Primary open-angle glaucoma (POAG) is a leading cause of blindness in the world. A primary risk factor for POAG is elevated intraocular pressure (IOP), caused by increased aqueous humor outflow resistance. Currently, lowering the IOP is the only effective way of treating glaucoma; however, the cause of increased outflow resistance remains unclear. This thesis will present a series of studies which investigated structures of the trabecular outflow pathway, including Schlemm’s canal endothelium, juxtacanalicular tissue, and trabecular beams, and their roles in regulating aqueous outflow resistance. The studies were conducted in both human and animal models using ex vivo ocular perfusion as well as in vitro microfluidic systems. In the first study, we investigated the effects of Y27632, a derivative of Rho-kinase inhibitor that is being developed as next generation glaucoma drug with unclear IOP lowering mechanism, on aqueous humor outflow dynamics and associated morphological changes in normal human eyes and laser-induced ocular hypertensive monkey eyes. In the second study, we developed and validated a novel three-dimensional microfluidic system using lymphatic microvascular endothelial cells. The microfluidic system can be used to study Schlemm’s canal endothelial cell dynamics and aqueous humor transport mechanism in the future. In the last study, we characterized the morphological structure, distribution, and thickness of the endothelial glycocalyx in the aqueous humor outflow pathway of human and bovine eyes. Together these studies will help define new directions for therapy that will help control IOP and preserve vision throughout a normal life span.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20277号 / 医博第4236号 / 新制||医||1021(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 渡邊 直樹, 教授 小池 薫, 教授 福田 和彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

No description available.

BACKGROUND: Netarsudil, a Rho kinase and norepinephrine transport (NET) inhibitor, is a new FDA approved drug used for decreasing raised intraocular pressure (IOP) in ocular hypertensive and primary open-angle glaucoma (POAG) patients. Previous studies reported that netarsudil increased outflow facility and lowered IOP by increasing active outflow areas around the circumference of the eye and dilating the episcleral veins (ESV; Kiel and Kopczynski, 2015; Ren et al., 2016). However, the mechanisms by which netarsudil increases outflow facility have not yet been fully elucidated. Moreover, the effects of netarsudil on the inner wall (IW) endothelium I-pores and B-pores of the Schlemm’s canal (SC) have also not been investigated yet. AIM: The goal was to determine if netarsudil-treatment increased the effective filtration areas (EFA) by increasing pore density in both high- and non-flow type areas, compared to untreated control eyes. METHODS: In this study, the effects of netarsudil on the pore densities on IW of SC were investigated by serial block-face scanning electron microscopy (SBF-SEM). Two pairs of eyes were perfused with green fluorescent tracers in order to determine the outflow pattern prior to treatment. Then, one eye of each pair was perfused with netarsudil, while the fellow eye of each pair was perfused with vehicle solution. All eyes were then perfused with red fluorescent tracers in order to determine the outflow pattern once they were treated with netarsudil. Both pairs of eyes were perfused and fixed at 15 mmHg. Global imaging was performed for all eyes to visualize high- and non- flow areas in the trabecular meshwork (TM) and ESV’s. A SBF-SEM was used to image eight wedges of tissue including the IW of SC and TM (high- and non-flow areas from four eyes) for a total of 16,378 images. The study analyzed the percentage of pore-types (GV-associated I-pores, Non-GV associated I-pores, B-pores), the median pore spans, the GV-associated I-pore locations, and the pore densities (per IW nuclei and IW area) between the equivalent control and netarsudil-treated flow areas. RESULTS: In global images, an increase in high-flow areas were observed in netarsudil-treated eyes due to recruitment from low-flow and non-flow areas. A greater percentage of GV-associated I-pores, B-pores, and total pores were found in high-flow in contrast to non-flow areas in both control and netarsudil-treated eyes (all P ≤ 0.05). However, the percentage of GV-associated I-pores in non-flow areas were significantly greater in treated compared to control eyes (P ≤ 0.05). Qualitative observations from two pairs of eyes showed a trend of greater I-pore, B-pore, and total pore density/per IW nucleus and density/per IW surface area in high-flow in contrast to non-flow areas for both treated and control eyes. No difference in I-pore, B-pore, and total pore density/per IW nucleus and density /per IW surface area were observed in equivalent flow-type areas when comparing control and netarsudil-treated eyes. In addition, there was a significant greater percentage of I-pores located on the side of GVs than the top of GVs in all cases (P ≤ 0.05). CONCLUSIONS: Netarsudil increased high-flow areas. A greater pore density was found in high-flow in contrast to non-flow areas. Netarsudil also significantly increased the proportion of GV-associated I-pores in non-flow areas when compared to control eyes. Our results suggests that one mechanism of netarsudil increasing outflow facility is acting through recruiting the high-flow areas around the circumference of the eye, which is associated with higher pore density and increasing the proportion of GV-associated I-pores in non-flow areas.

