Magíster en fisiología / La Hipertensión Pulmonar (HP) es una patología vascular compleja que conduce a falla cardíaca, menor expectativa de vida y tiene un impacto significativo en la sociedad, ya que produce la muerte de pacientes en edad productiva e implica altos costos de financiamiento en su tratamiento. Existe evidencia sobre la contribución de venas intrapulmonares a la Resistencia Vascular Pulmonar (RVP). La Hipertensión Venosa Pulmonar (HVP) puede producir un incremento en la RVP y aumentar la Presión Arterial Pulmonar (PAP), debido a transmisión pasiva de la presión venosa al territorio arterial. La familia de receptores purinérgicos se encuentra ampliamente expresada en diversos vasos sanguíneos y se ha descrito respuesta contráctil de arterias intrapulmonares a nucleótidos, en particular a Uridín Trifosfato (UTP). La investigación del rol de la señalización purinérgica en la circulación pulmonar venosa podría aportar información relevante para abordar otra vía involucrada en la fisiopatología de la HAP. Se realizaron rebanadas de pulmón de ratas Sprague Dawley controles y con HAP inducida por Monocrotalina (HAP-MCT), en un vibrátomo a 150 μm de espesor. La respuesta contráctil de Venas Intrapulmonares Pequeñas (VIP) inducida por UTP se registró mediante videomicroscopía de contraste de fases (VMCF), demostrando actividad del nucleótido como agente vasoconstrictor en VIP, con mayor respuesta en la VIP provenientes de ratas con HAP-MCT (EC50= 8,8±1,4 μM) que en las control (EC50= 16 μM) (p<0,05). Las imágenes de inmunofluorescencia muestran expresión de los receptores P2Y2 y P2Y4 en la capa media de VIP de ratas control y con HAP-MCT. Estos resultados sugieren un nuevo mecanismo involucrado en la HAP, orientando el estudio de nuevas terapias para combatir el curso de la enfermedad, mejorar la sobrevida y la calidad de vida de pacientes que padecen HAP. / Pulmonary Hypertension (HP) is a complex vascular condition that leads to heart failure, lower life expectancy and has a significant impact on society, because it involves the death of patients of working age and involves high costs of financing treatment. Pulmonary Venous Hypertension (HVP) may cause an increase in Pulmonary Vascular Resistance (RVP), Pulmonary Artery Pressure (PAP) is increased by the HVP, due to passive transmission of venous pressure to arterial territory. There is also evidence on the contribution of intrapulmonary veins to the RVP. Purinergic receptor family is widely expressed in various blood vessels and described intrapulmonary artery contractile responses to nucleotides, especially Uridine Triphosphate (UTP). The investigation of the role of purinergic signaling in the pulmonary venous circulation could provide relevant information to address another pathway involved in the pathophysiology of Pulmonary Arterial Hypertension (HAP). Lung slices from Sprague Dawley rats controls and rats with Pulmonary Hypertension monocrotaline induced (HAP-MCT), were made from vibratome to 150 μm thick . The contractile response of small intrapulmonary veins (VIP) was recorded by videomicroscopy phase contrast (VMCF), showing nucleotide activity as a vasoconstrictor agent in VIP, with greater response in the VIP from rats HAP-MCT (EC50 = 8.8 ± 1,4 μM) than in control (EC50 = 16 ± 1,1 μM (p<0,05). Immunofluorescence images show expression of P2Y2 and P2Y4 receptors in the middle layer of VIP of control and HAP-MCT rats. These findings suggest a new mechanism involved in PAH, directing the study of new therapies to combat disease course, improve survival and life quality of these patients.

The wound healing response within corneal epithelium cells is controlled by the purinergic P2X7 receptor, purinergic P2Y2 receptor, and Pannexin1 transmembrane proteins. The response is initiated by the release of ATP into the extracellular space by damaged cells and by the Pannexin1 channel protein and limited by the hydrolysis of extracellular ATP by ecto-ATPases. P2X7 and P2Y2 receptors are necessary for the initiation of cell migration, actin reorganization, and cell to cell communication required for the sheet like migration found within the corneal epithelium wound response. ARL 67156 Trisodium Salt Hydrate, an inhibitor for human and murine ecto-ATPases, was used to inhibit the hydrolysis of extracellular ATP during the wound response to prolong and further elevate extracellular ATP concentrations to increase the cellular activity and cell to cell communication of the wound response. The goal was to compare primary diabetic murine and human cells. However, there were issues with culturing these cells and diabetic murine and non-diabetic primary and established human cells were used for these experiments. Significant increases in cellular activity were found in both primary human cells with a 100 ARL to 10 ATP concentration ratio while no significant changes in cellular activity were found with the murine diabetic cells at any ARL concentration. In summary, with increased ATP in the wound response due to the inhibition of hydrolysis enzymes, human cells displayed increased levels and rates of cell-to-cell communication during the delayed wound response. / 2024-01-27T00:00:00Z

No description available.

Vários estudos têm apontado a sinalização e os receptores purinérgicos, representados em mamíferos pelos receptores ionotrópicos (P2X1 P2X7) e metabotrópicos (P2Y1,2,4,6, 11,12,14), como um sistema primitivo, envolvido não somente na sinalização neuronal, mas também em muitos outros processos vitais incluindo resposta imune, inflamação, dor, agregação plaquetária e nos processos de diferenciação, proliferação e morte celular, que ocorrem no desenvolvimento e na regeneração tecidual. Condizente com as descrições da literatura, dados do nosso laboratório, baseados na farmacologia dos receptores purinérgicos, sugeriram o envolvimento do subtipo P2Y2 na proliferação e na neurogênese in vitro de células de carcinoma embrionário P19. Tendo em vista a ausência de agonistas e antagonistas específicos para a maioria dos subtipos de receptores purinérgicos, o que vem dificultando a elucidação das funções exatas desses receptores em processos fisiológicos e patológicos, optamos para o screening de uma biblioteca combinatória de oligonucleotídeos para a identificação de ligantes de alta afinidade e especificidade para o receptor P2Y2 (procedimento de SELEX, Evolução Sistemática de Ligantes por Enriquecimento Exponencial). Essa abordagem envolve passos reiterativos de seleção in vitro de moléculas de RNA, estabilizadas por substituição do grupo 2´OH das pirimidinas por um átomo de flúor, que possuem afinidade pelo receptor, até que a mistura de RNAs, originalmente de 1013 diferentes seqüências que adotam uma gama de estruturas secundárias e terciárias, esteja purificada para uma população homogênea de ligantes de alta afinidade pelo receptor P2Y2. O processo envolve a transcrição in vitro da biblioteca de DNA para RNA, a apresentação desta ao alvo, a eluição dos ligantes específicos, denominados aptâmeros, e a regeneração da biblioteca de DNA por RT-PCR, a qual, após uma reação de transcrição in vitro, gera a mistura de RNAs para o próximo ciclo de seleção. Neste trabalho, nós utilizamos como alvo o receptor P2Y2 recombinante humano expresso na linhagem de células de astroglioma humano 1321N1. Ao final de nove ciclos de SELEX, nós isolamos 46 sequências que foram agrupadas em três classes estruturais, de acordo com a presença de regiões consensos. A mistura destas moléculas se ligou ao receptor P2Y2 humano com uma constante de dissociação de 164 nM. Um dos clones isolados, o aptâmero B7, se ligou preferencialmente ao receptor P2Y2 (Kd 184 nM), em relação aos receptores P2Y1 e P2Y4 recombinantes expressos em células 1321N1. A interação deste aptâmero não foi dependente da espécie, uma vez que ele foi capaz de se ligar tanto ao receptor P2Y2 de origem humana como murina. A atividade biológica do aptâmero foi avaliada em células P19 (sabidamente expressando receptores P2Y2 endógenos), na qual a proteção do ATP contra a apoptose, provavelmente interagindo com o receptor P2Y2, foi anulada na presença deste aptâmero em uma concentração mil vezes menor do que a do ATP. Além de confirmar a viabilidade da técnica SELEX para identificar ligantes subtipos-específicos dos receptores purinérgicos, o aptâmero anti-P2Y2 serve como ferramenta fundamental para definir demais funções fisiológicas deste receptor. Passos de otimização das suas propriedades como ligante e biodisponibilidade tornarão este aptâmero um composto de alta relevância farmacêutica. / Many published studies have focused on purinergic signaling and receptors, represented by ionotropic (P2X1 P2X7) and metabotropic (P2Y1,2,4,6,11,12,14) subtypes as a universal system which is not only involved in neuronal signaling, but also in various other vital processes including immune response, inflammation, platelet aggregation as well as differentiation, proliferation and cell death occurring during development and tissue regeneration. In agreement with other published reports, results of our laboratory based on overlapping purinergic receptor pharmacology suggest the participation of the P2Y2 subtype in proliferation and in vitro neurogenesis of P19 embryonal carcinoma cells. In view of the lack of availability of specific agonists and antagonists for most purinergic receptors making the elucidation of exact functions of these receptors in their cellular context almost impossible, we used a combinatorial oligonucleotide