Dissertations / Theses on the topic 'Capillary endothelial cells'

Doctor of Philosophy (Medicine)
Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model.
AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

The blood brain barrier (BBB), formed by endothelial cells lining the lumen of the brain capillaries, is a restrictively permeable interface that only allows transport of specific compounds into the brain. Cardiolipin (CL) is a mitochondrial- specific phospholipid known to be required for the activity and integrity of the respiratory chain. The current study examined the role of cardiolipin in maintaining an optimal mitochondrial function that may be necessary to support the barrier properties of the brain microvessel endothelial cells (BMECs). Endothelial cells have been suggested to obtain most of their energy through an-aerobic glycolysis based on studies of cells that were obtained from the peripheral vasculatures. However, here, we showed that the adult human brain capillary endothelial cell line (hCMEC/D3) appeared to produce ~60% of their basal ATP requirement through mitochondrial oxidative phosphorylation. In addition, RNAi mediated knockdown of the CL biosynthetic enzyme cardiolipin synthase (CLS), although did not grossly affect the mitochondrial coupling efficiency of the hCMEC/D3 cells, did seem to reduce their ability to increase their mitochondrial function under conditions of increased demand. Furthermore, the knockdown appeared to have acted as a metabolic switch causing the hCMEC/D3 cells to become more dependent on glycolysis. These cells also showed increase in [3H]-2-deoxyglucose uptake under a low glucose availability condition, which might have served as a mechanism to compensate for their reduced energy production efficiency. Interestingly, the increase in glucose uptake appeared correlated to an increase in [3H]-2-deoxyglucose glucose transport across the knockdown confluent hCMEC/D3 monolayers grown on Transwell® plates, which was used in our study as an in vitro model for the human BBB. This suggests that changes in the brain endothelial energy status may play a role in regulating glucose transport across the BBB. These observations, perhaps, also explain why the brain capillary endothelial cells were previously observed to possess higher mitochondrial content than those coming from non-BBB regions (Oldendorf et al. 1977).

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

In vitro, la formation de structures de type tubulaire avec des cellules endothéliales de veine ombilicale humaine (HUVEC) a été étudiée en combinant la fonctionnalisation de la chimie des matériaux et le développement de la géométrie tridimensionnelle. Le polycarbonate (PC) a été utilisé comme modèle pour le développement de l'échafaud. Le film de polysaccharide naturel, basé sur un dépôt alternatif couche par couche (LbL) d’acide hyaluronique (HA) et de chitosane (CHI), a d’abord été appliqué sur une surface PC et caractérisé en termes de croissance d’épaisseur microscopie à balayage lascar (CLSM). Cette première fonctionnalisation se traduit par un revêtement complet de la couche PC. Une biofonctionnalisation supplémentaire avec un peptide adhésif (RGD) et deux peptides angiogénétiques (SVV et QK) a été étudiée, immobilisant ces peptides sur le groupe carboxylique de HA précédemment déposé, en utilisant la chimie bien connue du carbodiimide. La version marquée de chaque peptide a été utilisée pour caractériser l’immobilisation et la pénétration des peptides dans les couches de polyélectrolytes, aboutissant à une greffe réussie avec une pénétration complète dans toute l’épaisseur du LbL. Des tests in vitro ont été effectués à l'aide de cellules HUVEC pour évaluer leur efficacité d'adhésion et leur activité métabolique sur la LbL avec et sans immobilisation de peptides, ce qui a permis d'améliorer l'activité préliminaire lorsque des combinaisons de peptides sont utilisées. Enfin, les micro-canaux PC (μCh) ont été développés et caractérisés pour la première fois, et les autres expériences ont été réalisées sur un micromètre de 25 μm de largeur, fonctionnalisé avec une architecture (HA / CHI) 12,5 (PC-LbL) avec des peptides RGD et QK -RGD + QK) ou avec des peptides RGD et SVV (PC-RGD + SVV). Notre première expérience de tubulogénèse a montré de manière surprenante la formation de structures de type tubulaire déjà après 2h d'incubation en utilisant la combinaison double-peptides, mais uniquement avec PC-RGD + QK. Les tubes étaient également présents après 3 et 4 heures de culture. L'expérience de co-culture avec des péricytes humains dérivés du placenta (hPC-PL) montre comment la stabilisation des tubes a été améliorée après 3 et 4 heures également pour l'échantillon de PC-RGD + SVV. Globalement, notre matériel bio-fonctionnel avec les peptides PC-RGD + QK et PC-RGD + SVV permet la formation d'une structure de type tubulaire à la fois dans une expérience de monoculture et de co-culture
In vitro, tubular-like structures formation with human umbilical vein endothelial cells (HUVECs) was investigated by combining material chemistry functionalization and three-dimensional geometry development. Polycarbonate (PC) was used as a template for the development of the scaffold. Natural polysaccharide’s film based on alternate layer-by-layer (LbL) deposition of hyaluronic acid (HA) and chitosan (CHI), was first applied to PC surface and characterized in terms of thickness growth both, in dry conditions using ellipsometry, and confocal lascar scanning microscopy (CLSM). This first functionalization results in a complete coating of the PC layer. Further biofunctionalization with one adhesive peptide (RGD) and two angiogenetic peptides (SVV and QK) was investigated, immobilizing those peptides on the carboxylic group of HA previously deposited, using the well-known carbodiimide chemistry. The labeled version of each peptide was used to characterize the peptides’ immobilization and penetration into the polyelectrolytes layers, resulting in a successful grafting with complete penetration through the entire thickness of the LbL. In vitro tests were performed using HUVECs to assess their adhesion efficiency and their metabolic activity on the LbL with and without peptide immobilization, resulting in a preliminary improved activity when peptide-combinations is used. Finally, PC micro-channels (μCh) were first developed and characterized, and the rest of the experiments were performed on μCh of 25μm width, functionalized with (HA/CHI)12.5 architecture (PC-LbL) with RGD and QK peptides (PC-RGD+QK) or with RGD and SVV peptides (PC-RGD+SVV). Our first tubulogenesis experiment surprisingly showed the formation of tubular-like structures already after 2h of incubation using the double-peptides combination but only using PC-RGD+QK the tubes were present also after 3 and 4 hours of culture. The co-culture experiment with human pericytes derived from placenta (hPC-PL) demonstrates how the stabilization of the tubes was improved after 3 and 4 hours also for the PC-RGD+SVV sample. Globally our bio-functional material with PC-RGD+QK and PC-RGD+SVV peptides allow the formation of tubular-like structure in both mono and co-culture experiment

