Músculos esqueléticos de ratos idosos apresentam uma reduzida capacidade de se regenerar após lesão. No presente estudo, nós lançamos a hipótese de que a estimulação farmacológica de adrenoceptores b2 em músculos de ratos idosos lesados poderia melhorar a regeneração destes. Ratos jovens e idosos foram tratados com injeção subcutânea do agonista b2-adrenérgico formoterol (2 mg/kg/dia) durante 10 e 21 dias após lesão do músculo sóleo. Os músculos de ratos idosos lesados e tratados com formoterol por 10 e 21 dias apresentaram menor processo inflamatório e fibras musculares em regeneração com maior calibre quando comparados aos músculos apenas lesados. O tratamento com formoterol preveniu a queda da força tetânica e aumentou a síntese de proteínas e a fosforilação de mTOR em músculos de ratos idosos lesados e avaliados após 10 dias. Nossos resultados sugerem que o formoterol melhora a capacidade regenerativa estrutural e funcional dos músculos esqueléticos de ratos idosos, e que esse efeito é mediado pelo aumento da síntese protéica através da ativação de mTOR. / Skeletal muscles from old rats fail to completely regenerate following injury. In the present work, we hypothesized that pharmacological stimulation of b2-adrenoceptors in aged muscles following injury could improve their regenerative capacity. Young and aged rats were treated with a subcutaneous injection of b2-adrenergic agonist formoterol (2 mg/kg/day) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days post-injury showed reduced inflammation and regenerating myofibers of greater caliber when compared to their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mTOR phosphorylation in old muscles at 10 days post-injury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mTOR activation.

A glândula pineal sintetiza o hormônio melatonina no período escuro. No rato, a ativação dos receptores a e b-adrenérgicos aumenta os níveis de AMPc, levando à síntese e ativação da enzima arilalquilamina-N-acetiltransferase (AANAT). A glândula recebe também inervação parassimpática, sendo inibitório o efeito da acetilcolina. A metilecgonidina (AEME) é o produto da pirólise da cocaína, quando esta é usada sob a forma de \"crack\". Neste trabalho estudamos os efeitos e os mecanismos de ação da AEME sobre a síntese da melatonina. Foram investigados a atividade da AANAT, o Ca2+, o AMPc e a viabilidade celular e a fragmentação do DNA. A AEME reduziu a síntese da melatonina in vivo e in vitro, sendo este efeito revertido pela atropina. A AEME induziu um aumento do Ca2+, não alterando o AMPc e a atividade da AANAT. A viabilidade celular e fragmentação do DNA não foram modificadas pela AEME. Em conclusão, a AEME reduz a síntese da melatonina, in vitro e in vivo, e a sua ação se dá por interferir com o sistema colinérgico muscarínico. / The pineal gland synthesizes the hormone melatonin in the dark. In rats, the activation of a and b-adrenergic receptors increases cAMP levels and the synthesis and activity of arylalkylamine-N-acetyltransferase enzyme (AANAT). The pineal gland is also innervated by parasympathetic fibers, being inhibitory the effect of acetylcholine. Methylecgonidine (AEME) is the pyrolysis product of cocaine when it is used as \"crack.\" In this work we studied the effects of AEME on the melatonin synthesis, in vitro and in vivo. We investigated AANAT activity, iCa2+, cAMP, cell viability and DNA fragmentation. AEME reduced melatonin synthesis in vivo and in vitro, and this effect was reversed by atropine. There was an increase in Ca2+, but not in cAMP or AANAT activity induced by AEME. Cell viability and DNA fragmentation were not affected by AEME. In conclusion, AEME reduced melatonin synthesis in vitro and in vivo, being this effect mediated by the muscarinic cholinergic system.

O núcleo retrotrapezóide (RTN) é uma região bulbar envolvida na respiração. Estudos prévios mostraram a presença de varicosidades catecolaminérgicas na região do RTN. O objetivo deste estudo foi investigar a fonte de catecolaminas e os efeitos promovidos pela ativação dos receptores adrenérgicos no RTN. Uma densa projeção neuronal do grupamento A7 para o RTN foi revelada usando o traçador retrógrado Fluorogold. Foi registrada a atividade eletromiográfica do diafragma (DiaEMG) e do abdominal (AbdEMG) de ratos Wistar anestesiados. A injeção de noradrenalina promoveu uma inibição da DiaEMG, sem alterar a AbdEMG; este efeito foi atenuado pela injeção prévia de ioimbina e não foi afetado pela injeção de prazosina e propranolol no RTN. A injeção de fenilefrina no RTN aumentou a DiaEMG e gerou AbdEMG; estes efeitos foram bloqueados por injeções prévias de prazosina no RTN. Os resultados deste estudo suportam a ideia de que o RTN recebe projeções adrenérgicas da ponte que modula a atividade dos neurônios do RTN por meio da ativação dos receptores adrenérgicos α -1 e α- 2. / The retrotrapezoid nucleus (RTN) is a medulla region involved in breathing. Previous studies showed the presence of catecholaminergic varicosities in the RTN region. The aim of this study was to investigate the source of cathecolamines and the effects produced by the activation of adrenergic receptors in the RTN. A dense neuronal projection from A7 to RTN was revealed using retrograde tracer FluorGold. In anaesthetized male Wistar rats, diaphragm (DiaEMG) and abdominal (AbdEMG) muscle activities were recorded. Injection of noradrenaline produced an inhibition of DiaEMG, but did not change AbdEMG; These effects was attenuated by pre-injection of yohimbine and were not affect by injection of prazosin and propranolol into the RTN. Injection of phenilephrine into the RTN increased DiaEMG and was also able to generate AbdEMG; these responses were eliminated by pre-injections of into the RTN. These results support the idea that RTN has pontine adrenergic inputs that modulate RTN neurons activity through activation of &#945 - 1 and - &#945 -2 adrenergic receptors.

Os hormônios tireoidianos (HT) executam um papel lipolítico importante no Tecido Adiposo Branco (TAB), sendo este efeito mediado por meio do aumento da expressão de receptores beta adrenérgicos na membrana do adipócito, o que aumenta a sensibilidade deste tecido as catecolaminas. Sabe-se que os principais efetores da ação lipolítica nesse tecido são a lipase hormônio sensível (LHS) e a lipase dos triglicerídeos dos adipócitos (ATGL), as quais hidrolisam os triglicerídeos em ácidos graxos e glicerol. Além disso, outros componentes estão envolvidos na atividade lipolítica, como as perilipinas, proteínas estas que envolvem a gota de gordura, formando uma barreira contra a ação da LHS e ATGL, de modo que precisam ser fosforiladas para que a LHS e ATGL possam exercer seu efeito lipolítico. Considerando: (a) a importância do tecido adiposo na homeostase energética e como fonte de citocinas, as quais estão relacionadas com a sensibilidade tecidual à insulina; (b) que a função e o metabolismo do tecido adiposo variam com a sua distribuição regional, e (c) que as ações lipolíticas dos HT, importantes reguladores da homeostase energética, têm sido muito pouco exploradas, pretendemos investigar, em ratos, (i) se os HT interferem na expressão da LHS, ATGL, perilipina A e dos receptores beta3 adrenérgicos no tecido adiposo branco, e (ii) se essas ações diferem nos distintos depósitos de gordura, o que poderia ampliar o campo de conhecimento sobre os efeitos lipolíticos destes hormônios e a nossa compreensão sobre a contribuição deles nas complicações associadas à obesidade e suas co-morbidades. / Thyroid hormones (TH) play an important lipolytic role in white adipose tissue (WAT). This effect is mediated by increased expression of beta-adrenergic receptors on adipocytes membrane, which increases the sensitivity of that tissue to catecholamines. It is known that the main effectors of the lipolytic action in WAT enzymatic activity, especially: hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which hydrolyze triglycerides into fatty acids and glycerol. In addition, other components are involved in the lipolytic activity, such as perilipin. These proteins support the fat droplet, forming a protective barrier against HSL and ATGL action. Considering: (a) the importance of adipose tissue in energy homeostasis and as a source of cytokines which are related to insulin tissue sensitivity; (b) function and metabolism of adipose tissue vary with their regional distribution; and (c) lipolytic actions of HT, important regulators of energy homeostasis, have been little explored, we investigated in rats with hypothyroidism and submitted to T3 treatment: (i) TH effects on the expression of hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL),perilipin A and beta-3 adrenergic receptors in WAT, and (ii) if this action are different on subcutaneous and visceral fat depot. This study has increased our understanding about the contribution of these hormones on WAT metabolism and metabolic disease as obesity.

La voie β-adrénergique (β-AR)/AMPc est cruciale pour l’adaptation de la fonction cardiaque. Dans l’insuffisance cardiaque (IC), cette signalisation est perturbée et une part importante des patients meurt de troubles du rythme. Classiquement, les effets inotrope et lusitrope positifs de l’AMPc sont attribués à la phosphorylation par la protéine kinase AMPc dépendante (PKA) des protéines clés du couplage excitation–contraction (CEC). L’AMPc active aussi le facteur d’échange Epac, impliqué dans l’hypertrophie cardiaque et le contrôle de l’homéostasie calcique. Une cible d’Epac est la CaMKII, une kinase modulée par le Ca2+ et la calmoduline qui phosphoryle aussi les protéines clés du CEC, et dont l’activation est pro-arythmique.Les phosphodiestérases (PDEs) de type 3 et 4 sont majeures pour dégrader l’AMPc et contrôler l’homéostasie calcique et le CEC. Les inhibiteurs de PDE3 sont de puissants cardiotoniques mais leur utilisation est limitée par leurs effets pro-arythmiques. De plus, l’invalidation de gènes codant pour PDE4 conduit à des arythmies ventriculaires. Mon travail a permis d’identifier les perturbations de l’homéostasie calcique responsables de la survenue d’arythmies lorsque l’activité des PDE3 et des PDE4 est diminuée. Mes résultats montrent que les inhibiteurs de PDEs exercent des effets inotropes via PKA, mais suscitent des vagues de Ca2+ pro-arythmiques impliquant la PKA et la CaMKII activée en partie via Epac. Ceci suggère l'utilisation potentielle d'inhibiteurs de CaMKII comme compléments aux inhibiteurs de PDEs pour limiter leurs effets délétères, une hypothèse que j’ai pu vérifier dans un modèle porcin plus proche du patient. / The β-adrenergic pathway (β-AR)/cAMP is crucial for the adaptation of the cardiac function upon stress. In heart failure (HF), this signaling pathway is disrupted and a significant proportion of patients dies of cardiac arrhythmias. Classically, the inotropic and lusitropic effects of cAMP are attributed to the phosphorylation by the cAMP-dependent protein kinase (PKA) of the key proteins of the excitation-contraction coupling (ECC). cAMP also activates the exchange factor Epac, which is involved in cardiac hypertrophy and controls intracellular Ca2+ homeostasis. Epac activates CaMKII, another kinase modulated by Ca2+ and calmodulin which phosphorylates the same key proteins of the ECC, and is involved in arrhythmogenesis.Phosphodiesterases (PDEs) type 3 and 4 are crucial enzyme to degrade cAMP and to control Ca2+ homeostasis, thus ECC. PDE3 inhibitors are potent cardiotonic drugs but their use is limited by their pro-arrhythmic effects. Furthermore, the invalidation of genes encoding PDE4 results in ventricular arrhythmias. My work allowed characterizing the perturbations of Ca2+ homeostasis which lead to arrhythmias when PDE3 and PDE4 activities are decreased. My results show that PDE inhibitors exert inotropic effects via PKA, but evoke pro-arrhythmic Ca2+ waves via both PKA and CaMKII, the latter being activated in part via Epac. Altogether, these results suggest the potential use of CaMKII inhibitors as adjuncts to PDEs inhibitors to limit their deleterious effects, a hypothesis I also tested in a porcine model closer to the patient.

