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.

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Indiana University-Purdue University Indianapolis (IUPUI) / HIV-associated cardio-pulmonary vascular pathologies such as coronary artery disease, pulmonary hypertension and emphysema remain a major issue in the HIVinfected population even in the era of antiretroviral therapy (ART). The continued production of HIV encoded pro-apoptotic protein, such as Nef in latently HIV-infected cells is a possible mechanism for vascular dysfunction underlying these diseases. HIVNef persists in two compartments in these patients: (i) extracellular vesicles (EV) of plasma and bronchoalveolar lavage (BAL) fluid and (ii) PBMC and BAL derived cells. Here I demonstrate that the presence of HIV-Nef protein in cells and EV is capable of stressing endothelial cells by inducing ROS production leading to endothelial cell apoptosis. HIV-Nef protein hijacks host cell signaling by interacting with small GTP binding protein Rac1 which activates PAK2 to promote the release of pro-apoptotic cargo containing EV and surface expression of pro-apoptotic protein Endothelial Monocyte Activating Polypeptide II (EMAPII). Using this mechanism, Nef protein robustly induces apoptosis in Human Coronary Artery Endothelial Cells and Human Lung microvascular endothelial cells. Endothelial specific expression of HIV-Nef protein in transgenic mice was sufficient to induce vascular pathologies as evidenced by impaired endothelium mediated vasodilation of the aorta and vascular remodeling and emphysema like alveolar rarefaction in the lung. Furthermore, EV isolated from HIV patients on ART was capable of inducing endothelial apoptosis in a Nef dependent fashion. Of therapeutic interest, EMAPII neutralizing antibodies to block EMAPII mediated apoptosis and statin treatment to ameliorate Nef induced Rac1 signaling was capable of blocking Nef induced endothelial stress in both in vivo and in vitro. In conclusion, HIV-Nef protein uses a Rac1-Pak2 signaling axis to promote its dissemination in EV, which in turn induces endothelial cell stress after its uptake.

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(医学) / 甲第19620号 / 医博第4127号 / 新制||医||1015(附属図書館) / 32656 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 森田 智視, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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20562号 / 医博第4247号 / 新制||医||1022(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 江藤 浩之, 教授 開 祐司 / 学位規則第4条第1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21003号 / 医博第4349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 山下 潤, 教授 木村 剛, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM