Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.

In normal adult physiology, intestinal smooth muscle cells (ISMC) are characterized as contractile and non-proliferative. Inflammation induces permanent changes to the intestine including hypertrophy of the smooth muscle layer largely due to smooth muscle cell (SMC) proliferation. While the consequences of this hyperplasia are largely unknown, increased muscularis mass may present permanent challenges to organ motility. Similar SMC hyperplasia is observed in other inflammatory pathologies including atherosclerosis and pulmonary arterial hypertension (PAH) where SMC de-differentiate into a ‘synthetic’ phenotype and the mitogens responsible for hyperplasia have been well studied. However, there are limited investigations of SMC mitogens in intestinal inflammation. The identification of these factors may be of critical importance in the case of intestinal strictures, whereby recurring inflammation can lead to bowel obstruction requiring surgical intervention. A novel, primary rat ISMC model was developed to identify the factors responsible for ISMC proliferation in vitro. Primary ISMC cultures are likely more representative of SMC in vivo than the commonly used late-passage cultures. As such, this primary ISMC model is valuable in the evaluation of mitogens involved in the onset of proliferation. This primary ISMC model was used to conduct a comprehensive evaluation of potential mitogens including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor-BB (PDGF-BB. This work identified IGF-1 and PDGF-BB as ISMC mitogens. However, multiple lines of evidence indicated that PDGF-BB was a more potent mitogen and the involvement of PDGF-BB was subsequently examined in vivo using the trinitrobenzenesulfonic acid (TNBS) model of rat intestinal inflammation. While control ISMC lacked expression of the PDGF-BB receptor (PDGF-Rβ), robust expression was observed within only 6 hr following the induction of TNBS inflammation. By Day 2, when ISMC proliferation in vivo is maximal, freshly isolated ISMC showed on-going PDGF-Rβ activation that was further increased by exogenous PDGF-BB. Taken together, the conclusions from this work in vitro identify PDGF-BB as a potent ISMC mitogen in vivo. Further, this work establishes PDGF-BB and its receptor as potential targets in the medical treatment of intestinal stricture formation. / Thesis (Ph.D, Biology) -- Queen's University, 2012-09-24 19:26:57.201

The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases. In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through an NF-κB-dependent signaling pathway. Functional studies reveal that NOR1 increases monocyte adhesion by inducing the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Transient transfection and chromatin immunoprecipitation assays identify VCAM-1 as a bona fide NOR1 target gene in endothelial cells. Finally, we demonstrate that NOR1-deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE-/- mice by decreasing the macrophage content of the lesion. In smooth muscle cells (SMC), NOR1 was previously established as a cAMP response element binding protein (CREB) target gene in response to platelet-derived growth factor (PDGF) stimulation. CREB phosphorylation and subsequent binding of phosphorylated CREB to the NOR1 promoter play a critical role in inducing NOR1 expression. In this dissertation, we further demonstrate that histone deacetylase (HDAC) inhibition potentiates and sustains PDGF-induced NOR1 mRNA and protein expression in SMC. This augmented NOR1 expression is associated with increased phosphorylation of CREB, recruitment of phosphorylated CREB to the NOR1 promoter, and trans-activation of the NOR1 promoter. Additionally, HDAC inhibition also increases NOR1 protein half-life in SMC. Collectively, these findings identify a novel pathway in endothelial cells underlying monocyte adhesion and expand our knowledge of the epigenetic mechanisms orchestrating NOR1 expression in SMC. Finally, we establish a previously unrecognized atherogenic role of NOR1 in positively regulating monocyte recruitment to the vascular wall.

