Pulmonary arterial hypertension (PAH) is a progressive disease that results in increased pulmonary vasculature resistance, causing right ventricular (RV) remodeling, which eventually progresses into right heart failure and mortality. New and emerging therapeutic strategies involve regenerative approaches to repair the underlying vascular pathology using regenerative cell therapy and methods to alleviate RV dysfunction in the setting of fixed RV afterload. In the first section of the thesis, we investigated the role of EPC paracrine mechanisms in the treatment of PAH. We characterized the paracrine function of EPCs by demonstrating that EPC conditioned medium enhances endothelial cell migration, survival and angiogenesis in vitro. We further examined the role of secreted extracellular vesicles in the paracrine function of EPCs, which played a minor role in promoting wound healing. However, using the monocrotaline rat model of PAH, we did not demonstrate a consistent benefit on RV pressures or remodeling with EPCs or EPC conditioned medium. The lack of effect may be related to the advanced phenotype observed in our model of PAH. Survival in severe pulmonary arterial hypertension (PAH) is related to the ability of the right ventricle (RV) to adapt to increased afterload. Therefore, we explored the effect of genetic background on right ventricular adaptation and survival in a rat model of severe (PAH). Compared to the conventional Sprague-Dawley rat strain, we observed high mortality in the Fischer SUHx model of severe PAH. This was related to a strain-dependent failure of RV adaptation, as evidenced by RV dilatation, RV contractile dysfunction, decreased cardiac ouptut and decreased exercise capacity. Further analysis by gene expression microarrays and fluorescence microangiography demonstrate that failure of RV adaptation is due at least in part due to lack of adequate microvascular angiogenesis in the hypertrophied RV. This work lays the foundation for the section on RV-specific therapy that follows. Using the Fischer model of maladaptive RV remodeling, we tested whether cardiotrophin-1 (CT-1), a pro-angiogenic and cardioprotective cytokine, could improve RV adaptation. We demonstrated that as a rescue treatment, CT-1 reduced RV dilatation and function without influencing RV afterload, which suggests improved RV adaptation. These changes were associated with an increase in RV capillary density. As an early-stage preventative treatment, in addition to improving RV remodeling, CT-1 also reduced pulmonary pressures. These hemodynamic changes suggest that CT-1 may also have a direct impact on vascular tone or the underlying pulmonary vascular pathology.

La dégénérescence vasculaire et l’incapacité l’organisme à produire des vaisseaux sanguins de façon adéquate lors d’une condition ischémique est un fait saillant des rétinopathies ischémiques telles que la rétinopathie du prématuré (ROP). La ROP demeure la principale cause de défaillance visuelle et dans les cas extrêmes, de cécité chez les nourrissons prématurés. Elle présente deux phases distinctes soit une phase initiale clef de vasooblitération (VO) rétinienne et choroïdale qui entraînent la deuxième phase de néovascularisation (NV) rétinienne désorganisée et excessive. Au cours du développement normal, la NV oculaire a recours au phénomène d’angiogenèse qui consiste en la formation de nouveaux capillaires à partir de vaisseaux préexistants et de vasculogenèse qui consiste en la formation de nouveaux capillaires à partir de cellules endothéliales progénitrices dérivées de la moelle osseuse (BM-EPCs). Cette vasculogenèse implique la mobilisation des EPCs de la moelle osseuse vers la circulation afin d’être recrutées au site de NV pour contribuer de façon directe, soit en intégrant directement les structures vasculaires pour former des néovaisseaux, ou bien de façon indirecte par leur activité paracrine en libérant différents facteurs de croissance vasculaires. Toutefois, les mécanismes moléculaires impliqués dans la dysfonction des EPCs lors de la ROP sont encore mal compris. Au cours de mon mémoire, mes travaux ont ciblé la première phase de VO rétinienne afin