O núcleo retrotrapezóide (RTN) é uma região bulbar envolvida na respiração. Estudos prévios mostraram a presença de varicosidades catecolaminérgicas na região do RTN. O objetivo deste estudo foi investigar a fonte de catecolaminas e os efeitos promovidos pela ativação dos receptores adrenérgicos no RTN. Uma densa projeção neuronal do grupamento A7 para o RTN foi revelada usando o traçador retrógrado Fluorogold. Foi registrada a atividade eletromiográfica do diafragma (DiaEMG) e do abdominal (AbdEMG) de ratos Wistar anestesiados. A injeção de noradrenalina promoveu uma inibição da DiaEMG, sem alterar a AbdEMG; este efeito foi atenuado pela injeção prévia de ioimbina e não foi afetado pela injeção de prazosina e propranolol no RTN. A injeção de fenilefrina no RTN aumentou a DiaEMG e gerou AbdEMG; estes efeitos foram bloqueados por injeções prévias de prazosina no RTN. Os resultados deste estudo suportam a ideia de que o RTN recebe projeções adrenérgicas da ponte que modula a atividade dos neurônios do RTN por meio da ativação dos receptores adrenérgicos α -1 e α- 2. / The retrotrapezoid nucleus (RTN) is a medulla region involved in breathing. Previous studies showed the presence of catecholaminergic varicosities in the RTN region. The aim of this study was to investigate the source of cathecolamines and the effects produced by the activation of adrenergic receptors in the RTN. A dense neuronal projection from A7 to RTN was revealed using retrograde tracer FluorGold. In anaesthetized male Wistar rats, diaphragm (DiaEMG) and abdominal (AbdEMG) muscle activities were recorded. Injection of noradrenaline produced an inhibition of DiaEMG, but did not change AbdEMG; These effects was attenuated by pre-injection of yohimbine and were not affect by injection of prazosin and propranolol into the RTN. Injection of phenilephrine into the RTN increased DiaEMG and was also able to generate AbdEMG; these responses were eliminated by pre-injections of into the RTN. These results support the idea that RTN has pontine adrenergic inputs that modulate RTN neurons activity through activation of &#945 - 1 and - &#945 -2 adrenergic receptors.

Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon. Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used. It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper. The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles. In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis. In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit.

Katecholamine beeinflussen durch direkte Stimulation über adrenerge Rezeptoren die Funktion von Immunzellen. Ziel der Untersuchungen an Patienten mit Rheumatoider Arthritis war es, das Expressionsprofil unterschiedlicher adrenerger Rezeptorsubtypen in CD4(+)T-Lymphozyten dieser Patienten zu bestimmen. Zur Quantifizierung der Expression wurden semiquantitative RT-PCR-Analysen durchgeführt. Die Untersuchung zeigte, dass alpha1-adrenerge Rezeptoren in CD4(+)-T-Lymphozyten von RA-Patienten exprimiert werden. Es scheint eine Korrelation zwischen bestimmten extraartikulären Organmanifestationen (z.B. Sicca-Sydrom und Tenosynovitis) und der Expression alpha1-adrenerger Rezeptoren zu bestehen. Die gefundene differenzielle Expression der Rezeptoren in CD4(+)-T-Lymphozyten von RA-Patienten legen vertiefende Untersuchungen zur Relevanz des adrenergen Systems bei der Lymphozytenfunktionsmodulation nahe.

