京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20797号 / 医博第4297号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 湊谷 謙司, 教授 齊藤 博英, 教授 Shohab YOUSSEFIAN / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Atherosclerosis and post-angioplasty neointimal proliferation, which are leading causes of cardiovascular morbidity and mortality, develop as a result of chronic or acute vascular injury producing inflammatory and proliferative responses in the vessel wall. Glucocorticoids, the stress hormones produced by the adrenal cortex, have anti-inflammatory and anti-proliferative characteristics and can also influence systemic cardiovascular risk factors. The systemic levels of these hormones are controlled by the hypothalamic pituitary adrenal axis. However, there is also a tissue-specific pre-receptor regulation of these hormones by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type 1 regenerates active glucocorticoids within the cells and type 2 inactivates glucocorticoids. Whilst it has been shown that the inhibition of 11β-HSD1 has favourable effect on cardiovascular risk factors and the inhibition of 11β-HSD2 results in hypertension; the effect of these enzymes on vascular lesion development is not known. The work described in this thesis tested the hypothesis that 11β-HSD1 inhibition reduces vascular lesion development due to improvement in cardiovascular risk factors, whereas 11β-HSD2 inhibition leads to adverse vascular remodelling. Apolipoprotein-E deficient (ApoE-/-) mice fed on western diet were used to study atherosclerosis, whereas neointimal proliferation was investigated using a well-established mouse model of wire-angioplasty. Vascular lesions were assessed using novel imaging and standard histological techniques. 11β-HSD1 inhibition reduced the size of atherosclerotic lesions and improved markers of plaque stability with a reduction in lipid content and increase in collagen content of the plaques. This was associated with a reduction in weight gain and blood pressure but without any effect on lipid profile. 11β-HSD1 inhibition did not produce any significant effect on neointimal proliferation in C57Bl/6J mice. However in ApoE-/- mice, 11β-HSD1 inhibition reduced neointimal proliferation with corresponding increase in size of patent lumen and with an associated reduction in macrophage content of neointimal lesions. 11β-HSD2 deletion produced an outward remodelling in un-injured vessels but there was no effect on neointimal proliferation after wire-angioplasty. Administration of a selective mineralocorticoid antagonist, eplerenone, reduced neointimal lesions significantly but to a similar degree in both C57Bl/6J and 11β-HSD2-/- mice, associated with a significant reduction in macrophage content of lesions but without any effect on blood pressure. Data in this thesis highlight the potential therapeutic application of 11β-HSD1 inhibition in reducing the size and vulnerability of atherosclerotic plaques and also reduction in neointimal proliferation (and hence post-angioplasty restenosis) in high risk patients with „metabolic syndrome‟ phenotype. The results also indicate that 11β-HSD2 has a limited, if any, role to play in the development of neointimal lesions.

INTRODUCTION: Specialized pro-resolving lipid mediators (SPM) such as resolvin-D1 (RvD1) act to resolve vascular inflammation and may guard against the progression of restenosis following cardiovascular interventions. Stimulating synthesis of these mediators directly in vascular cells may increase their local availability, and thus, protect against restenotic injury. However, the ability of endothelial (EC) and vascular smooth muscle cells (VSMC) to produce SPMs from their polyunsaturated fatty acid precursor decosahexaenoic acid (DHA) via lipoxygenase (LO) enzymatic transformation remains unknown. We sought to determine whether vascular cells produce SPMs from DHA and, if they do, how inflammation and mechanical injury of the vasculature alter biosynthesis. METHODS: Primary cultures of human saphenous vein endothelial and smooth muscle cells were treated with DHA in cell culture media (+ 10% serum) for 4h-24h. Freshly dissected rabbit aorta was incubated intact or following gentle endothelial denudation in cell culture media (+10% serum) with or without DHA for 48h. SPM levels in media were quantified by LC-MS/MS and ELISA and lipoxygenase expression and localization were assessed by western blotting and immunofluorescence staining, respectively. RESULTS: EC and SMC receiving media without DHA did not synthesize SPMs within the detection limits of the assay, whereas DHA treatment produced 17-HDHA, 14-HDHA, Mar1, RvD5, RvD2, and a dose and time-dependent increase in RvD1 production in EC (10.1 ±1.0 pg for 1000nM at 24h) and SMC (7.4 ± 0.2 pg for 1000nM at 24h). Intact rabbit aorta incubated in DHA+ media produced 0.24 ± 0.05 pg RvD1/mg tissue whereas aorta incubated in DHA− media produced 0.13 ± 0.007 pg RvD1/mg tissue. Moreover, EC-denuded aortas produced less RvD1/mg tissue than intact aortas. 5-LO was expressed in both cell types, however DHA induced 5-LO expression in EC (1.3 fold -DHA) but not in SMC. DHA promoted a nuclear to cytoplasmic shift of 5-LO in both EC and SMC. Finally, TNF-α stimulated an increase in RvD1 production in EC. CONCLUSIONS: Human vascular cells and rabbit vascular tissue can biosynthesize SPMs de novo from their precursor DHA, signifying a new source of SPMs in the vasculature.

Vascular access dysfunction is a cause of morbidity and mortality in chronic kidney disease (CKD) patients that require hemodialysis. The major cause of vascular access failure is venous stenosis due to neointimal hyperplasia (NH). Vascular smooth muscle cells (VSMC) are critical for the development of NH lesions, as they have the ability to modulate their phenotype from a "contractile" to a "synthetic" phenotype in the presence of uremia, through the regulation of sensor genes for uremia danger signals and VSMC-specific differentiation genes. Recent research indicates that Caspase-1 (casp-1) activation plays an essential role in sensing metabolic danger signal-associated molecular patterns and initiating vascular inflammation. Carbamylated LDL, a uremic toxin that has been shown to be found in higher levels in patients with CKD and in CKD murine models when compared to controls, and could play a role in casp-1 activation. Therefore, the goal of this project is to examine the role of cLDL/CKD-driven casp-1 activation in VSMC and CKD-related NH. We have established a CKD mouse model and published on CKD-associated vascular remodeling. We exposed wild type and caspase-1 knockout mice to our CKD model, analyzed and quantified the NH lesion formed. We also examined in vitro and ex-vivo changes in VSMC-specific differentiation genes when exposed to uremic serum and cLDL, in the presence or absence of caspase-1 inhibitor. We found that CKD serum induces with casp-1 activation and phenotypic changes in VSMCs from a "contractile" to a "synthetic" phenotype, which are reversed with casp-1 inhibition. In an ex-vivo model using relative quantification we found that VSMC contractile markers α -Actin, Calponin, SM-22, and Smoothelin gene expression of CKD mouse carotid VSMC were higher in casp-1 knockout mice when compared to wild-type (1.40, 1.28, 1.22, 1.41 respectively). Also using an in-vivo model, relative quantification of α-actin decreased from 1.0 to 0.329 when VSMCs were exposed to uremic serum and but increased back to 0.588 when Caspase-1 inhibitor is added. The relative quantification of Calponin also decreased from 1.0 to 0.394 when exposed to uremic serum and increased back to 0.601 with caspase-1 inhibitor. We also found that caspase-1 deficiency significantly reversed CKD-related vascular remodeling in casp-1 knockout mice and reduced NH volume by 50% from 1,440,023in wild-type mice to 71,069 µm2 in casp-1 knockouts (p-value 0.002). This evidence provides evidence that casp-1 plays a critical role in NH formation. Furthermore our results provide a novel insight over the therapeutic potential of casp-1 inhibitors for CKD induced NH and other inflammation induced vascular remodeling. / Public Health

Devido à sua alta taxa de mortalidade as doenças cardiovasculares vem sendo foco de pesquisas nos últimos anos. Estima-se que neste grupo, 42% das mortes foram consequência de doença coronariana (CHD) em 2012. Para auxiliar o acompanhamento de pacientes com CHD, este estudo teve como objetivo investigar um método simples e robusto que segmenta de maneira semi-automática a área da neoíntima em Tomografias Intravasculares por Coerência Óptica no Domínio da Frequência (FD-IOCT), a mais recente técnica de imagear vasos internamente. O método foi dividido em duas etapas. A primeira segmenta a área contida pelo lúmen através de operações morfológicas com valores de intensidade e aplicação da limiarização Otsu. Na segunda, o foco foi segmentar a área contida por partes de stent onde duas estratégias foram desenvolvidas e comparadas. Ambas as etapas (segmentação de área de lúmen e de stent) obtiveram resultados acurados com aproximadamente 98% de Verdadeiro Positivo, enquanto o Falso Positivo foi próximo de 3% para o lúmen e 5% para stent, onde uma das estratégias (para delimitar a área contida por stent) apresentou um tempo de execução 50 vezes maior que a outra. O método foi utilizado em 443 imagens com diferentes características e os resultados são encorajadores. / Due to its high mortality rate cardiovascular diseases have been the focus of research in recent years. It is estimated that among these, 42% of deaths were due to coronary heart disease (CHD) in 2012. To monitor patients with CHD, the goal of this study was to investigate a simple and robust method that segments semi-automatically the neointimal area on Frequency Domain Intravascular Optical Coherence Tomography (FD-IOCT), the latest technology to view vessels internally. The method was divided into two steps. The first one segments the area contained by the lumen through morphological operations on intensity values and the Otsu threshold. In the second one, the focus was to segment the area contained by stent struts where two strategies were developed and compared. Both steps (lumen and stent strut area segmentation) obtained accurate results with true positives approximately 98%, while the false positives were close to 3% for the lumen and 5% for the stent, where one strategy (to delimit the area contained by stent struts) showed run time of 50 times the other. The method was executed on 443 images with different characteristics and the results are encouraging.

