Hypoxia is one of the hallmarks of cancer. It was first postulated to occur in solid tumours by Thomlinson and Gray in 1955.1 The presence of hypoxia has been demonstrated in different types of solid tumours.2 Intratumoral hypoxia is caused by the lack of functional blood vessels in proliferating tumour tissue, resulting in low intratumoral oxygen concentrations. If hypoxia is severe or prolonged, cell death occurs.3 Malignant cells can undergo genetic and adaptive changes that allow them to escape from dying of oxygen deprivation. These changes are associated with a more aggressive malignant phenotype 4,5 conferring resistance to radiation 6,7 and chemotherapeutic agents.3,8,9 Hence hypoxia is known to be a key factor responsible for tumour resistance in humans. / Invasive polarographic oxygen sensor measurements have demonstrated hypoxia in solid tumours and it is generally defined to occur at an oxygen tension less than ten mmHg.10 Perhaps of more importance is that hypoxia has been demonstrated to be a prognostic indicator for local control after treatment with radiotherapy in glioma, head and neck and cervical cancers.11-13 It has also been able to predict for survival and the presence of distant metastases in soft tissue sarcomas.14 Finally, the significance of hypoxia in the activation and induction of functional molecules such as hypoxia inducible factors (HIFs) and VEGF, the modulation of gene expression (e.g. carbonic anhydrase IX), increased proto-oncogene levels, activation of nuclear factors and accumulation of other proteins (e.g. TP53) although progressing, is yet to be defined.15,16 / Thus, it is of clinical interest to understand the levels of hypoxia and numbers of hypoxic cell populations in tumours, particularly those resistant to radiation and chemotherapy. In doing so clinicians and researchers may formulate more accurate prognostic information and develop treatments targeting hypoxic cells. Renal cell carcinoma (RCC) is a tumour resistant to radiation and chemotherapy that is yet to have its oxygen status investigated. / Although the “gold standard” of oxygen tension measurement is the Polarographic Oxygen Sensor (POS or Eppendorf pO2 histograph), non-invasive means of measuring oxygen status via imaging, immunohistochemistry or serum tumour markers are more practical. As highlighted by Menon and Fraker, it is imperative that reliable, globally usable, and technically simplistic methods be developed to yield a consistent, comprehensive, and reliable profile of tumour oxygenation. Until newer more reliable techniques are developed, existing independent techniques or appropriate combinations of techniques should be optimized and validated using known endpoints in tumour oxygenation status and/or treatment outcomes.17 / Hanahan and Weinberg 18 surmised that the field of cancer research has largely been guided by a reductionist focus on cancer cells and the genes within them- a focus that has produced an extraordinary body of knowledge. Looking forward in time, they believe that progress in cancer research would come from regarding tumours as complex tissues in which mutant cancer cells have conscripted and subverted normal cell types (endothelial cells, immune cells, fibroblasts) to serve as active collaborators in their neoplastic agenda. The interactions between the genetically altered malignant cells and these supporting coconspirators will prove critical to understanding cancer pathogenesis and to the development of novel, effective therapies.18 / Essentially, the background outlined here not only highlights the core aim of this thesis: to better understand the oxygen status of renal cell carcinoma and the relationship of this to angiogenesis so that better targeted therapies may be pursued in the future; but it also places this research in the context of the future proposed by Hanahan and Weinberg,18 by clearly focusing on collaborators in the neoplastic agenda, rather than just tumour cells themselves, to better understand RCC.

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.

The central role of vascular endothelial growth factor (VEGF) in physiological and pathological angiogenesis makes it attractive both as a therapeutic target for anti-angiogenic drugs in cancer treatment and as a pro-angiogenic cytokine to treat ischemic disease. Currently, it is well established that the VEGF but also other growth factors exert their angiogenic effects partly through the activation of endothelial nitric oxide synthase (eNOS). A better understanding of the VEGF/NO signaling pathway could therefore lead to the identification of new therapeutic targets to impact on angiogenesis.This thesis is based on four different articles, the principal findings of which are summarized here below. First, we demonstrated that, chronologically, endothelial cell exposure to VEGF first led to eNOS dissociation from caveolin (a hallmark of the Ca2+/CaM-mediated activation of eNOS), and then to the interaction of eNOS with the heat shock protein hsp90. We also reported that eNOS-bound hsp90 could recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn could phosphorylate eNOS on the serine 1177. Finally, we found that although the VEGF-induced phosphorylation of eNOS led to a sustained production of NO independently of a maintained increase in intracellular [Ca2+], this late stage of eNOS activation was strictly conditional on the initial VEGF-induced Ca2+-dependent stimulation of the enzyme. These data established the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggested new ways of regulating the production of NO in response to this cytokine through the structural protein caveolin and the ubiquitous chaperone protein, hsp90. A second study identified caveolin and Hsp90 as key players in the proangiogenic action of statins and therefore as potential pharmacological targets to modulate NO-dependent angiogenesis. We found that atorvastatin stabilized endothelial tube formation from both outgrowing and isolated macrovascular ECs cultured in Matrigel through a decrease in caveolin abundance and in its inhibitory interaction with eNOS. In a similar angiogenic assay, microvascular endothelial cells appeared also responsive to statins, not through a decrease in the caveolin pool (which is (too) large in these cells) but via the increased recruitment of hsp90 in the eNOS complex and the associated eNOS phosphorylation on the serine 1177. These data provided new mechanistic insights into the NO-mediated effects of statins and underscored the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis. In a third study, we have reported a net decrease in the ability of cultured ECs expressing recombinant caveolin to migrate and to form capillary like networks (e.g. the crucial steps occurring during the angiogenic process) in presence of VEGF. We then exploited the propensity of cationic lipids to target EC lining tumor blood vessels to transfect tumor-bearing mice in vivo. A dramatic tumor growth delay associated to a decrease in tumor microvessel density in the central core of the tumor was observed in mice transfected with caveolin versus sham-transfected animals. Interestingly, we also found that in the early time after lipofection (e.g. when macroscopic effects on tumor growth were not yet detectable), caveolin expression also impaired NO-dependent tumor blood flow. These findings indicated that besides (before) acting as an anti-angiogenic agent, recombinant caveolin can modulate the endothelium phenotype and impact on the tumor blood flow, both effects leading to a decrease in tumor growth. Finally, we showed that the activation of the VEGF/NO signaling pathway led to the down-regulation of adhesion molecules and to the anergy of endothelial cells. We found, indeed, that the adhesion of human CD8+ lymphocytes on microvascular endothelial cells exposed to TNF-a was dramatically reduced in the presence of VEGF. Interestingly, we also documented that the co-administration of the NOS inhibitor L-NAME or the Hsp90 inhibitor geldanamycin could restore this adhesion to the level originally obtained with TNF-a alone. Finally, we confirmed the key role of NO in the VEGF-mediated effects on the CD8+ adhesion by tipping the balance towards more or less angiogenesis through the transfection of caveolin siRNA or caveolin plasmid, respectively. In these experiments, lymphocyte adhesion appeared directly correlated to the extent of caveolin expression, confirming that the so-called anti-angiogenic strategy can directly impact on the phenotype of (tumor) endothelial cells and instead of (before) killing them, be exploited to potentiate cancer immunotherapy. In conclusion, by dissecting the post-translational regulation of eNOS, we have identified major therapeutic targets, namely caveolin and hsp90, that may be exploited either to block or promote angiogenesis. More particulary, cDNA encoding for these proteins or their mutant form, when combined with adequate mode of delivery, appeared to exert profound effects on the vascular compartment of tumors but also of ischemic tissues.

Tumors need the development of new vessels from the pre-existing vasculature to bring nutrients and oxygen to the whole tumor mass. The tumor vascular network is known to be poorly functional due to architectural and functional abnormalities. The end result is an inadequate and heterogeneous tumor perfusion leading to the development of tumor hypoxia. From a therapeutic perspective, hypoxia is a source of radioresistance and the dysfunctional perfusion hampers drug delivery. Historically, tumor hypoxia refers to chronic hypoxia (or diffusion-limited hypoxia) that results from the increasing distance between O2-consuming cells and blood vessels due to the high metabolic rate of tumor cells. Many studies have demonstrated the impact of chronic hypoxia on the clonal selection of tumor cells resistant to conventional anti-cancer therapies. Growing evidence for the existence of another form of hypoxia caused by heterogeneities in tumor perfusion, namely acute or perfusion-limited hypoxia, plead however for a non-genetic source of phenotype conversion reaching not only tumor cells but also the tumor vasculature and in particular endothelial cells. In the cardiovascular field, the cyclic exposure to different pO2 levels is known to precondition cardiac myocytes to resist more prolonged ischemic insults. We hypothesized that this concept of myocardium preconditionning to promote the resistance vs pro-apoptotic stresses could be translated in tumors. Indeed, intermittent hypoxia in tumors is nothing else than cyclic changes in pO2 and radio- and chemotherapy can be viewed as pro-apoptotic stresses that the tumor can face. In particular, in the case of the tumor vasculature, the resistance could be a capacity to re-initiate angiogenesis after treatment. Radioresistance would be further potentiated since low pO2 is per se associated to reversibility of the damages. Also, since intermittent hypoxia is thought to be due in part to fluctuations in tumor blood flow (TBF), access of chemotherapy to the tumor could also further participate to chemoresistance. To address the above hypotheses, we first aimed to explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve the efficacy of chemotherapy. We then more directly examined whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. We also took advantage of this study to dissect the molecular mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. Finally, because VEGF plays a major role in hypoxia-mediated angiogenesis but also regulates major pro-survival pathways in endothelial cells, we evaluated the potential role of caveolin as a new therapeutic target to tackle EC resistance. Caveolin is, indeed, a key structural protein recently documented to interact with many downstream targets of VEGF. 1. To explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve chemotherapy. We focused this part of the work on the vascular tone modulator endothelin-1. Indeed, this peptide is over-expressed in many mouse and human tumors where it is documented to act as a mitogenic factor in both para- and autocrine manners. Endothelin-1 is also a potent vasoconstrictor acting through the ETA receptors located on VSMCs. In our lab, we previously showed that over-expression of endothelin-1 in tumors accounted for the development of a myogenic tone within the tumor vasculature. We have now documented that an ETA receptor antagonist induces the relaxation of microdissected tumor arterioles and selectively and quantitatively increases tumor blood flow in experimental tumor models. We also combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. We showed that administration of an ETA receptor antagonist reduces the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provided evidence that ETA antagonist could improve the access of cyclophosphamide to the tumor compartment and thereby induces a significant tumor growth delay. 2. To examine whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. To dissect the mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. This second part of our work, is a comprehensive investigation of the consequences of intermittent hypoxia, as caused by TBF heterogeneities, on the endothelial cell phenotype. First, we postulated that intermittent hypoxia (IH) favors endothelial cell (EC) survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We showed that exposing EC to cycles of hypoxia/re-oxygenation reduces radiation-induced cell death and promotes angiogenesis. In contrast, prolonged hypoxia failed to achieve such protection and even appeared deleterious. We also observed that although HIF-1£ is completely degraded during each re-oxygenation, its abundance is paradoxically found higher at each new hypoxic challenge. Moreover, the use of siRNA targeting HIF-1£ pointed out that HIF-1ƓÑ accumulation account for the increased resistance of EC to radiotherapy. Finally, we extended this concept in vivo by forcing IH in tumor-bearing mice and found that it is associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (vs normoxia or prolonged hypoxia). Next, we focused our work on the underlying mechanisms of EC phenotype conversion exposed to IH and particularly on potential actors that may favor HIF-1£ accumulation during IH. Prolylhydroxylases (PHD), MAPK and PI3K/Akt pathways as well as eNOS are known to regulate HIF-1£ abundance and transcriptional activity. We documented that PHD2 and PHD3 abundance are slightly decreased during IH, whereas prolonged hypoxia increases PHD3 expression in EC. We then showed that, ERK, Akt as well as eNOS were phosphorylated during reoxygenation periods of the IH protocol. We also used specific inhibitors of these cascades (i.e. PD98059, LY294002 and L-NAME, respectively), to evaluate their specific impact on HIF-1£ abundance and performed clonogenic assays to evaluate their consequences on EC survival. We showed that although, PD98059 and LY294002 sensitizes EC to pro-apoptotic stresses, only the PI3K/Akt inhibitor abrogates the HIF-1£ signal during IH. Conversely, L-NAME, a non-specific NOS-inhibitor, appears to potentiate the expression of HIF-1£ and to favor the EC survival. 3. To identify new therapeutic targets to prevent endothelial cell resistance by studying VEGF signaling, the major pro-survival and pro-angiogenic growth factor in endothelial cells. Because VEGF plays a central role in hypoxia-mediated angiogenesis and cell survival, the VEGF signaling cascade is a an obvious therapeutic target. To more specifically identify the pathways leading to cell survival and the resistance phenomena that we observed in response to intermittent hypoxia, a careful dissection of the downstream VEGF signaling cascades was performed. In this part of the work, we focused our attention on caveolin since it modulates the activity of eNOS, ERK and Akt that are major effectors acting downstream VEGF stimulation. We demonstrated the paradoxical role of caveolin-1 preventing signaling in basal conditions and ensuring the coupling between VEGFR2 and the downstream cascades upon VEGF stimulation. We used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice fails to recover a functional vasculature and actually loose part of the ligated limbs. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, endothelial tube formation is dramatically abrogated when compared with Cav +/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin are reached, VEGF exposure fails to activate ERK and eNOS. Altogether, these data identify caveolin as a new therapeutic target to alter VEGF signaling, in particular the cascades leading to angiogenesis and resistance to stresses.

Regulation of class IIa histone deacetylases (HDACs) phosphorylation is crucial because it provides the opportunity to control important developmental processes associated with these key enzymes. Indeed, the transcriptional repressor activity of class IIa HDAC is controlled via their phosphorylation-dependent nucleo-cytoplasmic shuttling. While a lot of efforts have been directed towards the identification of the inactivating kinases that phosphorylate class IIa HDACs, the identity of the antagonist phosphatase remained an open question. During this work, we found that protein phosphatase 2A (PP2A) is responsible for dephosphorylating the class IIa HDACs member HDAC7, thereby regulating its subcellular localization and repressor activity. In order to validate our model, functional consequences of these findings was illustrated during the two main biological processes involving HDAC7, i.e. T-cells apoptosis during negative selection and endothelial cells angiogenic activities during vascular network formation. Cellular PP2A represents a large population of trimeric holoenzymes containing a variable regulatory subunit, whose identity has a crucial role in determining the specificity of PP2A catalytic activity. In an effort to characterize the regulation of HDAC7 dephopshorylation, we identified the relevant PP2A holoenzyme regulating HDAC7 function during vasculogenesis and we found that, among diverse regulatory subunit isoforms, PP2A-Bα uniquely regulates endothelial cell angiogenic properties. PP2A-Bα silencing using small interfering RNAs results in a significant inhibition of endothelial cell tube formation and migration. These results establish PP2A, and more precisely the Bα containg PP2A holoenzyme, as an essential element in the regulation of the class IIa HDAC HDAC7 and unravel a first developmental function for the PP2A regulatory subunit Bα in the genesis of blood vessels.

A treatment strategy for cancer is the suppression of tumor growth by directing an immune response to the tumor vessels, which will destroy the tissue. In this thesis we describe the development of a vaccine that targets antigens expressed around angiogenic vasculature in most solid tumors. These antigens are alternative spliced extra domains of glycoproteins present in the extracellular matrix; e.g. the extra domain-B (ED-B) and extra domain-A (ED-A) of fibronectin and the C-domain of tenascin-C (TNCC). We show that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Furthermore, tumor growth was inhibited and the changes observed in the tumor tissue were consistent with an attack of the tumor vasculature by the immune system. For clinical development of therapeutic vaccines, targeting self-molecules like ED-B, a potent but non-toxic biodegradable adjuvant is required. The squalene-based Montanide ISA 720 (M720) in combination with CpG DNA fulfilled these requirements and induced an equally strong anti-self immune response as the preclinical golden standard Freund’s adjuvant. We have further characterized the immune response against ED-B generated with the adjuvant M720/GpG.  The ED-B vaccine also inhibited tumor growth in a therapeutic setting in a transgenic mouse model of pancreatic insulinoma in which tumorigenesis was already initiated. Furthermore, antibodies against ED-A and TNCC could be induced in mice and rabbits. We analyzed the expression of ED-A in breast tumors of transgenic MMTV-PyMT mice, a metastatic breast cancer model, with the aim to use this model to study the effect of an ED-A vaccine on metastasis. We also detected ED-B in canine mammary tumor tissue. Therefore vascular antigens might also represent potential therapeutic targets in dogs.  All together our preclinical data demonstrate that a vaccine targeting tumor blood vessels is a promising new approach for cancer treatment.

No description available.

Because engineered tissue constructs are inherently limited by their lack of microvascularization, which is essential to provide oxygen for cell survival, this thesis presents rationally designed materials and cell culture techniques capable of supporting functional tubule formation and stabilization. Combining a synthetic scaffold material with cells and their cell-secreted signals instigated tubule formation throughout the scaffold. Poly(ethylene glycol) (PEG) based hydrogels, biocompatible polymers which resist protein adsorption and subsequent nonspecific cellular adhesion, were modified to induce desired cell characteristics. Human umbilical vein endothelial cells were used as a reproducible and readily available cell type. Several tubule-stabilization signals, including platelet derived growth factor-BB (PDGF-BB) and ephrinA1, were covalently immobilized via conjugation to PEG to enable prolonged bioactive signaling and controlled local delivery. All hydrogels were further tested in a mouse cornea micropocket angiogenesis assay, a naturally avascular tissue for easy imaging in a reproducible and quantifiable assay. Hydrogels containing soluble growth factors induced vessel formation in the hydrogel, and the resulting vessel morphology was modulated using different growth factor concentrations. Immobilized PDGF-BB led to tubule formation in two dimensions, three dimensions, and in the mouse cornea while immobilized ephrinA1 stimulated secretion of extracellular matrix proteins laminin and collagen IV to stabilize the newly formed tubules. Finally, a co-culture of endothelial and pericyte cells encapsulated into hydrogels formed tubules that anastomosed to the host vasculature and contained red blood cells. PEG-based hydrogels represent a promising technique to induce microvascular formation in engineered constructs, leading to stable and functional vessel formation using covalently immobilized growth factors and encapsulated cells. These materials can be used for replacement of damaged or diseased tissues as the current supply of cadaveric donations cannot meet the demand of tissues for the 110,000 people awaiting an organ in the US.

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

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