Pathological angiogenesis, or new blood vessel formation, is involved in many pathologies, including cancer and serious eye diseases. While traditional anti-angiogenic therapies target vascular endothelial growth factor receptors to reduce or inhibit new vessel formation, this approach has several downsides, including unpleasant side effects and low efficacy over time. Therefore, identifying new targets to treat pathological angiogenesis is still needed. CMG2, one of the two identified anthrax toxin receptors, has been proposed as an alternative target to treat pathological angiogenesis. CMG2’s role as a cell surface receptor that mediates anthrax toxin internalization is very well documented. One physiological function for CMG2, not related to anthrax intoxication, is suggested by the observation that loss-of-function mutations in CMG2 cause hyaline fibromatosis syndrome (HFS), a genetic disease that results in accumulations of extra-cellular matrix (ECM) protein in different parts of the body. While the complete molecular mechanism for CMG2’s role in regulating angiogenesis has not been determined, this dissertation addresses multiple ways CMG2 regulates pathological angiogenesis. We have discovered that CMG2 plays a role in mediating ECM homeostasis via endocytosis of ECM proteins and protein fragments as a way to generate angiogenic signals from the cell. We have also demonstrated that a fragment from Col IV, S16, is endocytosed into the cells by interacting with CMG2, and S16 treatment to endothelial cells leads to a significant reduction in cell migration. Also, an endothelial cell migration assay with CMG2 knockout cells results in abolished directional migration, indicating that CMG2 is required for endothelial cell chemotaxis. Notably, we have identified that bFGF, VEGF, and PDGF are involved in CMG2 mediated chemotaxis but not insulin and sphingosine-1-phosphate (S1P). While recent literature reports show that CMG2 works closely with RhoA GTPase, which is commonly known to regulate cell migration, we have also observed that inhibition of RhoA also reduced cell chemotaxis towards VEGF but not S1P. These results could be leveraged to develop new classes of therapeutic molecules to treat pathological angiogenesis induced by multiple various growth factors via targeting CMG2.

