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No description available.

No description available.

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

Device anchoring of artificial cornea implants, through tissue integration of stromal tissue, is necessary to ensure long-term success. In this work, the delivery of basic fibroblast growth factor (FGF-2), a key modulator in corneal wound healing, via heparin modified materials was investigated as a means of sustained, soluble growth factor delivery for stimulation of device anchorage. Two materials types, commonly used for ophthalmic applications and currently under investigation for use in artificial cornea applications, were utilized. Poly (dimethyl siloxane) (PDMS) is currently under investigation as the base material for keratoprosthetic devices; dendrimer crosslinked collagen has been examined as the basis for use as a tissue engineered corneal equivalent. PDMS surfaces were modified directly or indirectly, through a poly (ethylene oxide) (PEO) spacer, to contain functionalized reactive NSC groups capable of binding heparin and FGF-2 Surface modifications were characterized with attenuated total reflection Fourier transform infrared spectrophotometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angles. Heparin coverage was assessed with metachromatic and bioactivity assays. Heparinized collagen gels were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and polypropyleneimine octaamine G2 dendrimers. Gel integrity was assessed with water uptake, differential sr::anning calorimetry, and heparin and dendrimer stability. Both materials were exposed to radiolabelled FGF-2 and growth factor immobilization and delivery were quantified. Heparinized PDMS surfaces were capable of binding on average 100 ng/cm2 ofFGF-2, while heparinized collagen gels had higher FGF-2 immobilization, 300 ng, likely attributed to their higher heparin densities and the fact that the bulk gel rather than the surface only was modified. Delivery of FGF-2 from the heparinized materials revealed a first order release profile, with an initial burst of FGF-2, followed by gradual growth factor release. Release rates, over a 2 week period, reached 6.5% and 50%, for 1 day and 3 day FGF-2 exposed heparinized PDMS modified surfaces, while hepruinized dendrimer crosslinked collagen gels released 40%. / Thesis / Master of Applied Science (MASc)

This thesis investigates the effects of low oxygen culture conditions and fibroblast growth factor-2 (FGF2) on adult human dermal fibroblasts. It was previously shown that low oxygen and FGF2 culture conditions lead to an extension of proliferative lifespan, low-level activation of stem cell genes, and global transcriptional changes in adult human dermal fibroblasts. Additionally, an increased in vivo tissue regenerative response can be observed when human muscle-derived fibroblasts grown with FGF2 and low oxygen are implanted into mouse muscle injury, leading to a decrease in collagen deposition and scar formation and increase of functional skeletal muscle regeneration, including formation of Pax7+ muscle stem cells. These findings led to an analysis of key cellular oxygen sensors, hypoxia inducible factors (HIFs) and their role in this regenerative response. Directly linking these factors with the regenerative response, I have shown, with knockdown experiments, that HIF-2α is required for the increased proliferative capability and decreased senescence of human dermal fibroblasts (hDFs) induced by hypoxia. I have also determined that low oxygen causes an early and transient increase of HIF-1α and late and sustained increase of HIF-2α protein accompanied by increased nuclear translocation. Using overexpression and knockdown approaches via lent-virus, I determined that HIF-2α appears to modulate FGF2 signaling through the FGF receptors. First, under low oxygen conditions, exogenous FGF2 led to downregulation of endogenous FGF2, which can be mimicked by overexpression of HIF-2α. In ambient oxygen we didn't see this effect. Second, HIF-2α overexpression appears to lead to increases in FGFR1 phosphorylation and consequently increased ERK1/2 phosphorylation, and increases in the expression of heparan sulfate modifying enzymes (NDST1, NDST2, and EXTL2). Lastly, sustained supplementation with FGF2 in low oxygen inhibits receptor-mediated FGF2 signaling. To understand these effects at the transcriptional level, using microarray technology, we identified oxygen-mediated FGF2 effects on genes involved in cell survival and proliferation. Through bioinformatics analyses, I determined that genes involved in wound healing (extracellular matrix genes, adhesion molecules, cytokines) are upregulated in FGF2 treated fibroblasts grown under low oxygen. By utilizing a gain-of-function approach, we were able to assess the effects of altered HIF-2α activity on the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 in adult hDFs. The results indicate that overexpression of the HIF-2α transcription factor increases Oct4 mRNA, but not Oct4 protein, levels, and had no effect on Nanog and Lin28 proteins. HIF-2α overexpression also mediated FGF2 induction of Sox2 and Rex1 proteins of higher molecular weight. This thesis expands our knowledge about effects of low oxygen and FGF2 on adult human dermal fibroblasts and explains in part, how FGF2 under low oxygen conditions may lead to increased proliferation, extended life span, regenerative competency and increased developmental plasticity of adult hDFs.

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京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13290号 / 論医博第2188号 / 新制||医||1039(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 浅野 雅秀 / 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・論文博士 / 博士(医学) / 乙第13380号 / 論医博第2214号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科外科系専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at http://onlinelibrary.wiley.com/doi/10.1002/term.2060/abstract;jsessionid=F0849D98381EEF9E83401A02B9042F4D.f04t02 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19602号 / 医博第4109号 / 新制||医||1014(附属図書館) / 32638 / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 伊佐 正, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM