Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Das Ziel der Arbeit war eine Charakterisierung der Angiogenese im unveränderten Stutenovar sowie in equinen Granulosazelltumoren (GZTt). In diesem Zusammenhang sollte das Vorkommen und die Bedeutung ausgewählter angiogener Faktoren und ihrer Rezeptoren in den genannten Geweben ermittelt werden. Darüber hinaus war zu klären, inwieweit histomorphologische und immunhistologische Untersuchungen bezüglich der Angiogenese einen Beitrag zur Charakterisierung der biologischen Wertigkeit equiner Granulosazelltumoren liefern können. Für die Untersuchungen standen 71 Granulosazelltumoren 70 einseitig ovariektomierter Tiere und einer euthanasierten Stute, bei der eine ausgedehnte abdominale Metastasierung des Tumors festgestellt wurde, zur Verfügung. Als Kontrolle dienten die unveränderten Ovarien von 20 Stuten (Sektion n=8, Ovariektomie n=2, Schlachthof n=10). Darüber hinaus wurden bei 46 Stuten mit GZTt die Serumhormonwerte (Östradiol, Progesteron und Testosteron) bestimmt. Die unveränderten Ovarien sowie die GZTt wurden zunächst pathologisch-anatomisch untersucht, in Formalin (4%) fixiert und routinemäßig für die Histologie aufgearbeitet. Mittels konventioneller Lichtmikroskopie (Hämalaun-Eosin-Färbung) erfolgte anschließend eine repräsentative Auswahl von verschiedenen Funktionskörpern (n=66) des unveränderten Ovars sowie von GZTt unterschiedlicher Wachstumsformen (n=21 sowie eine Metastase des malignen GZT), an welchen die immunhistolo-gischen Untersuchungen (vascular endothelial growth factor A (VEGF A), vascular endothelial growth factor B (VEGF B), vascular endothelial growth factor receptor 1 (VEGF-R1), vascular endothelial growth factor receptor 2 (VEGF-R2), Angiopoietin 1 (Ang1), Angiopoietin 2 (Ang2), Angiopoietinre-zeptor (Tie2), von Willebrand Factor) durchgeführt wurden. Es kann festgestellt werden, dass im unveränderten zyklischen Stutenovar die intensivste Koexpression der angiogenen Faktoren VEGF A, VEGF B, Ang1, Ang2 und ihrer Rezeptoren VEGF-R1, VEGF-R2 und Tie2 in den Granulosa- und Thekazellen sowie den vaskulären Strukturen der Thekazellschicht im periovulatorischen (Tertiär-/Graaf´sche Follikel bis Corpora haemorrhagica) Zeitraum, der Phase der am deutlichsten ausgeprägten Angiogenese im Ovar, nachgewiesen werden kann. Zum einen ist dies für die Erhaltung und Reifung eines Follikels bis zum sprungreifen Tertiärfollikel essentiell, zum anderen findet kurz nach der Ovulation eine so ausgeprägte Angiogenese statt, dass schließlich alle Luteinzellen des reifen C. luteum mindestens einen Kontakt zu den neugebildeten Gefäßen besitzen. Somit wird eine optimale Erfüllung der Funktion des C. luteum als temporär endokrine Drüse für die Progesteronsynthese gewährleistet. Im eigenen Untersuchungsgut können hinsichtlich des Expressionsmusters gewisse Ähnlichkeiten zwischen dem unveränderten zyklischen Ovar und den benignen GZTt festgestellt werden. Insbesondere die neoplastischen Granulosazellen und Leydig-like cells erinnern in ihrem Expressionsverhalten bezüglich der untersuchten angiogenen Faktoren/Rezeptoren an die Granulosazellen beziehungsweise die luteinisierten Thekazellen des periovulatorischen zyklischen unveränderten Ovars. Zum einen lassen diese Befunde den Schluss zu, dass die neoplastischen Granulosazellen und Leydig-like cells der untersuchten Tumoren, entsprechend den periovulatorischen Granulosazellen und Thekazellen des zyklischen Ovars, infolge der Expression der angiogenen Faktoren und ihrer Rezeptoren einen wesentlichen Beitrag zur Angiogenese und damit zur Durchblutung, zur Versorgung und zum Wachstum der GZTt leisten. Andererseits kann durch die Ähnlichkeit im Expressionsmuster der genannten Zellen auch mittels der durchgeführten Untersuchungen gezeigt werden, dass es sich bei den equinen Granulosazelltumoren um weitgehend gut differenzierte Neoplasien handelt. Trotzdem ist in Einzelfällen mit einer Metastasierung zu rechnen. Die konventionelle Histopathologie liefert diesbezüglich jedoch keine verwertbaren Anhaltspunkte. Mittels der immunhistologischen Untersuchungen kann aufgezeigt werden, dass zwischen benignen und malignen Granulosazelltumoren partiell ein verändertes Expressionsverhalten bezüglich der untersuchten angiogenen Faktoren und ihrer Rezeptoren existiert. Inwieweit diese Abweichung jedoch als hinweisend für eine potenzielle Malignität interpretiert werden kann, ist aufgrund der zu geringen Probenanzahl maligner Neoplasien abschließend nicht beurteilbar und Bedarf einer Konkretisierung der bisherigen Ergebnisse an einem größeren Untersuchungsgut.

