La protéine C-réactive (CRP) est aujourd’hui considérée comme un biomarqueur indispensable dans la prédiction de maladies cardio-vasculaires et de complications aiguës associées, et ce, par des mécanismes qui ne sont pas encore totalement élucidés. Nous avons étudié les effets de la CRP dans divers aspects de la biologie des cellules mononucléaires du sang périphérique (PBMC) et des monocytes humains ex vivo. En effet, ces cellules peuvent être impliquées dans la physiopathologie de ces maladies. Nous avons aussi utilisé la lignée promonocytaire THP-1. Ces trois types cellulaires ont été incubés avec la CRP purifiée ou recombinante et l’expression génique de cytokines pro-inflammatoires, de chimiokines et de MMP-9 a été analysée par PCR semi-quantitative en temps réel et l’expression protéique par test immunométrique ou par test ELISA. La CRP ne semble agir ni sur la synthèse de ces cytokines ni sur celle de MMP?9. Une approche globale par puce à protéines avec les surnageants de culture de monocytes a démontré que la CRP augmentait la synthèse du VEGF-A. Ce résultat a été confirmé au niveau transcriptionnel par RT-PCR et au niveau protéique par test ELISA. Une étude complémentaire avec la lignée THP-1 a permis de montrer l’implication des voies PI3?Kinase et MEK mais pas celle de la p38MAPKinase dans cette augmentation. Ces travaux ont ainsi permis la mise en évidence de plusieurs mécanismes permettant d’associer la CRP aux pathologies cardiovasculaires dans une relation de cause à effet. Ces mécanismes pourraient représenter des cibles thérapeutiques potentielles des maladies cardiovasculaires. / C-reactive protein is now considered as an essential biomarker for predicting the occurrence of cardiovascular diseases and their acute complications through mechanisms that are not fully elucidated. We investigated CRP effects on several aspects of the biology of ex vivo human peripheral blood mononuclear cells (PBMC) and monocytes. In fact, these cells can be involved in the pathophysiology of these diseases. Also, we used the promonocytic line THP-1. These three cellular types were incubated with purified or recombinant CRP and gene expression of pro-inflammatory cytokines, chemokines and (pro)MMP-9 was analysed by real time semi-quantitative PCR and protein expression by immunometric or ELISA tests. CRP doesn’t seem to act upon neither the tested cytokines synthesis nor the (pro)MMP-9 expression. A global approach by protein array with the cultured monocytes supernatants showed that CRP induced VEGF-A protein synthesis. This result was confirmed at transcriptional level by RT-PCR and at protein level by ELISA. A complementary study with the monocytic cell line THP-1 demonstrated the activation of PI3-Kinase and MEK pathways but not of p38MAPKinase pathway in this regulation. These results provide insight into several mechanisms that could transform the statistical association between CRP and cardiovascular diseases into a cause-to-effect relationship. Some of these mechanisms could represent therapeutic targets for cardiovascular diseases.

Beim humanen nichtkleinzelligen Bronchialkarzinom ist die schrittweise Progression vom gutartigen Tumor zur malignen Metastasierung weitestgehend ungeklärt. In einem transgenen Mausmodell für das humane nichtkleinzellige Bronchialkarzinom, in dem in Lungenepithelzellen eine onkogene Mutante der Proteinkinase C-RAF exprimiert wird, können einzelne Schritte im Prozess der malignen Progression entschlüsselt werden. Die durch C-RAF induzierten Adenome zeichnen sich durch eine hohe genomische Stabilität in den Tumorzellen, durch starke interzelluläre Adhäsionskontakte zwischen den Tumorzellen und das Fehlen einer malignen Progression aus. Hier wurde demzufolge untersucht, ob die Auflösung der E-Cadherin-vermittelten Zellkontakte zwischen den einzelnen Tumorzellen eine Metastasierung auslösen könnte. Es wurden zwei genetische Ansätze verfolgt, um die Rolle der Tumorzelladhäsion im C-RAF Modell zu bewerten, die konditionelle Eliminierung des E-Cadheringens Cdh1 sowie die regulierbare transgene Expression von dominant-negativem E-Cadherin. Die Auflösung der E-Cadherin-vermittelten Zelladhäsion führte zur Neubildung von Tumorgefäßen, welche in der frühen Phase der Gefäßbildung durch Wiederherstellung des Zellkontakts reversibel war. Die vaskularisierten Tumore wuchsen schneller, bildeten invasive Fronten aus und führten zur Ausbildung von Mikrometastasen. Es konnte gezeigt werden, dass Beta-Catenin für die Induktion der Angiogenesefaktoren VEGF-A und VEGF-C in Lungentumorzellinien des Menschen und der Maus essentiell war. Lungentumorzellen aus den in situ Tumoren mit aufgelösten E-Cadherin-vermittelten Zellkontakten exprimierten Gene endodermaler und anderer Zellabstammung, was epigenetische Reprogrammierung in Tumorzellen als den Mechanismus bei der malignen Progression vermuten lässt. / Steps involved in the progression of non-small cell lung cancer (NSCLC) to metastasis are poorly understood. Expression of oncogenic C-RAF in lung epithelial cells has yielded a model for non-small cell lung cancer (NSCLC). The induced adenomas are characterised by high genomic stability, a lack of tumor progression and pronounced cell-cell contacts raising the question whether disruption of E-cadherin complexes would promote progression to metastasis. Two genetic approaches were used to evaluate the role of adherens junctions in a C-RAF driven mouse model for NSCLC: conditional ablation of the Cdh1 gene and expression of dominant negative (dn) E-cadherin. Disruption of E-cadherin function caused massive formation of intratumoral vessels that was reversible in the early phase of induction. Vascularized tumors grew more rapidly, developed invasive fronts and gave rise to micrometastasis. ß-catenin was identified as a critical effector of E-cadherin disruption leading to up-regulation of angiogenic inducers (VEGF-A and VEGF-C) in mouse and human lung tumor cell lines. In vivo, lung tumor cells with disrupted E-cadherin expressed ß-catenin target genes of endodermal and other lineages suggesting that reprogramming may be involved in metastatic progression.

Preeclampsia (PE) remains one of the most enigmatic and pervasive conditions developed during pregnancy and is a leading cause of maternal and fetal morbidity and mortality throughout the world. Afflicting nearly 5-8% of pregnancies in the Unites States, PE is most commonly characterized by an increase in blood pressure and high protein excretion near or after the 20th week of gestation. Unfortunately, few effective treatments are available, and the only "cure" is delivery. While the molecular pathogenesis of PE remains undefined, an interruption in placental blood flow, or placental ischemia, is widely observed as a primary contributor to the syndrome progression. Furthermore, to investigate the role of both pharmacological and non-pharmacological modalities to prevent placental ischemia induced hypertension, we employed a robust model of reduced utero-placental perfusion pressure (RUPP) in the pregnant rat. First, in Chapter IV, exercise initiated during gestation was not effective in the prevention of RUPP-induced hypertension, whereas exercise training prior to and continued through gestation prevented the increase in blood pressure. Though the molecular contributions to this effect are undefined, the effects appear to be independent of angiogenic balance restoration. Finally, in Chapter V, administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was explored as a novel pharmacological modality to prevent the onset of hypertension and endothelial dysfunction in the RUPP model. As hypothesized, AICAR ameliorated the RUPP-induced hypertension, and the anti-hypertensive effect in the RUPP appears to be dependent on the restoration of angiogenic balance in the maternal plasma. This dissertation includes previously published and unpublished co-authored material.

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.

Reduction of cardiac myocyte loss and repair of the vasculature post myocardial infarction are important therapeutic goals because the potential for intrinsic repair is limited. Preclinical and limited clinical data support the possibility that bone marrow-derived mesenchymal stem cells may be a suitable cell type for cellular therapy. The goal of this research was to determine the effectiveness of using MSCs from aged mice in cellular therapy for the treatment of AMI. The central hypothesis for this research was that therapeutic potential of mesenchymal stem cells decreases with age. This research utilized global gene expression analysis to investigate molecular differences in MSCs harvested from three different age groups of mice. Microarray analysis was performed to investigate changes in gene expression with respect to aging. Furthermore, both in vitro and in vivo experiments were completed to analyze the functional and molecular characteristics of the MSCs. The data identified age-related defects in mouse MSCs as well as determined the molecular basis for these deficiencies. This study indicates that MSCs from 26m mice are severely deficient in the induction of angiogenesis and cardiac repair due to defective paracrine factor secretion caused by decreased expression of growth factor/cytokine genes. Hypoxia attenuates the deficiency in the aged mice, whereas in young mice low oxygen promotes secretion of paracrine growth factors. It was determined a dysfunction in HIF-1 alpha signaling was present in MSCs from 26m mice and is regulated by the PI3K/Akt signaling in MSCs. Furthermore, two novel and important and novel aspects of this study were the discovery that cell cycle regulation gene expression decreases with age and MSCs have increased insulin resistance with age. Increased insulin resistance in this cell type with aging is likely to have profound effects on the clinical outcomes of using these cells therapeutically. Likewise, loss of cell cycle regulation during proliferation could also lead to undesirable clinical effects. Gaining insight to the repair potential of these cells with respect to age will help to better define future trials of autologous stem cells not only for heart disease but for all of the many applications proposed for these cells.

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

During embryonic vasculogenesis, endothelial cells form in the mesoderm , assemble into cord-like structures and then undergo tube formation. Previous studies have shown that signaling by members of the hedgehog family of secreted growth factors is essential for normal development of embryonic blood vessels. Embryos lacking hedgehog function show the presence of abundant endothelial cells but the cells fail to assemble into vascular cords and lumenized endothelial tubes do not form. At present it is not known whether active hedgehog signaling is required for both cord and tube formation or only for the initial step. To address this question, we have used small molecule inhibitors and agonists to the alter activity of the hedgehog signaling pathway in the chick embryo. If development is allowed to proceed until endothelial cells of the future dorsal aortae have assembled into cords, subsequent inhibition of hedgehog signaling, using cyclopamine, does not prevent aortal cells from forming endothelial tubes, however, it does lead to a reduction in cross sectional area of the aorta and to a loss of density of the adjacent vascular plexus. In contrast, activation of the hedgehog pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. Very little, if any, of the observed effects are due to differences in number of endothelial cells in the treated embryos. Examination of endothelial cells during vascular plexus formation shows that inhibition of hedgehog signaling with cyclopamine inhibits formation of filopodia while treatment with SAG increases the number of filopodial extensions. These studies show that hedgehog signaling levels must be tightly regulated for normal vascular patterning to be achieved.

No description available.

Blood vessels deliver nutrients and oxygen to tissues. Importantly, the functions and growth of blood vessels are commonly altered in disease. The inside of all blood vessels are lined with endothelium, a thin specialized layer of endothelial cells that separate the blood from other tissues. This thesis deals with the identification and functional characterization of proteins and microRNAs that have key roles as modulators of growth factor signaling and directed cell migration of endothelial cells and other vascular cells. A previously uncharacterized protein of the exocyst complex, Exocyst complex component 3-like 2 (ExoC3L2) was identified and shown to be highly expressed in endothelial cells of sprouting vessels. Suppression of ExoC3L2 resulted in reduced VEGF-A signaling together with reduced chemotaxis in response to VEGF-A gradients. VEGF-A-signaling via its receptor VEGFR-2 is thus modulated by the exocyst complex and ExoC3L2. Expression profiling of highly vascularized tissues were used to identify several microRNAs selectively expressed in blood vessels. miR-145, targeting the transcription factor Fli1, was shown to be expressed in pericytes and mural cells. Elevated levels of miR-145 reduced chemotaxis of both endothelial cells and fibroblasts in response to growth factor gradients. miR-145 depletion in fibroblasts was shown to inhibit chemotaxis in response to PDGF-BB. The guanine nucleotide exchange factor FGD5 was shown to be selectively expressed in endothelial cells and to regulate Cdc42 activity. FGD5 was shown to regulate the turnover of activated VEGF-receptors. Suppression of FGD5 impaired endothelial cell chemotaxis, suggesting that FGD5 is required for efficient and sustained VEGF-A signaling. Inactivation of RhoD, a regulator of endosomal trafficking, resulted in an increased pool of acetylated and stable microtubules. Knockdown of RhoD in human fibroblasts resulted in a loss of cell polarity. A link between PDGFR-β and RhoD was implicated by the finding that PDGF-BB was shown to trigger formation of GTP-bound RhoD. Chemotaxis towards PDGF-BB was severely inhibited in cells with reduced RhoD expression, suggesting a role for RhoD in chemotaxis via its regulation of microtubule dynamics.