Dissertationen zum Thema „Atherosclerosis Drugs“

Cardiovascular disease secondary to atherosclerosis is the leading cause of death worldwide. The pathophysiology of atherosclerosis is multifactorial and complex. This thesis offers insight into the regulation of several potential biomarkers of cardiovascular disease and explores the effects of anti-platelet therapy in modulating these pathways. CD14highCD16+ monocytes are elevated in patients with atherosclerosis and may be predictive of future cardiovascular events. The initial study in this PhD project assessed the effects of anti-platelet drugs on the phenotype of circulating monocytes in 60 healthy volunteers in the presence of mild, systemic inflammation induced by the influenza immunisation. A significant rise in circulating CD14highCD16+ monocytes developed following immunisation and anti-platelet therapy was subsequently shown to exert an immunomodulatory action by counteracting this response. Netrin-1 is a laminin-like protein that is implicated in cardiovascular disease, including coronary artery disease. A series of experiments were performed to investigate the biomolecular mechanisms that regulate the synthesis of vascular netrin-1 in humans. Results showed that netrin-1 levels are directly modulated by changes in the production of vasoactive cyclooxygenase-derived molecules, such as prostaglandin E2, from the vascular endothelium. Oxidised low-density lipoprotein was the final biomarker that was investigated. Results indicated that CD16+ monocytes may regulate the clearance of oxidised lipoproteins and their systemic accumulation, possibly through the internalisation of circulating oxLDL/IgG immunocomplexes mediated by Fc γ receptors, including CD16.

Highly antiretroviral therapies (HAART) have been implemented to slow the progression of the human immunodeficiency virus (HIV). Although these new advances in the medications for HIV-positive patients have contributed in longer life expectancy, comorbidities, such as cardiovascular disease, still cause higher number of deaths among HIV-positive patients than in the regular population. Because of the intrinsic inflammation caused by the HIV virus, atherogenesis is more likely to occur and is driven by infected macrophages. These macrophages are known to secrete cathepsins, but infection causes the macrophages not to perform their function properly as an immune agent. I hypothesize that antiretroviral drugs play an important role in arterial remodeling by affecting cells within the artery and causing an alteration of cathepsin activity, leading to an increased risk of atherosclerosis in HIV patients. To test this hypothesis, we incubated THP-1 monocytes with antiretroviral drugs efavirenz and tenofovir individually to observe any changes in cathepsin activity. These lysates were analyzed through multiplex cathepsin zymography and quantified through densitometry. We found that our hypothesis held true for efavirenz and tenofovir in THP-1 monocytes, which caused decreased cathepsin K activity compared to vehicle controls. Still, stimulation of peripheral blood mononuclear cells (PBMCs) with efavirenz and tenofovir caused differential effects. Together, our data suggest that the HAART interaction with monocytes that are physiologically relevant to our system possibly contributes to the advancement of atherogenesis in HIV+ patients.

Abstract Chronic Chlamydia pneumoniae infections have been associated with cardiovascular diseases (CVD), but the treatment is difficult. Some drugs used for CVD have been found to have an inhibitory effect on the C. trachomatis infection, which is not considered to be associated with CVD. The purpose of this study was to investigate the effects of heparan sulfate-like glycosaminoglycans, COX inhibitors and rapamycin on the C. pneumoniae infection with cell culture methods. Almost any conceivable factors may affect the results of cell cultures. This study showed the complex interaction between temperature, time and medium during the pre-treatment before inoculation. The influences of these factors on the results overlapped and interlaced. The simple washing procedure could enhance the infectivity of C. pneumoniae although it is generally considered to cause the loss of chlamydial EBs and sequentially decrease the chlamydial infectivity. Although the detailed mechanisms were not studied, the results of this study showed that selective COX inhibitors and rapamycin can inhibit the infectivity of C. pneumoniae by inhibiting the growth and maturation, whereas heparan sulfate-like glycosaminoglycans perhaps inhibit the attachment of C. pneumoniae EBs onto the host cells. Recovery and repassage results showed that the growth can be only delayed by selective COX inhibitors, and it can recover to normal level once the drugs were removed. However, rapamycin inhibited the maturation of chlamydial EBs and therefore the infectivity fell down further even when the rapamycin was removed. This study also presented the variations of pathogenicity between different C. pneumoniae strains in vitro. This study is based on in vitro experiments with an acute infection model. Thus, any definite conclusions on the possible antichlamydial effects of the drugs tested in the treatment of cardiovascular diseases which are associated with chronic C. pneumoniae infections cannot be drawn on the basis of this study.

The overall goals of this project were to develop microstructurally based constitutive models to characterize the mechanical behavior of arteries and to investigate the effects of HIV proteins and antiretroviral drugs on the microstructure and mechanical behavior. To this end we created several constitutive models in aim 1 using a rule of mixtures approach, investigated the role of viral proteins in aim 2 through the use a transgenic mouse model, and studied the effects of the antiretroviral drug AZT administered to mice in aim 3. It is well known that the local mechanical environment which cells experience mediates growth and remodeling and that subsequent growth and remodeling can change that mechanical environment. This remodeling includes changes in the content and organization of the constituents of arteries (collagen, elastin, and smooth muscle cells). The first aim thus created models that incorporated the content and organization of these constituents using a rule-of-mixtures approach. The models we developed were able to capture the mechanical behavior of the arteries as well as previously developed phenomenological models while providing more physical meaning to the parameters, some which can be measured experimentally for incorporation into future models. Aims 2 and 3 investigated the mechanical and microstructural changes to murine arteries in response to HIV proteins or the drug AZT. While the development of antiretroviral therapy has greatly increased the life expectancy of patients with HIV, a number of other complications and co-morbidities including cardiovascular disease have become apparent. While clinical data has implicated both the virus and the antiretroviral drugs as playing roles, this work addressed the need of investigating these effects in a controlled manner. Specifically we used mouse models and focused on the two subclinical markers of increased intima-media thickness and arterial stiffening. Aim 2 used a transgenic mouse that expressed most of the human HIV proteins. We observed both intima-media thickening and arterial stiffening in alignment with clinical data. Other changes that also support a proatherogenic phenotype included decreased elastin content and changes in cathepsin activity. Aim 3 administered the antiretroviral drug AZT to healthy mice and we also observed the same subclinical markers of atherosclerosis including intima-media thickening and arterial stiffening as well as the other proatherogenic changes of decreased elastin and changes in cathepsin activity. Several other parameters including axial behavior, opening angles, collagen content, and collagen fiber angles were also quantified. These were important to fully characterize the vessel and may also be incorporated in the future into the constitutive models developed in aim1. In conclusion, in aim 1 we developed a microstructurally based constitutive model of arteries that effectively captures the mechanical behavior and includes parameters that have more physical meaning and some of which are experimentally tractable. Aims 2 and 3 both observed several subclinical markers of atherosclerosis in mice that express HIV proteins or were given AZT, providing a good model for future work and suggesting that both the HIV virus and antiretroviral drugs may play roles in the development of atherosclerosis in HIV.

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

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007.
Includes bibliographical references.
Drug eluting stents have revolutionized the practice of medicine and the landscape of medical devices. Yet, more than four years after introduction clinical trial data and clinical use have still not fully clarified what drives the safety and efficacy of these devices. The goal of this thesis was to help fill this void by describing the mechanisms by which stent-eluted drugs are distributed within healthy and atherosclerotic vascular models. In the first part of the thesis we investigated the effect of drug physicochemical properties on drug deposition, retention, and distribution in a healthy vascular model. We found that hydrophobic drugs are deposited to a far greater degree than hydrophilic drugs, with longer retention times, and distribution patterns that likely track with specific and general binding sites. The second part of the thesis investigated how arterial ultrastructure in health and disease modulates the arterial deposition and distribution of hydrophobic antiproliferative drugs used with drug-eluting stents. We tracked the distribution of radiolabeled and FITC-labeled compounds and demonstrated that macrostructural changes in arterial architecture led to profound changes in drug deposition. Paclitaxel in particular was sensitive to tissue state.
(cont.) This drug binds specifically to tubulin and to lesser extent in a general manner to elastic. Drug levels fell as paclitaxel, tubulin and elastin were displaced by lipid and collagen. These observations might well explain how drugs may partition within different arterial lesions as determined by lesion composition. Finally, we demonstrated that association with these binding sites was governed by association kinetics that reflects the different components of the arterial wall compartments. Slower release kinetics yielded up to 64% higher deposition of a drug from stents implanted in rabbit iliac arteries over a 28-day period. Mathematical modeling illustrates that the dependence of drug deposition on stent release kinetics is contingent on drug retention. Further model development is implicated for predicting drug deposition profiles for different types of drugs, arterial states, and stent release kinetics.
by Neda Vukmirovic.
Ph.D.

The deleterious effects of MPO make it a new target for medicinal research. The aim of our study is to find promising inhibitors of MPO for using them as starting point of new anti-inflammatory drugs. Depending on previous researches on MPO inhibitors, we selected 5-fluorotryptamine as starting compounds. Using docking experiments, we designed a series of compounds derived from 5-fluorotryptamine. Two modifications were proposed:

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Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Background: Calcification of atherosclerotic plaques is associated with vulnerability to rupture and increased risk of myocardial infarction. The mechanism of plaque calcification is unclear, but has been shown to be a cell-mediated process involving complex signaling pathways affecting the osteogenic transcription factor Runt-related transcription factor 2 (Runx2). The type-2 cannabinoid receptor (CB2) modulates processes involved in bone remodeling and our prior studies determined that CB2 alters the composition of early lesions in hyperlipidemic Ldlr-/- mice; however, the function of CB2 in plaque calcification is unknown. Therefore, we tested the hypothesis that CB2 modulates plaque calcification by evaluating the effects of systemic CB2 gene deletion on lesion calcification and aortic expression of Runx2 in Ldlr-/- mice. Results: Groups (n≥8) of 8-week old CB2+/+Ldlr-/- (WT) and CB2-/- Ldlr-/- (CB2-/-) mice were fed a high fat diet (HFD) for up to 24 weeks. Standard blood plasma analysis showed no difference in HFD-induced hyperlipidemia between WT and CB2-/- mice. Aortic levels of endocannabinoids, anandamide and 2-archidonylglycerol, were significantly elevated after 12 weeks of HFD feeding as determined by LC-MS/MS. En face analysis revealed the extent of atherosclerosis in the aortic arch and thoracic aorta did not differ between WT and CB2-/- mice, but was ~1.9-fold greater in the abdominal aortas of CB2-/- mice (17.0±1.3% vs 9.0±1.3%, p=0.002). Calcification of aortic root lesions was ~2.3 fold greater in CB2-/- mice compared to WT mice (12.9±1.1% vs 5.6±1.2%, p=0.002) as revealed by von Kossa staining. Western blot analysis showed significantly increased expression of Runx2 in aortas of WT mice compared to CB2-/- after 20 weeks of HFD (2.55±0.25 fold, p Conclusion: Systemic CB2 deficiency enhances lesion calcification and is associated with altered aortic expression of Runx2. These results provide novel mechanistic insights into the function of CB2 signaling in the pathogenesis of atherosclerosis and vascular calcification that may lead to the development of therapies aimed at stabilizing calcified plaque.

QUINDERE, Ana Luiza Gomes. Caracterização estrutural e atividade anti-inflamatória em modelo de aterosclerose em camundongos de uma galactana sulfatada da alga vermelha Acanthophora muscoides. 2015. 152 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015.
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Sulfated galactans from red marine algae are polysaccharides with heterogeneous structures that have presented a variety of potentially therapeutic biological effects including anticoagulant, anti-thrombotic and anti-inflammatory, however, their potential activity as anti-inflammatory agent in the treatment of atherosclerosis, a chronic inflammatory disease that culminates with thromboembolic disorders, has not been previously studied. Furthermore, experimental data from animal models and clinical studies support connections between the hemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. Herein, we determined the chemical structure of a novel sulfated galactan obtained from the marine alga Acanthophora muscoides (fraction AmII) and analysed its effect on a mice model of atherosclerosis in 10-week aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 4 or 11 weeks. Fraction AmII (10 mg/kg) or Vehicle were subcutaneously injected from week 2 until 4 of the diet or from week 6 until week 11 of the diet. In vitro assays of macrophage chemotaxis were also performed. The structure of the complex sulfated galactan was characterized by solution nuclear magnetic resonance and its molecular mass was determined by gel permeation chromatography and polyacrylamide gel electrophoresis. The sulfated galactan from A. muscoides presents a molecular mass of ~ 20kDa and an alternating 4-linked α-galactose and 3-linked β-galactose, substituted with sulfate esters and methyl ethers along with the occurrence of 3,6-anhydro-α-galactoses. In the 4 weeks diet model, treatment with fraction AmII did not alter the atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, macrophage, MMP-9 and collagen contents). In the 11 weeks diet model, treatment with fraction AmII reduced intraplaque macrophage and tissue factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability, such as lipid, neutrophil, MMP-9 and collagen contents, were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by AmII treatment. In vitro, treatment with AmII dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic AmII treatment was well tolerated. In conclusion, our results indicate that AmII treatment reduced intraplaque macrophage content, by impacting on cell recruitment, and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice.
As galactanas sulfatadas obtidas de algas marinhas vermelhas são polissacarídeos de estruturas heterogêneas que têm apresentado uma variedade de efeitos biológicos, potencialmente terapêuticos, incluindo anticoagulante, antitrombótico e anti-inflamatório. No entanto, a sua atividade potencial como agente anti-inflamatório para o tratamento de aterosclerose, uma doença inflamatória crônica que culmina com distúrbios tromboembólicos, não foi previamente estudada. Além disso, dados experimentais de modelos animais e estudos clínicos suportam conexões entre a hemostasia e inflamação na aterogênese. Estas interfaces entre inflamação e trombogênese têm sido sugeridas como alvos para intervenção farmacológica visando reduzir a progressão da doença. No presente trabalho, determinou-se a estrutura química de uma nova galactana sulfatada obtida da alga marinha Acanthophora muscoides (fração AmII) e analisou-se seu efeito sobre um modelo de aterosclerose em camundongos deficientes em apolipoproteína E (ApoE−/−) de 10 semanas submetidos a dieta de alto teor de colesterol durante mais 4 ou 11 semanas. A fração AmII (10 mg/kg) ou veículo (salina) foram injetados por via subcutânea durante a segunda até a quarta semana de dieta ou a partir da sexta até a décima primeira semana de dieta. Ensaios in vitro de quimiotaxia de macrófagos também foram realizados. A estrutura da galactana sulfatada complexa foi caracterizada por ressonância magnética nuclear em solução e a sua massa molecular foi determinada por cromatografia de permeação em gel e eletroforese em gel de poliacrilamida. A fração AmII apresentou uma massa molecular de ~ 20 kDa e uma alternância de α-galactose 4 ligada e β-galactose 3-ligada, substituído com ésteres de sulfato e éteres de metil, juntamente com a ocorrência de unidades de 3,6-anidro-α-galactoses. No modelo de dieta de 4 semanas, o tratamento com a fração AmII não alteraou o tamanho da placa aterosclerótica e demais características de vulnerabilidade intraplaca, tais como lípido, neutrófilos, macrófagos, metaloprotease de matriz (MMP)-9 e conteúdo de colágeno. No modelo de dieta de 11 semanas, o tratamento com a fração AmII reduziu os conteúdos de macrófago intraplaca e de fator tecidual (FT), em comparação com animais tratados com veículo. O FT intraplaca co-localizou e positivamente correlacionou com áreas ricos em macrófagos. Não foram observadas alterações no tamanho da placa aterosclerótica e nas outras características de vulnerabilidade intraplaca, tais como conteúdos de lipídios, neutrófilos, MMP-9 e do colágeno. Além disso, a expressão de mRNA de MMPs, quimiocinas e marcadores genéticos de polarização de linfócitos Th1/2/reg/17 nos arcos aórticos e nos baços dos camundongos não foi alterada pelo tratamento AmII. In vitro, o tratamento com AmII reduziu de forma dose-dependente a quimiotaxia de macrófagos sem afetar a produção de FT. No geral, o tratamento crônico com AmII foi bem tolerado. Em conclusão, nossos resultados indicam que o tratamento com AmII em camundongos ateroscleróticos reduziu o conteúdo de macrófagos intraplaca, agindo sobre o recrutamento celular, e, concomitantemente, o conteúdo de FT intraplaca de originado potencialmente de macrófagos.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Sulfated galactans from red marine algae are polysaccharides with heterogeneous structures that have presented a variety of potentially therapeutic biological effects including anticoagulant, anti-thrombotic and anti-inflammatory, however, their potential activity as anti-inflammatory agent in the treatment of atherosclerosis, a chronic inflammatory disease that culminates with thromboembolic disorders, has not been previously studied. Furthermore, experimental data from animal models and clinical studies support connections between the hemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. Herein, we determined the chemical structure of a novel sulfated galactan obtained from the marine alga Acanthophora muscoides (fraction AmII) and analysed its effect on a mice model of atherosclerosis in 10-week aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 4 or 11 weeks. Fraction AmII (10 mg/kg) or Vehicle were subcutaneously injected from week 2 until 4 of the diet or from week 6 until week 11 of the diet. In vitro assays of macrophage chemotaxis were also performed. The structure of the complex sulfated galactan was characterized by solution nuclear magnetic resonance and its molecular mass was determined by gel permeation chromatography and polyacrylamide gel electrophoresis. The sulfated galactan from A. muscoides presents a molecular mass of ~ 20kDa and an alternating 4-linked α-galactose and 3-linked β-galactose, substituted with sulfate esters and methyl ethers along with the occurrence of 3,6-anhydro-α-galactoses. In the 4 weeks diet model, treatment with fraction AmII did not alter the atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, macrophage, MMP-9 and collagen contents). In the 11 weeks diet model, treatment with fraction AmII reduced intraplaque macrophage and tissue factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability, such as lipid, neutrophil, MMP-9 and collagen contents, were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by AmII treatment. In vitro, treatment with AmII dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic AmII treatment was well tolerated. In conclusion, our results indicate that AmII treatment reduced intraplaque macrophage content, by impacting on cell recruitment, and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice.
As galactanas sulfatadas obtidas de algas marinhas vermelhas sÃo polissacarÃdeos de estruturas heterogÃneas que tÃm apresentado uma variedade de efeitos biolÃgicos, potencialmente terapÃuticos, incluindo anticoagulante, antitrombÃtico e anti-inflamatÃrio. No entanto, a sua atividade potencial como agente anti-inflamatÃrio para o tratamento de aterosclerose, uma doenÃa inflamatÃria crÃnica que culmina com distÃrbios tromboembÃlicos, nÃo foi previamente estudada. AlÃm disso, dados experimentais de modelos animais e estudos clÃnicos suportam conexÃes entre a hemostasia e inflamaÃÃo na aterogÃnese. Estas interfaces entre inflamaÃÃo e trombogÃnese tÃm sido sugeridas como alvos para intervenÃÃo farmacolÃgica visando reduzir a progressÃo da doenÃa. No presente trabalho, determinou-se a estrutura quÃmica de uma nova galactana sulfatada obtida da alga marinha Acanthophora muscoides (fraÃÃo AmII) e analisou-se seu efeito sobre um modelo de aterosclerose em camundongos deficientes em apolipoproteÃna E (ApoE−/−) de 10 semanas submetidos a dieta de alto teor de colesterol durante mais 4 ou 11 semanas. A fraÃÃo AmII (10 mg/kg) ou veÃculo (salina) foram injetados por via subcutÃnea durante a segunda atà a quarta semana de dieta ou a partir da sexta atà a dÃcima primeira semana de dieta. Ensaios in vitro de quimiotaxia de macrÃfagos tambÃm foram realizados. A estrutura da galactana sulfatada complexa foi caracterizada por ressonÃncia magnÃtica nuclear em soluÃÃo e a sua massa molecular foi determinada por cromatografia de permeaÃÃo em gel e eletroforese em gel de poliacrilamida. A fraÃÃo AmII apresentou uma massa molecular de ~ 20 kDa e uma alternÃncia de α-galactose 4 ligada e β-galactose 3-ligada, substituÃdo com Ãsteres de sulfato e Ãteres de metil, juntamente com a ocorrÃncia de unidades de 3,6-anidro-α-galactoses. No modelo de dieta de 4 semanas, o tratamento com a fraÃÃo AmII nÃo alteraou o tamanho da placa aterosclerÃtica e demais caracterÃsticas de vulnerabilidade intraplaca, tais como lÃpido, neutrÃfilos, macrÃfagos, metaloprotease de matriz (MMP)-9 e conteÃdo de colÃgeno. No modelo de dieta de 11 semanas, o tratamento com a fraÃÃo AmII reduziu os conteÃdos de macrÃfago intraplaca e de fator tecidual (FT), em comparaÃÃo com animais tratados com veÃculo. O FT intraplaca co-localizou e positivamente correlacionou com Ãreas ricos em macrÃfagos. NÃo foram observadas alteraÃÃes no tamanho da placa aterosclerÃtica e nas outras caracterÃsticas de vulnerabilidade intraplaca, tais como conteÃdos de lipÃdios, neutrÃfilos, MMP-9 e do colÃgeno. AlÃm disso, a expressÃo de mRNA de MMPs, quimiocinas e marcadores genÃticos de polarizaÃÃo de linfÃcitos Th1/2/reg/17 nos arcos aÃrticos e nos baÃos dos camundongos nÃo foi alterada pelo tratamento AmII. In vitro, o tratamento com AmII reduziu de forma dose-dependente a quimiotaxia de macrÃfagos sem afetar a produÃÃo de FT. No geral, o tratamento crÃnico com AmII foi bem tolerado. Em conclusÃo, nossos resultados indicam que o tratamento com AmII em camundongos aterosclerÃticos reduziu o conteÃdo de macrÃfagos intraplaca, agindo sobre o recrutamento celular, e, concomitantemente, o conteÃdo de FT intraplaca de originado potencialmente de macrÃfagos.