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

Branchial arches (BAs) are a series of transient structures that develop on the ventro-lateral surface of the head in vertebrate embryos. BAs initially appear as a series of similar segments; as development proceeds each BA will contribute to different structures. Here, it was investigated the transcriptional mechanisms that instruct the different fates of the BAs in development. Initially, each BA contains a blood vessel, known as aortic arch (AA) artery, that connects the dorsal aorta with the heart. Remodelling of the AAs is crucial to form the adult heart circulation. This process leads to regression of the anterior AAs, running though the first and second BAs (BA1 and BA2), and persistence of the AAs contained in more posterior BAs (PBA). To identify the mechanisms that control remodelling of the AAs, we compared the transcriptomes and epigenomic landscapes of different BAs. Using RNA-seq and H3K27Ac ChIP-seq, we uncovered the activation of a vascular smooth muscle cell (VSMC) differentiation transcriptional program exclusively in the PBAs (and not in BA1/BA2). In support of this finding, we show that VSMC differentiation occurs specifically in the PBAs, but not BA1-2 in mouse embryonic development. Despite the absence of VSMC differentiation in developing BA1-2, cells harvested from these tissues reveal a spontaneous tendency to differentiate towards VSMC fate when grown in vitro, and activate several VSMC-specific genes (Myocd, Acta2, Tagln, Jag1). Together, our results suggest that forming VSMCs is a key process for the persistence of AAs. We also showed that cells derived from all BAs have the potential to differentiate to VSMCs in vitro. However, only cells in the PBAs differentiate to VSMCs in vivo, resulting in the maintenance of posterior AAs. In this study, we also uncovered a novel transcriptional principle that specifies the fate of BA2. Using ChIP-seq, we found that binding of Meis transcription factors establish a ground pattern in the BAs. Hoxa2, which specifies BA2 identity, selects a subset of Meis-bound sites. Meis binding is strongly increased at these sites, which coincide with active enhancers, linked to genes highly expressed in the BA2 and regulated by Hoxa2. Thus, Hoxa2 modifies a ground state binding of Meis to instruct segment-specific transcriptional programs.

Os efeitos da obstrução uretral parcial na musculatura da bexiga urinária de coelhos foram investigadas usando as ferramentas estereológicas. Foram utilizadas 12 fêmeas de coelhos da raça Norfolk, com três meses de idade e peso corporal variando de 2,5-3,0 kg. O procedimento cirúrgico consistiu de celiotomia mediana retro-umbilical para exposição da bexiga urinária. A parede dorsal da uretra foi divulsionada de sua íntima associação com o útero e vagina, o suficiente para a passagem de fio nylon 2-0. Um pino de Steinmann (3 mm de diâmetro) foi interposto temporariamente entre a uretra e o fio para determinar indiretamente o grau de obstrução uretral. Após três, sete e doze semanas os animais foram ortotanasiados e comparados com o grupo de animais controle (não obstruídos). Os fragmentos da bexiga foram preparados para microscopia de luz. Cortes seriados foram realizados para o estudo morfométrico e estereológico. Os três eixos: crânio-caudal (CC), dorso-ventral (DV) e latero-lateral (LL) aumentaram em todos os grupos analisados: controle, 3, 7 e 12 semanas. Os valores para CC foram estatisticamente similares para 3, 7 e 12 semanas. O mesmo foi observado no eixo DV. Os valores para o eixo LL foram similares para os grupos de 7 e 12 semanas. O estudo morfométrico baseou-se em determinar o tamanho da fibra (área seccional) e comprimento da fibra muscular. Nos animais do grupo de 3, 7 e 12 semanas foi observado um aumento de 4,63x, 4,32x e 7,10x no tamanho celular e um decréscimo de 2,55x, 1,94x e 4,04x no comprimento da fibra muscular quando comparados ao grupo controle. O estudo estereológico baseou-se em estimar o volume referência (Vref), a densidade numérica (Nv), o número total de fibras musculares (N), a densidade de volume (Vv) e o volume da fibra muscular (Vn). O Vref apresentou um aumento de 11,07x, 7,98x e 31,7x quando comparado com o grupo controle. A densidade numérica (Nv) aumentou 0,06x e 0,05x para os grupos de 3 e 7 semanas, respectivamente, em relação ao grupo controle. O grupo de 12 semanas, no entanto, apresentou um decréscimo de 0,01x em comparação com o grupo controle. Os grupos de 3, 7 e 12 semanas apresentaram, respectivamente, um aumento de 0,81x, 12,56x e 38,43x em número total de células. A densidade de volume (Vv) para os grupos de 3, 7 e 12 semanas apresentou um aumento de 0,97x, 0,56x e 0,86x em relação ao grupo controle. E finalmente, o volume médio da fibra muscular apresentou um aumento de 0,62x, 0,81x e 0,82x, respectivamente para os animais de 3, 7 e 12 semanas. Os dois mecanismos: hipertrofia e hiperplasia ocorrem na bexiga urinária de coelhos, porém não sabemos a seqüência exata em que aparecem. / The effects of partial urethral obstruction on rabbit´s urinary bladder musculature were investigated using stereological designed methods. A total of 12 female Norfolk rabbits weighing from 2.5 to 3 kg were used. A retro-umbilical celiotomy was made to expose the urinary bladder. The urethra´s dorsal wall was isolated from its association with the uterus. A 3mm-Steinmann-pin was positioned on the urethra to produce a standard degree of obstruction and a ligature was tied up around it, using a 2-0 nylon silk. Three, seven and twelve weeks after the surgery procedures the rabbits were euthanised. Bladder fragments were prepared for light microscopy. Serial sections were performed to morphometric and stereological study. In relation to the bladder axis: cranio-caudal (CC), dorso-ventral (DV) and latero-lateral (LL) increased in all groups analysed: control, 3, 7 and 12 week-obstructed animals. Values for CC were statistically similar for 3, 7 and 12-week-obstructed groups. The same was observed for DV axis. The LL axis showed values statistically similar for 7 and 12-week-obstructed groups. The morphometric study was based on the muscle fibre size (sectional area) and the muscle fibre length. In 3, 7 and 12-week-obstructed animals, it was observed a 4.63, 4.32 and 7.10-fold cell size increase and a 2.55, 1.94 and 4.04-fold decrease in length, respectively, when compared to control group. As for the stereological study. Vref presented a 11.07, 7.98 and 31.7-fold increase when compared to control subjects. Numerical density (Nv) increased by 0.06 and 0.05 in 3 and 7-week-obstructed groups, respectively, in relation to control group. Twelve week-obstructed group. Presented however a 0.01x-decrease compared to control animals. Three, seven and twelve-week-obstructed groups presented, respectively, 0.81, 12.56 and 38.43-fold increase in total number of cells (N). Volume density presented a 0.97, 0.56 and 0.86-fold increase in 3, 7 and 12-week-obstructed groups, respectively. And finally, mean muscle cell volume (Vn) presented a 0.62, 0.81 and 0.82-fold in 3, 7 and 12-week obstructed groups, respectively. Both mechanisms: hypertrophy and hyperplasia happened to occur on rabbit´s urinary bladder, thought we do not know the exact sequence in which they appear altogether.

