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.

No description available.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Thesis (DScMedSc (Medical Sciences)--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: See full text for abstract / AFRIKAANSE OPSOMMING: Geen opsomming was ingehandig met tesis

Endothelial Progenitor Cells (EPCs) were first identified from human blood samples as a population of circulating mononuclear cells capable of displaying a mature endothelial cell phenotype in culture. Subsequent studies have established that EPCs arise from the bone marrow (BM) and incorporate into the endothelium at sites of blood vessel growth, suggesting a potential role for these cells in neovascularization. Furthermore, a decline in EPC count has been correlated to multiple vascular pathologies, indicating that EPC number could serve as a biomarker of cardiovascular disease. Unfortunately, due to the variability in techniques used for EPC isolation and identification, considerable heterogeneity exists within the population of cells commonly defined as EPCs. In order for the clinical potential of EPCs to be fully realized, thorough characterization of the BM-derived cell populations involved in neovascularization is required. The objective of our study was to determine the functional significance of circulating EPCs in postnatal vascular growth and repair. Two separate strategies were employed to achieve this objective. In the first, we attempted to generate a novel mouse model where the pool of bone marrow-derived endothelial precursors was drastically reduced or eliminated. Our overall approach was to deliver a "suicide" gene, under control of an endothelial cell-specific promoter, to bone marrow cells for use in bone marrow transplantation (BMT) experiments. Mice receiving BMTs would therefore lack the ability to deliver viable BM-derived EPCs to sites of neovascularization. Our central hypothesis for this study was that a reduction in EPC viability would hinder endogenous vascular repair mechanisms, thereby exacerbating cardiovascular disease. In the second strategy, we attempted to identify novel progenitor cell populations based on the transcriptional regulation of pro-angiogenic genes. Our overall approach was to transduce BM with a retrovirus containing a fluorescent reporter gene under control of pro-angiogenic promoters for use in transplantation experiments. Our central hypothesis for this study was that unique populations of BM-derived cells could be identified by expression of the fluorescent reporter gene directed by the Vascular Endothelial Growth Factor (VEGF), endothelial Nitric Oxide Synthase (eNOS) and Vascular Endothelial (VE) Cadherin promoters. The BMT strategy utilized to address our first hypothesis was unsuccessful due to the use of a truncated form of the pro-apoptotic Bax as our suicide gene target. A plasmid encoding GFP fused to the truncated Bax fragment (ΔN-Bax, consisting of amino acids 112-192 of the full length protein) was used in transfection experiments to assess ΔN-Bax function. The GFP:ΔN-Bax fusion protein formed distinct extranuclear aggregates (presumably due to mitochondrial translocation) but did not induce apoptosis in transfected cells. The ΔN-Bax fragment also did not induce cell death when targeted to endothelial cells with retoviral-mediated gene delivery or in a transgenic mouse setting. To address our second hypothesis, we generated retroviral vectors containing the fluorescent tdTomato reporter under control of the VEGF, eNOS and VE Cadherin promoters. Significant fluorescence was detected in cultured endothelial cells and ex vivo-expanded BM cells. Following transplantation of transduced BM cells into lethally irradiated recipient mice, we were able to identify circulating populations of tdTomato-positive cells using flow cytometry. With these results we have identified novel subpopulations of circulating BM-derived cells which may play a significant role in post-natal neovascularization in mice. Therefore, results acquired from these studies could lead to improved cell therapy techniques for treatment of vascular disease.

Spinal cord injury is a devastating source of suffering in the spectrum of human pathophysiology; advancement for clinical therapy in this area has been stagnant in comparison to modern medical development. Current treatments are palliative, and functional recovery is minimal. During the first two weeks after injury, dense glial scar forms that is impenetrable by regenerating axons. Intervention is imperative to minimize scar formation and provide a supportive environment for axonal regeneration. Oligodendrocytes are critical to maintain the health of growing axons during development and after injury. Obtaining these cells through differentiation of neural progenitor cells (NPCs) is a viable option, but current clinical trials involving stem cells are plagued by poor cell survival and undirected differentiation. Research indicates that local extracellular matrix (ECM) is vital to progenitor differentiation and tissue regeneration. During development, spinal cord ECM is comprised of high concentrations of laminin and hyaluronic acid (HA), which provide essential cues to direct NPC migration and differentiation. The purpose of this research is to create a biomaterial optimized to direct NPC differentiation to oligodendrocytes. Natural biomaterials were optimized from distinct combinations of collagen I, HA, and laminin I to model the native ECM signals found during oligodendrocyte maturation. Four material combinations (collagen, collagen-HA-laminin, collagen-HA, and collagen-laminin) were fabricated into injectable hydrogels to mimic the range of compressive and shear mechanical properties present in neonatal central nervous system (CNS) tissue. Differentiation was assessed by culturing rodent fetal NPCs in these materials without specific soluble factors to direct cellular behavior. The three-component hydrogel performed optimally and achieved a 66% oligodendrocyte differentiation rate compared to approximately 15% in the collagen alone hydrogel. An in vivo study was then conducted using a rat contusion model of spinal cord injury with intervention using the injectable, three-component hydrogel seeded with rat NPCs. Functional recovery was assessed using six behavioral tests. Significant recovery was observed using two behavioral tests six weeks post-treatment. Lesion size was measured and correlated well with behavioral outcomes. The data obtained in this research indicate that a multi-component hydrogel mimicking native, developmental CNS tissue may address problems associated with current clinical practice. / text

Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

No description available.