Chemotactic cytokines, also known as chemokines, direct the migration of leukocytes following their interaction with seven transmembrane domain receptors that are part of the chemokine receptor family (Bachelerie et al. 2014). Chemokines are key players in cancer progression and the regulation of cancer-related inflammation. Atypical chemokine receptors (ACKRs) represent a subset of proteins belonging to the family of chemokine receptors but unable to signal through conventional cascades. ACKRs have recently emerged as important molecular players in health and diseases (Massara et al. 2016). They affect chemokine availability and function and impact many pathophysiological events, including the tumorigenesis process (Sjöberg et al. 2020). Chemerin is a nonchemokine chemoattractant for dendritic cell subsets, macrophages, and natural killer cells (Valérie Wittamer et al. 2003). Chemerin is the natural ligand for the receptors CMKLR1 (ChemR23), GPR1, and CCRL2. Chemerin expression is frequently downregulated in human tumors. The chemerin/CMKLR1 axis has been linked to immunity and inflammation as well as to cancer and angiogenesis. However, the exact function of CCRL2 in physiological and pathological processes remains poorly characterized. CCRL2 shares up to 40% homology with other C-C chemokine receptors in addition to many structural and functional similarities with the family of ACKRs, such as the lack of conventional G protein-mediated signaling and the inability to induce functional responses. CCRL2 is expressed by different cell types, such as endothelial cells and various leukocyte populations, and its expression is strongly upregulated by inflammatory signals. CCRL2 acts as a chemerin presenting molecule to cells expressing functional chemerin receptors (CMKLR1 and possibly GPR1) (Zabel et al. 2008). We have demonstrated that the expression of bioactive chemerin by tumor cells delays the growth of tumors in vivo, and a similar tumor growth delay is observed when chemerin is expressed in the skin of the host mice. In these tumors, the neoangiogenesis process is impaired, resulting in hypoxia, necrosis, and growth delay. A similar phenotype is observed for tumor cells growing in CCRL2 KO mice. In contrast, in a chemical carcinogenesis model (DMBA/TPA), the development of papillomas is accelerated in CCRL2 KO mice. In the present study, we studied the role of chemerin in angiogenesis and further investigated the effect of CCRL2 on the chemerin/CMKLR1 axis in tumorigenesis by testing tumoral cell lines overexpressing or invalidated for CCRL2. Moreover, we investigated whether CCRL2 is involved in the proliferation, migration, clonogenicity, and spheroid formation capacity of B16 melanoma and LLC carcinoma cells. Firstly, our results showed that chemerin exerted a strong anti-angiogenic effect in a bead sprouting assay, whereas we could not detect pro- or antiangiogenic properties of chemerin in various other assays. We demonstrated that the overexpression of CCRL2 significantly inhibited tumor growth in vivo, partially dependant on chemerin/CMKLR1 axis and independent of GPR1 expression. Also we showed that CCRL2 invalidation restored the tumor growth delay observed in CCRL2 KO mice. Importantly, we validated that CCRL2 expression in tumors affected the proportion of blood vessels, and resulted in a larger hypoxic and necrotic areas. CCRL2 expression did not impact the proliferation, migration and clonogenicity of B16 and LLC cells, but it strongly affected the spheroid formation capacity of B16 melanoma cells with a potential effect on the adhesion processes. Taken together, these results indicate that chemerin/CMKLR1/CCRL2 axis is significantly affecting the tumor growth by regulating angiogenesis, and CCRL2 is considered as a negative regulator of tumorigenesis. / Les cytokines chimiotactiques, également appelées chimiokines, dirigent la migration des leucocytes suite à leur interaction avec des récepteurs à sept domaines transmembranaires (Bachelerie et al. 2014). Les chimiokines sont des acteurs clés dans la progression du cancer et la régulation de l'inflammation liée au cancer. Les récepteurs atypiques de chimiokines (ACKR) représentent un sous-ensemble de protéines appartenant à la famille des récepteurs de chimiokines mais incapables de signaler via les cascades conventionnelles. Les ACKRs ont récemment été reconnus comme des acteurs moléculaires importants en physiologie et physiopathologie (Massara et al. 2016). Ils affectent la disponibilité et la fonction des chimiokines, et ont un impact sur de nombreux événements physiopathologiques, y compris le processus de tumorigenèse (Sjöberg et al. 2020). La chémérine est une protéine chimioattractante non apparentée aux chimiokines, active sur différentes populations leucocytaires, dont les cellules dendritiques, les macrophages et les cellules natural killer (Valérie Wittamer et al. 2003). La chémérine est le ligand naturel des récepteurs CMKLR1 (ChemR23), GPR1 et CCRL2. L'expression de la chémérine est fréquemment diminuée dans les tumeurs humaines. Le rôle de l’axe chémérine/CMKLR1 a été montré dans l'immunité et l'inflammation ainsi que le cancer et l'angiogenèse. Cependant, la fonction exacte de CCRL2 dans les processus physiologiques et pathologiques reste mal caractérisée. CCRL2 partage jusqu'à 40 % d'homologie avec d'autres récepteurs de C-C chimiokines, en plus de nombreuses similitudes structurelles et fonctionnelles avec la famille des ACKRs, telles que l'absence de signalisation médiée par les protéines G et l'incapacité d’induire des réponses fonctionnelles. CCRL2 est exprimé par différents types cellulaires, tels que les cellules endothéliales et diverses populations de leucocytes, et son expression est fortement augmentée par les signaux inflammatoires. CCRL2 agit uniquement en régulant les concentrations locales de chémérine, et en présentant le ligand à des cellules exprimant des récepteurs fonctionnels de la chémérine (CMKLR1 et potentiellement GPR1) (Zabel et al. 2008). Nous avons démontré que l'expression de la chémérine bioactive par les cellules tumorales retarde la croissance des tumeurs in vivo, et un retard similaire de la croissance tumorale est observé lorsque la chémérine est exprimée dans la peau des souris hôtes. Dans ces tumeurs, le processus de néoangiogenèse est altéré, entraînant une hypoxie, une nécrose et un retard de croissance.Un phénotype similaire de croissance retardée de lignées tumorales est observé chez les souris CCRL2 KO. En revanche, dans un modèle de cancérogenèse chimique (DMBA/TPA), le développement des papillomes est accéléré chez les souris CCRL2 KO. Dans la présente étude, nous avons étudié le rôle de la chémérine dans l'angiogenèse et l'effet de CCRL2 sur l'axe chémérine/CMKLR1 dans la tumorigenèse en testant des lignées cellulaires tumorales surexprimant ou invalidées pour CCRL2. De plus, nous avons étudié si CCRL2 est impliqué dans la prolifération, la migration, la clonogénicité et la capacité de formation de sphéroïdes de cellules tumorales de mélanome B16 et de carcinome pulmonaire (LLC). Premièrement, nos résultats ont montré que la chémérine exerçait un fort effet anti-angiogénique dans un test d’angiogenèse sur billes (bead sprouting assay), alors que nous n'avons pas pu détecter les propriétés pro- ou anti-angiogéniques de la chémérine dans divers autres tests. Nous avons démontré que la surexpression de CCRL2 inhibait significativement la croissance tumorale in vivo, un effet partiellement dépendant de l'axe chémérine/CMKLR1 et indépendant de l'expression de GPR1. Nous avons également montré que l'invalidation de CCRL2 dans les cellules tumorales supprimait le retard de croissance tumorale observé chez les souris CCRL2 KO. Surtout, nous avons validé que l'expression de CCRL2 dans les tumeurs affectait la proportion de vaisseaux sanguins et résultait en des zones hypoxiques et nécrotiques plus grandes. L'expression de CCRL2 n'a pas eu d'impact sur la prolifération, la migration et la clonogénicité des cellules tumorales B16 et LLC, mais elle a fortement affecté la capacité de formation de sphéroïdes par les cellules de mélanome B16 avec un effet potentiel sur les processus d'adhésion cellulaire. En conclusion, notre étude a permis de mettre en évidence les effets inhibiteurs de l'axe chemerin/CMKLR1/CCRL2 sur la croissance tumorale tout en régulant l'angiogenèse, et de montrer que CCRL2 peut être considéré comme un régulateur négatif de la tumorigenèse. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Angiogenesis is a physiological process by which new blood vessels develop from preexisting vasculature. The process of converting endothelial cells into fully developed blood vessels involves multiple coordinated cellular events that occur through the collaboration that exists between a variety of growth factors, receptors and adhesion molecules. The immunoglobulin-containing and proline rich receptor-1 (IGPR-1) is an IgSF containing adhesion molecule that has been recently identified as a novel regulator of angiogenesis in vitro. In this study, we provide evidence that IGPR-1 promotes cell survival in porcine aortic endothelial cells (PAE) and plays a role in the inhibition of p38 MAPK in vitro. Deletion of the extracellular domain of IGPR-1 abolished IGPR-1’s ability to inhibit phosphorylation of p38 MAPK and promote the survival of endothelial cells. Likewise, mutation of serines 186 (A186-IGPR-1) and 220 (A220-IGPR-1) on the cytoplasmic domain of IGPR-1 was also found to reduce both the promotion of cell survival and inhibition of p38 MAPK. These findings suggest that both domains of IGPR-1 are important for endothelial cell survival and the activation p38 MAPK.

