by Keung Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-184). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xiv / List of Tables --- p.xvi / List of Figures --- p.xvi / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1. --- Events happened after brain injury --- p.1 / Chapter 1.2. --- Glial cells --- p.3 / Chapter 1.2.1. --- Microglia --- p.4 / Chapter 1.2.2. --- Oligodendrocytes --- p.5 / Chapter 1.2.3. --- Astrocytes --- p.5 / Chapter 1.2.3.1. --- Uptake of neurotransmitters --- p.7 / Chapter 1.2.3.2. --- Maintenance of extracellular homeostasis --- p.8 / Chapter 1.2.3.3. --- Induction of blood-brain-barrier --- p.8 / Chapter 1.2.3.4. --- Guidance of migrating neurons during development --- p.9 / Chapter 1.2.3.5. --- Immunocompetent cells of the brain --- p.9 / Chapter 1.2.3.6. --- Contribution to astrogliosis --- p.10 / Chapter 1.3. --- Cytokines and astrogliosis --- p.11 / Chapter 1.3.1. --- IL-6 and astrogliosis --- p.12 / Chapter 1.3.2. --- IL-1 and astrogliosis --- p.13 / Chapter 1.3.3. --- IFN-γ and astrogliosis --- p.14 / Chapter 1.3.4. --- TNF-α and astrogliosis --- p.14 / Chapter 1.3.4.1. --- General properties of TNF-α --- p.15 / Chapter 1.3.4.2. --- TNF receptors (TNFRs) --- p.17 / Chapter 1.3.4.3. --- NFkB induction --- p.18 / Chapter 1.3.4.4. --- Intermediate early genes --- p.19 / Chapter 1.3.4.5. --- iNOS is the target of NFkB and AP-1 --- p.20 / Chapter 1.4. --- β-Adrenergic receptors (P-ARs) --- p.21 / Chapter 1.4.1. --- β-ARs and astrogliosis --- p.22 / Chapter 1.4.2. --- General properties of β-ARs --- p.23 / Chapter 1.4.3. --- Interactions between β-adrenergic mechanism and TNF-α --- p.24 / Chapter 1.5. --- Aims and scopes of the project --- p.25 / Chapter CHAPTER 2. --- MATERIALS & METHODS / Chapter 2.1. --- Materials --- p.29 / Chapter 2.1.1. --- Rats for astrocyte culture --- p.29 / Chapter 2.1.2. --- Cell culture materials --- p.29 / Chapter 2.1.2.1. --- Complete Dulbecco's Modified Eagle Medium:F12 (DF12) --- p.29 / Chapter 2.1.2.2. --- Phosphate buffered saline (PBS) --- p.30 / Chapter 2.1.3. --- Drugs preparation --- p.30 / Chapter 2.1.3.1. --- Recombinant cytokines --- p.30 / Chapter 2.1.3.2. --- Modulators of protein kinase A (PKA) --- p.30 / Chapter 2.1.3.3. --- Modulators of protein kinase C (PKC) --- p.31 / Chapter 2.1.3.4. --- β-Agonists and -antagonists --- p.31 / Chapter 2.1.3.5. --- Antibodies used in western blot analysis --- p.31 / Chapter 2.1.4. --- Reagents for cell proliferation determination --- p.32 / Chapter 2.1.5. --- Reagents for RNA isolation --- p.32 / Chapter 2.1.6. --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.32 / Chapter 2.1.7. --- Reagents for Electrophoresis --- p.33 / Chapter 2.1.8. --- Reagents and buffers for western blotting --- p.35 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Preparation of primary astrocytes --- p.36 / Chapter 2.2.2. --- Preparation of cells for assays --- p.36 / Chapter 2.2.3. --- Determination of cell proliferation --- p.36 / Chapter 2.2.3.1. --- [3H]-Thymidine incorporation assay --- p.37 / Chapter 2.2.3.2. --- MTT assay --- p.37 / Chapter 2.2.3.3. --- Data analysis --- p.38 / Chapter 2.2.4. --- Determination of RNA expression by RT-PCR analysis --- p.38 / Chapter 2.2.4.1. --- RNA extraction --- p.38 / Chapter 2.2.4.2. --- Spectrophotometric Quantitation of DNA and RNA --- p.38 / Chapter 2.2.4.3. --- RNA gel electrophoresis --- p.39 / Chapter 2.2.4.4. --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.2.4.5. --- Separation of PCR products by agarose gel electrophoresis --- p.40 / Chapter 2.2.4.6. --- Quantification of band density --- p.41 / Chapter 2.2.5. --- Determination of protein expression by Western blotting --- p.41 / Chapter 2.2.5.1. --- Total protein extraction --- p.41 / Chapter 2.2.5.2. --- Western blotting analysis --- p.42 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1. --- Effects of pro-inflammatory cytokines on astrocyte proliferation --- p.43 / Chapter 3.1.1. --- Effects of TNF-α on astrocyte proliferation --- p.44 / Chapter 3.1.2. --- Effects of TNF-R1 and -R2 antibodies on astrocyte proliferation --- p.47 / Chapter 3.1.3. --- Effects of other cytokines on astrocyte proliferation --- p.50 / Chapter 3.1.4. --- Comparisons of the effects of cytokines on astrocyte proliferation --- p.53 / Chapter 3.2. --- Effects of β-agonist and -antagonist on astrocyte proliferation --- p.55 / Chapter 3.3. --- Effects of TNF-α on the expression of TNFR and endogenous TNF-α in astrocytes --- p.60 / Chapter 3.3.1. --- Effects of TNF-α on the expression of TNF-R1 and -R2 in astrocytes --- p.60 / Chapter 3.3.1.1. --- Effects of TNF-α on the expression of TNF-R1 and -R2 mRNA --- p.60 / Chapter 3.3.1.2. --- TNFR subtypes involved in the TNF-α-induced TNF-R2 mRNA expression --- p.62 / Chapter 3.3.1.3. --- Signaling pathways of the TNF-α-induced TNF-R2 mRNA expression --- p.67 / Chapter 3.3.1.4. --- Effects of TNF-α on the expression of TNF-R1 and -R2 --- p.68 / Chapter 3.3.2. --- Effects of TNF-α on the expression of endogenous TNF-α in astrocytes --- p.73 / Chapter 3.3.2.1. --- Effects of TNF-α on the expression of TNF-α mRNA --- p.73 / Chapter 3.3.2.2. --- TNFR subtypes involved in the TNF-α-induced TNF-α mRNA expression --- p.73 / Chapter 3.3.2.3. --- Signaling pathways of the TNF-α-induced TNF-α mRNA expression --- p.74 / Chapter 3.3.2.4. --- Effects of other cytokines on the expression of TNF-α mRNA --- p.75 / Chapter 3.4. --- Effects of TNF-α on the expression of β1- and β2-AR in astrocytes --- p.85 / Chapter 3.4.1. --- Effects of TNF-α on the expression of β1- and β2-AR mRNA --- p.85 / Chapter 3.4.2. --- TNFR subtypes involved in the TNF-a-induced β1 and β2-AR mRNA expressions --- p.88 / Chapter 3.4.3. --- Signaling pathways of the TNF-α -induced β1- and β2-AR mRNA expressions --- p.88 / Chapter 3.4.4. --- Effects of TNF-α on the expression of β1- and β2-AR protein --- p.100 / Chapter 3.4.5. --- Effects of other cytokines on the expression of β1- and β2-AR mRNA --- p.100 / Chapter 3.5. --- Interactions between TNF-α and β-adrenergic mechanism in astrocytes --- p.107 / Chapter 3.5.1. --- Effects of β-agonists and -antagonists on the TNF-α-induced endogenous TNF-α expression in astrocytes --- p.107 / Chapter 3.5.1.1. --- Effects of ISO and PROP on the expression of TNF-α mRNA --- p.107 / Chapter 3.5.1.2. --- β-AR subtypes involved in the TNF-α-induced TNF-α mRNA expression --- p.108 / Chapter 3.5.2. --- Effects of β-agonists and -antagonists on the TNF-α-induced TNFRs expression in astrocytes --- p.112 / Chapter 3.5.2.1. --- Effects of ISO and PROP on the expression of TNFRs mRNA --- p.112 / Chapter 3.5.2.2. --- β-AR subtypes involved in the TNF-α-induced TNF-R2 mRNA expression --- p.115 / Chapter 3.6. --- Effects of TNF-α on the expression of transcription factors in astrocytes --- p.117 / Chapter 3.6.1. --- "Effects of TNF-α on c-fos, c-jun and NFKB/p50 expression" --- p.118 / Chapter 3.6.2. --- Effects of other cytokines on the expression of NFKB/p50 mRNA --- p.119 / Chapter 3.6.3. --- "TNFR subtypes involved in the TNF-α-induced c-fos, c-jun and NFKB/p50 mRNA expression" --- p.125 / Chapter 3.7. --- Effects of TNF-α on the expression of iNOS in astrocytes --- p.130 / Chapter 3.7.1. --- Effects ofTNF-α the expression of iNOS mRNA --- p.130 / Chapter 3.7.2. --- TNFR subtypes involved in the TNF-α-induced iNOS mRNA expression --- p.131 / Chapter 3.7.3. --- Signaling pathways of the TNF-α-induced iNOS mRNA expression --- p.136 / Chapter 3.7.4. --- Effects of other cytokines on the expression of iNOS mRNA --- p.139 / Chapter 3.7.5. --- Effects of β-agonists and -antagonists on the TNF-α-induced iNOS expression --- p.142 / Chapter 3.7.5.1. --- Effects of ISO and PROP on the expression of iNOS mRNA --- p.142 / Chapter 3.7.5.2. --- β-AR subtypes involved in the TNF-α-induced iNOS mRNA expression --- p.143 / Chapter CHAPTER 4. --- DISCUSSIONS & CONCLUSIONS / Chapter 4.1. --- Effects of TNF-α on astrocyte proliferation --- p.148 / Chapter 4.2. --- Roles of endogenous TNF-α and TNFR in astrocyte proliferation --- p.150 / Chapter 4.3. --- Interactions between TNF-α and β-adrenergic mechanism in astrocytes --- p.154 / Chapter 4.4. --- Induction of transcription factors by TNF-α in astrocytes --- p.157 / Chapter 4.5. --- Possible source of β-agonists --- p.159 / Chapter 4.6. --- Conclusions --- p.160 / REFERENCE --- p.163

Sabe-se que a ativação do Sistema Nervoso Simpático (SNS) induz osteopenia via adrenoceptores b2 (b2-AR). Para investigar se o hormônio tireoideano (HT) interage com o SNS para regular a massa óssea, estudamos o efeito do HT em associação com isoproterenol ou propranolol (agonista e antagonista b-adrenérgicos) e avaliamos o efeito do HT em camundongos com elevado tônus simpático, devido à dupla inativação gênica do a2A-AR e a2C-AR (a2A/a2C-AR-/-), autorreceptores que inibem a liberação de noradrenalina. Vimos que esses animais apresentam um fenótipo de alta massa óssea, apesar do elevado tônus simpático e de intacta sinalização b2-adrenérgica, sugerindo que o a2A-AR e/ou a2C-AR, além do b2-AR, possam mediar ações do SNS no osso. O propranolol limitou e o isoproterenol acentuou os efeitos deletérios do HT no esqueleto, já os animais a2A/a2C-AR-/- apresentaram resistência à osteopenia induzida pela tireotoxicose, o que sugere que há interação entre SNS e o HT para regular a massa óssea, e que esta depende tanto do b2-AR como do a2A- e/ou a2C-AR. / It is known that the sympathetic nervous system (SNS) activation induces ostepenia, via b2-adrenoceptors (b2AR). To investigate if thyroid hormone (TH) interacts with the SNS to regulate bone mass, we studied the effect of TH in association with isoproterenol or propranolol (b-adrenergic agonist and antagonist) and evaluated the effect of TH in mice with a chronic elevated sympathetic tone, due to double disruption of a2A-AR and a2C-AR (a2a/a2c-AR-/-), autoreceptors that inhibit noradrenalin release. We showed that KO mice present a high bone mass phenotype in spite of an elevated sympathetic tone and of intact b2-adrenergic signaling, which suggests that a2A- and/or a2C-AR, besides b2-AR, may also mediate the SNS actions in the bone. Propranolol limited and isoproterenol accentuated the deleterious effects of TH in the skeleton, while a2A/a2C-AR-/- mice presented resistance to the T3-induced osteopenia, which suggest that there is an interaction between the SNS and TH to regulate bone mass, and that it is dependent on b2-AR and a2A-AR and/or a2C-AR signaling.

O intestino apresenta a maior área do corpo exposta ao ambiente, recebendo constantemente antígenos provenientes da alimentação e de microrganismos. A fim de manter a homeostase, evitando respostas inflamatórias desnecessárias e ao mesmo tempo respondendo apropriadamente à possíveis ameaças ao tecido, as células intestinas tem que ser capazes de perceber e responder apropriadamente a uma diversa gama de perturbações vindas do ambiente. Além do seu vasto sistema imune o intestino também abriga o maior número de neurônios fora do sistema nervoso central, os quais compõe o Sistema Nervoso Entérico (SNE). O SNE é composto por aproximadamente 100 milhões de neurônios que são capazes de regular autonomamente diversas funções fisiológicas do intestino e também de receber e enviar sinais para os sistemas nervoso autônomo simpático e parassimpático. Diversas evidências clínicas correlacionam inflamações intestinais com alterações no SNE, demonstrando a importância de se entender melhor as relações neuroimunes presentes no intestino. Os macrófagos são células essências da imunidade inata que residem tanto na área do intestino conhecida como lâmina própria como também na área conhecida como muscularis. Essas células fagocíticas desempenham um importante papel nas respostas antibacterianas e também na manutenção da homeostase do tecido, sendo capazes de adaptar rapidamente sua fisiologia em resposta aos sinais ambientais. Neste trabalho avaliamos a existência de uma comunicação bidirecional entre macrófagos e neurônios intestinais. Caracterizamos as duas populações de macrófagos presentes no intestino em homeostase e após um estimulo com SpiB um mutante de Salmonella, avaliando assim como estas células respondem à sinais de infecção provenientes do lúmen intestinal. Utilizando abordagens in vivo e in vitro, observamos que os macrófagos presentes na região da muscularis respondem rapidamente a uma possível infecção presente no lúmen regulando positivamente genes de proteção tecidual e reparo de danos, como Arg1 e Chi3l3. Nossos resultados indicam ainda que são os neurônios através da liberação de norepinefrina capaz de ativar os receptores β2 adrenérgicos presentes nos macrófagos intestinais que induzem a expressão dos genes relacionados com a proteção tecidual e reparo de danos. Observamos ainda que esta via induzida através da ativação dos receptores β2 adrenérgicos parece conferir também aos macrófagos um papel neuro-protetor em caso de danos teciduais. Em conjunto nossos resultados indicam a presença de uma comunicação neuroimune bidirecional entre os neurônios e macrófagos intestinais capaz de modular a resposta dos macrófagos a uma infecção entérica e de proteger os neurônios em caso de danos teciduais / The intestine is the largest area of the body exposed to the environment, which receives food and microbe antigens. In order to maintain homeostasis, avoiding unnecessary inflammation, and at the same time responding properly to potential treats to the tissue, intestinal cells must be able to sense and respond properly to this diverse set of environmental perturbations. In addition to a vast immune system, the intestine also harbors the largest collection of neurons outside the central nervous system, which constitute the enteric nervous system (ENS). The ENS is composed of approximately 100 million neurons that are capable of regulating the physiological functions of the gut autonomously and also receive input and send signals to the sympathetic and parasympathetic nervous system. Numerous clinical findings correlate gut inflammation with abnormalities in the ENS, revealing the importance of a better understanding of the neuro-immune interactions at this site. Macrophages comprise an essential innate immune cell residing both in the intestinal lamina propria and muscularis regions. These phagocytic cells play important roles in anti-microbial responses but also in tissue homeostasis, being able to quickly adapt their physiology in response to environmental cues. We evaluated the crosstalk between intestinal macrophages and surrounding enteric neurons, characterizing how these cells respond to possible infection in the intestinal lumen. Using in vivo and in vitro approaches, we found that macrophages in the intestinal muscularis quickly respond to a possible distal luminal infection, up regulating tissue-protective and wound repair genes, like Arg1 and Chi3l3. Also, our results indicate that the neurons trough norepinephrine release and subsequent activation of the β2 adrenergic receptor present on the intestinal macrophages are the ones up regulating tissue-protective and wound repair genes. We also observed that this pathway, trough the β2 adrenergic receptor activation seems to induce a neuro-protective role to these macrophages under tissue damage scenarios. All together our results indicate that a neuro-immune crosstalk between neurons and macrophages modulates macrophages response towards enteric infections and confers neuro-protection in case of tissue damage

O hormônio tireoidiano (HT) é essencial para o desenvolvimento, maturação e metabolismo ósseos, enquanto que o sistema nervoso simpático (SNS) é, também, um potente regulador do remodelamento ósseo. Demonstrou-se que SNS regula negativamente a massa óssea, agindo via receptores ?2-adrenérgicos (?2-AR), expressos em osteoblastos. O nosso grupo demonstrou que os receptores ?2 adrenérgicos (?2-AR) também medeiam ações do SNS no esqueleto e que são expressos em osteoblastos, osteócitos, condrócitos e osteoclastos. Considerando-se que o HT interage com o SNS para regular uma série de processos fisiológicos, e que o excesso de HT e a ativação do SNS causam perda de massa óssea, levantamos a hipótese de que há interação entre o HT com o SNS para regular a massa óssea. Estudos do nosso grupo vêm sustentando essa hipótese, uma vez que camundongos com inativação gênica dos receptores beta2-AR apresentam resistência à osteopenia induzida por doses tóxicas de HT. Considerando-se, ainda, que a interação do HT com o SNS em vários tecidos e/ou órgãos depende da sinalização beta2 adrenérgica, o presente estudo teve como objetivo avaliar se a interação do HT com o SNS para regular a morfofisiologia óssea envolve o beta2-AR. Para tanto, estudamos o efeito de 10x e 20x a dose fisiológica de triiodotironina (3,5ug ou 7.0ug de T3/100g de massa corporal/dia, respectivamente), por 90 dias, na microaquitetura óssea e em parâmetros biomecânicos do fêmur de camundongos com inativação gênica do beta2-AR (beta2-AR-/-), e nos seus respectivos Selvagens (Selv), os camundongos da linhagem FVB. Como esperado, o tratamento com T3 promoveu efeitos deletérios na microarquitetura trabecular das fêmeas Selv, enquanto alguns desses efeitos foram mais brandos ou inexistentes nos animais beta2-AR-/-, revelando resistência do osso trabecular dos animais knockout (KO) aos efeitos deletérios da tireotoxicose. Em contraste, a microarquitetura femoral dos camundongos machos beta2-AR-/- se mostrou mais sensível aos efeitos deletérios da tireotoxicose, em relação aos respectivos Selv. Quanto ao osso cortical femoral, vimos que o tratamento com T3 aumentou o perímetro endosteal e a área medular nos animais Selv machos e fêmeas, mas não nos animais beta2-AR-/-, o que sugere que o T3 promove reabsorção óssea endosteal no osso cortical, em um mecanismo que depende da via de sinalização do beta2-AR. Vimos, ainda, que o tratamento com T3 causou reduções significativas na carga máxima, tenacidade, rigidez e resiliência do fêmur dos camundongos fêmeas Selv. Em contraste, nenhum desses parâmetros biomecânicos foi afetado pelo tratamento com T3 no fêmur das fêmeas KO, evidenciando, mais uma vez, uma resistência desses animais aos efeitos deletérios da tireotoxicose no tecido ósseo. Por outro lado, os camundongos machos Selv e KO se mostraram resistentes aos efeitos deletérios do tratamento com T3 sobre os parâmetros biomecânicos do fêmur, sugerindo a participação de fatores sexuais na interação do HT com o SNS para regular a morfofisiologia óssea. Em conjunto, os achados do presente estudo corroboram a hipótese de que o HT interage com o SNS através da via dos receptores beta2 adrenérgicos para regular a morfofisiologia óssea, especialmente em fêmeas e no osso cortical / Thyroid hormone (TH) is essential for bone development, maturation and metabolism, while the sympathetic nervous system (SNS) is also a potent regulator of bone remodeling. SNS has been shown to negatively regulate bone mass, acting via beta2-adrenergic (beta2-AR) receptors expressed in osteoblasts. Our group demonstrated that alpha2-adrenergic (alpha2-AR) receptors also mediate SNS actions in the skeleton and are expressed in osteoblasts, osteocytes, chondrocytes and osteoclasts. Considering that TH interacts with the SNS to regulate a series of physiological processes, and that the excess of TH and the activation of the SNS cause loss of bone mass, we hypothesize that there is interaction between TH and the SNS to regulate the bone mass. Studies of our group have supported this hypothesis, since mice with gene inactivation of alpha2-AR present resistance to the osteopenia induced by toxic doses of TH. Considering that the TH-SNS interaction in various tissues and/or organs depends on beta2-adrenergic signaling, the present study aimed to evaluate whether the interaction of TH with the SNS to regulate the bone morphophysiology involves beta2- AR. Therefore, we studied the effect of 10x and 20x the physiological dose of triiodothyronine (3.5ug or 7.0ug of T3/100g body mass/day, respectively), for 90 days, in the bone microarchitecture and biomechanical parameters of the femur mice with beta2-AR gene inactivation (beta2-AR-/-), and of their respective Wild-type (WT) controls, the FVB lineage mice. As expected, T3 treatment promoted deleterious effects on the trabecular microarchitecture of the WT females, while some of these effects were milder or nonexistent in beta2-AR-/- animals, revealing trabecular bone resistance of knockout (KO) animals to the deleterious effects of thyrotoxicosis. In contrast, the femoral microarchitecture of the male beta2-AR-/- mice was more sensitive to the deleterious effects of thyrotoxicosis, in relation to the respective WT animals. Regarding to the femoral cortical bone, we saw that T3 treatment increased the endosteal perimeter and the medullary area both male and female WT animals, but not in the beta2-AR-/- mice, suggesting that T3 promotes endosteal bone resorption in the cortical bone, in a mechanism that depends on the alpha2-AR signaling pathway. We also found that treatment with T3 caused significant reductions in the maximum load, tenacity, stiffness and resilience of femurs of the WT female mice. In contrast, none of these biomechanical parameters was affected by T3 treatment in the KO females, demonstrating again resistance of these animals to the deleterious effects of thyrotoxicosis on bone tissue. On the other hand, WT and KO male mice were resistant to the deleterious effects of T3 treatment on the biomechanical parameters of the femur, suggesting the participation of sexual factors in the interaction of HT with the SNS to regulate bone morphophysiology. Taken together, the findings of the present study corroborate the hypothesis that TH interacts with the SNS through the beta2 adrenergic receptor pathway to regulate bone morphophysiology, especially in females and cortical bone

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

<p>Conclusion of this study: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.</p>

No description available.