Vascular stents have become a standard for treating atherosclerosis due to distinct advantages in trauma and cost with other surgical techniques. Unfortunately, the therapy is hindered by the risk of a new blockage (termed restenosis) developing in the treated artery. Clinical studies have indicated that stent design is a major risk factor for restenosis, with failure rates varying from 20 to 40% for bare metal stents. Subsequently, there has been a significant effort devoted to reducing failure rates by covering stents in polymer coatings in which anti-proliferative drugs are embedded, however complications have arisen (e.g. incomplete endothelization, lack of success in peripheral arteries, lack of long-term follow-up studies) that have limited the success of this technology. It has been thought that restenosis is directly related to the mechanical conditions that vascular stents create. Moreover, it has been hypothesized that stents that induce higher non-physiologic stresses result in a more aggressive pathobiological response that can lead to restenosis development. In this study, a combination of computational modeling and in vivo analysis were conducted to investigate the artery stent-induced wall stresses, and subsequent biological inflammatory response. In particular, variations in stent design were investigated as a means of examining specific stent design criteria that minimize the mechanical impact of stenting. Collectively, these data indicate that stent designs that subject the artery wall to higher stress values result in significantly more neointimal tissue proliferation, therefore, confirming the aforementioned hypothesis. Moreover, this work provides valuable insight into the role that biomechanics can play in improving the success rate of this percutaneous therapy and overall patient care.

Objetivo: Avaliar se a administração sistêmica de cilostazol reduz a hiperplasia neointimal nas artérias ilíacas de suínos submetidas a angioplastia com cateter balão. Métodos: O trabalho foi desenvolvido na Unidade de Experimentação Animal do Hospital de Clínicas de Porto Alegre. Vinte suínos foram submetidos a angioplastia com cateter balão 6 x 40 mm na artéria ilíaca comum direita, guiada por ultrassonografia com Doppler. Os animais foram randomizados em dois grupos: grupo 1 (n = 10), no qual foi administrado cilostazol 50 mg em duas doses diárias, e grupo 2 (n = 10), considerado controle. Após 30 dias, os animais foram sacrificados, e as artérias ilíacas preparadas para análise histológica. Os cortes histológicos foram digitalizados e analisados por morfometria digital. A análise estatística foi realizada com o teste t de Student e o de Mann-Whitney. Resultados: Comparando as artérias ilíacas submetidas a angioplastia com as não submetidas a angioplastia, houve hiperplasia neointimal significativa (0,228 versus 0,119 mm2; p = 0,0001). Nas artérias submetidas a angioplastia, não houve diferença entre o grupo 1 (cilostazol) e o grupo 2 (controle) na área do lúmen (2,277 versus 2,575 mm2; p = 0,08), área da íntima (0,219 versus 0,237 mm2; p = 0,64), área da média (2,262 versus 2,393 mm2; p = 0,53) e no percentual de obstrução neointimal (8,857 versus 9,257 %; p = 0,82). Conclusão: O uso de cilostazol 50 mg em duas doses diárias não reduziu a hiperplasia neointimal em artérias ilíacas de suínos submetidas a angioplastia com cateter balão. / Objective: To evaluate whether systemic administration of cilostazol reduces neointimal hyperplasia in the iliac arteries of pigs after balloon angioplasty. Methods: This study was conducted in the Experimental Animal Unit of Hospital de Clínicas de Porto Alegre, Brazil. Twenty pigs underwent angioplasty of the right common iliac artery under Doppler ultrasound guidance using 6x40-mm balloon catheters. The animals were randomized to one of two groups: group 1 (n = 10) received 50 mg cilostazol in two doses a day; and group 2 (n = 10) was the control group. After 30 days, the animals were killed and their iliac arteries were prepared for histological analysis. Histological images were digitalized and analyzed using digital morphometry. The Student t and the Mann-Whitney tests were used for statistical analyses. Results: Iliac arteries that underwent angioplasty had significantly more neointimal hyperplasia than those with no angioplasty (0.228 versus 0.119 mm2; p = 0.0001) Group 1 (cilostazol) and 2 (control) had no significant differences in lumen (2.277 versus 2.575 mm2; p = 0.08), intima (0.219 versus 0.237 mm2; p = 0.64) or media (2.262 versus 2.393 mm2; p = 0.53) area, or in percentage of neointimal obstruction (8.857 versus 9.257 %; p = 0.82). Conclusion: The use of 50 mg cilostazol in two doses a day did not reduce neointimal hyperplasia in iliac arteries of pigs that underwent balloon angioplasty.

Objetivo: Avaliar se a administração sistêmica de cilostazol reduz a hiperplasia neointimal nas artérias ilíacas de suínos submetidas a angioplastia com cateter balão. Métodos: O trabalho foi desenvolvido na Unidade de Experimentação Animal do Hospital de Clínicas de Porto Alegre. Vinte suínos foram submetidos a angioplastia com cateter balão 6 x 40 mm na artéria ilíaca comum direita, guiada por ultrassonografia com Doppler. Os animais foram randomizados em dois grupos: grupo 1 (n = 10), no qual foi administrado cilostazol 50 mg em duas doses diárias, e grupo 2 (n = 10), considerado controle. Após 30 dias, os animais foram sacrificados, e as artérias ilíacas preparadas para análise histológica. Os cortes histológicos foram digitalizados e analisados por morfometria digital. A análise estatística foi realizada com o teste t de Student e o de Mann-Whitney. Resultados: Comparando as artérias ilíacas submetidas a angioplastia com as não submetidas a angioplastia, houve hiperplasia neointimal significativa (0,228 versus 0,119 mm2; p = 0,0001). Nas artérias submetidas a angioplastia, não houve diferença entre o grupo 1 (cilostazol) e o grupo 2 (controle) na área do lúmen (2,277 versus 2,575 mm2; p = 0,08), área da íntima (0,219 versus 0,237 mm2; p = 0,64), área da média (2,262 versus 2,393 mm2; p = 0,53) e no percentual de obstrução neointimal (8,857 versus 9,257 %; p = 0,82). Conclusão: O uso de cilostazol 50 mg em duas doses diárias não reduziu a hiperplasia neointimal em artérias ilíacas de suínos submetidas a angioplastia com cateter balão. / Objective: To evaluate whether systemic administration of cilostazol reduces neointimal hyperplasia in the iliac arteries of pigs after balloon angioplasty. Methods: This study was conducted in the Experimental Animal Unit of Hospital de Clínicas de Porto Alegre, Brazil. Twenty pigs underwent angioplasty of the right common iliac artery under Doppler ultrasound guidance using 6x40-mm balloon catheters. The animals were randomized to one of two groups: group 1 (n = 10) received 50 mg cilostazol in two doses a day; and group 2 (n = 10) was the control group. After 30 days, the animals were killed and their iliac arteries were prepared for histological analysis. Histological images were digitalized and analyzed using digital morphometry. The Student t and the Mann-Whitney tests were used for statistical analyses. Results: Iliac arteries that underwent angioplasty had significantly more neointimal hyperplasia than those with no angioplasty (0.228 versus 0.119 mm2; p = 0.0001) Group 1 (cilostazol) and 2 (control) had no significant differences in lumen (2.277 versus 2.575 mm2; p = 0.08), intima (0.219 versus 0.237 mm2; p = 0.64) or media (2.262 versus 2.393 mm2; p = 0.53) area, or in percentage of neointimal obstruction (8.857 versus 9.257 %; p = 0.82). Conclusion: The use of 50 mg cilostazol in two doses a day did not reduce neointimal hyperplasia in iliac arteries of pigs that underwent balloon angioplasty.

