Dans l’espèce équine, les mécanismes de la fécondation sont mal connus. Afin de mieux les comprendre, nous avons développé une approche comparative entre les équins (faibles taux de fécondation in vitro : 0-60%) et les porcins (forts taux : 80–90%). Notre but était d’identifier les différences et les similitudes entre ces deux modèles opposés afin de mettre en évidence des éléments clés de la fécondation. Nous avons montré que 1) la zone pellucide de l’ovocyte est un élément clé dans l’interaction des gamètes, 2) sa composition et sa structure sont différentes entre les espèces équine et porcine, 3) chaque vertébré a son propre jeu de protéines permettant l’interaction des gamètes, 4) la ß-1,4-galactosyltransferase n’est pas nécessaire pour cette interaction dans les espèces équine et porcine, contrairement aux bovins, 5) les sécrétions de l’oviducte participent aux mécanismes de la fécondation équine, mais les protéines impliquées n’ont pas pu être identifiée. / In equine species, the mechanisms of fertilization remain largely enigmatic. In order to clarify these mechanisms, we have developped a comparative strategy between equine (low in vitro fertilization rates: 0- 60%) and porcine (high rates: 80-90%) species. Our objective was to identify differences and similarities between these two opposite models in order to highlight key components of fertilization. We showed that 1) the zona pellucida is a determining element in gamete interaction, 2) its composition and its structure are different between equine and porcine species, 3) each vertebrate has its own protein-set involved in gamete interaction, 4) the ß-1,4-galactosyltransferase is not necessary for gamete interaction in the horse and the pig, contrary to the bovine, 5) the secretions of oviduct cells take part in the mechanism of equine fertilization, but the proteins involved remain to be identified.

Ulm, Univ., Diss., 2001.

Vor etwas mehr als 20 Jahren wurden erstmals an das Myokard bindende beta1-Rezeptorantikörper im Zusammenhang mit der Chagas-Krankheit beschrieben. Die Arbeiten der beiden folgenden Jahrzehnte konnten zeigen, dass solche beta1-Rezeptorantikörper mit einer Prävalenz von ca. 30 bis 95% (in Abhängigkeit vom verwendeten Nachweisverfahren) v.a. auch bei Patienten mit dilatativer Kardiomyopathie (DCM) nachgewiesen werden können. Insbesondere Antikörper gegen die zweite extrazelluläre Domäne des beta1-adrenergen Rezeptors (beta1-ECII) scheinen dabei in der Lage zu sein, den Rezeptor funktionell zu beeinflussen und zu aktivieren. Beta1-ECII wurde in der Folge als potentes Autoantigen mit T- und B-Zell-Epitopen identifiziert. Erste klinische Untersuchungen an Patienten mit DCM haben gezeigt, dass das Vorhandensein zirkulierender b1-ECII-Antikörper mit einer schlechteren linksventrikulären Funktion, dem Auftreten ventrikulärer Arrhythmien sowie mit einer erhöhten kardiovaskulären Mortalität verbunden ist. Der Verdacht, dass beta1-Antikörper bei der DCM nicht nur ein Epiphänomen, sondern bei einem Teil der betroffenen Patienten auch mögliche Krankheitsursache darstellen könnten, verstärkten dann in jüngster Zeit die Bestrebungen, ein Tiermodell für die Antikörper-induzierte Genese einer Kardio-myopathie zu generieren. So fanden 1997 Matsui et al. im Kaninchenmodell nach Immunisierung mit einem beta1-ECII-homologen Peptid über 12 Monate eine biventrikuläre Dilatation und kompensatorische Hochregulation der beta-Adrenozeptoren im Myokard. Im Gegensatz dazu fanden Iwata et al. wenige Jahre später im gleichen Tiermodell nach 6-monatiger Immunisierung eine linksventrikuläre Hypertrophie bei beta1-ECII-Antikörper-positiven Tieren und eine Downregulation der beta-Rezeptoren. Diese Diskrepanzen ließen das Kaninchenmodell daher als fraglich geeignet erscheinen. Im Zusammenhang mit der vorliegenden Arbeit wurde die Lewis-Ratte zur Generierung eines Kardiomyopathiemodells gewählt, da die beta1-ECII-Sequenz bei Ratte und Mensch 100% homolog ist. Zur Immunisierung verwendeten wir ein Fusionsprotein aus GST und der humanen beta1-ECII-Sequenz. Der Schwerpunkt des Vorhabens lag auf der Charakterisierung der durch die Immunisierung induzierten morphologischen und funktionellen Veränderungen am Herzen der Versuchstiere. Echokardiographisch konnte bei immunisierten Tieren bereits nach 9 Monaten eine linksventrikuläre Erweiterung nachgewiesen werden, die sich nach 12 bzw. 15 Immunisierungsmonaten durch das Auftreten einer progredienten linksventrikulären Dysfunktion (Echokardiographie/Herzkatheteruntersuchung) auch funktionell manifestierte. Makroskopisch-morphometrisch ließ sich an Paraffinschnitten eine Erweiterung des linken Ventrikels bei relativer Abnahme der Wanddicke nachweisen und bestätigte somit histologisch die echokardiographischen Messungen. Die mikroskopische Analyse zeigte eine relative Vergrößerung der Kardiomyozytenkerne ohne signifikante Zellhypertrophie. Durch Radioligandenbindungsversuche konnte zudem in beta1-ECII-immunisierten Tieren eine Downregulation der kardialen beta1-Rezeptoren nachgewiesen werden. Durch die Immunisierung mit beta1-ECII-Antigen konnte der Phänotyp einer DCM somit erstmals in der Ratte induziert werden. Dieser Phänotyp ist vermutlich überwiegend auf die agonist-ähnliche funktionelle Aktivität der durch spezifische Immunisierung induzierten beta1-Rezeptorantikörper zurückzuführen. Das vorgestellte Tiermodell erfüllt dabei einerseits die immunologischen Kriterien des 1. Koch’schen Postulats für den „indirekten“ Nachweis einer Kardiomyopathieinduktion durch Antikörper, die gegen die zweite extrazelluläre Domäne des b1-adrenergen Rezeptors gerichtet sind. Andererseits lieferte es im Anschluss auch die direkte Evidenz für eine kausale Rolle solcher Antikörper bei der Induktion einer Autoimmunkardiomyopathie durch den Transfer induzierter Antikörper auf genetisch identische, herzgesunde Ratten (Nachahmen von Rezeptor-Autoantikörpern). Zukünftig könnte dieses Tiermodell auch dazu dienen, weitere immunologische Faktoren, die an der Entwicklung einer Autoimmunkardiomyopathie durch beta1-Rezeptor-antikörper beteiligt sind, zu untersuchen. Ferner können mit diesem Modell neue therapeutische Ansätze für die Behandlung der Rezeptorantikörper-positiven Kardiomyopathie entwickelt werden, mit dem Ziel einer späteren Anwendung im Tiermodell erprobter Strategien auch bei Antikörper-positiven Patienten. / About 20 years ago antibodies binding to the beta1-adrenergic receptor have been described in Chagas disease. In following decades different authors were able to prove, that these beta-1-adrenergic receptor antibodies are prevalent especially in patients with dilated cardiomyopathy (DCM) in 30 to 95%, depending on the proving method. Especially antibodies directed against the second extracellular loop of the beta-1-receptor (beta1-ECII) seemed to be able to modulate and to activate the receptor. In the past years the second extracellular loop could be identified as a potent (auto-)antigen containing T- and B-cell epitopes. Clinical studies in DCM-patients associated a significant poorer left ventricular function, a higher prevalence of serious ventricular arrhythmias and a higher cardiovascular mortality with the presence of anti-beta1-ECII-antibodies. In the following it was necessary to develop sufficient animal model proving that the presence of these antibodies might not be a side effect and furthermore to prove that these antibodies might causal in developing a DCM. In 1997 Matsui et al. could show a biventricular dilatation and a up-regulation of beta1-receptors in the myocard using rabbits immunised over 12 months with a synthetic peptides corresponding to beta1-ECII. Using the same animal model few years later Iwata et al. recognized a left-ventricular hypertrophy after 6 months of immunisation and a downregulation of beta-receptors. Actually it is not sure, if this animal model might be sufficient to answer question for the causal role of beta1-receptor-antibodies. Therefore, in the present sudy we attempted to create experimental immune-cardiomyopathy in the rat using human beta1-ECII (100% sequence identity human/rat) fused to bacterial GST as an antigen. The main aspect in our study was to characterize the different morphological und functional findings in the rat heart. Compared with the control animals, echocardiographic follow-up revealed significant left ventricular dilatation from the ninth study-month on, which slowly and steadily progressed in the course of the study. The final echocardiographic findings were furthermore supported by left ventricular pressure curves obtained in the left heart catheterization at study end (fifteenth month). The anatomic measurements, performed in hematoxylin an eosin-stained paraffin sections, showed a left ventricular dilatation and a relative increase in left ventricular wall-thickness which furthermore emphasized the echocardiographic results. Histomorphological analysis revealed significantly enlarged polymorphous nuclei, but not a cardiomyocyte-hypertrophy. The radioligand-binding studies showed a down-regulation of the beta1-adrenergic receptor in the cardiomyocyte membrane. We were able to show the first time the phenotype of DCM in the rat by immunization against the second extracellular loop of the beta1-adrenoceptor. It seems conceivable that the generated cardiomyopathic phenotype has to be attributed mainly to the sustained sympathomimetic activity of the produced activating anti-beta1-ECII-antibodies. Our animal model fulfills the first Witebsky postulate for autoimmune disease (indirect evidence) including antibodies against an identified corresponding (auto-)antigen. Furthermore it is the basis for the direct evidence proving a causal role of these beta1-ECII-antibodies in developing a autoimmune cardiomyopathy by transferring induced antibodies on healthy animals of the same strain. In the future this animal model might be used to detect further immunological elements that might be causal to develop a autoimmune cardiomyopathy associated with the evidence of beta1-adrenoceptor-antibodies. Furthermore this animal model might be useful to develop new strategies in the treatment of the beta1-adrenoceptor-positive DCM.

Muc4, a heterodimeric transmembrane mucin containing EGF-like domains, has been described as an ErbB2-binding protein which modulates signaling via the ErbB2-ErbB3 pathway. In Muc4-transfected MCF-7 cells, Muc4 expression resulted in alteration of both the time course and phosphorylation levels of NRG beta 1 induced phosphorylation and activation of both ErbB2 and ErbB3. Muc4 significantly enhanced the autophosphorylation of ErbB2 over the early (defined 0-30 min) and intermediate (30-120 min) NRG beta 1 treatment times at three sites, Y1248, Y1221 and Y1139. The sites displayed differential maximal phosphorylation times. At Y1248 and Y1139, maximal phosphorylation occurred entirely during the early treatment phase. However, Y1221/2 showed maximal phosphorylation during the intermediate phase with a smaller peak during the early phase. The ratio of phosphorylated ErbB3 and total receptor level was significantly enhanced (in cells that expressed Muc4 compared without Muc4) over both the early and intermediate NRG beta 1 treatment time at the Y1289 site. This motif is one of several similar ErbB3 motifs whose phosphorylation mediates the binding of PI3-kinase. This phospholipid kinase is a key modulator of numerous cellular pathways leading to proliferation, motility and survival. Aberrancies in the ErbB2-ErbB3 signaling pathway have been implicated in the aggressive behavior of tumor cells, and the identification and characterization of modulators of this pathway are being sought as targets of potential therapeutic interventions. Muc4 significantly enhanced activated ERK in the absence of NRG beta 1 treatment while a NRG beta 1 mediated activation of AKT was observed. At early NRG beta 1 treatment time phases, Muc4 co-localized with phosphorylated ErbB2 (pY1248) independent of NRG beta 1 treatment; co-localization of Muc4 and ErbB2 receptor (activated/receptor forms) was observed at the apical surface or around the cell surface membrane. These data provide evidence in the Muc4-transfected MCF-7 cells for the biological NRG beta 1 mediated ErbB2 and ErbB3 activation. Our data suggests that Muc4 affects steady state phosphorylation levels and duration of the phosphorylation signal of both ErbB receptors, and that NRG beta 1 might affect ErbB2 and ErbB3 signaling differently. Additionally, the results of the timing of phosphorylation studies suggest the possibility that temporal aspects of phosphorylation at different sites may determine the pathways activated preferentially in the subsequent signaling cascades.

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Tübingen, Univ., Diss., 2004.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

During cold acclimation the heat production is shifted from shivering to non-shivering thermogenesis, which is mediated by adrenergic signaling. It has also been observed, that cold acclimation may increase the organismal resistence to pathological stimuli and may affect functional parameters of cardiovascular system. However, acute exposure to sever cold is often associated with detrimental effects on the body. We have recently shown that chronic exposure to cold increases the heart's resistance to ischemia-reperfusion injury without negative side effects when mild temperatures are used, however the mechanism of protection is not yet known. The aim of this work was to determine whether: i) if the sensitivity of the heart to ischemia changes already after the first day of cold exposure and does not show any negative effects, ii) if β1-adrenergic signaling plays a role in chronic regimen of cold-induced cardioprotection. The results of this work showed that i) one day of exposure to mild cold did not change the sensitivity of the heart to ischemia and ii) metoprolol treatment reduced the infarct size in the control group, but did not affect the heart of cold-adapted rats. Key words : Heart, rat, beta 1 adrenergic receptors, cold acclimation