Η στεφανιαία νόσος αποτελεί ένα από τα πιο συχνά αίτια νοσηρότητας και θνητότητας στο γενικό πληθυσμό. Ένα μεγάλο ποσοστό από τους ασθενείς με στεφανιαία νόσο οδηγείται σε αγγειοπλαστική στεφανιαίων αρτηριών (PTCA) με ή χωρίς εμφύτευση stent, όταν η στένωση στο αγγείο είναι ≥ 70-75%. Παρά την πρόοδο στον τομέα της αγγειοπλαστικής, με τη χρήση των drug-eluting stents και την ελάττωση της επαναστένωσης σε ποσοστό <5-10%, το υψηλό ποσοστό (20-25%) επαναστένωσης παραμένει η Αχίλλειος πτέρνα στα συμβατικά, μεταλλικά(bare)stents. Η χρήση των drug-eluting stents περιορίζεται σε περιπτώσεις με επαναστένωση, σε σακχαροδιαβητικούς και σε υψηλού κινδύνου βλάβες για επαναστένωση. Τα μεγάλα ποσοστά όψιμης επαναστένωσης(≥ 9-10%) και το υψηλό κόστος τους, κάνουν ακόμα πιο επιτακτική την ανάγκη εντατικοποίησης της έρευνας προς την κατεύθυνση εντοπισμού παραγόντων σχετιζόμενων με την επαναστένωση. Σκοπός της εργασίας μας ήταν να διερευνήσει τον πιθανό ρόλο πολυμορφισμών γονιδίων στην υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών και εμφύτευση μεταλλικών stents. Συγκεκριμένα, εξετάσθηκε αναλυτικά ο γενετικός πολυμορφισμός των γονιδίων α2Β-αδρενεργικού υποδοχέα και της CETP(πρωτεΐνη μεταφοράς εστέρων χοληστερόλης). Η υπόθεση στηρίχτηκε στο γεγονός ότι σε μια σημαντική μελέτη 912 αρρένων Φινλανδών μέσης ηλικίας, αποδείχτηκε ότι ο D/D γονότυπος σε σχέση με τον I/D γονότυπο και τον I/I γονότυπο, εμφανίζει 2,5 φορές μεγαλύτερο κίνδυνο για οξέα στεφανιαία επεισόδια, συμπεριλαμβανομένου του εμφράγματος μυοκαρδίου. Η εκσεσημασμένη αγγειοσύσπαση, τόσο στην περιφέρεια, όσο και στις στεφανιαίες αρτηρίες μέσω του πολυμορφισμού του γονιδίου α2Β που θεωρείται πιθανό αίτιο για τα οξέα στεφανιαία επεισόδια, μαζί με το σημαντικό ρόλο του α2Β αδρενεργικού υποδοχέα στην υπερπλασία και μετανάστευση των λείων μυϊκών κυττάρων, πιθανόν να έχει μεγάλη συμβολή στη διεργασία της υποτροπής ισχαιμίας. Μελετήσαμε προοπτικά 96 Έλληνες που υπέστησαν επιτυχή PTCA και εμφύτευση stents, εκ των οποίων 81 ήταν άνδρες και 15 γυναίκες(μέση ηλικία ± σταθερά απόκλιση=57,7± 10,1 ετών, με όρια 37-76 ετών) που προσήλθαν με συμπτωματική στεφανιαία νόσο. Όλοι οι παραπάνω ασθενείς συμμετείχαν στη μελέτη μεταξύ των ετών 2001 και 2003 και παρακολουθήθηκαν κλινικά για 6-8 μήνες μετά από μια επιτυχή τεχνική διάνοιξης του αποφραγμένου αγγείου. Αμέσως μετά την PTCA και για ένα(1) μήνα οι ασθενείς έλαβαν ασπιρίνη(100-325mg/day) και κλοπιδογρέλη 75mg/day. Η εκτίμηση της υποτροπής ισχαιμίας βασίστηκε σε στατικό και δυναμικό σπινθηρoγράφημα θαλλίου στους 3 και 6-8 μήνες μετά την PTCA. Αιμοδυναμικά, σε όσους υπέστησαν νέα στεφανιογραφία μέχρι και τους 6-8 μήνες, η επαναστένωση ορίσθηκε ως ≥ 50% στένωση του αυλού του αγγείου στο σημείο όπου έγινε η αγγειοπλαστική. Εκτός από την ομάδα των ασθενών και μια ομάδα υγιών μαρτύρων 83 ατόμων, συμμετείχε στη μελέτη για σύγκριση της συχνότητας του γονότυπου. Το τελικό καταληκτικό σημείο για την παραπάνω μελέτη, ήταν η συχνότητα της υποτροπής ισχαιμίας στους 8 μήνες κλινικής παρακολούθησης. Υποτροπή ισχαιμίας και της επαναστένωσης ≥ 50% σε όσους υπεβλήθησαν σε νέα στεφανιογραφία, συνέβη σε 15 από τους 96 ασθενείς. Ας σημειωθεί ότι οι περισσότεροι ασθενείς (70/96) είχαν το φυσιολογικό γονότυπο με το αλληλόμορφο I, λιγότεροι ασθενείς (23/96) είχαν το Insertion/Deletion και μόλις 3/96 είχαν το Deletion/ Deletion γονότυπο. Από το γονοτυπικό group, υποτροπή ισχαιμίας παρουσιάσθηκε σε 11/70 για τον I/I, 3/23 για τον I/D γονότυπο και 1/3 για τον D/D γονότυπο. Δε βρέθηκε συσχέτιση μεταξύ πολυμορφισμού γονιδίου και υποτροπής ισχαιμίας στους ασθενείς μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Προηγούμενες μελέτες έχουν ερευνήσει τη συσχέτιση των πολυμορφισμών των γονιδίων της ACE, του AT1 υποδοχέα της αγγειοτενσίνης II και της CETP με την επαναστένωση μετά από αγγειοπλαστική. Εντούτοις, καμιά μελέτη δεν πραγματοποιήθηκε που να συγκρίνει τον α2Β-AR πολυμορφισμό και την υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Όπως αρχικά αναφέρθηκε, ο γονότυπος α2Β ευνοεί τη μετανάστευση των αγγειακών SMCs, επηρεάζει τη λειτουργία του Α.Ν.