Ο Λείος Μυϊκός Φαινότυπος (ΛΜΦ) χαρακτηρίζεται από σημαντική πλαστικότητα και παίζει κομβικό λειτουργικό ρόλο τόσο σε φυσιολογικές όσο και σε παθολογικές καταστάσεις. Στις αρτηρίες, η μεταβολή του ΛΜΦ στο φάσμα από φυσιολογικός-συσταλτός μέχρι συνθετικός-παθολογικός εμπλέκεται αιτιολογικά σε διάφορες ανθρώπινες ασθένειες όπως στην αθηροσκλήρωση, στην υπέρταση και στην επαναστένωση των αγγείων μετά από εγχείρηση. Η έκφραση γονιδίων ΛΜΦ έιναι επίσης μεγάλης σημασίας σε ασθένειες άλλων οργάνων, όπως η ίνωση των νεφρών και του ήπατος και η μετάσταση του καρκίνου. Συνεπώς, η μελέτη των μοριακών μηχανισμών μέσω των οποίων επηρεάζεται ο ΛΜΦ είναι πολύ σημαντική για την ανάπτυξη νέων τεχνικών περιορισμού της εξέλιξης αυτών των νόσων. Η λειτουργία πολλών ιστών όπως είναι τα αγγεία ελέγχεται σε σημαντικό βαθμό από το συμπαθητικό νευρικό σύστημα. Σκοπός λοιπόν της παρούσας εργασίας ήταν πρώτα-πρώτα να εξετάσουμε εάν in vitro η διέγερση των α και β αδρενεργικών υποδοχέων πιθανώς ρυθμίζει την έκφραση γονιδίων ΛΜΦ και προκαταρκτικά να διακριβώσουμε μέσω ποιών μοριακών μηχανισμών οι α1 (υπότυποι α1Α και α1Β) και οι β-ΑΥς επηρεάζουν και ελέγχουν το ΛΜΦ. Κατά δεύτερο λόγο θελήσαμε να εξετάσουμε εάν η εξωγενής έκφραση του μεταγραφικού παράγοντα Μυοκαρδίνη προκαλεί επιθηλιο-μεσεγχυματική μετάβαση (ΕΜΤ-Epithelial to Mesenchymal Transition) σε ενδοθηλιακά κύτταρα in vitro. Για τα πρώτα πειράματά μας χρησιμοποιήσαμε δύο διαφορετικούς κυτταρικούς πληθυσμούς: α) διαφοροποιημένα λεία μυϊκά κύτταρα αορτής αρουραίου της κυτταρικής σειράς A7r5, και β) κύτταρα ινοβλαστών της κυτταρικής σειράς ΝΙΗ3Τ3 προερχόμενα απο ποντικό, προκειμένου να δούμε αντίστοιχα πως η δράση των ΑΥ ελέγχει την έκφραση των γονιδίων αυτών σε διαφοροποιημένα ΛΜΚ (A7r5) και σε αδιαφοροποίητα κύτταρα που όμως μπορούν να μετατραπούν σε «ΛΜΚ» (ΝΙΗ3Τ3). Ως δείκτη για την έκφραση του ΛΜΦ, εξετάσαμε την έκφραση κυτταροσκελετικών, δομικών πρωτεϊνών-δεικτών, όπως η λείου μυϊκού τύπου α-Ακτίνη (SM-α-Αctin),, η λείου μυϊκού τύπου βαριά αλυσίδα της Μυοσίνης (SM-MHC), η λείου μυϊκού τύπου Καλπονίνη (SM-Calponin) και η λείου μυϊκού τύπου πρωτεΐνη 22α (τρανσγελίνη) (SM22α), σε δύο επίπεδα: α) είτε χρησιμοποιώντας αντισώματα, είτε β) με την χρήση πλασμιδίων αναφοράς όπου minimal υποκινητές των παραπάνω γονιδίων ελέγχουν την προσμετρούμενη έκφραση λουσιφεράσης. Η ενεργοποίηση της μεταγραφής αυτών των γονιδίων ρυθμίζεται, σε μεγάλο μέρος, από τις αλληλουχίες CArG (CArG boxes) στους υποκινητές τους, στις οποίες προσδένεται ο παράγοντας Serum Response Factor (SRF). Στα ΛΜΚ, ο μεταγραφικός παράγοντας SRF ενεργοποιεί τη μεταγραφή των γονιδίων-δεικτών μέσω δημιουργίας ενός απαραίτητου συμπλόκου με έναν μεταγραφικό παράγοντα της οικογένειας των Μυοκαρδινών, η οποία αποτελείται από 3 μέλη : τη Μυοκαρδίνη και τις συγγενείς πρωτεΐνες MRTF-A και MRTF-B. Στη μελέτη που πραγματοποιήσαμε, αρχικά παρατηρήσαμε ότι οι δύο αυτοί κυτταρικοί πληθυσμοί δεν εκφράζουν τους α1-ΑΥς (α1Α και α1Β υπότυποι) ενδογενώς, αλλά, με εισαγωγή του αντίστοιχου πλασμιδίου των α1Α ή α1Β ΑΥς, το σύστημα καθίσταται λειτουργικό. Επιπλέον ανακαλύψαμε ότι τα κύτταρα A7r5 εκφράζουν ενδογενώς τους β-ΑΥς. Από τα πειράματα που πραγματοποιήσαμε, καταλήξαμε στο συμπέρασμα ότι οι υποδοχείς αυτοί επηρεάζουν και καθορίζουν με διαφορετικό τρόπο το ΛΜΦ, ανάλογα με τον υπότυπο των ΑΥς και ανάλογα με τον τύπο των κυττάρων. Πιο