library approach, denominated SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the development of high-affinity and specificity ligands for the P2Y2 receptor. This approach is based on re-iterative steps of in vitro selection of 2´-fluoro-pyrimidine-modified RNA molecules for receptor-binding affinity from an RNA pool containing 1013 different sequences and secondary and tertiary structures until this pool is purified to a homogeneous population of ligands with high affinity to the P2Y2 receptor. The combinatorial DNA library is in vitro transcribed into RNA followed by target presentation of the RNA library and elution of the target-binders, denominated aptamers, and RT-PCR amplification in order to restore the DNA library used for in vitro transcription for next SELEX cycle. Following nine SELEX cycles using 1321N1 human astroglial cells expressing recombinant human P2Y2 receptors as target, we isolated 46 aptamer sequences which, based on consensus sequence motifs, were grouped in three structural groups. The mixture of the isolated sequences bound themselves to human P2Y2 receptors with a dissociation constant (Kd) of 164 nM. One of the isolated clones, the aptamer denominated B7, bound itself to P2Y2 receptors in preference to P2Y1 and P2Y4 recombinant receptors expressed in 1321N1 cells. The binding activity of the aptamer was not limited to P2Y2 receptors of human origin, as the aptamer also interacted with mouse P2Y2 receptors. The capability of the aptamer of affecting the biological activity of P2Y2 receptors was verified in P19 cells in which ATP-induced protection against apoptosis, mediated by P2Y2 receptors, was abolished in the presence of the aptamer. In addition to providing a proof of principle for the feasibility of developing purinergic subtype-specific ligands by using the SELEX technique, the anti-P2Y aptamer provides a fundamental tool for gaining insights of physiological functions of this receptor in various cellular contexts. Moreover, steps of optimization of receptor binding properties and aptamer half-life in vivo will turn this aptamer into a compound of high pharmaceutical relevance.

Vários estudos têm apontado a sinalização e os receptores purinérgicos, representados em mamíferos pelos receptores ionotrópicos (P2X1 P2X7) e metabotrópicos (P2Y1,2,4,6, 11,12,14), como um sistema primitivo, envolvido não somente na sinalização neuronal, mas também em muitos outros processos vitais incluindo resposta imune, inflamação, dor, agregação plaquetária e nos processos de diferenciação, proliferação e morte celular, que ocorrem no desenvolvimento e na regeneração tecidual. Condizente com as descrições da literatura, dados do nosso laboratório, baseados na farmacologia dos receptores purinérgicos, sugeriram o envolvimento do subtipo P2Y2 na proliferação e na neurogênese in vitro de células de carcinoma embrionário P19. Tendo em vista a ausência de agonistas e antagonistas específicos para a maioria dos subtipos de receptores purinérgicos, o que vem dificultando a elucidação das funções exatas desses receptores em processos fisiológicos e patológicos, optamos para o screening de uma biblioteca combinatória de oligonucleotídeos para a identificação de ligantes de alta afinidade e especificidade para o receptor P2Y2 (procedimento de SELEX, Evolução Sistemática de Ligantes por Enriquecimento Exponencial). Essa abordagem envolve passos reiterativos de seleção in vitro de moléculas de RNA, estabilizadas por substituição do grupo 2´OH das pirimidinas por um átomo de flúor, que possuem afinidade pelo receptor, até que a mistura de RNAs, originalmente de 1013 diferentes seqüências que adotam uma gama de estruturas secundárias e terciárias, esteja purificada para uma população homogênea de ligantes de alta afinidade pelo receptor P2Y2. O processo envolve a transcrição in vitro da biblioteca de DNA para RNA, a apresentação desta ao alvo, a eluição dos ligantes específicos, denominados aptâmeros, e a regeneração da biblioteca de DNA por RT-PCR, a qual, após uma reação de transcrição in vitro, gera a mistura de RNAs para o próximo ciclo de seleção. Neste trabalho, nós utilizamos como alvo o receptor P2Y2 recombinante humano expresso na linhagem de células de astroglioma humano 1321N1. Ao final de nove ciclos de SELEX, nós isolamos 46 sequências que foram agrupadas em três classes estruturais, de acordo com a presença de regiões consensos. A mistura destas moléculas se ligou ao receptor P2Y2 humano com uma constante de dissociação de 164 nM. Um dos clones isolados, o aptâmero B7, se ligou preferencialmente ao receptor P2Y2 (Kd 184 nM), em relação aos receptores P2Y1 e P2Y4 recombinantes expressos em células 1321N1. A interação deste aptâmero não foi dependente da espécie, uma vez que ele foi capaz de se ligar tanto ao receptor P2Y2 de origem humana como murina. A atividade biológica do aptâmero foi avaliada em células P19 (sabidamente expressando receptores P2Y2 endógenos), na qual a proteção do ATP contra a apoptose, provavelmente interagindo com o receptor P2Y2, foi anulada na presença deste aptâmero em uma concentração mil vezes menor do que a do ATP. Além de confirmar a viabilidade da técnica SELEX para identificar ligantes subtipos-específicos dos receptores purinérgicos, o aptâmero anti-P2Y2 serve como ferramenta fundamental para definir demais funções fisiológicas deste receptor. Passos de otimização das suas propriedades como ligante e biodisponibilidade tornarão este aptâmero um composto de alta relevância farmacêutica. / Many published studies have focused on purinergic signaling and receptors, represented by ionotropic (P2X1 P2X7) and metabotropic (P2Y1,2,4,6,11,12,14) subtypes as a universal system which is not only involved in neuronal signaling, but also in various other vital processes including immune response, inflammation, platelet aggregation as well as differentiation, proliferation and cell death occurring during development and tissue regeneration. In agreement with other published reports, results of our laboratory based on overlapping purinergic receptor pharmacology suggest the participation of the P2Y2 subtype in proliferation and in vitro neurogenesis of P19 embryonal carcinoma cells. In view of the lack of availability of specific agonists and antagonists for most purinergic receptors making the elucidation of exact functions of these receptors in their cellular context almost impossible, we used a combinatorial oligonucleotide library approach, denominated SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the development of high-affinity and specificity ligands for the P2Y2 receptor. This approach is based on re-iterative steps of in vitro selection of 2´-fluoro-pyrimidine-modified RNA molecules for receptor-binding affinity from an RNA pool containing 1013 different sequences and secondary and tertiary structures until this pool is purified to a homogeneous population of ligands with high affinity to the P2Y2 receptor. The combinatorial DNA library is in vitro transcribed into RNA followed by target presentation of the RNA library and elution of the target-binders, denominated aptamers, and RT-PCR amplification in order to restore the DNA library used for in vitro transcription for next SELEX cycle. Following nine SELEX cycles using 1321N1 human astroglial cells expressing recombinant human P2Y2 receptors as target, we isolated 46 aptamer sequences which, based on consensus sequence motifs, were grouped in three structural groups. The mixture of the isolated sequences bound themselves to human P2Y2 receptors with a dissociation constant (Kd) of 164 nM. One of the isolated clones, the aptamer denominated B7, bound itself to P2Y2 receptors in preference to P2Y1 and P2Y4 recombinant receptors expressed in 1321N1 cells. The binding activity of the aptamer was not limited to P2Y2 receptors of human origin, as the aptamer also interacted with mouse P2Y2 receptors. The capability of the aptamer of affecting the biological activity of P2Y2 receptors was verified in P19 cells in which ATP-induced protection against apoptosis, mediated by P2Y2 receptors, was abolished in the presence of the aptamer. In addition to providing a proof of principle for the feasibility of developing purinergic subtype-specific ligands by using the SELEX technique, the anti-P2Y aptamer provides a fundamental tool for gaining insights of physiological functions of this receptor in various cellular contexts. Moreover, steps of optimization of receptor binding properties and aptamer half-life in vivo will turn this aptamer into a compound of high pharmaceutical relevance.

Amongst respiratory diseases, inflammatory lung diseases constitute a major part of public <p>health problem. As a consequence, investigating the immune mechanisms that contribute to <p>the pathogenesis of these diseases is essential to identify candidate targets for the <p>development of new therapeutic drugs. Furthermore, over the past 20 years, the growing awareness <p>that purinergic signalling events shape the immune and inflammatory responses to infection and <p>allergic reactions warranted the development of animal models to assess their importance in vivo in <p>acute lung injury and chronic airway diseases. The field of purinergic inflammation formulated the <p>unifying concept that ATP is released as a «danger signal» to induce inflammatory responses upon <p>binding purinergic receptors.<p>According to these elements, we began in 2007 to evaluate lung inflammation in mice deficient for <p>the P2Y2 purinergic receptor in TH2 and TH1 models. The most convincing evidence that the P2Y2<p>receptor is engaged during alarm situations comes from studies related to cystic fibrosis and asthma. <p>Indeed, chronic respiratory diseases are commonly associated with elevated airway ATP <p>concentrations, as reported in cystic fibrosis, but also in idiopathic pulmonary fibrosis and chronic <p>obstructive pulmonary disease (COPD) patients, and they are raised by allergens in asthmatic <p>patients.<p>First, we demonstrated a significant role of the P2Y2R in a TH2-ovalbumin(OVA)-induced asthma <p>model. We observed that eosinophil accumulation, a distinctive feature of lung allergic inflammation, <p>was defective in OVA-treated P2Y2-deficient mice compared with OVA-treated wild type animals. <p>Interestingly, the upregulation of VCAM-1 was lower on lung endothelial cells of OVA-treated P2Y2 <p>knockout mice compared with OVA-treated wild type animals. Adhesion assays demonstrated that <p>the action of UTP on leukocyte adhesion through the regulation of endothelial VCAM-1 was <p>abolished in P2Y2-deficient lung endothelial cells. Additionally, the level of soluble VCAM-1, reported <p>as an inducer of eosinophil chemotaxis, was strongly reduced in the bronchoalveolar lavage fluid of <p>P2Y2-deficient mice.<p>Secondly, we studied the consequences of P2Y2R loss in lung inflammation initiated after pneumonia <p>virus of mice (PVM) infection in collaboration with the group of Pr. Daniel Desmecht (ULg). We <p>demonstrated here that P2Y2<p>-/-<p>mice display a severe increase in morbidity and mortality rate in <p>response to PVM. Lower survival of P2Y2<p>-/-<p>mice was not correlated with excessive inflammation <p>despite the higher level of neutrophil recruiters in their broncho-alveolar fluids. Interestingly, we <p>observed lower numbers of dendritic cells, CD4<p>+<p>T cells and CD8<p>+<p>T cells in P2Y2<p>-/-<p>mice compared to <p>P2Y2<p>+/+<p>infected lungs. Lower level of IL-12 and higher level of IL-6 in broncho-alveolar fluid support <p>an inhibition of Th1 response in P2Y2<p>-/-<p>mice. Quantification of DC recruiter expression revealed <p>comparable IP-10 and MIP-3& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The research of new therapeutic strategies for cardiovascular diseases has seen in the last 15 years the introduction of a new participant: pericardial adipose tissue (PAT). This tissue is able to modulate cardiac function and its volume was sometimes linked to risk of cardiovascular diseases. Moreover, adipose-derived stem cells (ASCs) isolated from PAT are considered as the best suitable for new regenerative strategies aiming at healing ischemic myocardium. Although the interests in understanding the functions and the formation of pericardial adipose tissue are high, the current knowledge on this tissue is still scarce. In this work, the starting point was the consideration that nucleotide receptors are established regulators of many biological functions, including the differentiation of adult mesenchymal stem cells, immunity and inflammatory process. The P2Y4 receptor has been recently recognized as a negative regulator of cardiac fat formation and ASCs differentiation. The purpose of this thesis was to analyze the involvement of the nucleotide receptor P2Y2 in the formation of pericardial adipose tissue (PAT) and its ASCs differentiation. We also investigated the possible contribution of this receptor to the functions of recently discovered fat-associated lymphoid clusters (FALCs). Our study analyzed the PAT of mice deficient for P2Y2 at basal conditions and in a model of myocardial infarction. P2Y2-null mice showed a lower mass of PAT compared to WT, which was correlated with decreased adipogenic differentiation and maturation potential of pericardial ASCs in vitro. PAT of basal P2Y2-deficient mice displayed a reduced density of FALCs due to a reduced number of B cells. RNA-sequencing experiments identified many P2Y2 target genes in PAT linked to immunomodulation. We identified a polarization of FALCs macrophages towards anti-inflammatory M2c subtype in P2Y2-null mice. We correlated it with a decreased number of follicular helper T cells, known to contribute to B cell expansion in germinal centers. These data could be correlated with increased apoptosis of B lymphocytes. The data obtained using the mouse infarct model confirmed an expected enlargement of pericardial FALCs in ischemic conditions. P2Y2-null mice were characterized by a reduced expansion of B cells and myeloid cells migration in PAT. These results suggested a participation of P2Y2 receptor in regulating the post-MI inflammatory response by modulating the leukocytes populations in the pericardial adipose tissue’s lymphoid clusters. The effect of P2Y2 on PAT post-ischemic inflammatory state could contribute to the P2Y2-mediated cardioprotective effect of UTP described in previous literature. Our study defines P2Y2 nucleotide receptor as a regulator of the formation of pericardial fat and its inflammatory status in ischemic conditions. P2Y2 receptor could represent an interesting therapeutic target for the regulation of PAT functions before and after MI. In general, a better comprehension of PAT and its consideration in the post-ischemic regeneration process could lead to the development of new therapeutic strategies for treating cardiovascular diseases and the adjustment of existing therapies. / Durant les 15 dernières années, un nouvel arrivant a fait son apparition dans la recherche de nouvelles approches thérapeutiques dans le domaine cardiovasculaire: le tissu adipeux cardiaque. Ce tissu est capable de moduler les fonctions cardiaques et son volume a pu être associé parfois à un risque de maladie cardiovasculaire. De plus, les cellules souches dérivées du tissu adipeux (ASCs) cardiaque sont considérées comme les mieux appropriées pour des stratégies thérapeutiques visant la réparation du myocarde ischémié. Bien que la compréhension de la fonction et de la formation du tissu adipeux cardiaque présente un intérêt majeur, la connaissance actuelle de ce tissu particulier est encore assez limitée. Pour le présent travail, le point de départ a été l’observation que les récepteurs nucléotidiques sont des régulateurs établis de nombreuses fonctions biologiques, incluant la différentiation des cellules souches mésenchymateuses et plus généralement la régulation de la réponse immune et inflammatoire. Le récepteur P2Y4 a été récemment reconnu comme un régulateur négatif de la formation du tissu adipeux cardiaque et de la différentiation des ASCs. Le but de cette thèse a été l’étude de l’implication du récepteur nucléotidiques P2Y2 dans la formation du tissu adipeux péricardique (TAP) et la différentiation des ASCs. Nous avons également investigué la contribution possible de ce récepteur dans la fonction des structures leucocytaires associées au tissu adipeux appelées FALCS pour fat-associated lymphoid clusters.Nous avons étudié le TAP de souris déficientes pour le récepteur P2Y2 à l’état de base et dans un modèle d’infarctus du myocarde. Les souris P2Y2 knock-out (KO) présentent une masse réduite du TAP corrélée avec le fait que l’absence du P2Y2 diminue la différentiation adipogénique et le potentiel de maturation des ASCs péricardiques in vitro. Le PAT des souris P2Y2 KO présentent une diminution de la densité de FALCs à l’état de base, principalement due à un nombre réduit de lymphocytes B, potentiellement corrélé à une apoptose accrue observée dans ces cellules. Nos expériences de RNA-sequencing ont identifié de nombreux gènes cibles du P2Y2 dans le PAT impliqués dans l’immunomodulation. Nous avons identifié une polarisation des macrophages de type M2c dans les FALCs de souris P2Y2 KO. Nous l’avons corrélée avec une diminution des lymphocytes T helper folliculaires connus pour contribuer à l’expansion des lymphocytes B dans les centres germinaux. Les données obtenues dans le modèle d’infarctus chez la souris ont confirmé une augmentation des FALCs péricardiques dans les conditions d’ischémie cardiaque. Les souris P2Y2 KO sont caractérisées par une expansion réduite des lymphocytes B et des cellules myéloïdes dans le TAP. Ces résultats suggèrent une participation du récepteur P2Y2 dans la régulation de la réponse inflammatoire post-infarctus par la modulation des populations leucocytaires dans les clusters lymphocytaires du tissu adipeux cardiaque. L’effet du P2Y2 sur l’état inflammatoire post-ischémique pourrait contribuer à l’effet cardioprotecteur de l’UTP médié par le P2Y2 et précédemment décrit dans la littérature.Notre étude définit le récepteur nucléotidique P2Y2 comme un régulateur de la formation du tissu adipeux péricardique et de son niveau inflammatoire dans des conditions ischémiques. Le récepteur P2Y2 pourrait représenter une cible thérapeutique intéressante pour la régulation des fonctions du PAT avant et après infarctus du myocarde. Plus généralement, une meilleure compréhension du tissu adipeux cardiaque et de son implication dans le processus de régénération cardiaque pourrait mener au développement de nouvelles stratégies thérapeutiques dans le traitement de maladies cardiovasculaires et à l’ajustement de thérapies déjà existantes. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Für die Ätiopathogenese der Atherosklerose spielen eine Vielzahl von Mediatoren eine Rolle. Dabei werden durch das Endothel sowohl protektive als auch schädliche Mediatoren sezerniert. High Density Lipoproteine (HDL) stellen einen bedeutenden protektiven Marker für das kardiovaskuläre Risiko dar, u.a. durch die Aktivierung der endothelialen NO-Synthase (eNOS). HDL besteht zu 50 % aus Proteinen und zu 50 % aus Lipiden. Welche Komponenten des HDL für die eNOS Aktivierung verantwortlich sind, ist nicht bekannt gewesen. Im ersten Abschnitt dieser Promotionsarbeit konnte erfolgreich gezeigt werden, dass die Lysophospholipide, Sphingosin-1-Phosphat (S1P) und Sphinsosylphosphorylcholin (SPC), die strukturelle Bestandteile der Lipidfraktion von HDL darstellen, für einen Teil der HDL induzierten eNOS Aktivierung durch Stimulation des S1P3-Rezeptors verantwortlich sind. Diese eNOS Aktivierung wird durch den intrazellulären Einstrom von Calcium und durch die Aktivierung der Akt-Kinase induziert. Im zweiten Abschnitt dieser Promotionsarbeit konnte nachgewiesen werden, dass das oral verfügbare Lysophospholipid-basierte Medikament, FTY720, das ein strukturelles Analogon des S1P ist, den HDL induzierten Signaltransduktionsweg der eNOS Aktivierung in gleicher Weise induziert. Im dritten Abschnitt dieser Promotionsarbeit konnte ein neuartiges endothelabhängig sezerniertes gemischtes Dinukleosidpolyphosphat, Uridin-Adenosin-Tetraphosphat (Up4A), identifiziert werden. Up4A ist ein Agonist an den P2X- und P2Y-Purinrezeptoren. Up4A induziert bei Applikation in eine isoliert perfundierte Rattenniere hauptsächlich über die Aktivierung des P2X1-Rezeptors und des P2Y2/P2Y4-Rezeptors eine starke Vasokonstriktion im renalen Perfusionsgebiet mit einhergehender Erhöhung des mittleren renalen Perfusionsdrucks. Die direkte Infusion von Up4A in vivo in eine WKY-Ratte führt zu einer signifikanten Erhöhung des mittleren arteriellen Blutdrucks. / In the pathogenesis of atherosclerosis many mediators are included. Therefore the endothelium plays a crucial part by secreting protective but also deleterious factors. High density lipoproteins are an established protective factor in the risk profile of cardiovascular events especially by activating the endothelial NO synthase (eNOS). HDL is composed of 50 % proteins and 50 % lipids. Which component of HDL is responsible for the eNOS activation was not known. In the first part of this dissertation it could be shown, that the lysophospholipids, sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), which are structural compounds of the lipid fraction of HDL, are responsible for a significant part of the HDL induced eNOS activation by stimulating the specific S1P3 receptor. In the signal transduction mechanism the activation of Akt kinase and an influx of calcium is involved. In the second part of this dissertation it could be shown, that the orally active lysophospholipide based drug FTY720, which is a structural analogue of S1P, is able to induce the same signal transduction mechanism activated by HDL including the stimulation of the S1P3 receptor. In the last part of this dissertation a new endothelium dependent vasoconstrictor, the dinucleoside polyphosphate uridine-adenosine-tetraphosphate (Up4A), could be for the first time identified. Up4A is a potent agonist of the P2X- and P2Y-purinoceptors. Via activating the P2X1 receptor and the P2Y2/P2Y4 receptor Up4A induce a strong vasoconstriction in the renal perfusion system in the model of the isolated perfused rat kidney with an adjacent increase of the mean perfusion pressure. By injection of Up4A in vivo in a Wistar Kyoto rat the mean arterial pressure also increase significantly.