<div> <div> <div> <p>Skeletal muscle occupies approximately 40 to 50 percent of body mass and is responsible for respiration, postural control, and locomotion and plays a pivotal role in regulating glucose, lipid, and protein metabolism. Acute muscle trauma and chronic disease conditions such as muscular dystrophies are associated with structural abnormalities, enhanced fatigability and impaired metabolism and consequently lead to exercise intolerance and poor quality of life. Despite the clinical importance and a number of studies on the treatment of muscle damage, few modalities have shown to elicit beneficial effects. Heat treatment has been used for a long time to treat soft tissue injuries in the field of physical therapy and sports medicine. However, the underlying mechanisms by which heat treatment accelerates muscle recovery following injury are not clear. </p> <p>The primary aim of my dissertation studies was to determine the impact of heat therapy on skeletal muscle structure and function in humans and animals. In Chapter 2, we report that a single session of local heat treatment promotes the expression of angiogenic and myogeneic mediators including vascular endothelial growth factor (VEGF) and angiopoietin 1(ANGPT1) in healthy human skeletal muscle. In Chapter 3, we report repeated exposure to heat therapy stimulates factors involved in muscle repair process and accelerates functional recovery from exercise-induced muscle damage. In Chapter 4, we show that 8 weeks of local heat therapy improves muscle strength of knee extensor and increases skeletal muscle capillarization in type II muscle fibers. In Chapter 5, we describe the effects of heat therapy in a mouse model of ischemia induced-muscle damage. Animals that were exposed to heat therapy at 39°C had improved maximal absolute force and relative muscle mass in the soleus muscle. These observations reveal that the beneficial effects of heat therapy are muscle fiber type specific and dependent on the treatment temperature. In Chapter 6, we review and summarize the outcomes described in Chapters 2-5 and provide a general conclusion as well the clinical implications of our findings. </p> </div> </div> </div>

Angiogenesis is the process of new blood vessel growth from preexisting vessels. This process relies on the activity of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and occurs in both normal and pathological tissues. Angiogenesis is often dysregulated in diseases such as cancer and many efforts have been made to treat such diseases by targeting the VEGFR-2 pathway. VEGFR-2 is activated upon ligand binding and subsequent autophosphorylation of tyrosine residues in the kinase domain, which leads to endothelial cell survival, proliferation, and growth – all of which are required for angiogenesis to occur. It was previously demonstrated that methylation of VEGFR-2 at Lys1041 enhanced its tyrosine autophosphorylation and is required for VEGFR-2 mediated angiogenesis in zebrafish and tumor growth in mouse. However, the Lysine Methyltransferase (KMT) involved in the methylation of VEGFR-2 remains unknown. This study aimed to identify the KMT involved in the methylation of VEGFR-2. We have identified Enhancer of zeste homolog 2 (EZH2) as the KMT likely responsible for catalyzing methylation of K1041 on VEGFR-2. Over-expression of EZH2 was found to increase phosphorylation of Tyr1054, one of the required phosphorylation sites for VEGFR-2 activation, in whole cell lysates and VEGFR-2 purified by immunoprecipitation. The effect of over-expression of EZH2 in the phosphorylation of VEGFR-2 at Tyr1054 was dose-dependent - increasing concentrations of EZH2 resulted in increasing phosphorylation of VEGFR-2 at Tyr1054. Moreover, we determined that EZH2 physically interacts with VEGFR-2 as demonstrated by co-immunoprecipitation in vitro GST-pulldown assays. The C-terminus of EZH2 (amino acids 371-746), physically interacted with VEGFR-2. Taken together, we have identified EZH2 as a candidate KMT involved in the methylation of Lys1041, which increases phosphorylation of VEGFR-2 at Tyr1054. / 2020-07-03T00:00:00Z

In the tumor microenvironment there are many different cell types present and among these, immune cells display a large proportion. Central players in the tumor immunity are macrophages that come in two different phenotypes, the M1 and M2 macrophages. M1 polarized macrophages are tumor suppressive while M2 polarized macrophages support tumor growth. The factors that contribute to the skewing of macrophages from one phenotype to another are under investigation. Interestingly, our lab has identified Immune Semaphorin 3A (Sema3A) as a participating plaer in regulating the accumulation of anti-tumoral M1 macrophages leading to a suppression of tumor growth.   In light of these data this thesis has focused on the role of endogenous Sema3A in the tumor microenvironment. A tumor cell line expressing shRNA against Sema3A mRNA was generated using lentiviral mediated gene therapy. This knockdoen cell line showed 72% lower mRNA expression compared to control and was evaluated in vivo by monitoring tumor progression in female BALB/c mice. The immune cell composition of the tumors was analysed using flow cytometry. The results from the in vivo experiment show that endogenous Sema3A has a limited effect on tumor progression. A slight shift to a more tumor supportive immune profile was observed in the knockdown tumors. Moreover, a virus for transducing cells to overecpress Sema3A under asuitable promoter for systemic delivery was generated. / Många olika sorters celler är närvarande i tumörers mikromiljö och immunceller utgör en stor andel av dessa. Makrofager är centrala spelare o tumörimmunförsvaret och dessa kan indelas i olika aktiveringsgrader eller fenotyper, M1 eller M2 makrofager. M1 polariserade makrofager är tumörsuppressiva medan M2 makrofager bidrar till tumörtillväxt. De faktorer som reglerar skiftningen mellan M1 och M2 fenotyperna är under utredning. Vårt labb har identifierat att Immunsemaforinen 3A (Sema3A) spelar en roll i att reglera ackumuleringen av antitumorala M1 makrofager vilket leder till hämmad tumörtillväxt.   Med denna information som bakgrund har detta examensarbete fokuserat på Sema3As roll i tumörmikromiljön. Med hjälp av lentivirusmedierad genterapi skapades en tumörcellinje som uttrycker shRNA mot Sema3AmRNA. Denna cellinjes visade 72% lägre Sema3A mRNA uttryck jämfört med kontorll och utvärderades sedan in vivo genom att följa tumörtillväxten i BALB/c mushonor. Immuncellsammansättningen i tumörerna analyserades sedan med hjälp av flödescytometri. Resultaten från in vivo experimentet visar att endogent Sema3A har en begränsad effekt på tumörutvecklingen. En något mer tumörgynnande immunprofil observerades i de tumörer där Sema3A uttryck var minskat. Utöver detta skapades också ett lentivirus för att transducera celler så att de överuttrycker Sema3A under en passande promotor för systemisk tillförsel.

Lung tumor progression is regulated by proangiogenic factors. Heat shock protein A12B (HSPA12B) is a recently identified regulator of expression of proangiogenic factors. However, whether HSPA12B plays a role in lung tumor growth is unknown. To address this question, transgenic mice overexpressing HSPA12B (Tg) and wildtype littermates (WT) were implanted with Lewis lung cancer cells to induce lung tumorigenesis. Tg mice showed significantly higher number and bigger size of tumors than WT mice. Tg tumors exhibited increased angiogenesis and proliferation while reduced apoptosis compared with WT tumors. Interestingly, a significantly enhanced upregulation of Cox-2 was detected in Tg tumors than in WT tumors. Also, Tg tumors demonstrated upregulation of VEGF and angiopoietin-1, downregulation of AKAP12, and increased eNOS phosphorylation compared with WT tumors. Celecoxib, a selective Cox-2 inhibitor, suppressed the HSPA12B-induced increase in lung tumor burden. Moreover, celecoxib decreased angiogenesis and proliferation whereas increased apoptosis in Tg tumors. Additionally, celecoxib reduced angiopoietin-1 expression and eNOS phosphorylation but increased AKAP12 levels in Tg tumors. Our results indicate that HSPA12B stimulates lung tumor growth via a Cox-2-dependent mechanism. The present study identified HSPA12B as a novel facilitator of lung tumor growth and a potential therapeutic target for the treatment of lung cancer.

Objective: Activation of PI3K/Akt signaling protects the myocardium from ischemia/reperfusion injury. MicroRNAs have been demonstrated to play an important role in the regulation of gene expression at the post-transcriptional level. In this study, we examined whether miR-130a will attenuate cardiac dysfunction and remodeling after myocardial infarction (MI) via PI3K/Akt dependent mechanism. Approaches and results: To determine the role of miR-130a in the proliferation and migration of endothelial cells, HUVECs were transfected with miR-130a mimics before the cells were subjected to scratch-induced wound injury. Transfection of miR-130a mimics stimulated the migration of endothelial cells into the wound area and increased phospho-Akt levels. To examine the effect of miR-130a on cardiac dysfunction and remodeling after MI, Lentivirus expressing miR-130a (LmiR-130a) was delivered into mouse hearts seven days before the mice were subjected to MI. Cardiac function was assessed by echocardiography before and for up to 21 days after MI. Ejection fraction (EF%) and fractional shortening (FS%) in the LmiR-130a transfected MI hearts were significantly greater than in LmiR-control and untransfected control MI groups. LmiR-130a transfection increased capillary number and VEGF expression, and decreased collagen deposition in the infarcted myocardium. Importantly, LmiR-130a transfection significantly suppressed PTEN expression and increased the levels of phosphorylated Akt in the myocardium. However, treatment of LmiR-130a-transfected mice with LY294002, a PI3K inhibitor, completely abolished miR-130a-induced attenuation of cardiac dysfunction after MI. Conclusions: miR-130a plays a critical role in attenuation of cardiac dysfunction and remodeling after MI. The mechanisms involve activation of PI3K/Akt signaling via suppression of PTEN expression.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and realtime PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

OBJECTIVE - HSPA12B is the newest member of HSP70 family of proteins and is enriched in atherosclerotic lesions. This study focused on HSPA12B expression in mice and its involvement in angiogenesis. METHODS AND RESULTS - The expression of HSPA12B in mice and cultured cells was studied by: (1) Northern blot; (2) in situ hybridization; (3) immunostaining with HSPA12B-specific antibodies; and (4) expressing Enhanced-Green-Fluorescent-Protein under the control of the HSPA12B promoter in mice. The function of HSPA12B was probed by an in vitro angiogenesis assay (Matrigel) and a migration assay. Interacting proteins were identified through a yeast two-hybrid screening. HSPA12B is predominantly expressed in vascular endothelium and induced during angiogenesis. In vitro angiogenesis and migration are inhibited in human umbilical vein endothelial cells in the presence of HSPA12B-neutralizing antibodies. HSPA12B interacts with multiple proteins in yeast 2-hybrid system. CONCLUSIONS - We provide the first evidence to our knowledge that the HSPA12B is predominantly expressed in endothelial cells, required for angiogenesis, and interacts with known angiogenesis regulators. We postulate that HSPA12B provides a new mode of angiogenesis regulation and a novel therapeutic target for angiogenesis-related diseases.

L’évaluation de la vascularisation tumorale par l’échographie de contraste ultrasonore a montré son intérêt pour déterminer l’efficacité des traitements anti-angiogéniques. Malgré tout, cette technique suscite de nombreux questionnements concernant la sensibilité des méthodes de quantification du signal ultrasonore. Pour répondre à cette problématique, il a été question dans cette thèse de développer la première modélisation numérique de l’écoulement du sang et des agents de contraste dans des réseaux vasculaires pour étudier les méthodes de quantification du signal ultrasonore et leurs sensibilités par rapport à des variations de volume du réseau tumoral et des vitesses du sang. Une première étape de la thèse a consisté à valider, par une comparaison expérimentale, les hypothèses faites pour la modélisation numérique et principalement la prise en compte du sang comme un fluide Newtonien homogène. La modélisation numérique a permis de mettre en évidence les paramètres les plus sensibles aux modifications du débit vasculaire tumorale que sont l’aire sous la courbe, le rehaussement maximal et la pente de la courbe de rehaussement du signal dans le cadre de la méthode semi-quantitative. Lorsqu’il s’agit de suivre les variations du volume vasculaire tumoral, il apparait que la méthode quantitative par deconvolution de la fonction artérielle est plus sensible. Les méthodes de quantification ont également été étudiées par le biais d’une étude in vivo sur 44 souris. Cette approche numérique de l’écoulement des agents de contraste est prometteuse et peut permettre à terme une évaluation plus large des autres méthodes de quantification développées à ce jour pour l’échographie de contraste. / Evaluation of tumor vascularization by dynamic contrast-enhanced ultrasonography showed interest for the assessment of the effectiveness of anti-angiogenic treatments. Nevertheless, this technique raises many questions about the sensitivity of quantification methods of the ultrasound signal. To address this issue, this thesis focused on the development of the first digital modeling of blood flow and contrast agents in vascular networks to study the methods of quantification of the ultrasound signal and theirs sensitivity according to variations of tumor network volume and blood velocity. A first step of the thesis was to validate by an experimental comparison, the assumptions of the digital modeling and mainly the taking into account of the blood as a homogeneous Newtonian fluid. Digital modeling allowed to highlight parameters sensitive to the modification of the blood flow which are in the case of the semi-quantitative method the area under the enhancement curve, the maximum of the enhancement curve and the slope of the enhancement curve. When it comes to follow variations of the tumor vascular volume, it appears that the quantitative method by deconvolution of the arterial function is more sensitive. The quantification methods have also been investigated throught an in vivo study of 44 mice. This digital approach of the flow of the contrast agents is promising and may eventually enable a more extensive evaluation of other quantification methods developed in dynamic contrast-enhanced ultrasonography to date.