碩士
國立彰化師範大學
化學系
100
A new analytical method involving capillary electrophoresis (CE) with magnetic nanoparticles was developed to analyze C-reactive protein (CRP) synthesized by human aortic endothelial cells. The surfaces of magnetic nanoparticles were modified with protein-G which bound to anti-CRP antibodies, and the particles were used to detect CRP. The complexes were then directly analyzed by CE. To optimize the CE conditions, we have examined several factors including magnetic particle size, phosphate buffer concentration and pH value, CE capillary temperature as well as applied voltage. The optimal magnetic particle size selected was 2.8 μm. The chosen separation buffer was 5 mM sodium phosphate buffer, pH 8.0. The chosen CE capillary temperature was 25 ℃, and the applied voltage was 25 kV. Previously, we have found that oxidized low-density lipoprotein (ox-LDL) induced the synthesis of CRP by HAECs. When the optimal conditions were used to determine CRP synthesized by HAECs, the observed mobilities were similar to that of CRP protein standard. Therefore, we have developed a simple, reliable and highly reproducible new CE method to detect CRP.

Nowadays, the biggest challenge in tissue engineering consists in developing structures and in the application of strategies to emulate the anatomical and cellular complexity and vascularization of native tissues to maintain cell viability and functionality. The presence of functional blood vessel networks is essential to ensure adequate nutrient flow and oxygen diffusion throughout the support structure, two key requirements for maintaining cell viability. This work aimed to develop a complex in vitro model that mimics the native vascular network. To this end, a multilayered membrane made of six bilayers of chitosan (CHI)/alginate (ALG) or CHI/ALG-RGD (tripeptide of Arginine (R)-Glycine (G)- Aspartic acid (D) responsible for the cellular adhesion to the extracellular matrix (ECM)) were produced via Layer-by-Layer (LbL) assembly technology on the ALG printed structures. The ALG structures coated with the multilayered membranes were embedded in xanthan gum, chemically modified with methacrylated groups in order to obtain a mechanically robust hydrogel structure after photocrosslinking by UV light exposure. The liquification of the ALG printed structures, coated with the CHI/ALG, CHI/ALG-RGD or without the multilayers membranes, with ethylenediaminetetraacetic acid (EDTA), led to the formation of microchannels in which human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours. The obtained results demonstrate that the microchannels encompassing CHI/ALG-RGD multilayered membranes contributed to a larger cellular adhesion, demonstrating their potential to be applied in tissue engineering and regenerative medicine strategies.
Atualmente, o maior desafio em engenharia de tecidos consiste no desenvolvimento de estruturas e aplicação de estratégias que visem mimetizar a complexidade anatómica e celular, assim como a vascularização de tecidos nativos, de forma a manter a viabilidade e funcionalidade das células. A presença de estruturas funcionais à base de vasos sanguíneos é essencial para garantir o fluxo adequado de nutrientes, assim como a difusão de oxigénio em toda a estrutura de suporte, dois requisitos essenciais para manter a viabilidade celular. Este trabalho teve como objetivo desenvolver um modelo complexo in vitro que mimetize a rede vascular nativa. Com esse intuito, membranas multicamadas compreendendo seis bicamadas de quitosana (CHI)/alginato (ALG) e CHI/ALG-RGD (tripéptido de Arginina (R)-Glicina (G)-Ácido aspártico (D) responsável pela adesão de células à matriz extracelular) foram produzidas, via tecnologia de deposição camada-a-camada (do inglês Layer-by-Layer assembly technology), em estruturas impressas de ALG. As fibras de ALG revestidas com os filmes multicamadas foram embebidas em goma xantana, quimicamente modificada com grupos metacrilatos, de modo a obter uma estrutura de hidrogel mecanicamente robusta após foto-reticulação por ação da luz UV. A liquefação das estruturas impressas de ALG, contendo as multicamadas de CHI/ALG ou CHi/ALG-RGD, com ácido etilenodiamino tetra-acético (EDTA), levou à formação de microcanais nos quais se cultivaram células endoteliais humanas, extraídas da veia umbilical durante 24 horas. Os resultados obtidos demonstraram que os microcanais compreendendo as membranas multicamadas à base de CHI/ALG-RGD contribuíram para uma maior adesão celular, demonstrando o seu potencial para estratégias de engenharia de tecidos e medicina regenerativa.
Mestrado em Biotecnologia

碩士
國立彰化師範大學
化學系
100
In this study, capillary electrophoresis with laser induced fluorescence detection and microplate reader in combination with magnetic particles have been used to analyze C-reactive protein (CRP). CRP was synthesized by human aortic endothelial cells (HAEC) which were stimulated with oxidized low-density lipoproteins to monitor oxidative stress in vivo. The surfaces of magnetic particles were modified by several steps. First, the magnetic particles were modified with protein G. Second, anti-CRP antibody-1 (1°Ab) was bound to protein G. Third, CRP was bound to 1°Ab. Fourth, anti-CRP antibody-2 (2°Ab/FITC) was bound to CRP. Two methods were used to analyze CRP: (a) non-elution (directly analyze the magnetic particle-1°Ab-CRP-2°Ab/FITC complex), and (b) elution (analyze the eluted analytes from magnetic particles). To optimize the binding conditions for magnetic particles and antibodies, several factors including the reaction time and concentration of 1°Ab as well as the concentration of 2°Ab/FITC have been examined. For 1°Ab, the optimal reaction time selected was 0.5 hour, and the optimal concentration was 0.035 mg/mL. For 2°Ab/FITC, the optimal concentration selected was 5 mg/mL. To optimize the conditions of capillary electrophoresis (CE), the diameter of magnetic particle and the additive in the CE separation buffer have been examined. The additive was used to prevent aggregations of magnetic particles. The optimal magnetic particle diameter selected was 100 nm, and the optimal CE separation buffer and additive selected was 20 mM PB (pH 7.4) with 0.1 mg/mL oleic acid.   Two peaks were observed for the magnetic particle-1°Ab-CRP-2°Ab/FITC complex when it was directly analyzed by CE. Two peaks were also observed when the eluted analytes from magnetic particles were analyzed by CE. However, no linear relationships were found between CRP concentration and fluorescence intensity from CE analysis. When the magnetic particle-1°Ab-CRP-2°Ab/FITC complexes and the eluted analytes were analyzed by a microplate reader, linear relationships were found between CRP concentration and fluorescence intensity for both methods. The linear ranges for non-elution and elution methods were 1×10-4 - 0.2 and 2×10-5 - 2×10-3 mg/mL, respectively. The concentration limits of detection for both methods were 2×10-5 mg/mL. When CRP released from human aortic endothelial cells under oxidative stress was analyzed by a microplate reader, the concentrations of CRP were 17.3×10-4 and 3.46×10-4 mg/mL for non-elution and elution methods, respectively.

碩士
中原大學
機械工程研究所
103
Part I To understand the impact of inhaled air pollution on cardiovascular diseases, Chao et al., in 2011, confirmed that PM2.5 air pollution particles have a 14% chance of containing <100 nm particles that could destroy junction adhesion sites of endothelial cells to blood circulation regions. There is not a standard method to quantify the rate of endothelial cell permeability. The units of or Darcy cannot represent the diffusion phenomenon of permeability at the cell junction and the change of permeability due to opening and closing of the blood vessels. Furthermore, blood to the tissue permeability changes easily over time and it makes difficult to obtain the transient measurement of cell permeability. This thesis has developed a standard methodology to quantify the rate of vascular permeability in order to understand the damage of endothelial junction and the change of endothelial permeability cased by the fine particles in the air pollution. Various concentrations of diesel PM2.5 particles were added to a HUVEC cell culture medium for 0, 24, 48 and 72 hours. Afterward, MTS reagent and Dextran-FITC were used to investigate the cell cytotoxicity and the permeation rates, respectively. In addition, Capillary Number (CA) was used to measure the permeability behavior in terms of viscous force over surface tension in order to build a standardized way to quantify the blood-to-tissue transport phenomena of PM2.5 at the junction between the endothelial cells. The experimental results showed cell permeability increases with the concentration of PM2.5 and exposure time. Moreover, increasing viscosity and decreasing surface tension caused by larger PM2.5 concentration and longer exposure time leading to a higher measure of CA. This thesis parametrically modeled the responses of cell cytotoxicity and CA with respect to PM2.5 concentration and exposure time. A mathematical function was determined to represent the safety region of PM2.5 exposure under the decision of critical CA. Part II An infant’s mood when subject to radioactive test or treatment is not easy to control. This often causes negligent operation of the radiographer such as overexposure, poor image, misjudgment, bodily harm to the baby and other adverse consequences. In addition to affecting the baby''s development this also increases the risk for cancer in the baby. This paper presents an innovative baby fixture for the purpose of restricting rapid, instantaneous movement in a baby and secures the range of movement during the consultation process. In addition, this design also focuses on baby comfort while being fixed, convenient adjustment to the desired position, effects on the clarity of the image, and the degree of protection of sensitive organs and limbs. In this study, a highly penetrative the acrylic material was used for the fixed base containing fixation holes and acrylic plugs with the active components (such as straps fixed cover, etc.) which can be securely fixed to the base to the baby. A fast rotary fixation design and Velcro fastening means that the size of infants may vary and any adjustments can be easily made which aides in the successful completion of a fast radioactive detection or treatment. To provide more comfort, a highly radiation penetrating foam was added to support the weight of the baby. The use of numerical analysis software was used to assess the fixation device, the supporting foam pad and the pin fixation capacity. The results of the numerical simulations show that the foam pad supporting a 50 kg baby only deformed 14.1397 mm which provides adequate support force. It can provide sufficient comfort for the baby without permanent deformation. The fixation pins subject to a 20 kilograms of downward bending force when fixed at the base is subject to 55.7268MPa bending stress. This stress is less than the yield strength of the material therefore it will not fail. The innovative baby fixation device developed this thesis can effectively avoid image blur, offset, appearance of visual artifacts and other issues which avoids the need to re-shoot, therefore reducing the infant’s exposure to radiation.