de promouvoir la revascularisation par une thérapie basée sur une supplémentation d’EPCs natives ou reprogrammées. Compte tenu du rôle capital des EPCs dans la NV, mon mémoire s’est d’abord intéressé au rôle de Nogo-A (une protéine de la famille de réticulon), connue pour son action anti-angiogénique, sur l'activité fonctionnelle des EPCs en condition de ROP. Pour ce faire, nous avons utilisé un modèle de rétinopathie induite par l’oxygène (OIR) simulant la ROP. L’objectif global de ce projet consiste à évaluer l’interrelation entre l’effet de l’hyperoxie (une condition clef de la ROP) sur la voie de signalisation Nogo-A et de son récepteur NgR1 sur la fonction des EPCs. Premièrement, les résultats obtenus montrent une augmentation de l’expression de Nogo-A et NgR1 chez les BM-EPCs soumis ex vivo à l’hyperoxie, mais aussi dans les EPCs extraites des rats OIR. En addition, l’augmentation de l’expression de Nogo-A/NgR1 par l’hyperoxie corrèle avec la dysfonction angiogénique des EPCs caractérisées par une diminution de leurs capacités de migration et de tubulogenèse. De façon intéressante, l’inhibition de Nogo-A (par un peptide neutralisant) améliore la capacité migratoire et tubulogénique des EPCs, et protège leur fonction contre l’hyperoxie. Également, l’inhibition de Nogo-A induit l’expression du facteur angiogénique et mobilisateur d’EPCs, SDF-1, suggérant que NgR1 régule négativement l’expression de SDF-1. Par ailleurs, nous avions également pour objectif final d’évaluer l’efficacité protectrice d’une supplémentation d’EPCs natives ou reprogrammées (Nogo-/-) pour améliorer la revascularisation rétinienne dans un modèle de rat OIR. Les résultats montrent qu’une supplémentation intrapéritonéale d’EPCs natives diminue significativement la VO rétinienne, mais que cet effet pro-angiogénique devient plus prononcé par le traitement d’EPCs préconditionnées (reprogrammées par l’inhibition de Nogo-A) chez les rats OIR. Collectivement, nos résultats démontrent que : 1) l’hyperoxie cause une dysfonction angiogénique des BM-EPCs en induisant Nogo-A ce qui contribue à la VO rétinienne chez les rats OIR, et que 2) une supplémentation d’EPCs conditionnées (reprogrammées par l’inhibition de Nogo-A) est plus efficace qu’une supplémentation d’EPCs natives pour améliorer la réparation vasculaire rétinienne. Pour conclure, nous mettons donc en évidence une cible potentielle qui est la protéine Nogo-A afin de préserver l’activité biologique des EPCs et ultimement, l’intégrité vasculaire chez les rats OIR. / Vascular degeneration and the inability of the body to produce adequate blood vessels during an ischemic condition is a salient feature of ischemic retinopathies such as retinopathy of prematurity (ROP). ROP remains the leading cause of visual impairment and in extreme cases, blindness in premature infants. It presents two distinct phases: a key initial phase of retinal and choroidal vasoobliteration (VO) which leads to the second phase of disorganized and excessive retinal neovascularization (NV). During normal development, ocular NV uses the phenomenon of angiogenesis which consists of the formation of new capillaries from pre-existing vessels and vasculogenesis which consists of the formation of new capillaries from progenitor endothelial cells derived from the marrow bone (BM-EPCs). This vasculogenesis involves the mobilization of EPCs from the bone marrow to the circulation in order to be recruited at the NV site to contribute directly, either by directly integrating the vascular structures to form new vessels, or indirectly by their paracrine activity by releasing different vascular growth factors. However, the molecular mechanisms involved in the dysfunction of EPCs during ROP are still poorly understood. During my thesis, my work targeted the first phase of retinal VO in order to promote revascularization by therapy based on supplementation of native or reprogrammed EPCs. Given the capital role of EPCs in NV, my thesis was first interested in the role of Nogo-A (a protein of the reticulon family), known for its anti-angiogenic action, on the functional activity of EPCs in ROP condition. To do this, we used an