Objetivo: Avaliar se a administração sistêmica de cilostazol reduz a hiperplasia neointimal nas artérias ilíacas de suínos submetidas a angioplastia com cateter balão. Métodos: O trabalho foi desenvolvido na Unidade de Experimentação Animal do Hospital de Clínicas de Porto Alegre. Vinte suínos foram submetidos a angioplastia com cateter balão 6 x 40 mm na artéria ilíaca comum direita, guiada por ultrassonografia com Doppler. Os animais foram randomizados em dois grupos: grupo 1 (n = 10), no qual foi administrado cilostazol 50 mg em duas doses diárias, e grupo 2 (n = 10), considerado controle. Após 30 dias, os animais foram sacrificados, e as artérias ilíacas preparadas para análise histológica. Os cortes histológicos foram digitalizados e analisados por morfometria digital. A análise estatística foi realizada com o teste t de Student e o de Mann-Whitney. Resultados: Comparando as artérias ilíacas submetidas a angioplastia com as não submetidas a angioplastia, houve hiperplasia neointimal significativa (0,228 versus 0,119 mm2; p = 0,0001). Nas artérias submetidas a angioplastia, não houve diferença entre o grupo 1 (cilostazol) e o grupo 2 (controle) na área do lúmen (2,277 versus 2,575 mm2; p = 0,08), área da íntima (0,219 versus 0,237 mm2; p = 0,64), área da média (2,262 versus 2,393 mm2; p = 0,53) e no percentual de obstrução neointimal (8,857 versus 9,257 %; p = 0,82). Conclusão: O uso de cilostazol 50 mg em duas doses diárias não reduziu a hiperplasia neointimal em artérias ilíacas de suínos submetidas a angioplastia com cateter balão. / Objective: To evaluate whether systemic administration of cilostazol reduces neointimal hyperplasia in the iliac arteries of pigs after balloon angioplasty. Methods: This study was conducted in the Experimental Animal Unit of Hospital de Clínicas de Porto Alegre, Brazil. Twenty pigs underwent angioplasty of the right common iliac artery under Doppler ultrasound guidance using 6x40-mm balloon catheters. The animals were randomized to one of two groups: group 1 (n = 10) received 50 mg cilostazol in two doses a day; and group 2 (n = 10) was the control group. After 30 days, the animals were killed and their iliac arteries were prepared for histological analysis. Histological images were digitalized and analyzed using digital morphometry. The Student t and the Mann-Whitney tests were used for statistical analyses. Results: Iliac arteries that underwent angioplasty had significantly more neointimal hyperplasia than those with no angioplasty (0.228 versus 0.119 mm2; p = 0.0001) Group 1 (cilostazol) and 2 (control) had no significant differences in lumen (2.277 versus 2.575 mm2; p = 0.08), intima (0.219 versus 0.237 mm2; p = 0.64) or media (2.262 versus 2.393 mm2; p = 0.53) area, or in percentage of neointimal obstruction (8.857 versus 9.257 %; p = 0.82). Conclusion: The use of 50 mg cilostazol in two doses a day did not reduce neointimal hyperplasia in iliac arteries of pigs that underwent balloon angioplasty.

Devido à sua alta taxa de mortalidade as doenças cardiovasculares vem sendo foco de pesquisas nos últimos anos. Estima-se que neste grupo, 42% das mortes foram consequência de doença coronariana (CHD) em 2012. Para auxiliar o acompanhamento de pacientes com CHD, este estudo teve como objetivo investigar um método simples e robusto que segmenta de maneira semi-automática a área da neoíntima em Tomografias Intravasculares por Coerência Óptica no Domínio da Frequência (FD-IOCT), a mais recente técnica de imagear vasos internamente. O método foi dividido em duas etapas. A primeira segmenta a área contida pelo lúmen através de operações morfológicas com valores de intensidade e aplicação da limiarização Otsu. Na segunda, o foco foi segmentar a área contida por partes de stent onde duas estratégias foram desenvolvidas e comparadas. Ambas as etapas (segmentação de área de lúmen e de stent) obtiveram resultados acurados com aproximadamente 98% de Verdadeiro Positivo, enquanto o Falso Positivo foi próximo de 3% para o lúmen e 5% para stent, onde uma das estratégias (para delimitar a área contida por stent) apresentou um tempo de execução 50 vezes maior que a outra. O método foi utilizado em 443 imagens com diferentes características e os resultados são encorajadores. / Due to its high mortality rate cardiovascular diseases have been the focus of research in recent years. It is estimated that among these, 42% of deaths were due to coronary heart disease (CHD) in 2012. To monitor patients with CHD, the goal of this study was to investigate a simple and robust method that segments semi-automatically the neointimal area on Frequency Domain Intravascular Optical Coherence Tomography (FD-IOCT), the latest technology to view vessels internally. The method was divided into two steps. The first one segments the area contained by the lumen through morphological operations on intensity values and the Otsu threshold. In the second one, the focus was to segment the area contained by stent struts where two strategies were developed and compared. Both steps (lumen and stent strut area segmentation) obtained accurate results with true positives approximately 98%, while the false positives were close to 3% for the lumen and 5% for the stent, where one strategy (to delimit the area contained by stent struts) showed run time of 50 times the other. The method was executed on 443 images with different characteristics and the results are encouraging.