Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Transforming growth factor-beta (TGFβ) is an important signaling molecule that regulates several cellular processes including angiogenesis. However, its effects on angiogenesis are complex, with it being pro-angiogenic only at low concentrations (Pepper et al., 1993). Evidence suggests that downstream signaling pathways of TGFβ may be activated in a dose-dependent fashion. In fact, previous work in our laboratory has shown that the non-canonical Par6 polarity pathway gets preferentially activated at low concentrations. Considering the different cellular effects of downstream signaling pathways, we propose that TGFβ may modulate its effects on angiogenesis via differential activation of the canonical Smad and non-canonical Akt, FAK, NFκB and Par6 polarity signaling pathways. Based on this premise, bovine aortic endothelial cells were treated with TGFβ1 and TGFβ2 at concentrations ranging from 0.05 to 5 ng/mL. The activation patterns of canonical and non-canonical signaling pathways were studied via western blotting; with the use of phospho-specific antibodies against Smad2, Akt, FAK and NFκB. Preliminary results reveal that high concentrations of both TGFβ1 and TGFβ2 (5 ng/mL) cause preferential activation of Smad2, while the Akt, FAK and NFκB signaling pathways do not appear to become activated in response to TGFβ1 or TGFβ2 at the concentrations and time points studied. These results suggest the effect of TGFβ on angiogenesis may not involve Akt, FAK or NFκB signaling, but may involve dose-dependent signaling of the Smad signaling pathway. / NSERC

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Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Worldwide, people are being exposed to natural and man-made sources of radiation. Epidemiological studies have shown an increased risk of vascular diseases in populations that have been exposed to ionizing radiation. Vascular endothelium is implicated as one of the targets for radiation leading to the development of cardiovascular diseases. However, the molecular mechanisms behind the development of radiation-induced cardiovascular disease in acute or chronic exposed people are not fully elucidated. The hypothesis that chronic low dose rate ionizing radiation accelerates the onset of senescence of primary human umbilical vein endothelial cells has been tested in papers I and II presented in this thesis. In vitro studies show that, when exposed to continuous low dose rate gamma radiation these cells enter premature senescence much earlier than non-irradiated control cells. Quantitative proteomic analysis using isotope coded protein labeling coupled to LC-ESI-mass spectrometry and followed by protein network analysis identified changes in senescence-related biological pathways including cytoskeletal organisation, cell-cell communication and adhesion, and inflammation influenced by radiation. Moreover, the role of PI3K/Akt/mTOR pathway was implicated during the senescence process. Thus, chronic low dose rated endothelial senescence may contribute to increased risk of radiation-induced cardiovascular disease. Paper III analyse the long-term effects of local high doses of radiation to the heart using a mouse model. The results from proteomic and bioinformatics analysis indicated that an impaired activity of the peroxisome proliferator-activated receptor-alpha (PPARA) is involved in mediating the radiation response. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPARA. This was reflected by the decreased expression of PPARA target genes involved in energy metabolism and mitochondrial respiratory chain. This in vivo study suggests that alteration of cardiac metabolism contributes to the impairment of heart structure and function after radiation. Taken together, these in vitro and in vivo studies provide novel information on the pathways in heart and endothelial cells that are affected over longer periods of time by ionizing radiation.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.