Σ. και συσχετίζει το α2Β-AR αλληλόμορφο D deletion με οξέα στεφανιαία επεισόδια. Όλα τα παραπάνω στοιχεία μπορεί να δικαιολογούν το ρόλο του α2Β-AR πολυμορφισμού στην υποτροπή ισχαιμίας και πιθανόν την επαναστένωση μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Βέβαια, η αρνητική συσχέτιση των πολυμορφισμών του α2Β-AR και της CETP ΤaqIB με την υποτροπή ισχαιμίας μετά από αγγειοπλαστική, μπορεί να θεωρηθεί προκαταρκτική, δεδομένου ότι συμμετείχε σχετικά μικρός αριθμός ασθενών συγκριτικά με μεγάλες πληθυσμιακές μελέτες και επειδή ο D/D γονότυπος δεν είναι ιδιαίτερα συχνός(για τη μελέτη των α2Β-AR). Όσον αφορά τη μελέτη με τη CETP, διερευνήσαμε τον πολυμορφισμό ΤaqIB που είναι μια σιωπηρή μετάλλαξη βάσης στο 277 νουκλεοτίδιο της CETP(η οποία μπορεί να αναγνωρισθεί με την περιοριστική ενδονουκλεάση ΤaqI), με την πιθανότητα υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Οι όροι Β1 και Β2 αντίστοιχα χρησιμοποιήθηκαν για να δηλώσουν την ύπαρξη ή μη της περιοριστικής περιοχής (site) της ΤaqIB. Το Β2 αλληλόμορφο σχετίζεται με αυξημένα επίπεδα HDL και ελαττωμένα επίπεδα CETP, τόσο σε υγιείς όσο και σε άτομα με στεφανιαία νόσο(μοιάζει με ήπιας μορφής ανεπάρκεια CETP). Αντίθετα το Β1 αλληλόμορφο σχετίζεται με ελαττωμένα επίπεδα HDL και με αυξημένα επίπεδα και δραστηριότητα CETP. Επειδή η ΤaqIB σχετίζεται με χαμηλά επίπεδα HDL και αυξημένο κίνδυνο για CHD(επηρεάζοντας το μεταβολισμό των λιποπρωτεϊνών), μπορεί να συμμετέχει στην παθοφυσιολογία της υποτροπής ισχαιμίας μετά από αγγειοπλαστική. Μελετήσαμε 204 ασθενείς από το έτος 2001 έως και το 2003 με την προοπτική να διερευνηθεί η συσχέτιση ΤaqIB στον Ελλαδικό πληθυσμό με την πιθανότητα υποτροπής ισχαιμίας μετά από αγγειοπλαστική σε άτομα που φέρουν τον παραπάνω γονότυπο. Η συχνότητα της ΤaqIB(54%) ήταν παρόμοια με τη συχνότητα του πολυμορφισμού σε μια ομάδα 35 υγιών μαρτύρων. Το αποτέλεσμα από αυτή τη μελέτη δεν αποδεικνύει ότι ο ΤaqIB πολυμορφισμός στο γονίδιο της CETP είναι σημαντικός προγνωστικός παράγων για εκτίμηση του κινδύνου υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Συμπερασματικά, η υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών οφείλεται σε έναν πολύπλοκο μηχανισμό και σε ένα πολυπαραγοντικό φαινόμενο. Μπορεί οι πολυμορφισμοί του α2Β και της CETP, να μην αναδείχθηκαν ως ανεξάρτητοι παράγοντες υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών, αλλά σε συνδυασμό με άλλους πολυμορφισμούς γονιδίων και υπό την επίδραση συγκεκριμένων περιβαλλοντικών παραγόντων, είναι πολύ πιθανόν να συμμετέχουν στην παραπάνω διεργασία της υποτροπής ισχαιμίας και κατ’επέκταση της επαναστένωσης μετά από PTCA. / Coronary heart disease is one of the most common causes of morbidity and mortality in the population. A great percent of the patients with coronary heart disease may undergo percutaneous coronary angioplasty (PTCA) with or without the implantation of stents, mainly when the stenosis of the vessel is ≥ 70-75%. Despite the progress made with the introduction of drug-eluting stents and the reduction of the restenosis rate up to 5%-10%, the Achillean heel of the angioplasty using bare metal stents, is the restenosis rate which is 20%-25% of all cases. The use of drug-eluting stents is limited in cases with restenosis, in patients with diabetes mellitus and in high-risk for restenosis lesions. The great percent of late restenosis(≥ 9-10%) and the high price of drug-eluting stents, make more urgent the necessity for more intense research on the identification of the exact factors involved in the pathophysiology of restenosis. The primary objective of our study is to define the role of gene polymorphisms in the recurrence of ischaemia after PTCA and the implantation of the stents. Virtually, we scrutinely examined the role of the genetic polymorhisms of α2Badrenergic receptor and the CETP(Cholesteryl Ester Transfer Protein)-TaqIB polymorphism. Our assumption was based on the conclusion drawn by a study conducted in Finnish patients, which showed that D/D genotype confers 2,5 increase in the risk for acute coronary events(including acute myocardial infarction). The intense vasoconstriction properties of the α2Badrenergic polymorphism both on the coronary arteries and the periphery is considered to be the primary cause of the acute coronary events. The aforementioned statement with the significant role of α2B adrenergic receptor α2B-AR on the hyperplasia and mainly the migration of smooth muscle cells, probably correlates well with the pathophysiology of the recurrence of ischaemia after PTCA. We conducted a genetic association/prospective follow-up study in 96 Greek coronary artery disease patients undergoing coronary angioplasty and stent implantation. 