Em estudos prévios, mostramos que uma nanoemulsão lipídica (LDE) é reconhecida e se liga aos receptores de LDL após sua injeção 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 utilizada como veículo para direcionar fármacos a essas células, diminuindo sua toxicidade e aumentando sua eficácia terapêutica. Anteriormente, reportamos que o tratamento com um derivado do paclitaxel, o oleato de paclitaxel, associado à LDE (PTX-LDE), reduziu em 60% a área lesionada de aortas de coelhos submetidos à dieta aterogênica, comparados a animais não tratados. No presente trabalho, avaliamos o efeito da associação de sinvastatina, medicamento hipolipemiante, e PTX-LDE, sobre a aterosclerose induzida por dieta em coelhos. Trinta e seis coelhos machos da raça Nova Zelândia foram submetidos à dieta enriquecida com 1% de colesterol durante oito semanas. A partir da quinta semana, os animais foram divididos em quatro grupos, de acordo com o tratamento: controle (solução salina EV), sinvastatina (2mg/kg/dia, VO), paclitaxel (PTX-LDE, 4mg/Kg/semana, EV), ou combinação de sinvastatina (2mg/Kg/dia, VO) com paclitaxel (PTX-LDE, 4mg/Kg/semana, EV). Após oito semanas, os animais foram sacrificados para análise das aortas. Em comparação aos controles, a área lesionada das aortas foi em torno de 60% menor, tanto no grupo paclitaxel, quanto no grupo da combinação, e em torno de 40% menor no grupo sinvastatina (p<0,05). A razão entre as camadas íntima/média foi menor nos grupos tratados, em relação ao grupo controle (controles, 0,35±0,22, sinvastatina, 0,10±0,07, paclitaxel, 0,06±0,16 e combinação, 0,09±0,05, p<0,0001). Os grupos combinação e sinvastatina apresentaram um aumento da porcentagem de colágeno nas lesões (combinação, 20% e sinvastatina, 22%), em comparação aos controles (11%) e ao grupo paclitaxel (12%), (p<0,0001). Houve uma diminuição da porcentagem de macrófagos na lesão em todos os grupos tratados (paclitaxel, 11%, sinvastatina, 8% e combinação, 5%), comparados ao grupo controle (30%), (p<0,0001). O grupo paclitaxel apresentou menor porcentagem de células musculares lisas na lesão (20%) em relação aos controles (33%), (p<0,0001), já na combinação, houve aumento dessa porcentagem (44%), (p<0,0001). A combinação com sinvastatina não aumentou a eficácia do tratamento com PTX-LDE na redução da área de lesões ateroscleróticas, porém, os efeitos adicionais sobre o perfil lipídico e na composição das lesões, observadas com o uso da combinação, são achados importantes, que sugerem benefícios no sentido de aumentar a estabilidade das placas ateroscleróticas, o que nos abre um caminho de pesquisa muito promissor.
In previous studies we have shown that a lipid nanoemulsion (LDE) is recognized and binds to LDL receptors after injection into the bloodstream. As those receptors are upregulated in cells with higher proliferation rates, as occurs in cancer and atherosclerosis, LDE can be used as a vehicle to direct drugs to those cells, diminishing toxicity and increasing therapeutic efficacy. Previously, we reported that treatment with antiproliferative agent paclitaxel derivative, paclitaxel oleate, associated with LDE (PTX-LDE), reduced by 60% the injured area of the aorta of rabbits subjected to atherogenic diet compared to untreated animals. In the current study we aim to test the effect of a combination of lipid-lowering drug simvastatin with PTX-LDE on diet-induced atherosclerosis in rabbits. Thirty-six male New Zealand rabbits were fed a 1% cholesterol diet for 8 weeks. Starting from week 5, animals were divided into four groups, according to the following treatments: controls (I.V. saline solution injections), simvastatin P.O. (2mg/kg/day), paclitaxel (PTX-LDE I.V. injections, 4mg/Kg/week), or paclitaxel-simvastatin combination (PTX-LDE I.V., 4mg/Kg/week + simvastatin P.O., 2mg/Kg/day). After 8 weeks, the animals were sacrificed for aorta evaluation. Compared to controls, the injured area was reduced by 60% in both paclitaxel and combination groups, and by 40% in simvastatin group (p<0,05). The intima/media ratio was reduced in treated groups, compared to control group (controls, 0,35±0,22, simvastatin, 0,10±0,07, paclitaxel, 0,06±0,16 and combination, 0,09±0,05, p<0,0001). Simvastatin and combination groups showed increased collagen content within the lesions (simvastatin, 22% and combination 20%), compared to controls (11%) and to paclitaxel group (12%), (p <0.0001). Macrophage content within the lesions was reduced in all treated groups (paclitaxel, 11%, simvastatin, 8% e combination, 5%), compared to controls (30%), (p <0.0001). The percentage of smooth muscle cells in the lesions was diminished in paclitaxel group (20%) compared to control group (33%), while the combination group showed increased percentage (44%) of smooth muscle cells in the lesions (p<0,0001). The combination of simvastatin did not improve the efficacy of the treatment with PTXLDE in reducing the area of atherosclerotic lesions, but the additional effects on lipid profile and lesion composition observed with the use of the combination are important findings that suggest benefits in order to enhance the stability of atherosclerotic plaques, which may lead us to a very promising research path.

With each heartbeat, major arteries experience circumferential expansion due to internal pressure changes. This pulsatile force is called cyclic strain and has been implicated in playing a pivotal role in the genetic regulation of vascular physiology and pathology. This dissertation investigates the hypothesis that in human umbilical vein endothelial cells (HUVEC), pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction. To determine expression and time-dependency of c-Myc in HUVEC, mRNA and protein expression of c-Myc under physiological (6-10% cyclic strain) and pathological conditions (20% cyclic strain) were studied. Both c-Myc mRNA and protein expression increased more than three-fold in HUVEC (P4-P5) cyclically-strained at 20%. This expression occurred in a time-dependent manner, peaking in the 1.5-2 hour range and falling to basal levels by 3 hours. Subsequently, the mechanism of c-Myc transcription was investigated by using specific inhibitors to modulate c-Myc transcriptional activation. These compounds, obtained from the University of Arizona Cancer Center, attenuated cyclic-strain-induced c-Myc transcription by about 50%. Having established this reduction in expression, it was investigated how these effects modulate downstream genes that are regulated by c-Myc. The results indicate that direct targeting of the c-Myc promoter may decrease stretch-induced gene expression of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA) and heat shock protein 60 (HSP60). These findings may help in the development of a novel therapeutic opportunity in vascular diseases.

INTRODUÇÃO: Sistemas nanométricos carreadores de fármacos, ao alcançarem a circulação sanguínea, se concentram em seus sítios de ação (\"drug-targeting\"), evitando tecidos saudáveis. O diâmetro médio e a polidispersidade de nanopartículas são parâmetros relevantes, pois podem influenciar no percurso pelo fluxo sanguíneo da partícula e na interação celular. A LDE, uma nanopartícula lipídica que mimetiza a lipoproteína de baixa densidade (LDL), é capaz de carrear fármacos como o quimioterápico paclitaxel para tecidos com alta taxa de proliferação celular, como por exemplo, lesões ateroscleróticas e tecidos neoplásicos. Assim, é relevante investigar a influência de diferentes tamanhos da LDE na captação celular e na eficácia terapêutica em aterosclerose experimental. OBJETIVO: Avaliar a influência de duas faixas de tamanhos da nanopartícula lipídica carreadora do quimioterápico paclitaxel (LDE-paclitaxel), na captação celular e na resposta terapêutica do tratamento da aterosclerose em coelhos submetidos à dieta rica em colesterol. MÉTODOS: A associação LDE-paclitaxel foi preparada por emulsificação por alta pressão. A separação da LDE-paclitaxel original em LDE-paclitaxel grande e LDE-paclitaxel pequena foi feita por ultracentrifugação por gradientes de densidade. Nos estudos com células endoteliais HUVEC foram avaliados citotoxicidade, internalização celular e detecção de apoptose/necrose. Para estudo em animal, foram utilizados coelhos New Zealand machos, com aterosclerose induzida por dieta, divididos em dois grupos: LDE-paclitaxel-grande (n=9) e LDE-paclitaxel-pequena (n=10). O tratamento com LDE-paclitaxel foi iniciado após 4 semanas da dieta. Aortas dos coelhos foram coletadas para análise macro e microscópica das lesões ateroscleróticas. RESULTADOS: A LDE-paclitaxel original foi caracterizada com diâmetro médio de 75nm; após a ultracentrifugação, a LDE-paclitaxel grande apresentou diâmetro médio de 83nm e a LDE-paclitaxel pequena de 40nm. Os ensaios de citotoxicidade mostraram que, após incubação por 24 horas, a LDE-paclitaxel pequena alcançou o IC50 com menor concentração que a LDE-paclitaxel grande. No ensaio de internalização, a LDE-paclitaxel pequena foi internalizada em menores concentrações e em menor tempo em comparação com as partículas da LDE-paclitaxel original ou LDE-paclitaxel grande. Nos ensaios para detecção de apoptose/necrose, LDE-paclitaxel, independentemente do tamanho, aumentou a porcentagem de células necróticas. A LDE-paclitaxel pequena também aumentou a percentagem de células apoptóticas, em comparação às outras partículas. No estudo in vivo, não houve diferença entre os tratamentos LDE-paclitaxel grande e LDEpaclitaxel pequena: a razão área de lesão/área total foi igual entre os grupos, assim como a quantificação de macrófagos e de células de músculo liso na íntima das aortas. CONCLUSÃO: O tamanho da LDE, apesar de ser um relevante parâmetro físico-químico, não influenciou no efeito antiaterosclerótico da associação LDE-paclitaxel. Portanto, em relação ao tamanho das partículas, a LDE-paclitaxel original, que possui ambas as populações, é eficienteno tratamento da aterosclerose experimental induzida por dieta rica em colesterol
INTRODUCTION: As nanometric drug carriers enter the blood circulation, they concentrate on their action sites, avoiding healthy tissue. The average diameter and polydispersity of nanoparticles are relevant parameters because they have influence on the particles course through the blood flow and on cell interaction. LDE, a lipid nanoparticle that mimics low-density lipoprotein (LDL), is capable of carrying chemotherapeutic drugs such as paclitaxel, for tissues with a high rate of cell proliferation, such as atherosclerotic lesions. Thus, it is important to investigate the influence of different sizes in the cellular uptake of LDE and therapeutic efficacy in experimental atherosclerosis. OBJECTIVE: To evaluate the influence of two size ranges of the lipid nanoparticle carrier of the chemotherapeutic drug paclitaxel (LDE-paclitaxel) in cellular uptake and therapeutic response in experimental atherosclerosis. METHODS: LDEpaclitaxel was prepared by emulsification by high energy. The separation of original-LDE-paclitaxel in large-LDE-paclitaxel particles and small-LDEpaclitaxel particles was performed by ultracentrifugation by density gradients. In studies with HUVEC endothelial cells, cytotoxicity, cell internalization and detection of apoptosis/necrosis were assessed. In in vivo study, New Zealand male rabbits, with atherosclerosis induced by cholesterol-rich diet, were divided into two groups: large-LDE-paclitaxel (n=9) and small-LDE-paclitaxel (n=10). Treatment with LDE-paclitaxel started after 4 weeks of diet. Aortas of the rabbits were collected for macroscopic and microscopic analysis of atherosclerotic lesions. RESULTS: The original-LDE-paclitaxel was characterized with an average diameter of 75nm. After ultracentrifugation, the large-LDE-paclitaxel showed average diameter of 83nm and small-LDE-paclitaxel 40nm. The cytotoxicity assay showed that, after incubation for 24 hours, small-LDE paclitaxel reached the IC50 in lower concentration than large-LDE paclitaxel. In internalization assays, small-LDE-paclitaxel was internalized in lower concentrations and shorter time as compared with the original and large particles. LDE-paclitaxel, independently of particle size, increased the percentage of necrotic cells. Small-LDE-paclitaxel was also able to increase the percentage of apoptotic cellsas compared with the original and large particles. In experimental study, there was no difference between large-LDE-paclitaxel and small-LDE-paclitaxel treatment: lesion area / total area ratio was similar between groups as well as the quantification of macrophages and smooth muscle cells of the intima of aortas.CONCLUSION: The size of the LDE, although an important physicochemical parameter, did not influence the antiatherosclerotic effect of LDE-paclitaxel. Therefore, with respect to particle size, the original-LDE-paclitaxel that has both populations is efficient to treat experimental atherosclerosis induced by a cholesterol-rich diet

Até o momento, pouco se sabe sobre a influência da modificação na composição do ateroma nas bordas dos stents e a ocorrência de alterações na geometria vascular. Este estudo objetiva correlacionar, utilizando de maneira seriada (pós-implante do stent e reestudo aos nove meses) o ultrassom monocromático e a Histologia Virtual®, as modificações na composição dos ateromas nas bordas proximais e distais de stents nãofarmacológicos e farmacológicos e as alterações ocorridas nas dimensões do vaso, luz e placa que possam explicar a ocorrência da reestenose nestes segmentos. Estudo prospectivo, de centro único, que randomizou (1:1) pacientes com síndrome coronária aguda para receberem stents nãofarmacológicos (Driver®, n=20 pacientes) ou farmacológicos (Cypher®, n=20 pacientes). Após a realização do procedimento, todos os pacientes submeteram-se a avaliação com ultrassom e Histologia Virtual®, que foi repetido ao final de nove meses de seguimento. O objetivo primário foi avaliar as modificações na área do vaso, luz e placa ao ultrassom e na composição do ateroma pela Histologia Virtual® no período entre o implante e o reestudo, buscando correlacionar as alterações no ateroma com as modificações na geometria vascular. Observou-se que na borda proximal, stents farmacológicos e não-farmacológicos tem um comportamento semelhante na avaliação ultrassonográfica, com tendência a remodelamento expansivo da área do vaso para compensar o crescimento na área da placa. Por outro lado, na borda distal, há menor crescimento da área da placa entre os pacientes tratados com stents farmacológicos, resultando em maior área da luz no reestudo de nove meses. Do ponto de vista da análise com Histologia virtual, nos dois grupos e em ambas as bordas houve redução do componente fibroso e núcleo necrótico com aumento no conteúdo fibrolipidico. Observou-se ainda importante correlação entre a variação do componente fibrótico e o aumento na área da placa (r=0.78, p=0.01). O uso de stents farmacológicos não se correlaciona com \"efeito de borda\". Ao contrário, parece haver menor crescimento da placa na borda distal destas endopróteses quando comparadas às sem fármaco. A modificação na composição do ateroma, com aumento do conteúdo fibro-lipídico pode explicar em parte estes achados.
To the present, little is known about the correlation between modifications in plaque composition at stent edges and the changes in vessel geometry. This study sought to evaluate, by serial grey-scale intravascular ultrasound (IVUS) and Virtual Histology(TM), the modifications in plaque composition at the edges of drug-eluting and bare-metal stents and the correlation of these findings with changes in the measuremntes of vessel, lumen and plaque area at those segments. Single-center, prospective and randomized (1:1) evaluation of 40 patients with acute coronary syndrome treated with bare-metal (Driver(TM), n=20 patients) or drug-eluting stents (Cypher(TM), n=20 patients). Following stent deployment, all individuals underwent gray scale IVUS and Virtual Histology(TM) evaluation, which were repeated at nine months. Primary endpoint included the modification in vessel, lumen and plaque area and in the composition of the plaque in the mean time between the baseline and follow-up procedure. Additionally, we tried to determine a correlation between plaque composition variation and changes in vessel geometry. At the proximal edge of both drug-eluting and bare-metal stents there was a trend to positive vessel remodeling which compensated the modest increase in plaque area. At the distal edge, patients treated with drug-eluting stents had less plaque growth resulting in a larger lumen area at follow-up. By Virtual Histology, there was a marked reduction in the % of fibrotic tissue and necrotic core in both edges of the two stents and a positive, strong correlation was seen between increase in % of fibrofatty component and augmentation in plaque area(r=0.78, p=0.01). The use of drug-eluting stents was not associated with \"edge effect\". On the contrary, patients treated with these devices experienced less plaque growth, especially at the distal edge of the stents. Modifications in plaque composition, with increase in fibrofatty content, might partially explain these findings.

Fundamentos: A caracterização precisa da interação da placa aterosclerótica no momento do implante do stent é crucial para o entendimento da complacência e da cicatrização vasculares. Objetivamos investigar se a composição da placa avaliada pela tomografia de coerência óptica (OCT), influencia as alterações agudas no procedimento índice do implante do stent e na cicatrização vascular no seguimento tardio. Métodos: Os pacientes tratados com um único tipo de stent eluidor de fármaco (cromo cobalto, eluidor de sirolimus e polímero bioabsorvível) foram incluídos prospectivamente, seguindo um protocolo com etapas de dilatações progressivas do vaso. As imagens de OCT sequenciais foram realizadas no procedimento índice (basal e a cada etapa do protocolo) e no seguimento tardio, co-registradas e analisadas a cada 0,6mm. A avaliação semiquantitativa da placa foi realizada dividindo-se secções transversas em 4 quadrantes, com cada quadrante rotulado de acordo com o seu componente mais prevalente (fibrótico, calcificado, lipídico, normal). A interação stent-vaso avaliada pela OCT foi utilizada como indicador substituto para lesão e cicatrização vasculares após o implante do stent. Resultados: Um total de 22 lesões (1stent/lesão) de 20 pacientes e 2298 seções transversas de OCT foram analisadas no procedimento índice. O reestudo com OCT foi realizado em 17 pacientes e 19 lesões (86%). O componente de placa predominante foi fibrótico (fibrótico = 46.84 ± 16%; lipídico = 17.63 ± 10.72%; calcificado = 4.63 ± 5.9%; normal = 29.16 ± 12.24; não analizável=1.74 ± 5.35%). Houve um aumento nas áreas da luz (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) e do stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001), com um aumento na área do prolapso tecidual (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segmentos com muito tecido fibrocalcificado tiveram áreas luminais menores ao longo das etapas da intervenção. Por outro lado, placas com muito conteúdo lipídico ou vaso normal tiveram maiores ganhos nas medidas das áreas luminais mínimas ao longo das dilatações sequenciais. Além disso, placas com muito tecido fibrocalcificado no momento basal apresentaram menor crescimento neointimal no seguimento tardio, enquanto que o grau de conteúdo lipídico e de vaso normal não tiveram impacto sobre a formação do tecido neointimal. Os indicadores substitutos de lesão vascular após o implante do stent correlacionaram-se significativamente com o crescimento neointimal no seguimento tardio. Conclusões: A composição tecidual das placas subjacentes influencia significativamente o comportamento mecânico agudo e a longo prazo dos vasos coronarianos submetidos ao implante de stent. Além disso, a lesão vascular após o implante do stent está potencialmente ligada ao futuro crescimento neointimal no seguimento tardio
Background Accurate characterization of atherosclerotic plaque interaction during stent deployment is crucial to understand vascular compliance and healing. We sought to determine whether plaque composition assessed by optical coherence tomography (OCT), influences acute changes at index procedure and vascular healing at follow up. Methods Patients treated with a single drug-eluting stent type (cobalt chromium with bioabsorbable polymer eluting sirolimus stent) were prospectively included, following a pre-defined step-by-step progressive vessel dilatation. Sequential OCT imaging were performed at the index procedure (baseline and at each time point of the protocol) and at follow up, co-registered and analyzed every 0.6mm for quantitative measurements. Semi-quantitative plaque assessment was performed at baseline by dividing cross-sections into 4 quadrants, with each quadrant labeled according to its most prevalent component (fibrotic, calcific, lipid). OCT assessments of stent-vessel interactions were used as a surrogate for vessel injury and healing after stent implantation. Results A total of 22 lesions (1stent/lesion) of 20 patients and 2298 OCT crosssections were analyzed at the index procedure. For an average of 19.7 months (591.88 ± 60.52 days), 17 of the patients and 19 lesions (86%) underwent OCT imaging at follow up. The predominant percentage plaque component was fibrotic (fibrotic = 46.84 ± 16%; lipid = 17.63 ± 10.72%; calcific = 4.63 ± 5.9%; normal = 29.16 ± 12.24; non-analyzable = 1.74 ± 5.35%). There was an increase in lumen (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) and stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001) areas, with an increase in tissue prolapse area (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segments with high fibrocalcific content tended to have decreased minimal luminal areas along the intervention time-points. Conversely, plaques with high lipid content had increased minimal luminal areas during sequential dilatations. Moreover, plaques with high fibrocalcific tissue at baseline had significantly smaller neointimal growth at follow-up, whereas the degree of lipid content or normal tri-layered vessel had no impact on neointimal formation. OCT surrogates of vessel injury after coronary stenting significantly correlated with neointimal growth at follow-up. Conclusions: Tissue composition of underlying plaques significantly influences the acute mechanical and the long-term behavior of coronary vessels undergoing stent implantation. In addition, vessel injury after coronary stenting is potentially linked to future neointimal growth at follow-up

博士
國立成功大學
基礎醫學研究所
95
Atherosclerotic vascular disease is the major cause of morbidity and mortality in many developed countries. Central to the pathogenesis of atherosclerosis is the inflammation in the arterial wall. New approaches to atherosclerosis-related diseases include novel uses of proven treatments and development of innovative agents. Statins are the most commonly prescribed agents for the treatment of hypercholesterolemia because of their efficacy in reducing low-density lipoprotein (LDL). Recent experimental and clinical evidence indicates the cholesterol-independent effects of statins. However, the genetic expression pattern changes in atherosclerotic lesions produced by statins are rarely studied. Cholesterol-fed apolipoprotein (apo) E-deficient mice were examined for the treatment effect of statin on aortic gene expression. The aortic gene expression affected by pravastatin was identified by the oligonucleotide microarray technology with Agilent gene chips. Microarray analysis of the expression of 20,281 murine genes in the aortas between the two groups indicated that 94 genes were significantly regulated. Thirty genes were up-regulated and 64 genes were down-regulated. Our study might provide insight into the clinical benefits of chronic statin treatment. Macrophages play an important role in the inflammatory process in atherosclerosis. Opioid peptides have been shown to modulate immunoresponse. Naloxone is a non-selective antagonist of the opioid receptors. Dextromethorphan (DM) is the d-isomer of the codeine analog levophanol, a dextrorotatory morphinan. It is widely used as a cough suppressant in cold and cough medications with a high safety profile. Both of them could inhibit activation of microglia, the resident macrophage in nervous systems. We investigated whether naloxone and DM could reduce macrophage activation and influence atherosclerotic lesion formation in mice. In our study, we found that naloxone and DM pretreatment significantly suppressed the production of proinflammatory factors and superoxide in macrophage and mice after stimulation. The novel anti-inflammatory effect of naloxone and DM reduced the spontaneous aortic atherosclerotic lesion formation in apoE-deficient mice and the carotid neointima formation in C57BL6 mice receiving carotid ligation. The study of naloxone and DM generate many new and exciting hypotheses to be tested in the future.

博士
國防醫學院
醫學科學研究所
92
Atherosclerosis is an inflammatory disease. Immune effector cells including monocytes and T lymphocytes have been shown to play important roles in the development of acute coronary syndrome. Moreover, these activated immune effector cells involved in the process of chronic inflammation, suggested to be a hallmark of atherosclerosis. It is a new subject to find out how Chinese and Western medicines modulate the inflammatory processes associated with atherosclerosis. Therefore, our studies pick out some drugs to evaluate how they can regulate immune effector cells. First, as known, interleukin-10 (IL-10) is an important mediator regulating the inflammatory responses. We wanted to recognize how carvedilol modulated in-vitro lipopolysaccharide (LPS)-induced IL-10 expression in monocytic cells. Both human peripheral blood monocytic cells and purified CD14+ monocytes belonging to primary cells were isolated from whole blood of healthy donors. U937 cells, one monocytic cell line, were cultured. Initially, these three monocytic cells were pre-treated with carvedilol (concentrations varied from 0.1-1.0 mM) for 2 hours and then LPS was added to induce IL-10 production for 3-24 hours before collecting supernatant. In the other hand, some U937 cells were long-term pretreated with both carvedilol and labetalol of varied concentrations for 1 to 2 months before LPS stimulation. We found that one-time carvedilol pretreatment at concentrations between 0.1 and 1.0 mM could not upregulate LPS-induced IL-10 production in human peripheral blood monocytic cells, purified CD14+ monocytes or U937 cells. Long-term labetalol pretreatment of U937 cells for 1-2 months at concentrations of 0.3-1.0 mM had no any effect on LPS-stimulated IL-10 production. However, long-term carvedilol pretreatment for U937 cells up to 1 to 2 months at concentrations of 0.1-1.0 mM could dose-dependently upregulate the LPS-stimulated IL-10 expression. Both carvedilol and labetalol pretreatment in concentrations of 0.1-1.0 mM showed no cytotoxic effects to U937 cells. Therefore, carvedilol could modulate the inflammation-based atherosclerosis via antioxidant effects to upregulate IL-10 production. Next, as reported, both granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-10 are important mediators regulating inflammatory responses associated with atherogenesis. Then, the effects of carvedilol on GM-CSF-induced IL-10 production were examined on human monocytic cell line, U937, and purified human monocytes. We showed that one-time carvedilol pretreatment at concentrations 0.3-10 mM dose-dependently inhibited GM-CSF-induced IL-10 production in U937 cells. In addition, we found carvedilol to be non-cytotoxic at concentrations equal to or less than 10 mM. However, at concentrations higher than 10 mM, carvedilol induced programmed cell death in U937 cells. The inhibition of GM-CSF-induced IL-10 production by carvedilol was also observed at the expression of mRNA. Furthermore, the down-regulated IL-10 production was demonstrated in GM-CSF-activated purified human peripheral blood monocytes. Second, long-term carvedilol pretreatment of U937 cells up to 1-2 months at concentrations of 1.0 mM mildly upregulated the production of IL-10 and IL-10 mRNA. Our observations that carvedilol modulated GM-CSF-induced IL-10 production may have some implication in understanding the broad-spectrum effects of carvedilol in regulating inflammatory reactions. The third part was to investigate the effect of Ginkgo biloba extract (GBE) on human peripheral T cells while activation. As known, GBE has antioxidant properties and has been applied clinically. However, it was unknown whether GBE can conduct its biological effects on human peripheral T cells. To gain insight into the mechanism of this drug’s action, we investigated the effects of GBE on activated human peripheral T lymphocytes. We found that GBE can dose-dependently inhibit cytokine production such as interleukin-2, interleukin-4, tumor necrosis factor-alpha and interferon-gamma from activated T cells. Further, we also showed that GBE suppressed the expression of IL-2 receptor-alpha (CD25), cell surface markers induced after T-lymphocyte activation. Molecular investigation further indicated that the inhibition of both human T-lymphocyte and Jurkat-cell activation correlated with the down-regulation of activator protein-1 (AP-1) DNA-binding activities, assessed by electrophoretic mobility shift assay. Results from transient transfections disclosed that GBE also inhibited AP-1 transcriptional activities in Jurkat cells. Finally, we revealed that GBE is unique in its ability to inhibit the activation of c-Jun NH2-terminal protein kinase (JNK). Our observations might extend a potential therapeutic mechanism of GBE with cell-mediated capacity, namely the inhibition of activated T lymphocytes via down-regulation of activator protein-1 activity, to prevent or treat some of disease processes associated with inflammatory pathology. Finally, irbesartan was used to study its effects on human T cells. It is known to be a promising antihypertensive drug with beneficial effects on atherosclerotic processes. In the progression of atheroslcerosis, human T lymphocytes play an important role. Nowadays, it is unknown how irbesartan modulates activated human T lymphocytes. To gain insight into the mechanisms of irbesartan, we investigated the effects of irbesartan on activated human T lymphocytes. Primary human T lymphocytes were isolated from whole blood. Cytokines were determined by ELISA. The AP-1 and related protein activities were determined by electrophoretic mobility shift assays, kinase assays, Western blotting and transfection assays. Obviously, irbesartan could inhibit production of both tumor necrosis factor-alpha and interferon-gamma from activated T cells, especially in therapeutic concentrations. Molecular investigation further indicated that the inhibition of activated human T-lymphocyte specifically correlated with the down-regulation of AP-1 DNA-binding activities. In-vivo study disclosed that irbesartan also inhibited AP-1 transcriptional activities in Jurkat T cells. Finally, we revealed that irbesartan was unique in its ability to inhibit the activation of both c-Jun NH2-terminal protein kinase and p38 MAPK. Our studies disclosed that irbesartan had cell-mediated capacity, namely by inhibiting activated T lymphocytes via down-regulation of AP-1 activity to attain its novel therapeutic effects on inflammation-based atherosclerotic diseases. From above studies, we know that these three kinds of drugs could attain their clinically beneficial effects in part through modulating the activation of immune effector cells.

Chan Yin Ling.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 147-167).
Abstracts in English and Chinese.
ABSTRACT --- p.III
ACKNOWLEDGEMENT --- p.X
TABLE OF CONTENTS --- p.XI
ABBREVIATIONS --- p.XV
LIST OF FIGURES --- p.XVII
LIST OF TABLES --- p.XXI
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Introduction to Cardiovascular Disease and Atherosclerosis --- p.1
Chapter 1.1.1 --- Cardiovascular Disease --- p.1
Chapter 1.1.2 --- A therosclerosis --- p.3
Chapter 1.1.2.1 --- Structure of Arteries --- p.4
Chapter 1.1.2.2 --- Pathophysiology of Atherosclerosis --- p.5
Chapter 1.1.2.3 --- Endothelial Dysfunction --- p.8
Chapter 1.1.3 --- Current Western Therapies --- p.11
Chapter 1.1.3.1 --- Surgery --- p.11
Chapter 1.1.3.2 --- Western Medications --- p.13
Chapter 1.1.4 --- Traditional Chinese Medicine --- p.17
Chapter 1.1.4.1 --- Long History --- p.17
Chapter 1.1.4.2 --- As Alternative Medicine --- p.18
Chapter 1.1.4.3 --- Modernization of Chinese Medicine --- p.19
Chapter 1.2 --- Introduction and Selection of Chinese Medicine --- p.20
Chapter 1.2.1 --- Selection ofTCM Formulation from Pharmacopoeia --- p.20
Chapter 1.2.1.1 --- Compound Formulation --- p.20
Chapter 1.2.2 --- Introduction to the Herbal Medicines --- p.21
Chapter 1.2.2.1 --- Danshen (Salvia miltiorrhiza) --- p.21
Chapter 1.2.2.2 --- Gegen (Puerariae thomsonii and Puerariae lobata) --- p.22
Chapter 1.2.2.3 --- Yanhu (Corydalis yanhusuo) and its Exclusion --- p.24
Chapter 1.2.3 --- Source and Authentication of the Herbal Medicines --- p.25
Chapter CHAPTER 2 --- OPTIMIZATION OF DANSHEN-GEGEN FORMULA --- p.26
Chapter 2.1 --- Project History --- p.26
Chapter 2.2 --- aims for the present study --- p.27
Chapter 2.3 --- Methods and Materials --- p.30
Chapter 2.3.1 --- Extracts --- p.30
Chapter 2.3.2 --- Extraction Process --- p.31
Chapter 2.3.3 --- In vitro Antioxidation Model --- p.33
Chapter 2.3.4 --- Ex vivo Vasodilation Model --- p.35
Chapter 2.3.5 --- Statistical Analysis --- p.38
Chapter 2.4 --- Results --- p.39
Chapter 2.4.1 --- Vasodilation Results --- p.39
Chapter 2.4.2 --- Antioxidation Results --- p.43
Chapter 2.5 --- Discussion --- p.46
Chapter 2.6 --- Further Modification of the Formula --- p.49
Chapter 2.6.1 --- Extracts --- p.49
Chapter 2.6.2 --- Results --- p.49
Chapter 2.7 --- discussion --- p.52
Chapter CHAPTER 3 --- MARKER CHEMICAL CONTENTS OF HERBAL EXTRACTS AND THEIR PHARMACOLOGICAL PROPERTIES --- p.56
Chapter 3.1 --- HPLC Analysis of Marker Contents --- p.56
Chapter 3.1.1 --- Methods --- p.57
Chapter 3.1.2 --- Results --- p.58
Chapter 3.1.2.1 --- HPLC Chromatograms --- p.59
Chapter 3.1.2.2 --- Content Percentage of Marker Compounds --- p.63
Chapter 3.1.3 --- Discussion --- p.64
Chapter 3.2 --- Studies on Marker Compounds --- p.65
Chapter 3.2.1 --- Introduction --- p.65
Chapter 3.2.2 --- Methods and Materials --- p.67
Chapter 3.2.2.1 --- Source of Pure Compounds --- p.67
Chapter 3.2.2.2 --- Purification and Identification of SAB --- p.68
Chapter 3.2.2.3 --- Vasodilation model --- p.70
Chapter 3.2.2.4 --- Antioxidation Model --- p.71
Chapter 3.2.2.5 --- Structures of Pure Compounds --- p.72
Chapter 3.2.3 --- Results --- p.73
Chapter 3.2.3.1 --- Vasodilation Results --- p.73
Chapter 3.2.3.2 --- Antioxidation Results --- p.76
Chapter 3.3 --- Discussion --- p.79
Chapter 3.4 --- Synergistic Effect Study --- p.85
Chapter 3.4.1 --- Introduction --- p.85
Chapter 3.4.2 --- Methods --- p.85
Chapter 3.4.3 --- Results --- p.86
Chapter 3.4.4 --- Discussion --- p.88
Chapter 3.5 --- STUDY ON 3'-HYDROXYPlIERARIN AND 3'-METHOXYPUERARIN PURIFIED FROM YFGE --- p.90
Chapter 3.5.1 --- 3 '-hydroxypuerarin and 3'-methoxypuerarin --- p.90
Chapter 3.5.2 --- Methods and Materials --- p.91
Chapter 3.5.2.1 --- Purification by HPLC semi-preparation --- p.91
Chapter 3.5.2.2 --- Bioassays --- p.93
Chapter 3.5.3 --- Results --- p.94
Chapter 3.5.3.1 --- Vasodilation Study --- p.94
Chapter 3.5.3.2 --- Antioxidative Effect of Yege --- p.95
Chapter 3.5.4 --- Discussion
Chapter CHAPTER 4 --- MECHANISTIC STUDY --- p.98
Chapter 4.1 --- Introduction --- p.98
Chapter 4.1.1 --- Nitric Oxide-mediated Vasodilation --- p.99
Chapter 4.1.2 --- Prostacyclin-mediated Vasodilation --- p.100
Chapter 4.1.3 --- EDHF-mediated Vasodilation --- p.101
Chapter 4.1.4 --- Endothelium-dependent and -independent Vasodilations --- p.103
Chapter 4.2 --- Methods and Materials --- p.104
Chapter 4.3 --- Results --- p.107
Chapter 4.3.1 --- Danshen-Gegen Formula (DY80) --- p.107
Chapter 4.3.2 --- Salvianolic acid B --- p.112
Chapter 4.3.3 --- Daidzein --- p.117
Chapter 4.4 --- Discussion --- p.121
Chapter CHAPTER 5 --- STUDY ON LIPID PEROXIDATION AND UPTAKE BY MACROPHAGES --- p.128
Chapter 5.1 --- Study of DY 80 and SAB on Copper-ion induced Low Density Lipoprotein Oxidation --- p.128
Chapter 5.1.1 --- Pathologic Role of oxidized Low Density Lipoprotein --- p.128
Chapter 5.1.2 --- Antioxidants in Low Density Lipoprotein and Role of Transition Metals --- p.129
Chapter 5.1.3 --- Methods and Materials --- p.130
Chapter 5.1.4 --- Results --- p.131
Chapter 5.1.5 --- Discussion --- p.133
Chapter 5.2 --- Study of Scavenger Receptor Regulation in Macrophages --- p.135
Chapter 5.2.1 --- Introduction --- p.135
Chapter 5.2.2 --- Methods and Materials --- p.136
Chapter 5.2.3 --- Results --- p.139
Chapter 5.2.4 --- Discussions --- p.140
Chapter CHAPTER 6 --- General Discussion --- p.143
REFERENCES --- p.147

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
No âmbito da unidade curricular de Estágio Curricular do Mestrado Integradoem Ciências Farmacêuticas da Faculdade de Farmácia da Universidade de Coimbra,o presente documento engloba dois relatórios de estágio, sob a forma de uma análiseSWOT (Strengths, Weaknesses, Opportunities and Threats). O primeiro é referenteao estágio realizado em Farmácia Comunitária, durante o período de 8 de Janeiro a13 de Abril, na Farmácia Gama, em Viseu. O segundo, realizado no INFARMED,Autoridade Nacional do Medicamento e Produtos de Saúde, I.P., durante o período deMaio a Julho de 2018, é referente ao estágio curricular realizado em AssuntosRegulamentares do Medicamento.Ainda neste documento é incluída a monografia intitulada “Alternativas àsestatinas: novos fármacos em ascensão no controlo da aterosclerose”. Aaterosclerose é o principal fator de risco para o desenvolvimento de doençascardiovasculares. Embora seja uma doença multifatorial, a dislipidémia e, emparticular, a hipercolesterolémia-LDL é um fator de risco crucial reconhecido. Sendoum problema crítico na sociedade atual e, uma vez que a terapêutica de primeira linhadirigida à dislipidémia falha numa grande parte da população, existe cada vez maisuma aposta clara na procura de novos alvos terapêuticos. Neste sentido, está emcurso o desenvolvimento de novas moléculas que têm apresentado resultadospromissores como linha de tratamento alternativa ou complementar às estatinas,fármacos estes considerados uma das pedras angulares da prevenção das doençascardiovasculares. Atuando em diferentes locais e apresentando diferentesmecanismos de ação, têm como objetivo comum a diminuição da concentraçãoplasmática das lipoproteínas aterogénicas, com a consequente diminuição do riscocardiovascular.
Within the curricular unit of the Integrated Master's degree in PharmaceuticalSciences of the Faculty of Pharmacy of the University of Coimbra, the presentdocument includes two internship reports, in the form of a swot analysis (strengths,weakness, opportunities and threats). The first one refers to internship in communitypharmacy from January 8 to April 13 2018 at the Farmácia Gama, in Viseu. The secondone was done in the INFARMED, National Authority of Medicines and Health Products,IP, from May to July 2018, and was concerned with regulatory affairs of medicines.Also included in this document, there is a monograph entitled "StatinAlternatives: New Rising Drugs in the Control of Atherosclerosis." Atherosclerosis isthe major risk factor for the development of cardiovascular disease. Althoughatherosclerosis is a multifactorial disease, dyslipidemia, in particular,hypercholesterolemia associated with LDL, is a crucial well recognized risk factor.Considering the relevance of this problem in today's society, and that statins, the firstlinetherapy for pharmacological lipid modification fail in a large part of the population,there is an increasingly interest on the search for new therapeutic targets. In this sense,the development of new promising molecules as alternative or complementarytherapeutics to statins is in course. Acting on different targets and presenting differentaction mechanisms, they have as their common goal the reduction of the plasmaticconcentration of atherogenic lipoproteins and consequently the reduction ofcardiovascular risk.

碩士
國立成功大學
藥理學研究所
92
Abstract 　　Calcium and cholesterol play an important role in the formation of atherosclerosis. Feeding the mixture of calciferol and cholesterol induces the atherosclerosis in rats. This atherosclerotic aorta was characterized by medial calcification and intimal cell proliferation. However, it is unknown whether the mixture of cholesterol and calciferol also causes gastric hemorrhage and mucosal damage. The aim of our study is observing the gastric oxidative stress and mucosal damage induced by the mixture of calciferol and cholesterol, and we evaluated the protective effects of several drugs. The effects of these drugs were significantly attenuated in the VD+CHOL as compared with the control. Male Wistar rats were challenged intragastrically with 1ml vegetable oil containing calciferol (4.5mg/kg) and cholesterol (22.5mg/kg), once daily and for 9 days. We fed normal rats with vegetable oil alone. During gastric surgery, rat stomachs were irrigated with physiological acid solution or normal saline for 3hrs. After that rats were sacrificed. We observed the increased level of acid back-diffusion, mucosal lipid peroxide content and hemorrhage ulcer area, and the decreased mucosal glutathione content in atherosclerotic rats. Besides, there was a high correlation between mucosal lipid peroxide levels and ulcer areas. Daily feeding clofibrate (300mg/kg), verapamil (20mg/kg), lovastatin (10mg/kg) or lysozyme (500mg/kg) effectively protected gastric mucosa in atherosclerotic rats. In conclusion, the mixture of calciferol and cholesterol may be produced gastric oxidative stress, which is an important factor in the formation of hemorrhagic ulcer in atherosclerotic rats.

博士
國立交通大學
應用化學系碩博士班
102
Atherosclerotic cardiovascular disease is a leading cause of death. Sudden rupture of a vulnerable atherosclerotic plaque triggers thrombosis and causes a deadly event such as ischemic stroke or myocardial infarction. Pathogenesis of atherosclerosis includes an accumulation of low-density lipoprotein (LDL) in the vascular wall and its oxidative modification by endogenous reactive oxygen species (ROS). Vascular inflammation induced by the accumulation of oxidatively modified LDL (oxLDL) plays a key role in the vulnerability of atherosclerotic plaque and increases the risk of cardiovascular thrombotic events. As a result, the development of methods for the assessment of such symptoms (i.e., vascular accumulation of LDL and its oxidative modification, and plaque inflammation) is important for the clinical diagnosis, and for the evaluation of the therapeutic efficacy of medications, targeting atherosclerosis. Toward this end, my PhD work focuses on the development of: (1) A quantum dots based molecular probe for the real-time and absolute quantifying ROS in vivo; (2) Fluorescence molecular imaging for the evaluation of plaque inflammation; (3) A platform (comprising confocal fluorescence imaging, Raman spectral analysis, and hypercholesterolemic zebrafish) for the direct assessment of vascular LDL deposition and oxidation in vivo and in situ, and its application for drug screening. Facilitated with these unique approaches, I demonstrated for the first time (1) Absolute quantification of endogenous HOCl in leukocyte; (2) Evaluation of the inflammation of individual atherosclerotic plaques in zebrafish in vivo; (3) Evaluation of the therapeutic activity of anti-atherosclerotic drugs (Atorvastatin and Ezetimibe) on individual plaques in zebrafish in vivo. I envisage that my approach will not only improve our understanding of atherogenesis but also expedite the screening of drugs and clinical diagnosis targeting atherosclerosis.

