Chronic inflammation is a complex immune response linked to several diseases. The first step in the inflammatory response is the recruitment of immune cells to the endothelium of the vascular wall. This process is mediated by E/P-Selectin, for which no antagonist efficiently interacts to limit the inflammatory response. Previous work identified transcription factor GATA5 as a key regulator of endothelial homeostasis and revealed an altered expression of inflammatory genes in human endothelial cells with loss of GATA5. The objective of my project is to understand the role of GATA5 in selectin-dependent vascular inflammation and to develop selectin inhibitors. I used biochemical, cellular and in vivo approaches to evaluate a series of novel small molecules for their ability to interfere with selectin binding to their ligand, PSGL-1. The work identified a new lead candidate, LCB 2248, for the development of new E/P-Selectin antagonists and contributed to the understanding of the role of GATA5 in cell recruitment and adhesion. The mechanistic insight gathered and the identification of an E/P-Selectin antagonist will hopefully pave the way for the development of effective treatments for patients with chronic inflammation.

Atherosclerosis, considered a chronic inflammatory disease, is the underlying mechanism for several cardiovascular diseases. Hyperlipidemia is the number one risk factor for atherogenesis. Caspase-1 is an inflammatory caspase, which can be activated by the metabolic stresses through pathogen associated molecular patterns (PAMPs)-recognition receptors, (PRR) recognition and inflammasome assembly. Activated caspase-1 can initiate inflammation in multiple ways. Thus, regulating inflammasome components expression is essential to control caspase-1 activation and its subsequent inflammatory processes. I hypothesized that the readiness of inflammasome component expression for caspase-1 activation in tissues is an index for inflammation privilege. Endothelial cells (EC) which are the innermost layer of the vessel and are the critical gatekeeper for monocyte migration. The first step of atherogenesis is activation of ECs, which allows monocyte adhesion and migration into the sub-endothelial layer. I also hypothesized that caspase-1 can sense hyperlipidemia and regulate EC activation and inflammation during early atherogenesis. I first determined the expression profiles of inflammasome components, pro-inflammatory caspases and PRRs is different among tissues, and cardiovascular tissues express relative less PRRs via a database-mining method. According to the readiness of inflammasome components, tissues could be classified into three tiers. The first tier consists of tissues with constitutively expressed inflammasomes. The second tier of tissues includes potentially inducible expression of one inflammasome component. The third tier of tissues has inducible expression of at least two inflammasome components. This three-tier model can be applied to determine the inflammation privilege of tissues in response to pro-inflammatory stimuli. I also demonstrated that hyperlipidemia induced caspase-1 expression and activation in aorta along with the atherogenesis in apolipoprotein E (ApoE)-/- mice with high fat (HF) diet, experimentally. We then generated the ApoE-/-/Casp-1-/- double knockout mice, and found that the ApoE-/-/Casp-1-/- mice contained significantly less atherosclerotic lesion in aortic sinus and less cytokine and chemokine expression in aortic tissues compared with ApoE-/- mice. ApoE-/-/Casp-1-/- mice also had less CD11b+/F4/80- neutrophil and CD11b+/F4/80+ monocyte recruitments into aorta compared with ApoE-/- mice. However, the percentage of monocyte subsets in peryphery blood remained at the same level in between ApoE-/- mice and ApoE-/-/Casp-1-/- mice. I then proposed that perhaps the caspase-1 activation in vascular cells, in ECs played the essential role of controling monocyte migraion. My in vitro data demonstrated that oxidized low density lipoprotein (ox-LDL) and its componnents could induced caspase-1 activation in human aortic ECs (HAECs) through ROS pathway which then led to EC activation and pyroptotic cell death. Deficiency of caspase-1 in aortic EC attenuated hyperlipidemia induced EC activation and inflammtion. Mechanically, I found that caspase-1 deficiency accumulated an anti-atherogenic protein, Sirt-1 in the aorta. Collectively, our data suggested that caspase-1/inflammasome in ECs can sense hyperlipidemia, become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis. / Pharmacology

No / Vascular inflammation is implicated in the pathogenesis of atherosclerosis and abdominal aortic aneurysm (AAA), and is thought to involve reactive species such as the nitric oxide-derived oxidant peroxynitrite. In the present study nitrotyrosine was measured as a stable marker of peroxynitrite production in vivo. Perioperative blood samples were obtained from patients undergoing elective open or endovascular repair of an AAA and from patients with intermittent claudication, smoking aged-matched controls, non-smoking aged-matched controls and non-smoking young healthy controls. Plasma nitrotyrosine was measured by an enzyme-linked immunosorbent assay. The median plasma nitrotyrosine concentration in patients with an AAA (0·46 nmol nitrated bovine serum albumin equivalents per mg protein) was significantly higher than that in patients with intermittent claudication (0·35 nmol; P = 0·002), smoking controls (0·36 nmol; P = 0·001), non-smoking controls (0·35 nmol; P = 0·002) and young healthy controls (0·27 nmol; P < 0·001). Nitrotyrosine concentrations increased during early reperfusion in open AAA repair, but not during endovascular repair. AAA exclusion from the circulation reduced levels to control values (P = 0·001). Patients with an AAA had raised levels of circulating nitrated proteins compared with patients with claudication and controls, suggesting a greater degree of ongoing inflammation that was not related to smoking. Copyright

Contraceptivos orais combinados (COCs) são largamente utilizados e bem aceitos para se obter a contracepção. Estima-se que mais de 100 milhões de mulheres ao redor do mundo fazem uso deste método contraceptivo. Embora seja alcançada a contracepção, estas usuárias estão predispostas ao desenvolvimento de tromboembolismo venoso (TEV) ou arterial (TEA), e ao desenvolvimento de aterosclerose, em virtude da alteração hemostática causada pela dose de estrógeno e tipo de progesterona utilizada nas formulações. Como forma de obter uma formulação dita \"ideal\" e na esperança de que os efeitos adversos que podem ocorrer em detrimento da composição da formulação contraceptiva sejam minimizados, a literatura aponta um contraceptivo recentemente lançado, cuja composição contém o Valerato de Estradiol (chamado de estrógeno natural pela indústria farmacêutica) como componente estrogênico e o Dienogest como progesterona sintética, integrando o grupo dos assim classificados novos progestágenos. OBJETIVO: Dentro do contexto que acima apresentamos, o projeto de pesquisa teve como objetivo avaliar duas formulações contraceptivas, através de um estudo experimental comparativo, onde como grupo controle utilizamos o clássico Levonorgestrel (LVG) e Etinilestradiol (EE), para compará-lo com o novo progestágeno lançado no mercado brasileiro em 2011, composto por Dienogest (DNG) e Valerato de estradiol (17? Estradiol - metabólito ativo) - ambos medicamentos isolados e em mistura, representados por MIX I (LVG+EE) e MIX II (DNG+17?), respectivamente. Pelo fato de pouco se saber sobre os efeitos deste novo progestágeno sobre os fatores vasoativos, hemostáticos e inflamatórios, propusemos aqui trabalhar com cultura de células endoteliais extraídas de cordão umbilical humano, estimulá-las com agente inflamatório LPS e/ou TNF? para posteriormente dosar os seguintes fatores: em Célula Endotelial de Veia Umbilical Humana (HUVEC) - moléculas de adesão VCAM-I, ICAM-I e E-Selectina (sobrenadante e superfície celular de HUVEC); fatores vasoativos derivados do endotélio (NO, Prostaglandina PGE2 e Endotelina ET-1 - sobrenadante) e citocinas pró-inflamatórias IL-1? e IL-6 no sobrenadante. Em Célula Endotelial de Artéria Humana (HAEC): Fator Tecidual. Os medicamentos reduziram a expressão de moléculas de adesão no sobrenadante e na superfície celular, regularam a produção de fatores vasoativos derivados do endotélio, reduziram a produção de citocinas pró-inflamatórias e reduziram também a expressão de fator tecidual, de maneira preventiva e terapêutica. A mistura dos contraceptivos representada por MIX I e MIX II apresentou diferenças significativas quando comparadas aos demais grupos de estudo e MIX II apresentou maior efetividade na redução de alguns fatores aqui estudados, conferindo uma proteção endotelial. Desta maneira, podemos concluir que o novo progestágeno lançado no mercado brasileiro apresentou efeitos antitrombóticos e anti-inflamatórios, por reduzir de maneira significativa os níveis de moléculas de adesão, fatores vasoativos, fatores pró-inflamatórios e fator hemostático / Combined oral contraceptives (COCs) are widely used and well accepted to provide contraception. It\'s estimated that over 100 million women around the world make use of this contraceptive method. Although contraception is achieved, these users are predisposed to developing venous thromboembolism or arterial and development of atherosclerosis due to the change caused by the hemostatic dose of estrogen and progestin type used in the formulations. In order to get a said formulation \"correct\" and hoping that the adverse effects that may occur at the expense of the contraceptive formulation composition are minimized, the literature indicates a contraceptive recently launched, whose composition contains Estradiol valerate (called natural estrogen by the pharmaceutical industry) as estrogen component and the Dienogest as synthetic progesterone, integrating the group of so classified new progestins. OBJECTIVE: Within the context that we present above, the research project aimed to evaluate two contraceptive formulations, through a comparative experimental study, where as the control group used the classic Levonorgestrel (LVG) and ethinylestradiol (EE), to compare it with the new progestin released in Brazil in 2011, composed of Dienogest (DNG) and estradiol valerate (17? Estradiol - active metabolite) - both alone and in combination medications, represented by MIX I (LVG plus EE) and MIX II (DNG plus 17?), respectively. \'Cause little is known about the effects of the progestogen on the new vasoactive factors, hemostatic and inflammatory proposed here work with cultured endothelial cells derived from human umbilical cord stimulus them with LPS inflammatory agent and / or TNF? to further quantitate the following factors: HUVEC (Human Umbilical Vein Endothelial Cell) - adhesion molecules VCAM-I, ICAM-I and E-selectin (supernatant and cell surface); endothelium-derived vasoactive factors (NO, PGE2 and Endothelin ET-1 - culture supernatant) and pro-inflammatory cytokines as IL-1? and IL-6 in the supernatant. HAEC (Human Arterial Endothelial Cell): Tissue factor on primary cell culture of umbilical cord artery. The drugs reduced the expression of adhesion molecules in the supernatant and the cell surface reduced the production of endothelium-derived vasoactive factors, regulated the production of cytokines proinflammatory and also reduced the expression of tissue factor, preventive and therapeutic way. The mixture of contraceptives represented by MIX MIX I and II showed significant differences when compared to the other study groups and MIX II showed greater effectiveness in reducing some factors studied here, giving an endothelial protection. Thus, we can conclude that the new progestogen released in Brazil showed antithrombotic effects and anti-inflammatory, to reduce significantly the levels of adhesion molecules, vasoactive factors, proinflammatory factors and hemostatic factor.

Contraceptivos orais combinados (COCs) são largamente utilizados e bem aceitos para se obter a contracepção. Estima-se que mais de 100 milhões de mulheres ao redor do mundo fazem uso deste método contraceptivo. Embora seja alcançada a contracepção, estas usuárias estão predispostas ao desenvolvimento de tromboembolismo venoso (TEV) ou arterial (TEA), e ao desenvolvimento de aterosclerose, em virtude da alteração hemostática causada pela dose de estrógeno e tipo de progesterona utilizada nas formulações. Como forma de obter uma formulação dita \"ideal\" e na esperança de que os efeitos adversos que podem ocorrer em detrimento da composição da formulação contraceptiva sejam minimizados, a literatura aponta um contraceptivo recentemente lançado, cuja composição contém o Valerato de Estradiol (chamado de estrógeno natural pela indústria farmacêutica) como componente estrogênico e o Dienogest como progesterona sintética, integrando o grupo dos assim classificados novos progestágenos. OBJETIVO: Dentro do contexto que acima apresentamos, o projeto de pesquisa teve como objetivo avaliar duas formulações contraceptivas, através de um estudo experimental comparativo, onde como grupo controle utilizamos o clássico Levonorgestrel (LVG) e Etinilestradiol (EE), para compará-lo com o novo progestágeno lançado no mercado brasileiro em 2011, composto por Dienogest (DNG) e Valerato de estradiol (17? Estradiol - metabólito ativo) - ambos medicamentos isolados e em mistura, representados por MIX I (LVG+EE) e MIX II (DNG+17?), respectivamente. Pelo fato de pouco se saber sobre os efeitos deste novo progestágeno sobre os fatores vasoativos, hemostáticos e inflamatórios, propusemos aqui trabalhar com cultura de células endoteliais extraídas de cordão umbilical humano, estimulá-las com agente inflamatório LPS e/ou TNF? para posteriormente dosar os seguintes fatores: em Célula Endotelial de Veia Umbilical Humana (HUVEC) - moléculas de adesão VCAM-I, ICAM-I e E-Selectina (sobrenadante e superfície celular de HUVEC); fatores vasoativos derivados do endotélio (NO, Prostaglandina PGE2 e Endotelina ET-1 - sobrenadante) e citocinas pró-inflamatórias IL-1? e IL-6 no sobrenadante. Em Célula Endotelial de Artéria Humana (HAEC): Fator Tecidual. Os medicamentos reduziram a expressão de moléculas de adesão no sobrenadante e na superfície celular, regularam a produção de fatores vasoativos derivados do endotélio, reduziram a produção de citocinas pró-inflamatórias e reduziram também a expressão de fator tecidual, de maneira preventiva e terapêutica. A mistura dos contraceptivos representada por MIX I e MIX II apresentou diferenças significativas quando comparadas aos demais grupos de estudo e MIX II apresentou maior efetividade na redução de alguns fatores aqui estudados, conferindo uma proteção endotelial. Desta maneira, podemos concluir que o novo progestágeno lançado no mercado brasileiro apresentou efeitos antitrombóticos e anti-inflamatórios, por reduzir de maneira significativa os níveis de moléculas de adesão, fatores vasoativos, fatores pró-inflamatórios e fator hemostático / Combined oral contraceptives (COCs) are widely used and well accepted to provide contraception. It\'s estimated that over 100 million women around the world make use of this contraceptive method. Although contraception is achieved, these users are predisposed to developing venous thromboembolism or arterial and development of atherosclerosis due to the change caused by the hemostatic dose of estrogen and progestin type used in the formulations. In order to get a said formulation \"correct\" and hoping that the adverse effects that may occur at the expense of the contraceptive formulation composition are minimized, the literature indicates a contraceptive recently launched, whose composition contains Estradiol valerate (called natural estrogen by the pharmaceutical industry) as estrogen component and the Dienogest as synthetic progesterone, integrating the group of so classified new progestins. OBJECTIVE: Within the context that we present above, the research project aimed to evaluate two contraceptive formulations, through a comparative experimental study, where as the control group used the classic Levonorgestrel (LVG) and ethinylestradiol (EE), to compare it with the new progestin released in Brazil in 2011, composed of Dienogest (DNG) and estradiol valerate (17? Estradiol - active metabolite) - both alone and in combination medications, represented by MIX I (LVG plus EE) and MIX II (DNG plus 17?), respectively. \'Cause little is known about the effects of the progestogen on the new vasoactive factors, hemostatic and inflammatory proposed here work with cultured endothelial cells derived from human umbilical cord stimulus them with LPS inflammatory agent and / or TNF? to further quantitate the following factors: HUVEC (Human Umbilical Vein Endothelial Cell) - adhesion molecules VCAM-I, ICAM-I and E-selectin (supernatant and cell surface); endothelium-derived vasoactive factors (NO, PGE2 and Endothelin ET-1 - culture supernatant) and pro-inflammatory cytokines as IL-1? and IL-6 in the supernatant. HAEC (Human Arterial Endothelial Cell): Tissue factor on primary cell culture of umbilical cord artery. The drugs reduced the expression of adhesion molecules in the supernatant and the cell surface reduced the production of endothelium-derived vasoactive factors, regulated the production of cytokines proinflammatory and also reduced the expression of tissue factor, preventive and therapeutic way. The mixture of contraceptives represented by MIX MIX I and II showed significant differences when compared to the other study groups and MIX II showed greater effectiveness in reducing some factors studied here, giving an endothelial protection. Thus, we can conclude that the new progestogen released in Brazil showed antithrombotic effects and anti-inflammatory, to reduce significantly the levels of adhesion molecules, vasoactive factors, proinflammatory factors and hemostatic factor.

Yes / Non-invasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5- methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F- FEDAA1106 and 18F-FDG (a marker of glucose metabolism) for PET imaging of vascular inflammation. This was tested using a murine model where focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomical data generated by CT/CT angiography. Standardized uptake values (SUV) were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p<0.01) or FDG (p<0.05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the non-inflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 correlated with vascular inflammation more specifically than FDG uptake. / : This study was funded by the British Heart Foundation and through a grant from the Swiss National Science Foundation (310030_143343/1 to B.R.K.)

