Haematopoietic stem cells (HSCs) are capable of self-renewing and multi-lineage reconstitution of the haematopoietic system of irradiated recipient mice. In the mouse embryo, HSCs originate in a step-wise manner from the haematogenic endothelium. The first HSC precursor has been detected at E9.5 in the dorsal aorta, while HSCs emerge in the aorta-gonad-mesonephros (AGM) region around E11. To date, the molecular mechanisms regulating these events are poorly characterized. Through the activating role of Angiopoietin1 (Ang1) on Tie2 receptor, the Ang-Tie signalling pathway plays a critical role in HSC maintenance in the adult bone marrow niche. Tie2 ligand Angiopoietin2 (Ang2) is described as being a Tie2 inhibitor, however its role is unknown. The aim of this thesis was to characterise the role of Ang-Tie signalling pathway in HSC formation in the mouse embryo. First, I used an ex vivo aggregate system to culture with angiopoietins cells derived from the AGM region at stages of development preceding HSC formation (E9.5-E11). Ang2- treated cells were able to reconstitute the peripheral blood of recipient mice to a higher extent compared to control, indicating a role for Ang2 in promoting HSC maturation. Then, I characterized the expression pattern of Ang-Tie molecules in the AGM region. Ang2-expressing cells were identified as perivascular and sub-aortic mesenchymal cells located in the ventral side of the aorta and in proximity of intra-aortic haematopoietic clusters. Finally, I performed an RNA-seq analysis with the aim of unravelling the molecular mechanisms involved in Ang2-mediated HSC maturation. Pre-HSC-I were cultured in presence or absence of Ang2 and their transcriptional profiles were compared, revealing a number of genes and pathways up-regulated or down-regulated in presence of Ang2, which might indicate a role for Ang2 in increasing cell proliferation, favouring cell migration, and regulation of other signalling pathways involved in HSC development. All together, these data support Ang2 as a novel regulator for HSC formation.

An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future. / Ph.D. / Traumatic brain injuries (TBIs) impact millions of individuals each year in the United States, making it a significant cause of death and disability. Furthermore, TBI has been linked to other comorbidities such as Alzheimers Disease, mood disorders, and epilepsy. Since the primary impact of a TBI cannot be predicted or prevented, research focuses on the secondary injury response as a therapeutic target to improve the outcomes following brain insult. Blood brain barrier (BBB) disruption is a well described consequence of TBI and has been correlated to a worse prognosis. The BBB normally provides a barrier between the circulating blood and the brain as protection and to maintain homeostasis. It is understood that decreased BBB integrity leads to subsequent edema, inflammatory response, and glial excitotoxicity, however, the mechanisms regulating this response remain to be investigated. Recent focus has been on a family of receptor tyrosine kinases, Eph receptors, that are unregulated following brain injury. Utilizing a mouse model, we can manipulate the temporal and spatial expression of Eph receptors to understand their role in the secondary injury cascade. Findings indicated that ablation of Eph receptors specifically on endothelial cells (ECs) resulted in preservation of BBB integrity at 1-, 4-, and 7- days following injury. Based on these results, we hypothesize that Eph receptor signaling on ECs negatively mediates BBB function and recovery following TBI. To test this hypothesis, we performed a comparative analysis between wild type (WT) and knockout (KO) mice on the expression of genes integral to BBB integrity, functional motor deficits, and loss of tissue in the lesion site following injury. We discovered significant decreases in lesion volume correlating with improvements in motor function in the KO mice compared to the WT. Moreover, KO mice showed increased expression of genes important for BBB maintenance such as Occludin and Tie2. To further discern the mechanism for these effects, we blocked Tie2 in the KO mice and observed similar negative prognostic indicators as in the WT. Future studies are warranted to understand the downstream signaling of Eph receptors on the Tie2 pathway. This data provides evidence that Eph signaling influences the BBB negatively following TBI through the Tie2 pathway and may be exploited for therapeutic means in the future.

Abstract Venous malformations (VMs) are localized defects in vascular morphogenesis which can seriously impede or even threaten the patient’s life. VMs are characterized by enlarged, torturous vein-like channels lined by unevenly distributed smooth muscle cells. A large number of mutations in the endothelial TIE2 receptor tyrosine kinase have been found from more than half of the lesions screened, thus providing a common genetic cause. TIE2 has a crucial role in vascular development, remodeling and quiescence. However, the molecular and cellular abnormalities caused by TIE2-mutations in endothelial cells and how they relate to VM formation have been unknown. The aim of this study was to examine how VM-specific mutations affect the molecular characteristics of TIE2-receptor downstream signaling and cellular functions. Because no effective treatment has been available for VMs, a better understanding of the molecular basis of their pathology should enable the development of more potent and non-invasive treatments as well as provide a better understanding of vascular morphogenesis in general. The results demonstrate that the TIE2-VM forms have both common and specific effects on TIE2 and the endothelial cells (ECs) expressing them. Mutation-induced TIE2 autoactivation leading to loss of normal EC monolayer organization due to extracellular matrix (ECM) fibronectin deficiency was found to be a common change. This was shown to occur through chronic activation of the mitogen-activated protein kinase (MAPK) pathway, which also caused activation of the proteolytic plasminogen system. Also, most mutations altered TIE2 trafficking and angiopoietin ligand regulated TIE2 functions, albeit through different mechanisms. Using RNA-screening we showed that the most common sporadic TIE2-VM mutation dysregulates genes affecting vascular development, cell migration and ECM remodeling. PDGFB, a major attractant of vascular mural cells, was found to be strongly attenuated due to chronic activation of Akt, which also increases EC survival, by the TIE2 mutant receptors. To conclude, the results in this thesis reveal genetic, molecular and cellular alterations which may potentiate VM formation. This data provides new information on the pathological mechanisms behind abnormal vascular morphogenesis and should assist the development of new molecular treatment strategies for VM patients. / Tiivistelmä Laskimoepämuodostumat ovat paikallisia verisuoniston kehityksen häiriöitä. Riippuen niiden koosta ja anatomisesta sijainnista ne voivat aiheuttaa merkittävää haittaa. Epämuodostumat koostuvat laajentuneista, laskimonkaltaisista verisuonista, joissa sileiden lihassolujen kerros on puutteellisesti järjestäytynyt. Yli puolessa tutkituista laskimoepämuodostumista havaitaan mutaatioita verisuonten sisäpinnan endoteelisoluissa ilmenevässä TIE2 reseptorityrosiinikinaasissa, joka säätelee verisuonten kehitystä, muokkausta ja fysiologista toimintaa. TIE2-mutaatioiden aiheuttamia molekyyli- ja solutason muutoksia tai niiden yhteyttä epämuodostumien syntyyn ei ole aikaisemmin tunnettu. Tämän tutkimuksen tarkoituksena oli selvittää, miten laskimoepämuodostumista löydetyt mutaatiot vaikuttavat TIE2-reseptorin toimintaan molekyyli- ja solutasolla sekä TIE2-reseptorista alkavaan solunsisäiseen viestintään. Koska pysyvää hoitomuotoa laskimoepämuodostumille ei tunneta, voisi tieto niiden taustalla olevista patologisista mekanismeista edesauttaa parempien, ei-kajoavien hoitomuotojen kehittämisessä ja antaa myös yleisesti uutta tietoa verisuoniston kehityksestä. Väitöskirjan tulokset osoittavat, että mutaatiot vaikuttavat TIE2-reseptoriin ja sitä ilmentäviin endoteelisoluihin mutaatioille yhteisillä sekä mutaatiokohtaisilla tavoilla. Mutaatioille tyypillinen TIE2-reseptorin ligandista riippumaton aktivaatio aiheutti aktivaation nousun myös TIE2:sta alavirtaan olevissa viestinvälittäjissä. Tämä puolestaan johti fibronektiini-proteiinin häviämiseen soluväliaineesta, sileitä lihassoluja säätelevän PDGFB-kasvutekijän ilmenemisen laskuun ja solujen ohjelmoidun solukuoleman vähenemiseen. Useimmat tutkitut mutaatiot muuttivat myös TIE2-reseptorin sijaintia soluissa häiriten TIE2:n angiopoietiini-ligandien säätelemiä toimintoja usean eri mekanismin kautta. Transkriptomin laajuiset RNA-tutkimukset osoittivat monien verisuonten kehitykseen, solujen liikkumiseen ja soluväliaineen muokkaukseen liittyvien geenien ilmentymisen muuttuneen. Lopputuloksena tutkimus paljasti geeni-, molekyyli-, ja solutason muutoksia, jotka saattavat vaikuttaa laskimoepämuodostumien syntyyn. Tulokset antavat lisätietoa sairautta aiheuttavista mekanismeista verisuoniston kehityksen häiriöiden taustalla ja ovat hyödyksi kehitettäessä uusia lääkkeitä laskimoepämuodostumien molekulaarisia hoitoja varten.

