Angiogenesis is the formation of new blood vessels by sprouting from pre-existing ones. Transforming growth factor-beta (TGFβ) promotes angiogenesis and is a known inducer of endothelial-mesenchymal transition (EndMT), a process whereby endothelial cells become fibroblastic and motile. We hypothesize that TGFβ-induced EndMT enables endothelial cells to detach from the mature vessel and migrate to form the sprout that becomes a new vessel during angiogenesis. This study characterized EndMT in response to TGFβ +/- vascular endothelial growth factor (VEGF). Bovine aortic endothelial cells (BAEC) were stimulated with TGFβ +/- VEGF for prolonged periods. Confocal imaging and immunoblotting analyses revealed the strongest EndMT response at 5 ng/ml of TGFβ after 144 hours of exposure. A three-dimensional collagen model of angiogenesis revealed a potential relationship between EndMT and blood vessel sprouting. These results suggest that EndMT induction in BAECs requires high concentrations and prolonged exposure to TGFβ and is not significantly influenced by VEGF. / NSERC

During normal placental development, a primitive vascular network develops through vasculogenesis and angiogenesis, and is then remodelled through maturation and regression. The mechanism behind this regression is unknown, but data from other systems suggests that it could be due to an endothelial-mesenchymal transition (EndMT). If this is the case, then dysregulated EndMT could lead to increased vascular regression, which could result in placental hypovascularisation. As the placental vasculature is the area of exchange between maternal and fetal circulations, a reduction in its surface area could result in fetal growth restriction (FGR). The hypothesis of this thesis is that EndMT occurs during normal placental development, but is increased during FGR and contributes to placental hypovascularisation. A primary cell model consisting of endothelial and mesenchymal cells was isolated from human first trimester placental villous stroma. These cells were shown to lose CD31 mRNA (n = 1-3) and protein (n = 15) over 4 passages, with no loss of cell viability (n = 8). EndMT-associated transcription factors were also present in these cells at all 4 passages (n = 2-4). When cells were isolated from this mixed cell model based on their CD31-positivity and examined immediately after isolation, a small proportion also expressed αSMA (n = 5). Co-expression of endothelial and mesenchymal markers suggests that an EndMT was occurring. After 24 hours in culture, the proportion of these cells expressing αSMA increased (n = 5), and some cells co-expressed vWF and αSMA, while others lost their CD31-positivity, indicating that these cells had undergone EndMT. Cells isolated based on their CD31-positivity were treated with factors shown to inhibit EndMT in other systems. However, culture with 10µM SB431542 (TGFβ receptor inhibitor; n = 6), 10µM Dorsomorphin (BMP receptor inhibitor; n = 3), or 0.1µM PDGFR-β Tyrosine Kinase Inhibitor IV (n = 3) did not inhibit gain of αSMA by these cells. Culture on Matrigel in endothelial growth medium containing VEGF and FGF also failed to stabilise the endothelial phenotype (n = 3). The possibility that EndMT occurs in placenta in vivo was examined; genes associated with EndMT were shown to be present in placenta (n = 5), and there was limited evidence of CD31 or vWF co-expression with αSMA in tissue. Preliminary evidence was obtained to suggest that expression of EndMT-associated genes was altered in FGR placentas compared to normal. In summary, the data presented in this thesis demonstrate that an EndMT occurs in primary placental microvascular endothelial cells in vitro. Furthermore, these studies provide evidence to suggest that this transition also occurs in vivo and could be altered in placentas from pregnancies complicated by FGR.

Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.