A aterosclerose é considerada, hoje, doença inflamatória e com intensa proliferação celular, daí o racional de se usar medicamentos antiproliferativos e com ação anti-inflamatória como o paclitaxel (PTX) e o metotrexato (MTX) no tratamento dessa condição. A nanoemulsão lipídica (LDE), de composição semelhante à da lipoproteína de baixa densidade (LDL), se liga a receptores de LDL após sua injeção endovenosa na corrente sanguínea. Como tais receptores estão superexpressos em células com altas taxas de proliferação, como ocorre no câncer e na aterosclerose, a LDE pode ser usada como veículo para direcionar agentes antiproliferativos a essas células, aumentando a sua eficácia e diminuindo a toxicidade. O paclitaxel é um quimioterápico com ação antiproliferativa usado em vários tipos de câncer e recobrindo stents farmacológicos, trabalhos anteriores, usando coelhos submetidos a uma dieta aterogênica, nos animais tratados com LDE-PTX houve redução de 60% da área lesionada. O metrotexato, além de ser usado em vários esquemas quimioterápicos, possui, também, ação anti-inflamatória, sendo usado em doenças inflamatórias crônicas, como a artrite reumatoide. Em outro estudo envolvendo, coelhos hipercolesterolêmicos, o uso de MTX comercial por 4 semanas demonstrou uma redução de 75% na área de placa aterosclerótica. Esse estudo tem por objetivo avaliar macroscopicamente a eficácia das terapias quimioterápicas combinada, composta de PTX-LDE com MTX-LDE, e monoterapia, apenas com PTX-LDE, na regressão da aterosclerose experimental. No presente trabalho, vinte e oito coelhos machos da raça New Zealand receberam dieta rica em colesterol a 1% durante 8 semanas. Depois desse período, foram divididos em quatro grupos: grupo CONTROLE, que foi sacrificado e as aortas fixadas para análise posterior, grupo DIETA, que apenas teve a ração enriquecida com colesterol a 1% suspensa, PTX, que recebeu tratamento com injeções endovenosas semanais de LDE-paclitaxel na dose de 4 mg/kg por 8 semanas, e PTX+MTX, que recebeu LDE-paclitaxel e LDE-metotrexato na dose de 4 mg/kg/semana por 8 semanas. Foram avaliados perfil hematológico, lipídico, bioquímico, ponderal e o consumo de ração. Após a eutanásia, foram medidas as lesões ateroscleróticas macroscópicas nas aortas dos coelhos. Em seguida, o arco aórtico foi analisado por morfometria e por imuno-histoquímica. Os marcadores inflamatórios foram analisados no plasma, por ELISA e por meio de expressão gênica por Qrt-pcr. Observou-se que não houve diferença no perfil ponderal e no consumo de ração entre os grupos de estudo. Não houve toxicidade hematológica, hepática e renal relacionada ao tratamento. No perfil lipídico, ao final do estudo, as concentrações de colesterol total, não HDL-C e triglicerídeos aumentaram significativamente em todos os grupos. Houve uma marcante regressão na área de placa aterosclerótica nos coelhos tratados com LDE-paclitaxel, da ordem de 64% e mais marcante no grupo LDE-metotrexato de 71%, quando comparados ao grupo CONTROLE. Na comparação com o grupo DIETA, houve, também, regressão, de 49% nos coelhos do grupo PTX e de 59% no grupo PTX+MTX. O tratamento quimioterápico também mostrou ação antiaterosclerótica nos outros parâmetros avaliados, destacando a intensa redução na relação íntima-média das aortas, na expressão proteica de MMP-9 e da redução na expressão gênica de TNF-? em relação ao grupo DIETA. Portanto, o tratamento quimioterápico com PTX e MTX associado à LDE possui potencial para uso clínico em pacientes com doença aterosclerótica, sendo muito eficaz e com boa tolerabilidade / Atherosclerosis is nowadays considered as an inflammatory disease with intense cell proliferation, hence the rationale of using antiproliferative drugs with an anti-inflammatory action such as paclitaxel (PTX) and methotrexate (MTX) in the treatment of this condition. The lipid nanoemulsion (LDE), with a similar composition to low density lipoprotein (LDL) binds to LDL receptors after their intravenous injection into the bloodstream. Since such receptors are overexpressed in cells with high proliferation rates, such as occurs in cancer and atherosclerosis, LDE can be used as a vehicle to direct antiproliferative agents to these cells, increasing their efficacy and reducing toxicity. Paclitaxel is a chemotherapeutic drug with an anti-proliferative action used in various types of cancer and drug-eluting stents. In previous studies using rabbits subjected to an atherogenic diet, animals treated with LDE-PTX had a 60% reduction in the injured area. Methotrexate in addition to being used in various chemotherapy regimens also has an anti-inflammatory action and is used for chronic inflammatory diseases such as rheumatoid arthritis; another study involving hypercholesterolemic rabbits using commercial MTX for 4 weeks showed a 75% reduction of the atherosclerotic plaque area. This study aims to evaluate the effectiveness of combined chemotherapy treatments, composed of PTX-LDE with MTX-LDE, and PTX-LDE in monotherapy, on the regression of experimental atherosclerosis. In this study, twenty eight male New Zealand breed rabbits received a diet enriched with 1% cholesterol for 8 weeks. After that time, they were divided into four groups: the CONTROL group, which was sacrificed and the aortas kept for later analysis, the DIET group, which only had the diet supplemented with 1% cholesterol suspended; the PTX group which received treatment with weekly intravenous injections of LDE paclitaxel, at a dose of 4 mg/kg for 8 weeks and the PTX+MTX group which received PTX - LDE + MTX-LDE at a dose of 4 mg/kg per week for 8 weeks. The hematological, lipid, biochemical, weight and food intake profiles were evaluated. After euthanasia, macroscopic atherosclerotic lesions in the aortas of the rabbits were measured. Then, the aortic arch was analyzed by morphology and immunohistochemistry. The inflammatory markers were analyzed in the plasma by ELISA and gene expression by qRT-PCR. There was no difference in weight profile and feed intake among the study groups. There was no hematological, hepatic or renal toxicity related to treatment. The lipid profiles of all the groups at the end of the study showed significantly increased concentrations of total cholesterol, non-HDL-C and triglyceride levels. There was a marked regression of 64% in the atherosclerotic plaque area, in the LDE-paclitaxel treated rabbits, and an even more striking 71% in the LDE-methotraxate group compared to the CONTROL group. There was also regression when compared to the DIET group, 49% in rabbits from the PTX group and 59% in the PTX+MTX group. The chemotherapy also showed an anti-atherosclerotic action in the other evaluated parameters, especially notable were the intense reduction in the intima-media ratio of the aortas in protein expression of MMP-9 and the reduction in gene expression of TNF-alpha compared to the DIET group. Therefore, chemotherapy with PTX and MTX associated with LDE, has potential for clinical use in patients with atherosclerotic disease, as it is very effective and well tolerated

Aims: LDL oxidation plays an important role in the initiation and progression of atherosclerosis. HDL impedes oxidation, glycation and glycoxidation in vitro and there is evidence to suggest paraoxonase-1 (PON1) plays an important role in this. 1. In patients with dyslipidaemia treated with statins, I assessed the relationship of serum PON1 activity with in vitro HDL antioxidant capacity, susceptibility of LDL to oxidation and the protection offered by HDL. 2. I studied the effect of the presence and severity of obstructive sleep apnoea (OSA) in morbidly obese patients on HDL anti-oxidant and anti-inflammatory functions. 3. I investigated the influence of extended release niacin/ laropiprant (ERN/LRP) versus placebo in patients who had persistent dyslipidaemia despite receiving high doses of potent statins. I assessed the effect of ERN/LRP on mediators of vascular inflammation and HDL's in vitro anti-oxidant function. Methods: 1. LDL isolated from dyslipidemic patients was incubated with and without HDL, in the presence of Cu2+. Similarly isolated HDL was incubated alone. Lipid peroxides (LPO) generated over 3 hours were measured. Patients were divided into 2 groups based on median serum PON1 activity. 2. 41 morbidly obese patients were divided into two groups based on the presence or absence of OSA ("OSA" and "no OSA" group) or on severity of OSA (high or low apnoea-hypoapnoea index (AHI) groups). I studied HDL's ability to protect itself from in vitro oxidation and measured serum PON1 activity, tumor necrosis factor alpha (TNFalpha) and intercellular adhesion molecule 1 (ICAM1). 3. This was a randomised double blind cross over trial, where I studied the effect of ERN/LRP compared to placebo in 27 patients who had high LDL-C inspite of maximum tolerated doses of statins. I measured lipid profile, apolipoproteins, cholesteryl ester transport protein (CETP) activity, paraoxonase 1 activity (PON1), oxidised LDL (oxLDL) and related mediators of vascular inflammation. I also examined the capacity of HDL to protect LDL from in vitro oxidation. Results and conclusion: 1. In statin treated dyslipidemic patients the capacity of HDL to protect itself and LDL from oxidation in vitro is significantly better in individuals with higher serum PON1 activity. 2. The capacity of HDL to protect itself from in vitro oxidation in morbidly obese patients is reduced with onset and severity of OSA. The differences in TNFalpha and ICAM1 levels may suggest endothelial dysfunction due to OSA. Oxidative damage of PON1 attributable to OSA could be a mechanism for HDL and endothelial dysfunction. 3. Treatment with ERN/LRP resulted in a significant improvement in HDL-C but did not affect HDL's in vitro anti-oxidant function in patients who had persistent dyslipidaemia despite high doses of potent statins. For the first time I have shown that ERN/LRP reduces mediators of vascular inflammation.

Abnormal leukocyte adhesion (i.e. leukostasis) to retinal vascular endothelial cells occurs in early diabetes. The processes of leukostasis have been clearly demonstrated in the vascular endothelium of patients with diabetes. In non-proliferative DR, clinical outcomes are manifested by excessive permeability from inflammatory progression leading to inner blood retinal barrier disruption, endothelial cell damage and widespread capillary nonperfusion. Diabetes promotes vascular leakage in DR by upregulation of adhesion molecules. Moreover, many of the pathological changes in NPDR are related to abnormalities in retinal blood flow. Studies have shown that specific circulating markers of inflammatory activity and endothelial dysfunction are associated with clinical signs of diabetic retinopathy. However, few have found an association between circulating levels of inflammatory and endothelial dysfunctional markers and abnormal retinal hemodynamics in patients with non-proliferative DR. The specific aims of this thesis are as follows: (Chapter 3)To correlate baseline levels of inflammatory and endothelial dysfunction markers and 1) baseline retinal arteriolar hemodynamics and 2) any disturbance in retinal hemodynamics over 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow. In Chapter 4: To correlate circulating levels of inflammatory and endothelial dysfunction markers and 1) baseline vascular reactivity and 2) any disturbance in vascular reactivity after 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow in patients with increasing non-proliferative diabetic retinopathy (NPDR) severity. Methods for Chapter 3: Diabetes subjects were stratified into either mild-to-moderate (Group 2) or moderate-to-severe (Group 3) NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls (Group 1). Forearm blood sample was collected to determine baseline levels of inflammatory and endothelial dysfunctional markers. At visit 1, baseline retinal hemodynamics was acquired using Canon Laser Blood Flowmeter. Patients returned for a visit 2 (6 month follow-up visit) and retinal hemodynamics was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both baseline and change in retinal hemodynamic parameters over 6-month time. For Chapter 4: Diabetes subjects were stratified into either mild-to-moderate NPDR or moderate-to-severe NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls. At visit 1, forearm blood sample was collected to determine levels of inflammatory and endothelial dysfunctional markers and baseline vascular reactivity response was acquired. Retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to isocapnic hyperoxic stimuli. At visit 2 (6 month follow-up), retinal vascular reactivity was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both magnitude of baseline and change in vascular reactivity in terms of retinal hemodynamics. Results of Chapter 3: Maximum-to-minimum velocity ratio (max: min) was found to be significantly elevated in the group 3 compared to group 1 at baseline (0.72 vs. 0.49, after Bonferroni correction P<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p=0.04). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (234.0 vs. 151.5 ng/ml, P=0.02 and 53.4 vs. 27.6 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (7.9 vs. 5.6 % , P<0.01). There were no significant associations found between baseline markers of inflammation and baseline retinal hemodynamics across all groups. The Δ velocity was correlated with the baseline sICAM-1 (r=0.42, p=0.02) and A1c levels (r=0.37, p=0.04) in patients with NPDR. After adjustment for all other variables (A1c, hsCRP and vWF), Δ velocity, sICAM-1 and A1c were found not to be reliable predictors of baseline retinal hemodynamics. For Chapter 4: There were no significant differences in magnitude of retinal vascular reactivity in hemodynamic parameters between groups at visit 1 or visit 2. Over 6 months time, compliance was found to be significantly reduced in patients of Group 3 compared to Group 2 (-0.4 vs. 0.1, t-test p<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (243.4 vs. 157.3ngml, P<0.01 and 57.0 vs. 29.3 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (8.8 vs. 5.6 % , P<0.01). Baseline VR in blood velocity weakly correlates with sE-selectin (r=0.31, p=0.04) across all groups while sVCAM-1 was associated with VR in terms of blood flow (r=-0.62, p<0.01) in patients with mild-to-moderate NPDR. The ∆ blood flow after 6 months was found to be weakly associated with sE-selectin (r=0.46, p=0.03) across all groups. Finally, the ∆ blood velocity after 6 month time was found to be moderately correlated with baseline vWF Ag level (r=-0.78, p=0.02). Multiple regression analysis found that vascular inflammatory and endothelial function markers had weak predictive power for Δ hemodynamic parameters. Conclusions Chapter 3: We found weak associations between circulating markers and baseline or the disturbance in retinal hemodynamics after 6 months time. Overall, we found both an increase in rigidity of the arteriolar circulation and elevated inflammatory adhesion markers (sICAM-1 and sE-selectin) within the same population sample. Change in velocity over the follow-up period was correlated with sICAM-1 and A1c levels in patients with NPDR but the level of association was such that neither sICAM-1 nor A1c proved to reliably predict retinal hemodynamics. Finally, in Chapter 4 we demonstrated two important characteristics in early NPDR; 1) a disturbance in vascular reactivity in terms of compliance and 2) an increase in systemic markers of inflammation were found in patients with NPDR. Although systemic markers of vascular inflammation and endothelial dysfunction are not predictive of hemodynamic parameters, our study found moderate associations between baseline and disturbances in VR after 6 months time. Therefore, there is evidence that inflammation and vascular function may be related with respect to their development in NPDR.

