Background: Currently, the optimal long-term therapy for end stage heart failure is heart transplantation. Cardiac allograft vasculopathy contributes to a significant number of deaths following transplantation. This vasculopathy is related to early endothelial injury sustained at the time of organ transplantation and to persistent endothelial injury as a result of cytotoxic immunosuppression, as well as chronic rejection. Epidermal growth factor-like domain 7 (Egfl7), is expressed in endothelial cells upon arterial injury and may have a role in maintaining vascular endothelial integrity and regeneration following injury. Similarly, novel pharmacologic agents such as Bosentan, an endothelin-1 antagonist, and Cilostazol, a phosphodiesterase 3 inhibitor, have been demonstrated to attenuate calcineurin inhibition induced endothelial dysfunction and neointimal hyperplasia, respectively. We hypothesized that, 1) Egfl7 will attenuate endothelial activation, cell adhesion molecule expression and neutrophil adhesion following simulated ischemia-reperfusion injury or exposure to calcineurin inhibition and that, 2) Bosentan and Cilostazol will inhibit neointimal hyperplasia following endothelial injury in a mouse model of vascular injury. Methods: Human coronary artery endothelial cells were subjected to hypoxia-reoxygenation injury or the calcineurin inhibitors Cyclosporine A and Tacrolimus to examine the effects of Egfl7 on these injury mechanisms. Cell adhesion molecule expression, neutrophil adhesion to endothelial cells, and NF-κB activation were measured. Cell adhesion molecule and Egfl7 expression were also examined in a mouse model of neointimal. This model was used to examine the effects of Bosentan and Cilostazol on neointimal hyperplasia. Results: Egfl7 had potent anti-inflammatory properties including inhibition of NF-κB pathway activation, ICAM-1 expression and neutrophil adhesion to injured endothelium. Within vessels exhibiting neointimal hyperplasia, Egfl7 was expressed in regions lacking ICAM-1 expression. Both cilostazol and bosentan attenuated neointimal hyperplasia in isolation as well as during co-treatment with CNI therapies. Conclusions: Egfl7 is an endothelial protective signaling protein with anti-inflammatory properties effective against simulated ischemia-reperfusion injury and calcineurin inhibition mediated injury. Cilostazol and Bosentan are pharmacologic strategies demonstrating efficacy against the development of neointimal hyperplasia. These observations provide a novel therapeutic target and strategies that may be relevant to endothelial protection and prevention of cardiac allograft vasculopathy following heart transplantation.

In Caenorhabditis nematodes, the androdioecious, self-fertilizing reproductive strategy has evolved relatively recently from gonochoristic, outcrossing ancestors. Transitions in mating system impact how genes are vertically transmitted between generations and horizontally between populations through gene flow. To date, species-wide studies have targeted selfing species, and we have limited understanding about how population genetic processes have shaped the genome structure and evolutionary history of outcrossing species of Caenorhabditis. To fill this gap, I investigate patterns of genetic variation and population genetic processes focusing on two outcrossing species in the genus, C. brenneri and C. remanei, using a survey of nucleotide polymorphisms in a multipopulation, multilocus context. I discover extensive genetic diversity in Caenorhabditis brenneri, termed hyperdiversity, giving this species the highest known levels of nucleotide polymorphism for any multicellular eukaryote. Genetic crosses between populations, extensive intra-locus recombination, and evidence of weak selection on codon usage all suggest that this is due to a large effective population size in the species and not an artifact of cryptic species divergence. I demonstrate that C. remanei also is characterised by high genetic variation globally, albeit not as extreme as in C. brenneri, and within each local population. Despite geographic barriers, considerable gene flow occurs between inter-continental locations. While exploring genetic diversity in C. remanei, I discovered C. sp. 23, a new, gonochoristic species reproductively isolated and highly divergent from it. Subsequently, taking advantage of this newly discovered species pair, I explore the patterns of postzygotic reproductive isolation between C. remanei and C. sp. 23. I find evidence of partial F1 hybrid inviability, strong F1 hybrid male sterility (Haldane’s rule) and strong F2 hybrid breakdown between the two. The findings from this thesis, especially the notion of hyperdiversity, can be taken advantage of to answer key questions on testing limits of natural selection, of evolution of genome complexity as well as identifying and characterising functional, non-coding regulatory elements. Discovery of a new species pair in Caenorhabditis helps establish an emerging age of speciation genetics in the worm.

The goal of my M.Sc. thesis research was to investigate the link between divergent adaptation and reproductive isolation in speciation, the process by which one species becomes two over time. Previous studies followed the evolution of 12 replicate experimental populations of the yeast Saccharomyces cerevisiae in either of two non-optimal environments, high-salt or low-glucose, over 500 generations. In this research, I re-sequenced the whole genomes of six diploid strains of these populations, three from high salt and three from low glucose. Among the replicates, there was remarkable degree of parallelism in the underlying determinants of adaptation including multiple mutations in PMA1, encoding the main proton exporter, in high-salt and in several genes in the ras signalling pathway in both environments. A variety of positive and negative epistatic interactions were found among mutations from within and among populations, emphasizing the importance of epistasis in adaptation and potentially in speciation.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however, its mechanism of action remains elusive. Our results demonstrate a novel finding wherein IVIg treatment induces a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and that the liver is the organ of increased IL-11 transcription. Furthermore, we show that IL-11Rα knockout mice, although initially protected, become resistant to protection by IVIg during experimental autoimmune encephalomyelitis (EAE) and develop disease with a similar incidence and severity as control-treated IL-11Rα-/- mice. The inability of IVIg to prevent EAE in IL-11Rα-/- mice correlated with a failure of this agent to decrease IL-17 production by myelin-reactive T-cells in the draining lymph nodes. Finally, we show that IL-11 can directly inhibit IL-17 production by lymph node cells in culture. Together, these results implicate IL-11 as an important immune effector of IVIg in the amelioration of EAE.

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however, its mechanism of action remains elusive. Our results demonstrate a novel finding wherein IVIg treatment induces a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and that the liver is the organ of increased IL-11 transcription. Furthermore, we show that IL-11Rα knockout mice, although initially protected, become resistant to protection by IVIg during experimental autoimmune encephalomyelitis (EAE) and develop disease with a similar incidence and severity as control-treated IL-11Rα-/- mice. The inability of IVIg to prevent EAE in IL-11Rα-/- mice correlated with a failure of this agent to decrease IL-17 production by myelin-reactive T-cells in the draining lymph nodes. Finally, we show that IL-11 can directly inhibit IL-17 production by lymph node cells in culture. Together, these results implicate IL-11 as an important immune effector of IVIg in the amelioration of EAE.

The goal of my M.Sc. thesis research was to investigate the link between divergent adaptation and reproductive isolation in speciation, the process by which one species becomes two over time. Previous studies followed the evolution of 12 replicate experimental populations of the yeast Saccharomyces cerevisiae in either of two non-optimal environments, high-salt or low-glucose, over 500 generations. In this research, I re-sequenced the whole genomes of six diploid strains of these populations, three from high salt and three from low glucose. Among the replicates, there was remarkable degree of parallelism in the underlying determinants of adaptation including multiple mutations in PMA1, encoding the main proton exporter, in high-salt and in several genes in the ras signalling pathway in both environments. A variety of positive and negative epistatic interactions were found among mutations from within and among populations, emphasizing the importance of epistasis in adaptation and potentially in speciation.