Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerosis is a chronic disease that happens mostly in aged people, and recently studies have showed many similarities between Hutchinson Gilford Progeria Syndrome (HGPS) cells and aging cells, implicating dysfunctions of lamin A/C in aging process and atherosclerosis, as HGPS is caused by a mutated form of lamin A/C. Blood flow in arteries is generating shear stress that is mostly applied on endothelial cells that align along inner blood vessel wall. At the same time, endothelial cells are also under stretch by periodic arterial pulses. Considering the fact that atherosclerosis is prone to developing at arterial branches with disturbed shear and increased stretch, it is highly possible that laminar flow and proper stretch force are regulating endothelium to function appropriately. In this thesis, the investigation of what effects laminar flow or cyclic stretch can bring to endothelial cells was conducted, and examination of lamin A/C expression under mechanical forces were elaborated and incorporated with cell senescence. Results showed that laminar shear stress and stretch force can regulate lamin A/C expression in different patterns, which were impaired in senescent cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Biofilm-related infections account for more than 75% of all microbial infections in humans. Several studies argued that the biofilm-dispersal process initiates systemic infections by causing bacteria to be released into the host. Although our knowledge of the characteristics of dispersed bacteria is still limited, it is recognized that these bacteria have different characteristics, such as higher virulence and adhesion factors, in contrast to their planktonic and sessile counterparts. Streptococcus mutans (S. mutans), which is the major pathogen in the formation of dental caries has also been detected in atherosclerotic plaques, and heart valve specimens from patients with cardiovascular diseases. In oral isolates, the frequency of S. mutans strains positive for the collagen binding protein (CBP) cnm+ gene has been estimated to be 10-20%. Tobacco use is considered to be an independent risk factor for both atherosclerosis and dental caries. Knowledge about S. mutans biofilm dispersal is lacking. Thus, studying the characteristics of dispersed bacteria is crucial to fill that gap of knowledge. We began our investigation by conducting a review of the literature on current findings about biofilm formation and dispersion of several oral and extraoral pathogens, in addition to methodologies for analyzing the dispersion phase. For this study, we identified and chose three dispersion-inducing compounds: adenosine triphosphate (ATP), cis-2-deconoic acid (CDA), and nicotine (NIC). Subsequently, the dispersion, adhesion to collagen type IV, and invasion of bovine aortic endothelial cells (BAEC) were studied using two S. mutans strains, UA159 (Cnm-) and TLJ60a (Cnm+). Both strains showed increased dispersion, adherence rates to collagen type IV, and invasion percentages of BAEC when treated with dispersion inducers compared to their control. In the ATP and NIC groups, TLJ60a (Cnm+) demonstrated greater dispersion and adherence to collagen type IV than UA159 (Cnm-). Harboring the cnm encoding gene appears to enhance S. mutans invasion of BAEC in both biofilm and dispersed cells. In the Cnm+ strain, ATP-induced dispersed cells demonstrated a consistent increase in type IV collagen adhesion and BAEC invasion rates. Therefore, it is imperative to investigate the impact of ATP secretion by damaged endothelial cells in determining S. mutans role in atherogenesis. / 2023-12-28

Cultured bovine aortic endothelial (BAE) cells were found to synthesize and secrete heparan sulfate proteoglycans (HSPG), which bound basic fibrobalst growth factor (bFGF). bFGF is a known mitogen for vascular smooth muscle cells, and is indicated to have a role in some proliferative vascular disorders. In the present study, we have purified proteoglycans from BAE cells conditioned media (BAE PG), and further separated the PG into two fractions, PG-I and PG-II, by ion exchange chromatography on a Q-Sepharose column using a linear salt gradient (0.15 M to 1.2 M). PG-I and PG-II elute at 0.85M salt and 0.1M salt respectively. BAE PG is primarily composed of heparan sulfate, which is accessible to the digestion of Heparinase I/III and nitrous acid treatment; and a small amount of chondroitin sulfate, which can be digested by Chondroitinase ABC. Gel filtration chromatography (Sepharose CL-2B and CL-4B columns) showed that BAE PG consisted of two different sized peaks, and had an average molecular weight of approximately 5 x 10⁵ Da. SDS-PAGE with silver staining indicated that BAE PG had two core proteins with estimated sizes of 300kDa and 320kDa, which corresponded to the core protein of PG-I and PG-II respectively. Western blotting with anti-perlecan primary antibody recognized the core proteins of BAE PG. Size exclusion chromatography (Sepharose CL-6B column) following β-elimination showed that BAE PG had GAG chains with an estimated size less than 2 x 10⁵ Da. A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, K_D, of (1.6 ± 0.8) x 10⁻¹⁰ M; the dissociation rate constant, k_r, of 0.01 min⁻¹; the association rate constant, k_f, of 6.2 x 10⁷ M⁻¹min⁻¹ and the total binding sites of the proteoglycan, R_T, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds. / Master of Science

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19865号 / 生博第346号 / 新制||生||46(附属図書館) / 32901 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 松田 道行, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13085号 / 論医博第2126号 / 新制||医||1021(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 渡邊 直樹, 教授 岩田 想, 教授 富樫 かおり / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21009号 / 医博第4355号 / 新制||医||1028(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 羽賀 博典, 教授 前川 平, 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

No description available.

>Magister Scientiae - MSc / Cancer is the most common leading cause of death worldwide. Glioblastoma and breast cancer are the most aggressive solid tumour. The survival rate of these tumours depends on their ability to progress and spread. These cancers use their high proliferative capabilities for survival, increasing their malignancies. Glioblastoma is considered the most aggressive tumour initiated in the brain, whereas breast cancer is the most common metastatic cancer in the brain, both types of cancer are known as high infiltrated cancer and their invasiveness due to their capability to release factors that can alter the neighbouring cells to facilitate their progression.

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.