Abnormal leukocyte adhesion (i.e. leukostasis) to retinal vascular endothelial cells occurs in early diabetes. The processes of leukostasis have been clearly demonstrated in the vascular endothelium of patients with diabetes. In non-proliferative DR, clinical outcomes are manifested by excessive permeability from inflammatory progression leading to inner blood retinal barrier disruption, endothelial cell damage and widespread capillary nonperfusion. Diabetes promotes vascular leakage in DR by upregulation of adhesion molecules. Moreover, many of the pathological changes in NPDR are related to abnormalities in retinal blood flow. Studies have shown that specific circulating markers of inflammatory activity and endothelial dysfunction are associated with clinical signs of diabetic retinopathy. However, few have found an association between circulating levels of inflammatory and endothelial dysfunctional markers and abnormal retinal hemodynamics in patients with non-proliferative DR. The specific aims of this thesis are as follows: (Chapter 3)To correlate baseline levels of inflammatory and endothelial dysfunction markers and 1) baseline retinal arteriolar hemodynamics and 2) any disturbance in retinal hemodynamics over 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow. In Chapter 4: To correlate circulating levels of inflammatory and endothelial dysfunction markers and 1) baseline vascular reactivity and 2) any disturbance in vascular reactivity after 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow in patients with increasing non-proliferative diabetic retinopathy (NPDR) severity. Methods for Chapter 3: Diabetes subjects were stratified into either mild-to-moderate (Group 2) or moderate-to-severe (Group 3) NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls (Group 1). Forearm blood sample was collected to determine baseline levels of inflammatory and endothelial dysfunctional markers. At visit 1, baseline retinal hemodynamics was acquired using Canon Laser Blood Flowmeter. Patients returned for a visit 2 (6 month follow-up visit) and retinal hemodynamics was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both baseline and change in retinal hemodynamic parameters over 6-month time. For Chapter 4: Diabetes subjects were stratified into either mild-to-moderate NPDR or moderate-to-severe NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls. At visit 1, forearm blood sample was collected to determine levels of inflammatory and endothelial dysfunctional markers and baseline vascular reactivity response was acquired. Retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to isocapnic hyperoxic stimuli. At visit 2 (6 month follow-up), retinal vascular reactivity was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both magnitude of baseline and change in vascular reactivity in terms of retinal hemodynamics. Results of Chapter 3: Maximum-to-minimum velocity ratio (max: min) was found to be significantly elevated in the group 3 compared to group 1 at baseline (0.72 vs. 0.49, after Bonferroni correction P<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p=0.04). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (234.0 vs. 151.5 ng/ml, P=0.02 and 53.4 vs. 27.6 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (7.9 vs. 5.6 % , P<0.01). There were no significant associations found between baseline markers of inflammation and baseline retinal hemodynamics across all groups. The Δ velocity was correlated with the baseline sICAM-1 (r=0.42, p=0.02) and A1c levels (r=0.37, p=0.04) in patients with NPDR. After adjustment for all other variables (A1c, hsCRP and vWF), Δ velocity, sICAM-1 and A1c were found not to be reliable predictors of baseline retinal hemodynamics. For Chapter 4: There were no significant differences in magnitude of retinal vascular reactivity in hemodynamic parameters between groups at visit 1 or visit 2. Over 6 months time, compliance was found to be significantly reduced in patients of Group 3 compared to Group 2 (-0.4 vs. 0.1, t-test p<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (243.4 vs. 157.3ngml, P<0.01 and 57.0 vs. 29.3 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (8.8 vs. 5.6 % , P<0.01). Baseline VR in blood velocity weakly correlates with sE-selectin (r=0.31, p=0.04) across all groups while sVCAM-1 was associated with VR in terms of blood flow (r=-0.62, p<0.01) in patients with mild-to-moderate NPDR. The ∆ blood flow after 6 months was found to be weakly associated with sE-selectin (r=0.46, p=0.03) across all groups. Finally, the ∆ blood velocity after 6 month time was found to be moderately correlated with baseline vWF Ag level (r=-0.78, p=0.02). Multiple regression analysis found that vascular inflammatory and endothelial function markers had weak predictive power for Δ hemodynamic parameters. Conclusions Chapter 3: We found weak associations between circulating markers and baseline or the disturbance in retinal hemodynamics after 6 months time. Overall, we found both an increase in rigidity of the arteriolar circulation and elevated inflammatory adhesion markers (sICAM-1 and sE-selectin) within the same population sample. Change in velocity over the follow-up period was correlated with sICAM-1 and A1c levels in patients with NPDR but the level of association was such that neither sICAM-1 nor A1c proved to reliably predict retinal hemodynamics. Finally, in Chapter 4 we demonstrated two important characteristics in early NPDR; 1) a disturbance in vascular reactivity in terms of compliance and 2) an increase in systemic markers of inflammation were found in patients with NPDR. Although systemic markers of vascular inflammation and endothelial dysfunction are not predictive of hemodynamic parameters, our study found moderate associations between baseline and disturbances in VR after 6 months time. Therefore, there is evidence that inflammation and vascular function may be related with respect to their development in NPDR.