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

Αξιοσημείωτη πρόοδος έχει σημειωθεί τα τελευταία χρόνια στην αποσαφήνιση των μορίων που πρωταγωνιστούν στην αγγειογένεση, με τον αγγειακό ενδοθηλιακό αυξητικό παράγοντα (VEGF) να παίζει πρωταγωνιστικό ρόλο. Ο VEGF προκαλεί αρκετές αγγειογενετικές λειτουργίες στα ενδοθηλιακά κύτταρα διαμέσου των υποδοχέων του VEGFR-1 και VEGFR-2 ή KDR. Η προκαλούμενη από τον VEGF μετανάστευση των ενδοθηλιακών κυττάρων φαίνεται ότι μεσολαβείται από τον KDR, πιθανά μέσω δέσμευσης του με την ιντεγκρίνη αvβ3. Η πλειοτροπίνη (PTN) είναι ένας εκκρινόμενος αυξητικός παράγοντας με συγγένεια δέσμευσης ηπαρίνης και διαμέσου του υποδοχέα της με δράση φωσφατάσης τυροσίνης β/ζ (RPTPβ/ζ) και την ανβ3 ιντεγκρίνη προάγει τη μετανάστευση στα ανθρώπινα ενδοθηλιακά κύτταρα. Πρόσφατα δείξαμε ότι εξωγενώς χορηγούμενη PTN αναστέλλει την προκαλούμενη από τον VEGF μετανάστευση των ενδοθηλιακών κυττάρων. Στην παρούσα εργασία μελετήσαμε την επίδραση της ενδογενούς PTN και του υποδοχέα της RPTPβ/ζ στη προκαλούμενη από τον VEGF μετανάστευση των ενδοθηλιακών κυττάρων. Βρήκαμε ότι η ενδογενής PTN δε συμμετέχει στη μετανάστευση που προκαλεί ο VEGF. Αντίθετα, μείωση της έκφρασης του ενδογενούς RPTPβ/ζ με siRNA είχε ως αποτέλεσμα πλήρη αναστολή της επαγόμενης από VEGF κυτταρικής μετανάστευσης. Η αποσιώπηση του γονιδίου του RPTPβ/ζ δεν επηρέασε την προκαλούμενη από τον VEGF ενεργοποίηση των ERK1/2, αλλά μείωσε την επαγόμενη από VEGF αλληλεπίδραση ανάμεσα στον VEGFR-2 και την ιντεγκρίνη ανβ3, αλληλεπίδραση σημαντική για την προκαλούμενη από τον VEGF κυτταρική μετανάστευση. Τέλος, βρέθηκε ότι ο VEGF αλληλεπιδρά άμεσα με τον RPTPβ/ζ, αφού τα δύο μόρια συγκατακρημνίζονται σε εκχυλίσματα κυττάρων που εκφράζουν τόσο VEGF όσο και RPTPβ/ζ. Συμπερασματικά, ο RPTPβ/ζ φαίνεται να δρα ως υποδοχέας του VEGF και να είναι απαραίτητος για την προκαλούμενη από τον VEGF μετανάστευση των ενδοθηλιακών κυττάρων διαμέσου του VEGFR-2. / A considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis, with vascular endothelial growth factor (VEGF) representing the major inducer of angiogenesis up to date. VEGF induces several angiogenic functions of endothelial cells through its receptors VEGFR-1 and VEGFR-2 or KDR. VEGF-induced endothelial cell migration seems to be mediated by KDR, possibly via engagement of integrin αvβ3. Pleiotrophin (PTN) is a secreted heparin-binding growth factor that through its receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) and ανβ3 integrin induces human endothelial cell migration. We have previously shown that exogenous PTN inhibits VEGF-induced endothelial cell migration. In the present work we studied the effect of endogenous PTN and its receptor RPTPβ/ζ in VEGF-induced endothelial cell migration. We found that endogenous PTN is not involved, while RPTPβ/ζ is required for VEGF-induced endothelial cell migration. Although VEGF may directly interact with RPTPβ/ζ, down-regulation of the latter by siRNA does not affect VEGF-induced ERK1/2 activation but seems to affect the interaction of KDR with ανβ3, which is important for VEGF-induced cell migration. Collectively, RPTPβ/ζ seems to be required for VEGF-induced endothelial cell migration through its receptor KDR.