Abstract Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol. Ethanol causes changes in the concentration and composition of plasma lipoproteins and it also influences the enzymes and transfer proteins that modify lipoproteins in plasma. PEth might be one of these changes brought on by ethanol in the circulation. The present study was designed to investigate whether qualitative changes in high density lipoprotein (HDL) phospholipids caused by ethanol can mediate the beneficial effects of alcohol on atherosclerosis, and to investigate the transfer of PEth between lipoproteins and the effects of PEth on the charge of lipoprotein particles. PEth was shown to be transferred from low density lipoproteins (LDL) to HDL particles mainly by transfer proteins other than cholesteryl ester transfer protein (CETP). The transfer of PEth between lipoproteins enables the redistribution of PEth between lipoproteins in plasma. The results of this study provide evidence that PEth in HDL particles stimulates the vascular endothelial growth factor (VEGF) secretion from vascular wall cells. The increase in the secretion was mediated through protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signalling pathways. PEth-containing HDL particles were able to increase the VEGF secretion in rats in vivo. Similar effects were also observed when rats were given HDL particles isolated from the plasma of alcoholics. The PEth-induced change in the electrical charge of lipoproteins may affect the binding of lipoproteins to their receptors and binding proteins. The effects of PEth on the secretion of VEGF from the endothelial cells were shown to be mediated through HDL receptor. The changes in HDL particles caused by phosphatidylethanol may modify the metabolism of lipoproteins and lipid-mediated signalling pathways regulating VEGF in vascular wall cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22641号 / 医博第4624号 / 新制||医||1044(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 山下 潤, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM