Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation. CAV is caused by damage to the allograft endothelium, resulting in occlusive intimal lesions. Administration of ex vivo cultured early endothelial progenitor cells (eEPCs) enhances endothelial repair and inhibits intimal hyperplasia. However, engraftment rates of eEPCs remain low. We examined changes in eEPC adhesion molecule expression during ex vivo cultivation, and how these changes affect their ability to adhere. Compared to their parent cell population (freshly isolated peripheral blood mononuclear cells, PBMCs), eEPCs had decreased expression of integrins necessary to form firm adhesions with endothelial cells. Despite this eEPCs showed an enhanced ability to adhere under static conditions compared to PBMCs. However, under conditions of physiological flow, eEPC rolling adhesion was reduced compared to PBMCs. We hypothesize that low eEPC retention rates observed in vivo may be due to impaired eEPC rolling resulting from ex vivo culture.

Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation. CAV is caused by damage to the allograft endothelium, resulting in occlusive intimal lesions. Administration of ex vivo cultured early endothelial progenitor cells (eEPCs) enhances endothelial repair and inhibits intimal hyperplasia. However, engraftment rates of eEPCs remain low. We examined changes in eEPC adhesion molecule expression during ex vivo cultivation, and how these changes affect their ability to adhere. Compared to their parent cell population (freshly isolated peripheral blood mononuclear cells, PBMCs), eEPCs had decreased expression of integrins necessary to form firm adhesions with endothelial cells. Despite this eEPCs showed an enhanced ability to adhere under static conditions compared to PBMCs. However, under conditions of physiological flow, eEPC rolling adhesion was reduced compared to PBMCs. We hypothesize that low eEPC retention rates observed in vivo may be due to impaired eEPC rolling resulting from ex vivo culture.

Percutaneous coronary intervention (PCI) acutely improves coronary blood flow and myocardial perfusion but at the expense of endovascular laceration and endothelial denudation. PCI associated vascular injury is associated with intense inflammation and a loss of vascular function that may lead to significant in-stent restenosis (ISR), and the potentially catastrophic, acute stent thrombosis. Reendothelialisation is essential to the restoration of normal homeostasis and facilitating vascular healing. Attention has recently focused on a novel mechanism of reendothelialisation mediated by bone marrow-derived precursor or stem cells: endothelial progenitor cells (EPC). EPC are thought to home to, and reendothelialise sites of endothelial denudation, and therefore offer the potential to provide exciting new developments in the management of cardiovascular disease. Understanding the role of EPC following vascular injury may help us to enhance vascular repair following PCI. The following studies were performed to clarify the relationships between putative EPC and vascular injury associated with PCI. In studies of patients undergoing elective PCI for stable anginal symptoms I found that concentrations of traditional circulating phenotypic EPC expressing CD34+VEGFR-2+ were unaffected, unlike CD34+CD45- cell concentrations, which were transiently increased six hours following PCI, subsequently returning to normal by 24 hours, notably without an increase in CD34+ adhesion molecule expression or VEGF-A production. However, the purported progeny of CD34+VEGFR-2+ cells, endothelial cell-colony forming units (EC-CFU), were mobilised at 24 hours, commensurate with a systemic inflammatory response. Interestingly the concentration of circulating CD34+VEGFR-2+ cells and EC-CFU were unrelated to each other, emphasising the distinction between these two cell populations. Although EC-CFU contained proliferating cells and exhibited some endothelial characteristics, EC-CFU predominantly expressed the leukocyte antigen CD45 in addition to the lymphocyte markers CD4 and CD8, and most intensely, the surface markers CD68 and CD105, epitopes commonly expressed on macrophages. Notably, EC-CFU were a potent stimulus for the migration of mononuclear cells. However, despite being mobilised in the context of an acute systemic inflammatory response and being composed of leukocytes, isolated systemic inflammation in healthy volunteers (induced by Salmonella Typhus vaccination) in the absence of vascular injury did not cause selective mobilisation of EC-CFU or indeed of putative phenotypic EPC. It is therefore likely that EC-CFU mobilisation is a relatively specific inflammatory response to cardiovascular injury. In a cohort of 201 patients undergoing coronary angiography, traditional circulating phenotypic EPC (CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+) were very rare indeed and were not increased in response to an acute coronary syndrome (ACS). Furthermore traditional EPC concentrations bore no relation to atheroma burden or clinical outcome. In contrast, concentrations of CD34+CD45- cells were increased in patients with coronary artery disease compared to those with normal coronary arteries and were increased in association with more severe coronary disease. Increased concentrations of circulating CD34+CD45- cells were also associated with a shorter cumulative event-free survival. Both EC-CFU and angiogenic monocytes expressing Tie-2 and VEGFR-2 were increased following acute myocardial infarction but did not relate to coronary atheroma or clinical outcome. These studies examine the behavior of putative EPC in response to both discrete vascular injury and myocardial infarction, and isolated inflammation in the absence of vascular injury. I have identified novel characteristics of the EC-CFU assay and determined that specific factors associated with cardiovascular injury likely trigger EC-CFU mobilisation. The clinical relevance of the traditional phenotypic EPC population is uncertain, but a novel CD34+CD45- population is mobilised acutely following discrete vascular injury and is significantly associated with coronary atheroma and clinical events. It is probable that the circulating CD34+CD45- concentration reflects vascular injury and atheroma burden, and I suggest that CD34+CD45- cells are released directly from the vessel wall following PCI, and do not reflect a reparatory response. In order to determine the impact of EPC populations on vascular healing, prospective studies examining the impact of periprocedural EPC concentrations on vascular healing following PCI are required.

Cultures of peripheral blood mononuclear cells (MNCs) give rise to at least two different variants of endothelial progenitor cells (EPCs), early and late outgrowth EPCs. We investigated whether microRNAs in early and late EPCs could serve as markers of internal processes that can be exploited to distinguish cell identity and functional capacity. We hypothesized that as MNCs give rise to early and late EPCs, there is a gradual change in total microRNA profile, reflecting a total change in processes within the predominant cell population. Using a candidate microRNA array, early and late EPCs showed vastly different microRNA expression profiles. MiR-146a expression increased progressively as early EPCs emerged around 5-7 days (p<0.05). Through targeting TRAF6 and IRAK1, miR-146a conferred inflammatory tolerance in early EPCs, likely contributing to their purported ability to suppress inflammation. MiR-146a knock down (KD) in endotoxin-stimulated early EPCs reduced anti-inflammatory cytokine IL-1RA (p<0.001), and increased expression of pro-inflammatory cytokines IL-1 (p<0.001) and IL-8 (p<0.01). Interestingly, the microRNA expression profile of late EPCs was highly congruent to mature endothelial cells, with 100-fold greater miR-126 expression than monocytes and early EPCs (p<0.01). MiR-126KD in late EPCs abolished matrigel-network formation (p<0.05); while overexpression (OE) in early EPC augmented network formation (p<0.05) and chemotactic migration (p<0.001). We also found that the melanoma cell adhesion molecule or MCAM (CD146) identified late EPC precursors. Only MCAM+MNCs from adult blood (<5% of total MNCs) yielded late EPC-like colonies. Robust miR-126 expression in these cells predicted the generation of late EPCs. Overall, our results suggest that miR-146a in early EPCs likely contributes to repair by suppressing inflammation during cardiovascular injury; while in late EPCs, miR-126 directly promotes angiogenesis and vascular repair. Finally, we highlight a unique method for the efficient generation of late EPCs by using MCAM selection and screening for miR-126.

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.

Endometriosis is a common cause of subfertility and pelvic pain, affecting up to 10% of women of reproductive age. It is characterised by the presence of endometrial-like tissue outside the uterus. The development of the disease is still poorly understood and, currently, the diagnosis relies on visualisation of typical lesions during surgery. There is great interest in identifying biomarkers to assist in diagnosis and disease management. Blood vessel development is known to be a crucial feature of endometriosis, but the mechanisms involved in angiogenesis are not well described for this disease. Most vessel development relies on the proliferation and migration of pre-existing endothelial cells. However, there may also be roles for cells derived from peripheral blood (circulating angiogenic cells) and surrounding stromal cells. In this thesis, the contribution of these different cell types to vessel development in endometriosis is assessed. In chapter 2, a robust protocol was optimised to identify circulating angiogenic cells (CACs) with flow cytometry. The reliability of the protocol was verified, and the level of these cells was found not to fluctuate with the menstrual cycle in healthy women (P=0.279, F=1.359, 3 d.f.). In chapter 3, levels of CACs in women with and without endometriosis were found to be equivalent (0.0835% ± 0.0422 compared to 0.0724% ± 0.0414), demonstrating that they have no use as a disease biomarker. In chapter 4, isolation and culture of endothelial cells from the endometrium was attempted. However, a pure culture of endometrial endothelial cells could not be obtained, which may be due to contamination by other cell types or cellular transdifferentiation. Finally, in chapter 5, the contribution of endometrial stromal cells to vessel development was considered. Stromal cells were found not to differentiate towards an endothelial cell phenotype, but were able to participate in tube formation assays. However, the presence of endometriosis did not influence this behaviour.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.