Retinopathy of prematurity, a disease characterised by aberrant retinal vascular development in premature neonates, is a leading cause of blindness and visual impairment in childhood. This work sought to examine differences in the susceptibility of inbred rat strains to oxygen-induced retinopathy, a model of human retinopathy of prematurity. The overriding aim was to identify genetic factors in rats that might be generalisable to humans. Newborn rats of six different strains were exposed to alternating cycles of hyperoxia and relative hypoxia for fourteen days. Rats were removed to room air and killed for analysis immediately, to assess oxygen-induced retinal vascular attenuation, or four days later to evaluate the extent of hypoxia-induced vasoproliferation. Whole flat-mounted retinae were stained with fluorophore conjugated isolectin GS-IB4, and measurement of vascular area was conducted using fluorescence microscopy and video-image analysis. A hierarchy of susceptibility to the inhibitory effects of cyclic hyperoxia and relative hypoxia on postnatal retinal vascularization was identified for the rat strains studied. Susceptibility to vascular attenuation was predictive of the subsequent risk of vascular morphological abnormalities. Cross-breeding experiments between susceptible and resistant strains demonstrated that the susceptible phenotype was dominantly inherited in an autosomal fashion. These studies confirmed an association between ocular pigmentation and retinopathy risk, however the finding of differential susceptibility amongst albino rat strains implicated factors in addition to those associated with ocular pigmentation. Quantitative real-time reverse transcription-polymerase chain reaction was used to compare the retinal expression of angiogenic factor genes in susceptible and resistant strains with the aim of identifying a genetic basis for the strain difference. Eight angiogenic factor genes were selected for study: vascular endothelial growth factor (VEGF); VEGF receptor 2; angiopoietin 2; Tie2; pigment epithelium-derived factor; erythropoietin; cyclooxygenase-2 and insulin-like growth factor-1. The most notable difference between strains was the expression of vascular endothelial growth factor (VEGF) during the cyclic hyperoxia exposure period - higher VEGF expression was associated with relative resistance to retinopathy. Other differences in retinal angiogenic factor gene expression between strains, such as higher expression of VEGF receptor 2 and angiopoietin 2 in resistant strains, appeared to be secondary to those in VEGF. Following cyclic hyperoxia, the expression pattern of angiogenic factor genes changed - messenger RNA levels of hypoxia-induced genes, including VEGF, VEGF receptor 2, angiopoietin 2 and erythropoietin, were significantly higher in those strains with larger avascular areas, than in those strains that were relatively resistant to retinopathy. These findings provide firm evidence for hereditary risk factors for oxygen-induced retinopathy in the rat. Differences in the regulatory effects of oxygen on VEGF expression appear to be central to the risk of retinopathy. The potential relevance of these hereditary factors is discussed in the context of the human disease.

Dans l'épilepsie du lobe temporal (ELT), l'hyperexcitabilité est attribuée à la mort neuronale, la gliose et la plasticité synaptique. Un remodelage vasculaire n'a jamais été recherché dans le tissu épileptique, bien que des données récentes suggèrent que la perméabilité de la barrière hémato-encéphalique (BHE) est épileptogène.<br />Nous avons observé dans l'hippocampe de patients atteints d'ELT réfractaire une dégradation de la BHE, une néo-vascularisation et la surexpression du vascular endothelial growth factor (VEGF) et de son récepteur VEGFR-2. <br />Pour comprendre cette angiogenèse, nous avons modélisé l'épilepsie in vivo et in vitro chez le rat. In vivo, la néo-vascularisation, la surexpression de VEGF/VEGFR-2 et la rupture de BHE sont présentes dans un modèle avec lésions et gliose, mais transitoires dans un modèle non lésionnel. <br />In vitro, nous avons observé que des crises déclenchées sur des cultures organotypiques d'hippocampe (COHs) induisent une angiogenèse et une dégradation de BHE qui ne persiste qu'en présence de lésions.<br /> Nous avons étudié le rôle du VEGF in vitro, en le neutralisant ou en inhibant des voies de signalisation de VEGFR-2 dans les COHs, confirmant l'importance de PKC et src dans l'angiogenèse et la dégradation de la BHE. De plus, nous avons montré un déséquilibre des angiopoiétines en faveur d'Ang2 qui potentialise les effets du VEGF.<br />Chez l'homme et l'animal, la rupture de la BHE persiste dans les foyers chroniques entretenant l'induction des crises. En ciblant des facteurs angiogéniques pour réparer la BHE, nous espérons réduire l'épileptogenèse et donc proposer de nouvelles stratégies pour les épilepsies réfractaires.

Abstract Angiopoietins 1 and 2 (Ang1 and Ang2) are the ligands of the Angiopoietin/Tie signalling system, which is a binary pathway offering mechanisms for healthy vessels to reach and maintain their quiescence but also to rapidly respond to activating stimuli leading to a remodelling of endothelium. The latter is linked to disease settings such as inflammation and cancer where endothelial cell (EC) integrity is compromised and is often related to an increase in Ang2 expression. This study focused on the mechanisms enabling Ang1 to mediate both EC stability and migration and molecular and cellular determinants for ligand-specific functions of Ang2 and its isoform Ang2443. The findings revealed that Ang1 induces differential signalling depending on whether it anchors and activates Tie2 in cell-cell junctions in quiescent ECs, or in cell-matrix contacts in mobile ECs, thus leading to cellular phenotypes characteristic of resting and mobile ECs, respectively. In the second part of the thesis Ang2-Tie2 specific cell-extracellular matrix (ECM) contact sites were studied. Formation of Ang2/Tie2 EC-ECM contact sites was dependent on the collagen I and IV matrices, low Ang2 oligomerization state, α2β1-integrins, and intact microtubules. In the third part of the thesis the comparison of Ang2 mRNA splice variant Ang2443 with full length Ang2 (Ang2FL) revealed both redundant and ligand form–specific effects, expression of Ang2443443 increased the amount of monomeric ligand forms due to proteolytic processing and promoted transendothelial migration of cancer cells in vitro. On the other hand, both Ang2443 and Ang2FL were stored in endothelial Weibel-Palade bodies (WPBs), similarly induced Ang2-specific Tie2 cellular redistribution, and were mostly comparable in developmental angio- and lymphangiogenesis. / Tiivistelmä Angiopoietiinit 1 ja 2 (Ang1 ja Ang2) ovat Ang/Tie signalointireitin kasvutekijöitä. Ang1 kasvutekijää tarvitaan sydämen ja verisuoniston sikiöaikaiseen kehittymiseen, se vähentää Tie2 reseptorin kautta verisuonten läpäisevyyttä, mutta edistää myös yksittäisten endoteelisolujen liikkumista. Saman Tie2 signalointireitin toisen kasvutekijän Ang2:n ilmeneminen johtaa verisuonten läpäisevyyden kasvuun tulehduksessa, uusien verisuonten muodostumiseen syöpäkasvaimissa ja syöpäsolujen leviämiseen elimistössä. Väitöskirjatutkimuksessa selvitettiin niitä solutason mekanismeja, joilla Ang1 kykenee välittämään sekä endoteelisolujen tiiviyttä että liikkumista. Lisäksi tutkittiin niitä molekyyli- ja solutason mekanismeja, joilla Ang2 ja sen isomuoto Ang2443 välittävät kasvutekijäspesifisiä vaikutuksiaan. Väitöskirjassa osoitettiin että Tie2 reseptori paikantuu verisuonten endoteelisoluissa Ang1 sitoutumisen seurauksena joko solu-soluliitoksiin, tai yksittäisissä endoteelisoluissa solu-soluväliaine rajapinnalle. Tie2:n siirtyminen solu-soluliitoksiin aktivoi soluissa signalointireittejä, jotka ovat tyypillisiä normaaleille tiiviille verisuonille ja solu-soluväliaineliitoksissa liikkuville endoteelisoluille tyypillisiä piirteitä. Väitöskirjatyön toisessa osassa tutkittiin Ang2:lle ominaisia vaikutuksia ja Ang2-Tie2 kompleksin paikantumista erityisiin solu-soluväliaineliitoksiin. Tämä oli riippuvaista Ang2:n oligomerisaatiosta, kollageenisoluväliaineesta, α2β1-integriinistä ja normaalista mikrotubulusverkostosta. Väitöskirjatyön kolmannessa osassa osoitettiin että Ang2443 isomuodolla on sekä yhteisiä että isomuotospesifisiä piirteitä verrattuna kokopitkään Ang2:een (Ang2FL). Liukoinen Ang2443, mutta ei Ang2FL, esiintyi yleisesti monomeerisenä ligandimuotona proteiinin multimerisaatio-osan pilkkomisen seurauksena. Ang2443 lisäsi myös syöpäsolujen liikkumista endoteelisolujen läpi. Toisaalta sekä Ang2443 että Ang2FL varastoitiin endoteelisoluissa Weibel-Palade varastokappaleisin, ne välittivät samanlaista Tie2 reseptorin paikantumista endoteelisoluissa ja toimivat pääsääntöisesti samanlaisina kasvutekijöinä veri- ja imusuonten kehityksen aikana hiiressä.