81 patients were men and 15 women(mean age ± standard deviation=57,7± 10,1 years, ranges 37-76 years old) who presented with symptomatic CAD. All patients were enrolled in the study between 2001 and 2003 and were followed-up clinically for 6-8 months after an initially successful procedure. Post-angioplasty and for one (1) month following the procedure, all the patients received aspirin(100-325mg/day) and clopidogrel(75mg/day). Assessment of recurrence of ischaemia was based on positive thallium stress testing(at least moderate defect to the distribution of the culprit lesion of the vessel which was revascularised). Hemodynamically, restenosis was defined as ≥50% narrowing of the vessel at the point where angioplasty was performed. In addition to the patient group, a control group of totally 83 asymptomatic individuals were included in the study for comparison of the frequency of the genotype. The final end-point of the current study was the incidence of restenosis at 8 months of clinical follow-up. Recurrence of ischaemia (including restenosis rate ≥50% to the patients who underwent coronary angiography) occurred in 15 of the 96 patients. In note, the majotiy of patients (70/96) had the Insertion/Insertion genotype, fewer patients (23/96) had the Insertion/Deletion genotype and only 3/96 had the Deletion /Deletion genotype. With regard to to the genotype groups , restenosis was found in 11/70 for I/I, 3/23 for I/D and 1/3 for the D/D genotype. No association between gene polymorphisms and recurrence of ischaemia was detected to the patients who underwent coronary angioplasty. Previous studies have investigated the association between gene polymorhisms of angiotensin-converting enzyme(ACE), the AT1 receptor for angiotensin II and cholesteryl ester transfer protein (CETP) with restenosis in patients after coronary angioplasty. However, no study has been performed to involve the α2B-AR polymorphism with recurrence of ischaemia after percutaneous angioplasty of coronary vessels. As it was initially mentioned, α2B genotype promotes the migration of vascular SMCs, influences the function of autonomic nervous system and the α2B-AR deletion variant is associated with acute coronary events. All these data might correlate the role of α2B-AR polymorphism with the recurrence of ischaemia and probably with the restenosis after an angioplasty of coronary vessels. Nevertheless, the negative findings of our study might be considered preliminary, taking into account the small number of patients that were studied and the rarity of the Deletion/Deletion(D/D) genotype. As far as the CETP study, we investigated the TaqIB polymorphism, which is a silent base change affecting the 277th nucleotide and can be identified by the restriction endonuclease TaqI, with the chance of recurrence of ischaemia after PTCA. The terms B1 and B2 are used to denote the presence and absence, respectively, of the TaqI restriction site. The B2 allele has been associated with increased HDL levels and decreased CETP levels and activity in both patients with CHD and healthy subjects(resembling a mild form of CETP deficiency). On the other hand, the B1 allele has been associated with decreased HDL levels and increased CETP levels and activity. Due to the fact that TaqIB is associated with decreased HDL levels and increased risk for CHD, affecting the lipoprotein metabolism might be involved in the pathophysiology of reccurence of ischaemia after PTCA. We studied 204 patients between 2001 and 2003 with the primary objective to investigate the frequency of TaqIB and possible association with reccurence of ischaemia after PTCA in the patients who have this genotype. The frequency of TaqIB was 54% similar to the frequency of the polymorphism in a group of 35 healthy controls. The results from this study does not indicate that the TaqIB polymorphism at the CETP gene locus is a significant predictor for assessing the risk of reccurence of ischaemia after PTCA. As a conclusion, reccurence of ischaemia after PTCA is due to a complicated mechanism and to a multifactoral phenomenon. Virtually, we didn’t find any correlation of α2ΒAR polymorphism and CETP TaqIB with reccurence of ischaemia, especially as causitive factors, but based on their role in the pathophysiology, under certain circumstances, especially with the cooperation of other genes, these polymorphisms can not be definitely excluded in the reccurence of ischaemia and the restenosis after PTCA.

No description available.

<p>Conclusion of this study: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.</p>