Determining how coordinated gene expression changes direct embryonic heart development is paramount to understanding the genetic causes and developmental origins of congenital cardiomyopathies. Towards this goal, I present an optimized protocol for mouse thiouracil tagging (TU-tagging), a novel transcriptomics methodology for defining dynamic and cell specific gene expression programs, and validate TU-tagging for cardiovascular research. I then apply related and additional high-resolution approaches to characterize how vascular endothelial growth factor (VEGF) signaling coordinates cell and gene expression dynamics underlying heart ventricle development. TU-tagging is a powerful approach to study dynamic gene expression programs of defined cell types while they are natively embedded within a complex organ. TU-tagging integrates genetic and chemical approaches to provide temporally controlled in vivo covalent labeling of cell type–specific RNA. Here, I describe two significant optimizations of the TU-tagging molecular biology and bioinformatics workflows that improve the method’s ability to identify differentially expressed genes and expand the technology’s utility. Next, using chemical inhibition of VEGF signaling in combination with high-resolution imaging and transcriptomic methods, I show that VEGF signaling directly promotes formation of the trabeculae that uniquely develop in the heart ventricle. By RNA-Seq, I identify VEGF-dependent target genes, including Gpr126 and Bmp10, which encode additional signaling proteins. I further show that myocardial Bmp10 expression and resulting endocardial Bmp10-driven pSMAD1/5/8 signaling is under sustained control by endocardial VEGF signaling. This continuous VEGF-BMP signaling interplay between endocardial and myocardial cells led me to examine the dynamic tissue arrangements between the two cell types during early stages of trabeculation. By extensive staining and high resolution imaging, I show that endocardial cells can be subdivided into three classes; 1) quiescent cavity cells that are well-separated from the myocardium, 2) proliferative and signal responsive transition zone/stalk cells that directly interact with myocardium to coordinate both cell types’ activities, and 3) CD34-expressing migratory tip-like cells uniquely found at the base of forming trabeculae. VEGF promotes trabeculation by 1) driving proliferation of endocardial transition zone/stalk cells and, secondarily, neighboring myocardium, and 2) directing the outward migration of endocardial tip cells that causes myocardial tissue to accumulate within individual and extensive ridge-like trabeculae. Defining these multiple roles of VEGF signaling during ventricle development reveals a novel conceptual framework for understanding trabeculation mechanisms and therefore processes likely to be disrupted in common congenital cardiomyopathies. This dissertation includes previously published and unpublished co-authored material. / 10000-01-01

Purely diffusive O2 transport typically is insufficient to sustain aerobic metabolism in most multicellular organisms. In small animals, however, a high surface-to-volume ratio may allow passive diffusion alone to supply sufficient O2 transfer. The purpose of this thesis was to explore the impacts of internal convection on the exchange of respiratory gases in a small complex organism, the larval zebrafish (Danio rerio). Thus, I tested the hypothesis that internal convection is required for the normal transfer of the respiratory gases O2 and CO2 and maintenance of resting aerobic metabolic rate. Use of morpholino knockdown of the VEGF-A and TNNT2 proteins allowed examination of two independent models lacking internal convection. Using micro-respirometry, I demonstrated that loss of internal convection reduces resting rates of O2 consumption and CO2 excretion in larvae at 4 days post fertilization. I also used the scanning micro-optrode technique to demonstrate that acute loss of internal convection resulted in reduced rates of cutaneous O2 flux, a trait that was reversed upon the restoration of internal convection. Finally, I demonstrated that in larval zebrafish, loss of internal convection resulted in decreased hypoxic performance and loss or severe reduction of the hypoxic cardiorespiratory responses. The results from these experiments showed that internal convection is i) required to maintain resting rates of respiratory gas transfer in the larval zebrafish, ii) important in facilitating the hypoxic cardiorespiratory responses in larval zebrafish and iii) augments O2 extraction capacity in the face of progressive hypoxia.

Abstract Fetal alcohol syndrome (FAS) is a pattern of anomalies in affected children due to maternal alcohol administration at vulnerable stages of fetal development. Intrauterine growth restriction and facial malformation are the presenting phenotypes of FAS. In this investigation, novel pathomechanisms of intrauterine growth restriction and facial malformation were the primary aims. We found by a FAS mouse model that AceCS1 gene expression and polyamines are the immediate targets of fetal alcohol exposure. The AceCS1 product is a precursor for lipid synthesis and protein acetylation and possibly, for polycation acetylation. We cloned the Mus musculus nuclear-cytosolic AceCS1 gene, and showed that its expression is developmentally regulated with a dynamic localization in the cytosolic and nuclear compartment. The enzyme plays an essential role in de novo synthesis of acetyl Coenzyme A. Fetal alcohol administration targets nutrient supplying networks, which are localized at critical barriers. The main findings were reduced surface of the labyrinthine zone, destruction of gap junctions in the hemotrichorial placenta, reduced syncytiotrophoblastic cell layers and loosening of interaction between cell layers and embryo endothelial cells, reduced Reichert’s membrane thickness with discontinued Reichert’s trophoblast and loss of interaction by Reichert’s-parietal cells, reduction of capillary network and reduced vascularization in the brain area, and perturbed neural crest migration and formation of neural tube defect. Alteration of angiogenesis -regulating proteins such as VEGF, PlGF, PECAM was detected in FAS, with no significant changes in placental angiogenesis of the labyrinthine zone, but up-regulation of VEGF/PlGF caused permeability changes in the placenta and yolk sac. On the other hand, the PECAM pool in embryos’ brain was reduced, which in turn led to decreased angiogenesis and vascularization. / Tiivistelmä Sikiön alkoholisyndrooma (engl. Fetal alcohol syndrome, FAS) on joukko muutoksia, joita esiintyy äidin raskaudenaikaisen alkoholin käytön seurauksena, kun käyttö osuu sikiökehityksen kannalta kriittiseen vaiheeseen. Kohdunsisäisen kasvun rajoittuminen ja kasvojen epämuodostumat ovat FAS:n tyypillisimpiä ilmentymiä. Tässä tutkimuksessa pyrittiin löytämään uusia patomekanismeja kohdunsisäisen kasvun rajoittumiselle ja kasvojen epämuodostumille. Hiiren FAS-mallin avulla selvisi, että sikiön altistuminen alkoholille vaikuttaa suoraan AceCS1-geenin ilmentymiseen ja polyamiinien pitoisuuteen. AceCS1-geenin tuote on esiaste lipidien synteesissä ja proteiinien asetylaatiossa sekä mahdollisesti myös polykationien asetylaatiossa. Työssä myös kloonattiin hiiren (Mus musculus) AceCS1-geeni, jonka tuotetta esiintyy sekä tumassa että solulimassa. Lisäksi osoitettiin, että geenin ekspressio oli kehityksen aikana säädelty tuottamaan entsyymiä dynaamisesti eri paikkoihin solussa. Entsyymillä on lisäksi merkittävä osuus asetyyli-koentsyymi-A:n de novo–synteesissä. Sikiön altistuminen alkoholille kohdistuu sellaisten ravintoaineiden saatavuuteen, jotka sijaitsevat kriittisesti tärkeissä kudosrajapinnoissa. Päälöydöksinä olivat vähentynyt labyrinttikudoksen pinta-ala, gap-liitosten tuhoutuminen istukan veriesteessä (hemotrichorial?), ohentunut trofoblastisolujen kerros ja Reichertin kalvon paksuus, harventunut hiusverisuonten verkosto sekä verisuonitus aivojen alueella sekä hermopienan solujen siirtymishäiriö ja hermostoputken sulkeutumishäiriö. Verisuonten muodostumista (angiogeneesiä) säätelevien proteiinien (kuten VEGF, PlGF, PECAM) muutoksia todettiin FAS:ssa, mutta merkittäviä muutoksia ei havaittu istukan verisuonten muodostumisessa. VEGF/PlGF-suhteen suureneminen muutti istukan ja ruskuaispussin verisuonten läpäisevyyttä. Toisaalta sikiöiden aivojen PECAM-määrä pieneni, mikä johti verisuonten ja verisuoniverkoston muodostumisen vähenemiseen.

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.