αναλυτικά, όσον αφορά στα κύτταρα A7r5 παρατηρήσαμε ότι: α) η ενεργοποίηση των α1Α-ΑΥ επάγει την έκφραση και των τεσσάρων γονιδίων-δεικτών που καθορίζουν το ΛΜΦ τόσο σε μεταγραφικό όσο και σε πρωτεϊνικό επίπεδο, β) η ενεργοποίηση του υποκινητή της SM22α από τους α1Α-ΑΥς εξαρτάται από τα στοιχεία CΑrG ενώ η ενεργοποίηση του υποκινητή της SM-Calponin δεν εξαρτάται από τα στοιχεία αυτά, γ) η ενεργοποίηση των α1Β-ΑΥ επάγει τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM22α και SM-Calponin ενώ δεν επηρεάζει την μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM-α-Αctin και της SM-MHC, δ) η ενεργοποίηση των υποκινητών της SM22α και της SM-Calponin από τους α1Β-ΑΥς εξαρτάται από τα στοιχεία CΑrG, ε) οι β-ΑΥς, σε αντίθεση με τους α1-ΑΥς, επηρεάζουν με διαφορετικό τρόπο τη μεταγραφή των γονιδίων-δεικτών που καθορίζουν το ΛΜΦ, κυρίως ελαττώνοντας τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM-Calponin, SM-α-Αctin και SM-MHC ενώ δεν επηρεάζουν τη μεταγραφική ενεργότητα του SM22α. Επίσης, όταν παρόμοια πειράματα έγιναν στα κύτταρα ΝΙΗ3Τ3 παρατηρήσαμε ότι: α) οι α1Α-ΑΥs επάγουν τη μεταγραφή των υποκινητών και των τεσσάρων γονιδίων-δεικτών που καθορίζουν το ΛΜΦ, β) στα κύτταρα αυτά η ενεργοποίηση των υποκινητών της SM22α και της SM-Calponin από τους α1Α-ΑΥς δεν εξαρτάται από τα στοιχεία CΑrG, γ) η ενεργοποίηση των α1Β-ΑΥs επάγει τη μεταγραφική ενεργότητα των υποκινητών των γονιδίων SM22α, SM-Calponin και SM-MHC ενώ δεν επηρεάζει την ενεργότητα του υποκινητή της SM-α-Αctin. Από τα παραπάνω λοιπόν, καταλήξαμε στα εξής συμπεράσματα: 1) Η διέγερση των α1-ΑΥ οδηγεί σε άυξηση της έκφρασης των γονιδίων-δεικτών του ΛΜΦ, και άρα οι α1-ΑΥς λειτουργούν, τουλάχιστον in vitro, ως παράγοντες προώθησης του ΛΜΦ. 2) Η δράση των α1-ΑΥ εξαρτάται μόνο μερικώς απο την ύπαρξη στοιχείων CArG (SRE), και διαφέρει ανάλογα με τον υποκινητή, άρα οι διάφοροι υποκινητές χρησιμοποιούν διαφορετικά τα στοιχεία CArG που περιέχουν, πράγμα που ενισχύει την υπόθεση συνδυαστικής χρήσης μεταφραφικών παραγόντων για την «πλαστική» έκφραση του λειτουργικού ΛΜΦ. 3) Οι δύο υπότυποι των α1-ΑΥ που εξετάστηκαν, α1Α-ΑΥς και α1Β-ΑΥς, έχουν προφανείς διαφορές ως πρός την διέγερση της έκφρασης των ΛΜ γονιδίων, και άρα πιθανώς θα παίζουν διαφορετικό λειτουργικό ρόλο στα αγγεία και στους άλλους ιστούς όπου εκφράζονται. 4) Αντίθετα με τους α1-ΑΥς, οι β-ΑΥς λειτουργούν ανασταλτικά στην έκφραση των γονιδίων του ΛΜΦ, και άρα το τελικό προϊόν της συμπαθητικής διέγερσης στα αγγεία θα είναι η συνισταμένη των δράσεων α και β ΑΥ, που θα διαφέρει στα διάφορα αγγεία ανάλογα με την σχετική έκφραση των υποδοχέων αυτών. Παράλληλα, εξετάζοντας ενδοθηλιακά κύτταρα φλέβας ανθρώπινου ομφάλιου λώρου (HUVEC), είδαμε ότι η εξωγενής έκφραση της Μυοκαρδίνης προκαλεί αλλαγές στο φαινότυπό τους, τόσο σε μορφολογικό επίπεδο όσο και μέσω de novo έκφρασης της SM-α-Ακτίνης και της SM-Καλπονίνης σε πρωτεϊνικό επίπεδο. Παρατηρήσαμε δηλαδή αλλαγές που είναι συμβατές με την επιθηλιο-μεσεγχυματική μετάβαση (ΕΜΤ-Epithelial to Mesenchymal Transition), διαδικασία με πολύ σπουδαίο ρόλο στην εμβρυογένεση, στη διαμόρφωση ιστών και οργάνων, αλλά επίσης και στην ίνωση, στη μετάσταση του καρκίνου και στην παθολογική αγγειογένεση. Συμπερασματικά, τα ενδοθηλιακά κύτταρα των αγγείων συμμετέχουν στη μετάβαση αυτή και μπορεί να συμβάλλουν σε διεργασίες κυτταρικής διαφοροποίησης και ιστικής δόμησης. Συμπεραίνουμε ότι η έκφραση της Μυοκαρδίνης επαρκεί για να προκαλέσει ΕΜΤ σε ανθρώπινα ενδοθηλιακά κύτταρα, με πιθανές συνέπειες για τη δυνατότητα trans-διαφοροποίησης και συνεισφοράς αυτών των κυττάρων, σε ανακατασκευή και αναδιοργάνωση ιστών, όπως πχ. στην αθηροσκλήρωση και την καρδιακή ανεπάρκεια. / The Smooth Muscle Cell (SMC) Phenotype is characterized by important plasticity and it plays crucial functional role in physiological and pathological conditions. Modulation of SMC Phenotype in arteries is a key etiological feature of some major human pathologies, including atherosclerosis, hypertension and vessel restenosis. Expression of SMCs marker-genes is also important in chronic diseases of other organs, such as in kidney or hepatic fibrosis and in cancer metastasis. So, study of the molecular mechanisms which affect SMC phenotype is necessary in order to develop new therapeutic approaches to combat to these diseases. Function of many tissues such as the vasculature is regulated by the sympathetic nervous system. The aim of this study was first, to examine in vitro whether the stimulation of alpha (α) and beta (β) adrenergic receptors (ARs) can control the expression of SMCs marker-genes in vitro and in case it did, to probe the molecular mechanisms via which α1-ARs (subtypes α1A and α1B) and β-ARs affect and regulate SMC Phenotype. Secondly, we wanted to investigate if the exogenous expression of Myocardin can cause Epithelial to Mesenchymal Transition (ΕΜΤ)-like changes in endothelial cells in vitro. For our experiments, we used two different cell populations : a) A7r5, which are differentiated Smooth Muscle Cells isolated from rat embryonic aorta, and b) ΝΙΗ3Τ3, mouse fibroblasts, in order to examine how stimulation of ARs modulates the expression of these marker-genes in differentiated SMCs (A7r5) and in mesenchymal cells which can convert to SMCs (ΝΙΗ3Τ3), respectively. As a marker for the expression of SMC Phenotype, we monitored the expression of cytoskeletal, structural protein-markers, such as Smooth Muscle-α-Actin (SM-α-Actin), SM-Myosin Heavy Chain (SM-MHC), h1-Calponin (SM-Calponin) and SM22α (transgelin) at two levels: a) using specific antibodies or b) using reporter plasmids in which the minimal promoters of the above genes drive luciferase gene transcription and hence activity. The coordinate transcriptional activation of these genes is, in major part, regulated by the function of CArG boxes in their promoters, which bind Serum Response Factor (SRF). In SMCs, SRF mediates its transcriptional effects via essential complex formation with members of the Myocardin family, which includes Myocardin (Myocd), Myocardin-Related Transcription Factor-A (MRTF-A) and MRTF-B. In our study, we initially noticed that these two cell populations do not express α1-ARs (subtypes α1Α and α1Β) endogenously, but when we transfect them with the plasmids expressing α1Α and α1Β ARs, the cells respond to α1-ARs agonist stimulation. In addition, we discovered that A7r5 cells express endogenous β-ARs. From our experiments, we concluded that these receptors can modulate SMC Phenotype in distinct way. This depends on both the specific subtype of receptor as well as on the cellular background (cell type). More specific, we observed that in A7r5 cells: a) activation of α1A-ARs by phenylephrine induces the expression of all four marker-genes at a transcriptional and at a protein level, b) activation of the SM22α minimal promoter by α1A-ARs depends on CΑrG boxes, while activation of the SM-Calponin minimal promoter does not depend on the presence of CΑrG boxes, c) activation of α1B-ARs induces the transcriptional activity of the minimal promoters of SM22α and SM-Calponin but does not affect the transcriptional activity of the minimal promoter of SM-α-Αctin and SM-MHC, d) activation of both the SM22α and the SM-Calponin minimal promoters by α1B-ARs depends on the presence of CΑrG boxes, e) on the contrary, β-ARs affect the transcription of SMCs marker-genes in an opposite way to α1-ARs reducing the transcriptional activity of the minimal promoters of SM-Calponin, SM-α-Αctin and SM-MHC genes, without affecting the transcriptional activity of the SM22α promoter. Additionally, we noticed that in ΝΙΗ3Τ3 cells: a) α1Α-ARs induce transcriptional activity of minimal promoters of SMC marker-genes, b) activation of minimal promoters of SM22α and SM-Calponin by α1A-ARs does not depend on CΑrG boxes, c) activation of α1B-ARs induce the transcriptional activity of the minimal promoters of SM22α, SM-Calponin και SM-MHC but does not affect the transcriptional activity of the minimal promoter of SM-α-Αctin. Based on the above findings, we conclude that: 1) Stimulation of α1-ARs drives an increased expression of SMC marker genes and consequently α1-ARs function, at least in vitro, as factors which have the ability to induce/maintain the SMC Phenotype. 2) The activity of α1-ARs depends variably on the presence of CArG boxes (SREs) and differs between these minimal promoters. In essence, different promoters use their CArG boxes in a different way. This is in support of the hypothesis that combinational use of transcriptional factors is essential for «plastic» expression of the SMC Phenotype. 3) The two subtypes of α1-ARs examined, α1Α and α1Β, display obvious differences in stimulating SM-specific gene expression and consequently these subtypes may play different functional role in vessels and in other tissues in which they are expressed. 4) On the contrary, β-ARs inhibit the expression of SMC marker-genes. Therefore, the final result of vascular sympathetic stimulation would depend on the combined action of α και β ARs. This action will differ in different vessels, depending on the relative expression of these receptors and their subtypes. In addition, adenoviral expression of Myocardin in human umbilical vein endothelial Cells (HUVECs) induced phenotypic alterations, evidenced by morphological changes and by de novo expression of SM-α-Αctin and SM-Calponin at the protein level. These observations are compatible with an Epithelial to Mesenchymal Transition (EMT), a process which plays an important role in embryogenesis, tissue and organs formation and angiogenesis, but also participates, in fibrosis and cancer metastasis. Consequently, vascular endothelial cells can undergo in EMT and may contribute in cellular differentiation and in tissue formation. We conclude that the expression of Myocardin is sufficient to cause EMT-like changes in human endothelial cells. This may lead to cellular trans-differentiation and contribution of these cells in active tissue remodeling such as in atherosclerosis and in cardiac failure.