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The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia. / Doctor of Philosophy / Every tissue and organ in the human body receives blood from the heart via the extremely complex network of "highways" known as the vasculature. These vessels oversee moving nutrients, oxygen, hormones, and waste materials out of the body. At the capillary level, endothelial cells and pericytes, as well as the surrounding protein milieu known as the extracellular matrix (ECM), are required for the development of a functional vascular system. If the vascular network fails to develop and operate properly because of poor protein and cellular coordination, it can eventually lead to the failure of organ systems. The study for this dissertation focuses on how vascular development is impacted by insufficient ECM deposition. We used several strategies to modify the composition of the ECM, including 1) hypoxia exposure, 2) pharmaceutical ECM reduction, and 3) lentiviral-mediated delivery of shRNA to silence Type IV Collagen (Col-IV, gene Col4a1) production. We noticed alterations in the coordination between endothelial cells and pericytes as vascular networks were being formed in these experimental environments. In summary, this dissertation work contends that, in contrast to reduced ECM deposition, excess ECM deposition, and specifically that of Col-IV, has distinct detrimental consequences on the developing vasculature. The results of this study offer methods by which diseases associated with hypoxia, such preeclampsia, may cause the vasculature to become unstable.

Indiana University-Purdue University Indianapolis (IUPUI) / The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.

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No description available.

Every year millions of individuals suffer from traumatic brain injury (TBI) leading to permanent disabilities and even death. Mild TBI (mTBI) is the most common form of TBI comprising about 80-90% of all occurrences. Following a CNS insult like an mTBI, astrocytes can undergo activation resulting in the transformation into reactive astrocytes (RAs). RAs also play an important role in brain remodeling following an mTBI. Research on the mechanical complexity of the brain has important implications for understanding brain function and dysfunction, as well as for the development of new diagnostic and therapeutic tools for neurological disorders. This study aimed to develop and utilize an emph{in vitro} mTBI platform to investigate the intricate mechanical interplay between the extracellular matrix (ECM) and astrocytes following a simulated mTBI. Cellular mechanisms underlying mTBI and the contribution of mechanical forces that result in prolonged brain damage are yet to be comprehensively understood. Successfully devised mechanical characterization techniques for tissue-engineered models were developed utilizing atomic force microscopy and rheology. Astrocyte exposure to high-rate overpressure revealed altered mechanical properties of the surrounding matrix and decreased expression of laminin and collagen IV, which are critical for brain function and may contribute to pathologies associated with mTBI. The developed platform and methods provide new insights into the mechanistic complexity underlying ECM-astrocyte interactions following an mTBI. / Master of Science / Every year, millions of people suffer from traumatic brain injury (TBI), which can lead to permanent disabilities or even death. The most common form of TBI is mild TBI (mTBI), which accounts for 80-90% of all cases. After a mTBI, astrocytes, the most common cell type in the brain, can become activated and turn into reactive astrocytes (RAs). RAs play an important role in the brain's recovery following a mTBI. Understanding the mechanical complexity of the brain is crucial for developing new diagnostic and therapeutic tools for neurological disorders. This study aimed to investigate the mechanical interplay between the modeled tissue and astrocytes following a simulated mTBI using an emph{in vitro} platform. Development of mechanical characterization techniques allowed for any alterations caused by the astrocytes to their environment to be detectable. The astrocyte exposure to the simulated mTBI revealed altered mechanical properties of the surrounding environment and decreased expression of proteins laminin and collagen IV, which are critical to brain function and may contribute to pathologies associated with mTBI. This study provides new insights into the mechanistic complexity underlying the interaction between astrocytes and their environment, which could lead to the development of new treatments.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24198号 / 医博第4892号 / 新制||医||1060(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 川口 義弥, 教授 松田 秀一, 教授 小濱 和貴 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. With gene knock-down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Additionally, studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. Hemocyte-pericardial cell association could indicate that hemocytes take up proteins that are produced by pericardial cells and deliver them to other locations or that there might be a previously unidentified hematopoietic site at the Drosophila larval heart. / Thesis / Master of Science (MSc)

The cardiac extracellular matrix (ECM) is a dynamic protein scaffold that is required to support cardiac function. Regular remodelling of the matrix involves protein turnover and deposition and is a highly regulated process. In disease states the normal balance of the ECM is disrupted and aberrant protein deposition and crosslinking can occur. This process, termed fibrosis, causes stiffening of the cardiac ECM, which in turn impairs organ function. Fibrosis is a hallmark of cardiovascular disease, is a progressive condition that can contribute to adverse clinical outcomes, and currently has no available treatments. One of the leading causes of cardiovascular disease is obesity and fibrosis is known to occur in this context. In order to investigate the development of fibrotic remodelling in the context of obesity I have developed a dietary obesity model in the fruit fly Drosophila melanogaster. Additionally, I developed a genetic overgrowth model as increased cardiac load is also known to trigger fibrotic remodelling. Dietary obesity models reveal altered ECM organization, as well as impaired cardiac contractility, while overgrowth models demonstrate a remarkable ability to appropriately scale heart morphology with increased body size. The overgrowth model does have extremely elevated expression levels of the crosslinking enzyme LOXL2, suggesting a major contributor to impaired function is increased crosslinking rather than altered protein deposition. However, inhibition of crosslinking caused only minor ECM organizational defects but was able to rescue the elasticity of the overgrowth model. Overall, this thesis raises intriguing questions for treatment of cardiovascular disease, where tissue dynamics are often overlooked in a clinical setting. / Thesis / Doctor of Science (PhD) / The cardiac extracellular matrix (ECM) is a protein scaffold that supports heart function. Cardiovascular disease often involves increased levels of ECM proteins, a condition called fibrosis, which causes increased tissue stiffness and functional impairment. There is no cure for fibrosis and developing treatments requires an understanding of how the ECM responds to disease. I developed a dietary obesity model and a genetically triggered overgrowth model to examine how the ECM responds to disease states. I found that obesity causes ECM reorganization and functional defects, but that overgrowth models scale their hearts remarkably well with increased body size. Overgrowth models were found to have elevated levels of matrix crosslinking enzymes, which contributed to a stiffer matrix in these individuals. This was rescued by inhibition of crosslinking. Overall, this thesis reveals a connection between cardiac ECM organization, tissue elasticity, and heart function, and how these are altered in disease.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13573号 / 論医博第2299号 / 新制||医||1069(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 妹尾 浩, 教授 藤田 恭之, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM