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19397号 / 医博第4048号 / 新制||医||1012(附属図書館) / 32422 / 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 小泉 昭夫, 教授 木村 剛 / 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・課程博士 / 博士(医学) / 甲第20236号 / 医博第4195号 / 新制||医||1019(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 木村 剛, 教授 松原 和夫, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hypertension, a risk factor for aortic valve stenosis, increases transvalvular load and can elicit extracellular matrix (ECM) remodeling. Elevated cyclic pressure and the vasoactive agent angiotensin II (Ang II) both promote collagen synthesis, an early hallmark of aortic sclerosis. It was hypothesized that increased collagen production induced by elevated pressure conditions or the presence of Ang II would affect the mechanical properties of leaflet tissue by decreasing extensibility. Porcine aortic valve leaflets were exposed to pressure conditions of increasing magnitude with and without Ang II. Biaxial mechanical testing was performed to determine peak stretch. Collagen content was determined using a quantitative dye-binding method. The results demonstrated Ang II and elevated pressure decrease the extensibility of leaflet tissue and increase the collagen content in the ECM. In conclusion, the results demonstrated that both elevated pressure and Ang II play a role in altering the biomechanical properties of aortic valve leaflets.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13494号 / 論医博第2259号 / 新制||医||1060(附属図書館) / (主査)教授 石見 拓, 教授 大鶴 繁, 教授 近藤 尚己 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24484号 / 医博第4926号 / 新制||医||1063(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 森信 暁雄, 教授 YOUSSEFIAN Shohab, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p> Various areas of studies on the natural and the prosthetic aortic valves are reviewed. </p> <p> A microtensile technique devised to investigate the inhomogeneous and anisotropic material properties of a porcine aortic valve's leaflets is described. Also, the theory and apparatus of a new stereophotogrammetric technique to define points in space by their Cartesian coordinates is introduced. The technique is used to investigate the local surface strains and curvatures of a porcine aortic valve's leaflets from 0 to 120 mm. Hg. in-vitro. </p> <p> It is found that the valve leaflets display marked inhomogeneity and anisotropy (orthotropy is assumed) in the elastic moduli and transition strains. For the non-coronary leaflet, the radial post-transition moduli vary from 42 to 215 gm/mm² with a mean of 111 gm/mm² (s.d. = 43 gm/mm²); and the radial transition strains vary from 30% to 70% with a mean of 58% (s.d. = 7%). Areas nearer the leaflet's coaptation edge tend to exhibit lower radial transition strains than the annulus edge. The central region of the leaflet is found to be the stiffest. For the same non-coronary leaflet, the circumferential post-transition moduli vary from 220 to 590 gm/mm² with a mean of 342 gm/mm² (s.d. = 118 gm/mm²); and the circumferential transition strains vary from 22% to 47% with a mean of 33% (s.d. = 3%). </p> <p> Inhomogeneity between leaflets is also observed; preliminary results seem to suggest that the non-coronary leaflet is the stiffest in the radial direction and the least stiff in the circumferential direction. In comparison, the right coronary leaflet exhibits the largest radial transition strains (~80% ) and the smallest circumferential transition strains (~25%). </p> <p> For the diastolic valve in-vitro, the circumferential strains are less than 10% at all pressures; therefore , this suggests pre-transition behaviour during diastole which is contrary to the general belief. Radial strains at diastole vary from 10% to well over 100% and show a definite tendency to increase from the sinus-annulus edge to the coaptation edge. The non-coronary leaflet is the least strained of the leaflets (10% to 60% at diastole). </p> <p> The determination of pre-or post-transition state at diastole is discussed and the implications of the results on stress analyses and trileaflet valve designs are noted. </p> / Thesis / Master of Engineering (ME)

No description available.