oxygen-induced retinopathy (OIR) model simulating ROP. The overall objective of this project is to assess the interrelationship between the effect of hyperoxia (a key condition of ROP) on the Nogo-A signaling pathway and its NgR1 receptor on the function of EPCs. First, the results obtained show an increase in the expression of Nogo-A and NgR1 in BM-EPCs subjected to hyperoxia ex vivo, but also in EPCs extracted from OIR rats. In addition, the increase in the expression of Nogo-A / NgR1 by hyperoxia correlates with the angiogenic dysfunction of EPCs characterized by a decrease in their capacity for migration and tubulogenesis. Interestingly, inhibition of Nogo-A (by a neutralizing peptide) improves the migratory and tubulogenic capacity of EPCs, and protects their function against hyperoxia. Also, inhibition of Nogo-A induces expression of the angiogenic and mobilizing factor of EPCs, SDF-1, suggesting that NgR1 negatively regulates the expression of SDF-1. In addition, our final objective was also to evaluate the protective efficacy of supplementation of native or reprogrammed EPCs (Nogo - / -) to improve retinal revascularization in an OIR rat model. The results show that intraperitoneal supplementation of native EPCs significantly decreases retinal VO, but that this pro-angiogenic effect becomes more pronounced by treatment of preconditioned EPCs (reprogrammed by inhibition of Nogo-A) in OIR rats. Collectively, our results demonstrate that: 1) hyperoxia causes angiogenic dysfunction of BM-EPCs by inducing Nogo-A which contributes to retinal VO in OIR rats, and that 2) supplementation of conditioned (reprogrammed by inhibition of Nogo-A) is more effective than supplementation of native EPCs in improving retinal vascular repairs. To conclude, we therefore highlight a potential target which is the Nogo-A protein in order to preserve the biological activity of EPCs and ultimately, vascular integrity in OIR rats.

<p>Asthma involves a systemic element that includes the mobilization and lung-accumulation of bone marrow-derived endothelial progenitor cells (EPC). This traffic may be driven by the stromal cell derived factor-1α (SDF-1α)/CXCR4 axis, where SDF1-α is a potent progenitor cell chemoattractant.</p> <p>Interfering with EPC lung-accumulation by administering AMD3100, a CXCR4 antagonist, was previously shown to be associated with the modulation of airway angiogenesis and airway hyperresponsiveness. However, since eosinophils express CXCR4, it is unknown whether AMD3100 acted directly on EPC or indirectly through its anti-inflammatory effects on eosinophils.</p> <p>We investigated the role that eosinophilic inflammation plays in the lung-homing of EPCs and airway angiogenesis in allergic asthmatic response by utilizing eosinophil deficient (PHIL) mice.</p> <p>Wild-type BALB/c (WT) and PHIL mice underwent a chronic house dust mite (HDM) exposure protocol. Treatment groups were administered AMD3100. Outcome measurements were made 24hrs post final exposure and included: flow cytometry to enumerate lung-extracted EPCs, immunostaining for von Willebrand factor to assess bronchial vascularity, bronchoalveolar lavage for airway inflammation, haematoxylin and eosin stain to enumerate eosinophils, picrosirius red stain to assess collagen deposition, and measurement of airway resistance to increasing intranasal doses of methacholine.</p> <p>HDM exposed mice had a significant increase in EPC lung accumulation, bronchial vascularity, airway inflammation, collagen deposition and airway hyperresponsiveness (AHR) in both WT and PHIL groups, with some indices at lower levels in PHIL mice. Concurrent treatment with AMD3100 significantly attenuated EPC lung homing, bronchial vascularity, eosinophil numbers in lung tissue and AHR, but not collagen deposition in WT mice. AMD3100 treatment significantly attenuated all indices in PHIL mice.</p> <p>The findings of this study show that, EPC-driven angiogenesis and the development of AHR in allergic airway responses are independent of eosinophils, the presence of these cells, however, may have a role in worsening of the pathology of allergic airways disease.</p> / Master of Science (MSc)