No description available.

No description available.

No description available.

No description available.

Endothelial cells form the inner lining of a blood vessel and their structure and functional integrity are important in maintenance of the vessel wall and circulatory function. These cells play key roles in immune and inflammatory reactions by regulating lymphocyte and leukocyte movement into tissues; they are also main targets for antivascular agents in cancer therapy. Endothelial cell responses to different stimuli have been previously investigated using conventional approaches, 'HUVEC in vitro culture system'. In this study an ex vivo perfusion model was constructed and utilized using whole human umbilical cords, in attempts to replicate a more accurate in vivo microenvironment. Assessment of the proportion and duration of endothelial cell viability in the ex vivo model was undertaken using a MTT 3, (4,5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide viability assay. A time baseline was successfully established for all experimental perfusions. Endothelial cell immune response was assessed through intravenous perfusion of the endotoxin, Lipopolysaccharide (LPS). Gene expression profiles revealed a significant increase in expression levels of E-Selctin (E-Sel), Intracellular adhesion molecule (I-CAM) and Tissue Factor (TF) relative to the housekeeping gene Beta 2 Microglobulin. When LPS was administered in combination with Hypertonic Saline Solution (HSS), expression levels declined indicating HSS interferes with the activation pathway of LPS ultimately suppressing its effectiveness on endothelial cells. HSS impact was also recognized from perfusion experiments on resting endothelial cells. All identified genes were suppressed by HSS apart from inducible nitric oxide synthase (iNOS). As a potential target for antivascular agents, HUVEC were then stimulated with DMXAA and gene responses of Tumour Necrosis Factor-α (TNF-α) was analysed. DMXAA demonstrates excellent antivascular acivity in experimental tumours, so tumour conditioned media (TCM) was administered intravenously through umbilical cord segments to replicate a tumour microenvironment prior to DMXAA addition. When cells were stimulated with Tumour conditioned media then administered DMXAA, TNF-α expression was significantly upregulated; relative to the housekeeping gene Human Proteosome subunit Y, however, when the cells were exposed to tumour conditioned media in absence of DMXAA gene expression significantly decreased. Thus, the antitumour action of DMXAA is capable of inhibiting the gene response of TNF-α replicated in a tumour environment.

In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

Pancreatic islets are heavily vascularized micro-organs containing insulin secreting beta-cells coupled with endothelial cells (EC). These EC slowly deteriorate in static culture, precluding long term study of beta-cell-EC interaction, and likely limiting tissue revascularization post-transplantation. We postulate this EC deterioration is due to an absence of hemodynamics, blood movement. We created a microfluidic device to mimic aspects of hemodynamics, delivering a range of media flow to ex vivo islets. With our resulting desk-top system, we have conducted long term incubations (72 hrs), fixed tissue treatments (maintaining endothelial cell morphology) and real-time live tissue imaging (glucose-stimulated Ca2+-response). Our data show that flow in a microfluidic device maintains EC morphology in ex vivo islets better than non-flowing culture, providing an improved platform to study ex vivo islets and to examine the interaction between beta-cells and EC. Our data also suggest an opportunity to prime islet EC for revascularization using microfluidic flow prior to transplantation.

Pancreatic islets are heavily vascularized micro-organs containing insulin secreting beta-cells coupled with endothelial cells (EC). These EC slowly deteriorate in static culture, precluding long term study of beta-cell-EC interaction, and likely limiting tissue revascularization post-transplantation. We postulate this EC deterioration is due to an absence of hemodynamics, blood movement. We created a microfluidic device to mimic aspects of hemodynamics, delivering a range of media flow to ex vivo islets. With our resulting desk-top system, we have conducted long term incubations (72 hrs), fixed tissue treatments (maintaining endothelial cell morphology) and real-time live tissue imaging (glucose-stimulated Ca2+-response). Our data show that flow in a microfluidic device maintains EC morphology in ex vivo islets better than non-flowing culture, providing an improved platform to study ex vivo islets and to examine the interaction between beta-cells and EC. Our data also suggest an opportunity to prime islet EC for revascularization using microfluidic flow prior to transplantation.

Organic nitrates have been effective treatment for ischemic heart disease for over 100 years. However, there is limitation in their clinical utility since prolonged use of these drugs results in rapid development of nitrate tolerance, which is associated with increased arginase activity and production of reactive oxygen species. Quercetin, a flavonol abundantly found in fruits and vegetables, has been shown to reduce nitrate tolerance in vitro. The objective of this study is to investigate the potential effect of quercetin and three other flavonols (namely, kaempferol, myricetin and rutin) on the development of nitrate tolerance and the activity of arginase in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated with or without nitroglycerin (GTN) and/or flavonols followed by an acute stimulation with GTN. Level of cyclic guanosine monophosphate (cGMP) released into the cell medium was measured by an enzyme immunoassay. Activity of arginase in cell lysate was measured by a quantitative colorimetric arginase determination assay. Prior treatment with GTN at 〖10〗^(-5)M for either one or 24 hours did not affect the level of cGMP released from HUVECs induced by subsequent stimulation with GTN. On the other hand, arginase activity was significantly decreased in HUVECs pre-treated with GTN at 〖10〗^(-5)M alone for 24 hours and the decrease was not affected by the concomitant presence of the flavonols during the incubation period. However, the data obtained in HUVECs pre-treated with GTN for 24 hours is questionable due to the lack of a corresponding control (i.e. cells incubating with medium for 24 hours) for proper comparison. Pre-treatment for one hour with myricetin (〖10〗^(-5) M) and rutin (〖10〗^(-5) M), alone but not in combination with GTN (〖10〗^(-5) M), appears to increase the release of cGMP to subsequent stimulation by GTN (〖10〗^(-5) M). Rutin pre-treatment for one hour also seems to decrease the activity of arginase in HUVECs. However, these effects of myricetin and rutin were significant only when compared to the control group (without pre-treatment with GTN) but not when comparison was made to the vehicle-treated group, while there is no significant difference between the control and the vehicle group in both the cGMP release and arginase activity. As such, these potential effects of myricetin and rutin are inconclusive. The inability to induce nitrate tolerance in the present experimental condition does not allow further investigation on the potential effect of flavonols on nitrate tolerance. In addition, there are limitations in the present study [namely, lack of corresponding control for the 24 hour incubation groups and small sample size (n = 2-3)]. Therefore, the findings need to be interpreted with cautions; improvements of the present experimental design and increasing the number of experiments are needed in order to obtain more conclusive findings. Future experiments should also be performed in other vascular cells in addition to endothelial cells, as flavonols may exert their beneficial effect in an endothelium-independent manner. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

The objective of this study was to develop a fluorescent near-infrared endothelial cell binding conjugate using Lycopersicon esculentum lectin and Cy5.5 N-hydroxysuccinimide ester for the purpose of imaging the microvascular network in mouse hearts under in vivo and ex vivo conditions. Cy5.5-lectin conjugate was synthesized with a dye/protein ratio of 2.90 ± 1.54 (n=6). Mouse hearts were successfully labelled in both in vivo and ex vivo and showed similar labelling patterns. Cy5.5-lectin labelling patterns and that of ICAM2 and FITC-lectin co-localized, indicating binding to endothelial cells. Finally, it was shown that Cy5.5-lectin is capable of visualizing, in real-time, areas of normal and abnormal heart perfusion at resolutions of 76.8 pixels/mm. Areas of the heart that were not perfused post-ligation displayed no Cy5.5 staining on histological sections and during real-time cardiac imaging of intact hearts showed minimal fluorescent signal (~35 a.u.) compared to areas where normal perfusion occurred (~150 a.u.).