L'ingénierie tissulaire est reconnue comme une méthode potentielle pour réparer ou régénérer les tissus endommagés. Malgré de grandes avancées dans l'ingénierie tissulaire, la réussite de la construction de tissus complexes avec des réseaux vascularisés reste un défi. Dans les modèles d'angiogenèse actuels, les cellules endothéliales sont ensemencées au hasard, n'offrant pas de structure organisée. La technologie de bioimpression par laser offre une résolution d'impression précise. Par cette technique, les structures microvasculaires peuvent être construites pour la fabrication d'organes complexes, ou pour modéliser la progression de la maladie ou les modèles de réponse aux médicaments. Dans cette étude, des techniques de bio-impression au laser ont été utilisées pour étudier le guidage de l'angiogenèse in vitro. Deux techniques basées sur le laser, le transfert direct induit par laser (LIFT) et le transfert latéral induit par laser (LIST) sont utilisées. Comparée à LIFT, la technologie LIST offrait des conditions idéales pour l'impression cellulaire telles que la concentration cellulaire requise pour la formation du tubes endothéliaux et l'uniformité du motif désiré. Nous avons réalisé le modelage de la formation de structures de type capillaire dans des motifs organisés via l'impression LIST. Les constructions de type capillaire formées présentent des motifs uniformes. Les structures formées ont été analysées par microscopie confocale et reconstruction d'images 3D. Bien que le développement de la lumière endothéliale soit incomplet, la technique développée possède le potentiel d'atteindre une stabilisation et un développement de la lumière si l'on recrute un deuxième type de cellule tel que les fibroblastes ou les péricytes.
Tissue engineering has been well acknowledged as a potential method to repair or regenerate damaged tissues in the human body, fulfilling the limitations and shortage in autologous and organ transplantations. Despite great advances in engineering tissues with simple geometry and low requirement for oxygen and blood supply such as cartilage, skin and cornea, success in constructing 3D complex tissues with vascularized networks remains a major challenge. Angiogenesis plays an important role in vascular development in vivo. In current angiogenesis models, endothelial cells are seeded randomly not offering precise and desired patterning. Laser-based bioprinting technology offers precise and high cell printing resolution. By using laser-based bioprinting technology, microvascular structures can be constructed as a platform for complex organ fabrication, disease progression and drug response models. In this study, laser-based bioprinting techniques are employed to study angiogenesis guidance in vitro by patterning endothelial cells. Two laser-based techniques, Laser-Induced Forward Transfer (LIFT) and Laser-Induced Side Transfer (LIST) are used as patterning tools. Compared to LIFT, LIST technology provided ideal conditions for cell printing such as required cell concentration for endothelial tube formation and pattern uniformity. In this study, we achieved the guidance of capillary-like structure formation in desired patterns via LIST printing. The formed capillary-like constructs featured precise patterns and uniformity. The structures were analyzed by confocal microscopy, 3D image reconstruction and frozen section procedure. Though lumen development was incomplete, it possesses the potential to attain further stabilization and lumen development if recruiting a second cell type such as fibroblast or pericyte.