A vast majority of heart attacks occur due to rapid progression of plaque buildup in the coronary arteries that supply blood to the heart muscles. The diseased arteries can be treated with drugs delivered locally to vulnerable plaques—ones that may rupture and release emboli, resulting in the formation of thrombus, or blood clot that can cause blockage of the arterial lumen. In designing these local drug delivery devices, important issues regarding drug distribution and targeting need to be addressed to ensure device design optimization as physiological forces can cause the local concentration to be very different from mean drug tissue concentration estimated from in vitro experiments and animal studies. Therefore, the main objective of this work was to develop a computational tool-set to support the design of a catheter-based local drug delivery system that uses nanoparticles as drug carriers by simulating drug transport and quantifying local drug distribution in coronary artery walls. Toward this end, a three dimensional mathematical model of coupled transport of drug and drug-encapsulated nanoparticles was developed and solved numerically by applying finite element based isogeometric analysis that uses NURBS-based techniques to describe the artery wall geometry. To gain insight into the parametric sensitivity of drug distribution, a study of the effect of Damkohler number and Peclet number was carried out. The tool was then applied to a three-dimensional idealized multilayered model of the coronary artery wall under healthy and diseased condition. Preliminary results indicated that use of realistic geometry is essential in creating physiological flow features and transport forces necessary for developing catheter-based drug delivery design procedures. Hence, simulations were run on a patient-specific coronary artery wall segment with a typical atherosclerotic plaque characterized by a lipid pool encased by a thin fibrous cap. Results show that plaque heterogeneity and artery wall inhomogeneity have a considerable effect on drug distribution. The computational tool-set developed was able to successfully capture trends observed in local drug delivery by incorporating a multitude of relevant physiological phenomena, and thus demonstrated its potential utility in optimizing drug design parameters including delivery location, nanoparticle surface properties and drug release rate.
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Η οπτική συνεκτική τομογραφία με τη χρήση συχνοτήτων ( FD-OCT) είναι μια ενδαγγειακή απεικονιστική μέθοδος που χρησιμοποιεί εγγύς στο υπέρυθρο φως, για να παράγει υψηλής ανάλυσης εικόνες του τοιχώματος του αυλού του αγγείου. Όπως και στην τεχνολογία υπερήχων, εκπέμπεται φωτεινή ενέργεια η οποία ανακλάται και εξασθενεί, σύμφωνα με την υφή του προσπιπτομένου ιστού. Το OCT μπορεί να απεικονίσει, με ανάλυση από 10 έως 20 μm, μικροδομές του αγγειακού τοιχώματος με εξαίσια λεπτομέρεια. Μέχρι σήμερα, η δυνατότητα εφαρμογής της μεθόδου είχε περιοριστεί σε μικρές αρτηρίες διαμέτρου έως 4mm και δεν είχε εφαρμοστεί in vivo στα αγγεία των κάτω άκρων, κάτωθεν του επιπέδου των βουβώνων. Σκοπός της παρούσας μελέτης είναι να αναφερθεί για παγκοσμίως πρώτη φορά η ασφάλεια και η σκοπιμότητα της απεικόνισης με Οπτική Συνεκτική Τομογραφία του αρτηριακού άξονα των κάτω άκρων , κάτωθεν του επιπέδου της βουβώνας ( μηροϊγνυακός άξονας και κνημιαία αγγεία), καθώς και οι σχετιζόμενες με το FD-OCT επιπλοκές. Επιπρόσθετα, να διερευνηθούν για πρώτη φορά, με τη χρήση FD-OCT, τα χαρακτηριστικά του αγγειακού τοιχώματος του ανωτέρω άξονα (τόσο πριν όσο και μετά από αγγειοπλαστική ή/και τοποθέτηση stent), η μορφολογία της αθηρωματικής πλάκας, η μορφολογία και η ποσοτικοποίηση της υπερπλασίας του νέου εσωτερικού χιτώνα (neointima) εντός του stent, η επαναστένωση εντός του stent (ISR) και η κακή εναπόθεση (malapposition) των stent struts σε μια σειρά από ασθενείς που πάσχουν από περιφερική αρτηριοπάθεια (PAD). Μελετήθηκαν, με ποσοτική ανάλυση του αυλού τους (Quantitative vascular analysis), αρτηρίες με διάμετρο έως 7 χιλιοστά. Μικτά χαρακτηριστικά από περιοχές πλούσιες σε λιπίδια, εναποθέσεις ασβεστίου και ασβεστοποιημένες πλάκες, νεκρωτικές περιοχές και ίνωση εντοπίστηκαν σε όλες τις απεικονιζόμενες αθηροσκληρωτικές βλάβες. Ωστόσο, με βάση το επικρατέστερο από τα παραπάνω απεικονιστικά χαρακτηριστικά, οι βλάβες στο πλαίσιο της έρευνας ταξινομήθηκαν ως αμιγώς ινωτικές, ως ινοασβεστοποιημένες, ως πλούσιες σε λιπίδια και τέλος ως νεκρωτικές/ασβεστοποιημένες. Συσσώρευση των μακροφάγων εντός της αθηρωματικής πλάκας σημειώθηκε σε μικρό ποσοστό των de novo αθηρωματικών αλλοιώσεων. Ποικίλοι βαθμοί υπερπλασίας του νέου έσω χιτώνα απεικονίσθηκαν σε όλες τις περιπτώσεις ISR αλλοιώσεων, με καθαρά ινωτικά χαρακτηριστικά και σημαντική νεοαγγείωση σε κάποιες από αυτές. Η νεοαγγείωση συνέπεσε με το επίπεδο της μέγιστης στένωσης του αγγειακού αυλού. Σημαντικού βαθμού διαχωρισμός με μεγάλο περιορισμό του αγγειακού αυλού, τέτοιος ώστε να απαιτηθεί να τοποθετηθεί ενδοαυλικό stent, ανιχνεύθηκε σε αρκετές περιπτώσεις της de novo αθηρωμάτωσης. Η ψηφιακή αφαιρετική αγγειογραφία παρέλειψε να προσδιορίσει μεγάλο ποσοστό των σοβαρών διαχωρισμών μετά από αγγειοπλαστική. Η νεοαθηροσκλήρυνση εντός του νέου έσω χιτώνα των κνημιαίων φαρμακευτικών stents (DES), είναι ένα συχνό εύρημα τόσο στους συμπτωματικούς όσο και στους ασυμπτωματικούς ασθενείς. Μπορούμε να υποθέσουμε ότι, κατά αναλογία με τα εμφυτευμένα DES στα στεφανιαία αγγεία, η ελαττωματική ενδοθηλιοποίηση που προκαλείται από την εκλυόμενη φαρμακευτική ουσία, μαζί με την νεοαγγείωση που αναπτύσσεται μεταξύ των stent struts, μπορούν να υποδαυλίσουν την νεοαθηροσκλήρυνση εντός του νέου έσω χιτώνα των κνημιαίων DES, η οποία μπορεί να οδηγήσει σε επαναστένωση εντός του stent (ISR) και απώλεια του εμβαδού του αυλού των περιφερικών αρτηριών. Οι παρατηρήσεις της μελέτης αυτής θέτουν σε αμφισβήτηση το παραδοσιακό τρόπο κατανόησης της περιφερειακής επαναστένωσης εντός του stent ως μιας απλής υπερπολλαπλασιαστικής απάντησης στο βαρότραυμα. Η απεικόνιση με FD-OCT είναι ένα βέλτιστο πειραματικό εργαλείο για την αξιολόγηση της εξέλιξης της αθηροσκληρωματικής νόσου και την επαναστένωση του αγγείου. Μπορεί να παρέχει υψηλής ευκρίνειας ενδοαγγειακή απεικόνιση κατά τη διάρκεια αγγειοπλαστικών επεμβάσεων στα κάτω άκρα και θα μπορούσε να αποδειχθεί κλινικά χρήσιμο για τον προσδιορισμό της εντός του stent πρόπτωσης ιστού και του strut malapposition. Παρ 'όλα αυτά, δεν πρέπει να χρησιμοποιηθεί ως εργαλείο για τη συνήθη κλινική πρακτική μέχρι να προκύψουν στοιχεία από περαιτέρω κλινικές δοκιμές για τον καθορισμό των ειδικών ενδείξεων της απεικόνισης με FD-OCT στις περιφερικές αρτηρίες.
Optical coherence tomography (OCT) is a catheter-based imaging method that employs near-infrared light to produce high-resolution intravascular images. OCT can readily visualize vessel microstructure at a 10- to 20-μm resolution with exquisite detail. To date, however, applicability of the method has been limited to small diameter arteries (≤4 mm). To the best of the author’s knowledge, this study is the first worldwide that demonstrates the safety and clinical feasibility of frequency domain Optical Coherence Tomography (FD-OCT) imaging of infrainguinal vessels in vivo during infrainguinal angioplasty procedures. It is also the first study that reports the use of intravascular FD-OCT to detect and characterize in-stent neointimal tissue following infrapopliteal drug eluting stent (DES) placement in patients suffering from critical limb ischemia. Quantitative lumen analysis of arteries with diameter up to 7 mm was performed. High-resolution OCT images provided exquisite two-dimensional axial and longitudinal views of the infrainguinal arteries and allowed thorough investigation of a variety of angioplasty sequela, including and not limited to intimal tears and dissection flaps, white and red thrombus, stent mesh malapposition, and intrastent plaque prolapse. Of interest, OCT identified cases of suboptimal postangioplasty outcome that single-plane subtraction angiography did not recognize and accounted. Mixed features of lipid pool areas, calcium deposits and calcified plaques, necrotic areas, and fibrosis were identified in all of the imaged atherosclerotic lesions. However, based on the predominant baseline imaging findings, lesions under investigation were classified as purely fibrotic, fibrocalcific, mostly lipid-laden and necrotic/calcified. Intraplaque accumulation of macrophages was noted in some of de novo atheromatic lesions. Varying degrees of neointimal hyperplasia were demonstrated in all cases of in stent restenosis (ISR) lesions with purely fibrotic features and considerable neovascularization in some of them. The latter finding coincided with the level of maximum vessel stenosis in all cases. Neoatherosclerosis following infrapopliteal DES placement is a frequent finding in both symptomatic and asymptomatic patients. Our preliminary observations allow us to speculate that analogous to coronary implanted DES, defective endothelialization induced by the eluted drug, along with neovascularization developing between the stent struts, may incite neointimal neoatherosclerosis, which may result in ISR and lumen loss of the peripheral arteries. It also seems that infrapopliteal neoatherosclerosis may be a significant contributing factor for ISR rather than a minor and sporadic process, highlighting the clinical significance of the phenomenon. Our observations put in dispute the traditional way of understanding peripheral in-stent restenosis as a simple hyperproliferative response to barotraumas and may explain the paramount importance of aggressive risk factor modification strategies. Neointimal neoatherosclerosis as identified by FD-OCT may have a role in the development of below-the-knee restenosis and thus warrants further investigation by larger controlled studies. Moreover, it may prove clinically useful for the determination of intrastent tissue prolapse and strut malapposition. FD-OCT should not be utilized as a tool for routine clinical practice until evidence from further clinical trials emerge to determine the specific indications for OCT imaging of the peripheral arteries.