No description available.

Osteoarthritis is an inflammation-related disease that progressively destroys joint cartilage. This disease causes pain and stiffness of the joints, and at advanced stages, limitations to the movement or bending of injured joints. Therefore, it often restricts daily activities and the ability to work. Currently, there is no cure to prevent its progression, although certain damaged joints, such as fingers, knees and hips, can be treated with joint replacement surgeries. However, joint replacement surgeries of larger joints are very invasive operations and the joint replacements have a limited lifetime. Cell-based therapies could offer a way to treat cartilage injuries before the ultimate damage of osteoarthritis on articular cartilage. The development of novel treatments needs both a good knowledge of articular cartilage biology and tissue engineering methods. This thesis primarily investigates the effects of mechanical cyclic stretching, a 5% low oxygen atmosphere and the Rho-kinase inhibitor, Y-27632, on protein responses in chondrocytic human chondrosarcoma (HCS-2/8) cells. Special focus is placed on Rho-kinase inhibition, relating to its potential to promote and support extracellular matrix production in cultured chondrocytes and its role in fibroblast cells as a part of direct chemical cellular differentiation. The means to enhance the production of cartilage-specific extracellular matrix is needed for cell-based tissue engineering applications, since cultured chondrocytes quickly lose their cartilage-specific phenotype. A mechanical 8% cyclic cell stretching at a 1 Hz frequency was used to model a stretching rhythm similar to walking. The cellular stretching relates to stresses, which are directed to chondrocytes during the mechanical load. The stretch induced changes in proteins related, e.g., to certain cytoskeletal proteins, but also in enzymes associated with protein synthesis, such as eukaryotic elongation factors 1-beta and 1-delta. Hypoxic conditions were used to model the oxygen tension present in healthy cartilage tissue. Long-term hypoxia changed relative amounts in a total of 44 proteins and induced gene expressions of aggrecan and type II collagen, in addition to chondrocyte differentiation markers S100A1 and S100B. A short-term inhibition of Rho-kinase failed to induce extracellular matrix production in fibroblasts or in HCS-2/8 cells, while its long-term exposure increased the expressions of chondrocyte-specific genes and differentiation markers, and also promoted the synthesis of sulfated glycosaminoglycans by chondrocytic cells. Interestingly, Rho kinase inhibition under hypoxic conditions produced a more effective increase in chondrocyte-specific gene expression and synthesis of extracellular matrix components by HCS-2/8 cells. The treatment induced changes in the synthesis of 101 proteins and ELISA analysis revealed a sixfold higher secretion of type II collagen compared to control cells. The secretion of sulfated glycosaminoglycans was simultaneously increased by 65.8%. Thus, Rho-kinase inhibition at low oxygen tension can be regarded as a potential way to enhance extracellular matrix production and maintain a chondrocyte phenotype in cell-based tissue engineering applications.

The longitudinal muscle layer in gut is the functional opponent to the circular muscle layer during the peristalsis reflex. Differences in innervation of the layers allow for the contraction of one layer that corresponds with the simultaneous relaxation of the other, enabling the passage of gut contents in a controlled fashion. Differences in development have given the cells of the two layers differences in receptor populations, membrane lipid handling, and calcium handling profiles/behaviors. The kinase signaling differences between the two layers is not as well characterized. Upon activation of cells from the circular muscle layer, it is known that Rho kinase and ERK1/2 promote contraction, while CaMKK/AMPK and CaMKII perform inhibitory/self-inhibitory roles. Such behaviors are poorly understood in the longitudinal muscle layer. In longitudinal muscle strips, we measured muscarinic receptor-mediated contraction following incubation with kinase inhibitors. Upon comparison to control, contributions of Rho Kinase and ERK1/2 were similar to those seen in circular muscle. Inhibition of both of these enzymes leads to diminished contraction. However, CaMKK/AMPK and CaMKII have effects in longitudinal muscle opposite to their regulation in circular muscle – their inhibition also diminishes the contractile response. These contractile data from strips were supported by immunokinase assay measurements of MLCK activity from strip homogenates with and without kinase inhibition. Therefore, we suggest that the activities of CaMKK/AMPK and CaMKII in longitudinal muscle are indeed different from their regulatory roles in circular muscle, perhaps a consequence of the different calcium handling modalities of the two muscle types.

LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo. / LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.