Il est reconnu que la protéine filamenteuse intermédiaire Nestine est exprimée lors du processus de cicatrisation et du remodelage fibrotique. De plus, nous avons identifié l’expression de la Nestine au sein de deux populations distinctes qui sont directement impliquées dans les réponses de fibroses réparative et réactive. Ainsi, une population de cellules souches neurales progénitrices résidentes du coeur de rat adulte exprime la Nestine et a été identifiée à titre de substrat de l’angiogenèse et de la neurogenèse cardiaque. Également, la Nestine est exprimée par les myofibroblastes cicatriciels cardiaques et il a été établi que la protéine filamenteuse intermédiaire joue un rôle dans la prolifération de ces cellules. Ainsi, l’objectif général de cette thèse était de mieux comprendre les évènements cellulaires impliqués dans la réponse neurogénique des cellules souches neurales progénitrices résidentes cardiaques Nestine(+) (CSNPRCN(+)) lors de la fibrose réparative cardiaque et d’explorer si l’apparition de fibroblastes Nestine(+) est associée avec la réponse de fibrose réactive secondaire du remodelage pulmonaire. Une première publication nous a permis d’établir qu’il existe une régulation à la hausse de l’expression de la GAP43 (growth associated protein 43) et que cet événement transitoire précède l’acquisition d’un phénotype neuronal par les CSNPRCN(+) lors du processus de cicatrisation cardiaque chez le rat ayant subi un infarctus du myocarde. De plus, la surimposition de la condition diabétique de type 1, via l’injection unique de Streptozotocine chez le rat, abolit la réponse neurogénique des CSNPRCN(+), qui est normalement induite à la suite de l’ischémie cardiaque ou de l’administration de 6-hydroxydopamine. Le second article a démontré que le développement aigu de la fibrose pulmonaire secondaire de l’infarctus du myocarde chez le rat est associé avec une augmentation de l’expression protéique de la Nestine et de l’apparition de myofibroblastes pulmonaires Nestine(+). Également, le traitement de fibroblastes pulmonaires avec des facteurs de croissances peptidiques pro-fibrotiques a augmenté l’expression de la Nestine par ces cellules. Enfin, le développement initial de la condition diabétique de type 1 chez le rat est associé avec une absence de fibrose réactive pulmonaire et à une réduction significative des niveaux protéiques et d’ARN messager de la Nestine pulmonaire. Finalement, la troisième étude représentait quant à elle un prolongement de la deuxième étude et a alors examiné le remodelage pulmonaire chronique chez un modèle établi d’hypertension pulmonaire. Ainsi, les poumons de rats adultes mâles soumis à l’hypoxie hypobarique durant 3 semaines présentent un remodelage vasculaire, une fibrose réactive et une augmentation des niveaux d’ARN messager et de la protéine Nestine. De plus, nos résultats ont démontré que la Nestine, plutôt que l’alpha-actine du muscle lisse, est un marqueur plus approprié des diverses populations de fibroblastes pulmonaires activés. Également, nos données suggèrent que les fibroblastes pulmonaires activés proviendraient en partie de fibroblastes résidents, ainsi que des processus de transition épithélio-mésenchymateuse et de transition endothélio-mésenchymateuse. Collectivement, ces études ont démontré que des populations distinctes de cellules Nestine(+) jouent un rôle majeur dans la fibrose réparative cardiaque et la fibrose réactive pulmonaire. / It is well established that the intermediate filamentous protein Nestin is expressed during wound healing and fibrotic remodeling. Furthermore, we have identified Nestin expression in two distinct populations directly implicated in reparative and reactive fibrosis. The adult rodent heart contains a resident population of neural progenitor/stem cells that express Nestin and identified as a cellular substrate of cardiac angiogenesis and neurogenesis. Moreover, Nestin is also expressed in cardiac scar myofibroblasts and the intermediate filament protein plays a direct role in proliferation. Thus, the general aim of the present thesis was to better understand the cellular events implicated in the neurogenic response of neural progenitor/stem cells during cardiac reparative fibrosis and to explore whether the appearance of Nestin(+)-fibroblasts was associated with reactive fibrotic response secondary to pulmonary remodeling. The first study revealed that the transient upregulation of growth associated protein 43 (GAP43) represents a transition event during the acquisition of a neuronal-like phenotype by cardiac resident neural progenitor/stem cells in the scar of the infarcted rat heart. Furthermore, the superimposition of a type 1 diabetic environment, via the single injection of streptozotocin in rats, abrogated the neurogenic response of cardiac resident neural progenitor/stem cells to ischemia and 6-hydroxydopamine, respectively. The second study has demonstrated that the development of acute pulmonary fibrosis secondary to myocardial infarction of the adult rat heart was associated with the increased expression of Nestin protein levels and appearance of Nestin(+)-myofibroblasts. Furthermore, the treatment of pulmonary fibroblasts with putative pro-fibrotic peptide growth factors increased Nestin protein levels. Lastly, in the lungs of type 1 diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of Nestin protein/mRNA levels. Finally, the third study represented an extension of the second study and examined chronic lung remodeling in an established model of pulmonary hypertension. The lungs of adult male rats subjected to 3 weeks of hypobaric hypoxia were associated with vascular remodeling, reactive fibrosis and increased Nestin protein and mRNA levels. Moreover, Nestin, rather than smooth muscle α-actin expression was identified as a more relevant marker of activated pulmonary fibroblasts. Furthermore, the appearance of activated pulmonary fibroblasts may be derived in part from resident fibroblasts and secondary to endothelial-mesenchymal transition and epithelial-mesenchymal transition. Collectively, these studies have demonstrated that distinct populations of Nestin-expressing cells play a seminal role in cardiac reparative fibrosis and pulmonary reactive fibrosis.