induced the CXCL1 reporter activity, indicating that the CXCL1 promoter was regulated by YAP/TAZ/TEAD complex. The EMSA analysis further demonstrates that constitutively active YAP and TAZ induced the binding between TEAD1 and the CXCL1 promoter, suggesting a direct association between TEAD1 and CXCL1 promoter.
To further explore whether the Hippo pathway activation could regress atherosclerosis development, TAZ knockdown adenovirus was generated and administered to ApoE⁻/⁻ mice fed on Western diet. The formation and sizes of atherosclerotic plaques were visualized using oil red O staining. Monocyte infiltration was estimated using monocyte marker CD68. The expression of CXCL1 was detected using immunohistochemistry. The present results demonstrated that TAZ gene silencing significantly suppressed the expression of CXCL1 and reduced the sizes of atherosclerosis plagues as well as monocyte infiltration in the vascular wall of ApoE⁻/⁻ mice.
To further identify whether Hippo pathway could be a drug target for the treatment of atherosclerosis, several known atherosclerosis risk factors and beneficial factors were tested for their effects on Hippo pathway. Four out of nine atheroprotective factors were found to suppress the TEAD reporter activity. On the other hand, four out of six risk factors were identified to increase the TEAD reporter activity. The present results suggest that atheroprotective LSS activates while atherogenic OSS inhibits the Hippo pathway.
The Hippo pathway could be activated by atheroprotective LSS, while it was inhibited by atherogenic OSS. Activation of the Hippo pathway inhibits the development of atherosclerosis at least in part through reducing the expression of adhesion molecules and decreasing macrophage accumulation in the vascular wall. The present new findings suggest that targeting this Hippo pathway is potentially useful for therapeutic intervention of atherosclerosis and associated vascular diseases.
血流動力學（即血液在心血管系統中流動的模式），包括層流剪切應力（LSS）和振盪剪切應力（OSS）。血流動力學在血管穩態和動脈粥樣硬化的發展中起重要作用。LSS主要發生在血管系統的直線部分，可防止動脈粥樣硬化斑塊形成，而OSS是血管系統中分支和彎曲部分的主要血流模式，大部份粥樣硬化斑塊發生在此。最近的研究表明，Hippo通路在機械刺激傳導中起重要作用。然而，Hippo信號通路在血流動力學和動脈粥樣硬化發展中的確切作用目前尚不清楚。因此，本論文的目的是研究Hippo通路如何響應血流動力學刺激以及Hippo通路對動脈粥樣硬化的發展的影響。
Hippo通路主要由一系列激酶鏈和下游YAP/ TAZ效應器組成，其中的激酶鏈包括絲氨酸/蘇氨酸－蛋白激酶1/2（LATS1/ 2）。激活Hippo通路可導致YAP和TAZ磷酸化，促進其出核運輸並抑制其轉錄激活的功能。本研究中，我們首次證明了在LSS條件下，人臍靜脈內皮細胞（HUVEC）和人主動脈內皮細胞（HAECs）中的Hippo通路被激活。Western雜交結果表明，Hippo通路的激活很可能是受YAP激酶的磷酸化調節。細胞核質分離結果顯示在LSS條件下，人臍靜脈內皮細胞中YAP/ TAZ的細胞核部分的量顯著減少。LSS处理后，YAP/ TAZ靶基因CTGF和CYR61的表達降低，其TEAD告基因受到抑制，这進一步證明激活Hippo通路可抑制YAP/ TAZ下游基因的表達。動物實驗結果顯示YAP磷酸化和YAP/ TAZ出核在C57小鼠的胸主動脈直的區域顯著高於彎的區域，這也表明在生理條件下，LSS可激活Hippo通路。為了進一步了解不同的血流模式是否對Hippo通路產生不同的影響，我們研究了OSS條件下的Hippo通路。正如所期，OSS通過誘導YAP的去磷酸化，促進YAP/ TAZ的核內分佈，從而抑制Hippo通路的活性。OSS誘導YAP/ TAZ靶基因（CTGF和CYR61）產生轉錄活性，證明了OSS可抑制Hippo通路。
不利的血流模式是動脈粥樣硬化斑塊產生的最重要因素之一，因此我們推測抑制Hippo通路或許可導致動脈粥樣硬化斑塊的形成。粘附分子是一類調節細胞間相互作用的細胞表面蛋白。在動脈粥樣硬化形成的初始階段，粘附分子會在內皮細胞的表面高表達，從而提高單核細胞附著和聚集於血管壁上。OSS上調粘附分子的表達，但機制仍不清楚。為了識別Hippo通路在粘附分子表達中的作用，本文用實時定量PCR檢測了YAP/ TAZ敲低了的人臍靜脈內皮細胞的基因表達水平。實驗結果顯示四種粘附基因，包括ICAM1，E-Selectin，MCP1和CXCL1通過YAP和TAZ的基因沉默而被下調。為了進一步研究此中機制，本文擴增了臍靜脈內皮細胞的基因組DNA中的四個靶基因啟動子區，並將其克隆到PGL3報告質粒。通過共轉染不同濃度的組成型激活的YAP和TAZ，本文測定了這四個啟動子的活性。結果顯示激活的YAP和TAZ能夠劑量依賴性上調這四個基因的啟動子活性。由於最近有報導說氧化低密度脂蛋白可誘導單核細胞附著於內皮細胞，而CXCL1在此誘導過程中具有重要作用，因此本文選擇CXCL1作進一步研究。通過分析CXCL1的啟動子，本文發現CXCL1的啟動子區域內有兩個TEAD1結合位點。報告基因檢測結果顯示TEAD1可誘導CXCL1報告活性，這表明CXCL1啟動子是通過YAP/ TAZ/ TEAD複合體來調節。EMSA分析進一步揭示了組成型激活的YAP和TAZ可誘導TEAD1和CXCL1啟動子之間的結合，表明TEAD1直接結合到CXCL1啟動子。
為進一步開拓Hippo通路是否是動脈粥樣硬化消退潛在的治療靶點，本文構建了TAZ敲低的腺病毒並將其轉入餵以西方飲食的ApoE⁻/⁻小鼠。通過油紅O染色來觀察動脈粥樣硬化斑塊的形態和大小。用單核細胞表面標記物CD68測定單核細胞對血管的浸潤。用免疫組化鑑定CXCL1的表達。結果表明TAZ基因沉默顯著抑制ApoE⁻/⁻小鼠血管壁CXCL1的表達，降低動脈粥樣硬化斑塊的大小及單核細胞浸潤。
為了進一步確定Hippo途徑是動脈粥樣硬化的藥物治療靶點，本文對幾個已知的動脈粥樣硬化的危險因子和有利因子對Hippo通路活性的影響進行了研究。出人意料的是，九個動脈粥樣硬化的保護因子中，有四個被確定為抑制TEAD報告基因活性。另一方面，六個危險因子中，有四個被鑑定為可提高TEAD報告活性。這些結果與上面的研究發現共同證明了LSS降低動脈粥樣硬化，激活Hippo通路，而OSS促進動脈粥樣硬化，抑制Hippo通路。
本研究確定了層流剪應力（LSS）激活Hippo通路，抑制動脈粥樣硬化；振盪剪切應力（OSS）抑制Hippo通路，導致動脈粥樣硬化。Hippo通路被激活能夠抑制動脈粥樣硬化的機制是通過降低粘附分子的表達，減少巨噬細胞在血管壁上的附著。這一新發現表明，靶向Hippo通路對介入治療動脈粥樣硬化以及相關的血管疾病具有潛在的應用意義。
Wang, Li .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 129-138).
Abstracts also in Chinese.
Title from PDF title page (viewed on 08, November, 2016).
Wang, Li.
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.

碩士
國立臺灣大學
免疫學研究所
84
Atherosclerosis is a chronic and multifactorial process commen- cing during childhood and becoming clinically manifest later in life. The atherosclerotic lrsion is characterized by intima thi- ckening,lipid accumulation,and increase in connective disease, and disease of the aorta and peripheral arterial circulation. Nevertheless,the pathogenesis of atherosclerosis is still not yet fully understood.Serveral lines of evidence have suggest that both humoral and cellular immunity are involved in athor- genesis of atherosclerosis,the present study was divided into two parts:In part I,study was conducted using fresh samples of human aortic aneurysm obtained from patients during surgery.His- tological examination confirmed the presence of advanced athero- sclerosis associated with variable aortic adventitial chronic inflammatory cell infiltrates.Indirect immunofluorescent stain- ing was performed to identify the CD3 T lymphocytes present in aortic media and adventitia.Using highly sensitive transcription -polymerase chain reaction (RT-PCR) amplification method,the T cell receptor(TCR)V.beta.gene expression in the adventitial in- filtrates of aortic aneurysm was shown to be polyclonal,and no compartmentalized TCR V.beta.gene usage was observed in the in- filtrates.In addition,the preferential utilization of any TCR V .BETA.gene expression in the blood of aneurysmal patients was also not observed compared with that in normal individuals.In part II,the apoE-knockout mice was used as a murine model of atherosclerosis for the study.It was shown that different sta- ges of atherosclerotic lesions were found throughout aorta,and autoantibodies against oxidized LDL were present in circulation of the animals.By RT-PCR analysis,both mRNAs encoding IFN-.gamma .and IL-12p40 were detected in aorta with atherosclerosis.Fur- thermore,the expression of IL-12p40 mRNA was localized within foam cells in atherosclerotic lesions by in situ hybridization analysis.

博士
國立臺灣大學
藥理學研究所
95
Atherosclerosis and pressure-overload cardiac hypertrophy are common diseases in cardiovascular medicine. A plethora of gene expressions and complicated signaling pathways are involved in the pathogenesis. In this study, we tried to identify the major receptor that was involved in the early stage of atherosclerosis and cardiac hypertrophy. We used siRNA for investigating the role of these receptors and downstream signaling pathways. We also experimentally attenuated the receptors up-regulation by specific siRNA, statins or antagonist. In the first part of the study, we investigated the relationship between C-reactive protein (CRP) and atherosclerosis and the exact receptor involved in CRP-induced endothelial changes. Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were used for the experiments. After incubation with CRP, immunobltting showed a significant NF-κB activation and vascular cell adhesion molecule-1 (VCAM-1) expression. The mRNA level of CD32, the major binding protein for CRP in HUVEC, increased significantly as measured by Northern blot. When these HUVEC were transfected with siRNA directed against CD32 (siCD32), the mRNA of CD32 decreased significantly. The IκB degradation, NF-κB nuclear translocation and VCAM-1 upregulation induced by CRP were all blocked by treatment with siRNA against CD32. SB203580, a P38 inhibitor, significantly attenuated the CRP induced responses while SP600125 (c-jun kinase inhibitor) did not. CRP induced IκB degradation, NF-κB nuclear translocation and VCAM-1 protein expression in HUVEC and HAEC. CRP also increased CD32 expression in HUVEC and HAEC. All the above changed were dependent on CD 32, which might serve as the receptor for CRP and mediated the effects of CRP on HUVEC and HAEC. In the second part of the study, we compared the effects of simvastatin and siRNA against CD32 in CRP-induced pro-inflammatory changes in endothelial cells. We tested the hypothesis that simvastatin inhibited CRP-induced pro-inflammatory changes in endothelial cells by decreasing mevalonate pathway products. HUVEC were incubated with CRP and measurement of CD32, NF-κB activation, VCAM-1 expression and monocyte adhesion were performed. The effects of simvastatin, siCD32 and mevalonate pathway products were also examined. After incubation with CRP, there was a significant increase of CD32 in HUVEC. IB degradation and NF-κB nuclear translocation were also observed. Pre-treatment with simvastatin significantly attenuated the CRP-induced CD32 expression and NF-κB activation. Simvastatin also decreased CRP-induced VCAM-1 expression and reduced monocyte adhesion on endothelial cells. The inhibitory effects of simvastatin were significantly reversed by adding mevalonate, geranylgeranyl pyrophosphate but not by adding farnesyl pyrophosphate. Pre-treatment with siCD32 also decreased CRP induced CD32 expression and IκB degradation. However, neither mevalonate nor geranylgeranyl pyrophosphate reversed the effects of siCD32. CRP induced CD32 expression, IκB degradation, NF-κB nuclear translocation and VCAM-1 protein expression in HUVEC. All the above changes were attenuated by simvastatin. A decrease of mevalonate and subsequent geranylgeranyl pyrophosphate contributes to the inhibitory effects of simvastatin. In the third part of the study, we identified the role of serotonin 2B receptors (SR2BR) in the pathogenesis of cardiac hypertrophy in vivo and in vitro. Recent data suggested that SR2BR might be involved in some cardiac physiopathological situations. Wistar rats of aortic banding and neonatal cardiomyocyte with mechanical stretch were used for cardiomyopathy models. After two weeks of aortic banding surgery, serum serotonin, mRNA and protein expression of SR2BR increased significantly. Selective SR2BR antagonist, SB215505 (SB), significantly reduced the increase in heart weight, the ratio of heart weight and body weight, interventricular septum thickness, left ventricular posterior wall thickness, left ventricular mass and brain natriuretic peptide (BNP) protein but did not significantly attenuate the up-regulation of SR2BR protein expression in rats after aortic banding for three weeks. Down-regulation of nerve growth factor-β in aortic banding rat was also reversed by SB. When cultured cardiomyocytes were subjected to mechanical stretch and serotonin 1 μM, the level of SR2BR and BNP protein increased time-dependently. When transfected with specific siRNA against SR2BR or pretreated with caffeic acid phenethyl ester, the increase of nuclear factor-κB (NF-κB) translocation and BNP protein in cardiomyocytes were both reversed. These results suggested that SR2BR expression was involved in excess of pressure-induced cardiomyopathy and its downstream signaling may through NF-κB to modulate BNP expression in cardiomyocyte. In conclusion, our study confirmed that CD32 mediate the CRP-induced inflammation in endothelial cells. Treatment with siRNA against CD32 and simvastatin both reduced the up-regulation of CD32 and the effects of CRP. On the other hand, SR2BR expression on cardiomyocyte played an important role in pressure-induced cardiac hypertrophy. Therefore, CD32 and SR2BR might serve as therapeutic targets for atherosclerosis and cardiac hypertrophy in the nearly future.

博士
國立成功大學
基礎醫學研究所
105
Atherosclerosis is an inflammatory disease driven by hyperlipidemia. There are accumulating evidences to support that inflammation and lipid homeostasis are closely linked. Macrophages mediate innate immune responses and lipid homeostasis and act as a key player in atherosclerosis. However, the cross talk among these processes in the development and progression of atherosclerosis are not fully defined. For the past few years, our recent studies successfully demonstrated the important role of the transcription factor CCAAT/enhancer-binding protein delta (CEBPD) in inflammation and cancer microenvironment over macrophages. In this current study, we aimed to dissect whether CEBPD functions at the junction of inflammation and macrophage lipid homeostasis. We found that CEBPD colocalized with macrophages in human and mouse atherosclerotic plaques and that Cebpd deficiency in bone marrow cells suppressed atherosclerotic lesions in hyperlipidemic Apoe-/- mice. In response to modified LDL, the p38MAPK/CREB pathway contributed to CEBPD activation which promoted lipid accumulation in M1 macrophages but not in M2 macrophages. The underlying mechanisms involved in this process included an increase in pentraxin 3 (PTX3)-mediated macropinocytosis of LDL and a reduction in ATP-binding cassette subfamily A member 1 (ABCA1)-mediated cholesterol efflux. Also, we found that ZNF202 mediates CEBPD-repressed ABCA1 gene transcription. In addition, we found that simvastatin (a HMG-CoA reductase inhibitor) can target CEBPD to block lipid accumulation in M1 macrophages. In conclusion, this study underscores the importance of cross talk between inflammation and lipid homeostasis and provides new insight into targeting macrophage phenotypes and functions in cardiovascular diseases.