The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts. / May 2008

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts.

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts.

Les malformations obstructives congénitales de l’uretère sont parmi les défauts les plus fréquents à la naissance chez l’Homme mais leur étiologie reste peu comprise au niveau moléculaire.L’uretère assure la fonction essentielle d’acheminer l’urine du rein à la vessie. Mon projet porte sur l’analyse d’un modèle murin d’obstruction fonctionnelle de l’uretère pour lequel le gène Teashirt 3(Tshz3) a été inactivé. J’ai montré que le phénotype d’hydro uretère (dilatation de l’uretère) de ce mutant résulte d’un défaut de différenciation des progéniteurs des cellules musculaires lisses pendant le développement et par voie de conséquence de l’absence de muscle lisse (ML). Chez le mutantTshz3, les cellules mésenchymateuses de l’uretère n’expriment pas le gène myocardin (Myocd) ni ses gènes cibles codant pour les protéines marqueurs du ML. Le facteur de transcription TSHZ3 est donc nécessaire à l’expression de Myocd et à la mise en place du ML. Afin de comprendre le contrôle moléculaire de la différenciation du ML, un crible double hybride a permis d’identifier parmi les partenaires de TSHZ3, la protéine à domaine HMG : SOX9. Au cours du développement de l’uretère,SOX9 s’exprime de manière similaire à TSHZ3 dans le mésenchyme jusqu’à l’apparition des marqueurs du ML, étape qui voit la perte d’expression de Sox9. Pendant l’étape précoce d’initiation dela différenciation TSHZ3, SOX9 et MYOCD sont co-exprimés. Un modèle cellulaire a permis de mieux comprendre les mécanismes mis en jeu pendant cette étape. Dans ce modèle, TSHZ3 et SOX9répriment l’activité promyogénique de MYOCD en l’empêchant de former un complexe activateur avec SRF. Dans l’uretère, la présence de TSHZ3 et SOX9 simultanément avec MYOCD suggère que ces facteurs participent au contrôle temporel de l’initiation du programme myogénique à un moment précis du développement de l’uretère. L’ensemble de ces résultats démontre que Tshz3 remplit des fonctions successives au cours du développement de l’uretère / Congenital obstructives disorders of the ureter are amongst the most common human birthdefects but the molecular networks underlying these defects remain unknown. Ureter plays a pivotalrole in propelling urine from kidney down to the bladder. My project aims at understanding a mouse model of functional obstructive ureter which possesses a null allele for the gene Teashirt3 (Tshz3). Ishowed that the hydroureter phenotype (dilated ureter) in the mutant embryos results of a failure ofureteric smooth muscle (SM) formation occurring since early embryonic steps of SM differentiation.Indeed, mutant ureteric mesenchymal cells do neither express the myocardin (Myocd) gene nor theMyocd targeted genes, encoding for contractile proteins of the SM. Thus, the transcription factorTSHZ3 is necessary for the expression of Myocd and subsequent SM differentiation. To understandthe molecular networks underlying the differentiation of SM, we performed a yeast two-hybrid screenthat allowed us to identify the HMG domain protein SOX9 as a TSHZ3 partner. During ureter development, SOX9 is expressed in an overlapping expression pattern with TSHZ3 until expression ofSM markers genes, a step whereby Sox9 is downregulated. An early initiation step of SM differentiation corresponds to the simultaneous expression of MYOCD with TSHZ3 and SOX9. A cellular model allowed to mimic this stage. TSHZ3 and SOX9 act as repressing factors of the myogenic activity of MYOCD by disrupting the MYOCD/SRF complex activator. These results suggest that TSHZ3 and SOX9 influence MYOCD activity and participate to the temporal control ofthe on set of ureteric myogenesis in the developing ureter. Together, these results demonstrate thatTSHZ3 fulfills successive functions during ureter development

No description available.

Ο έλεγχος της έκφρασης των πρωτεϊνών που χαρακτηρίζουν τον Λείο Μυικό Φαινότυπο (ΛΜΦ) είναι εξαιρετικής σημασίας για την κατανόηση, σε μοριακό επίπεδο, διεργασιών που σχετίζονται με πολλές φυσιο-παθολογικές καταστάσεις στον άνθρωπο. Μεταξύ των ασθενειών όπου ο ΛΜΦ είναι καθοριστικής σημασίας για την ανάπτυξη και εξέλιξή τους, είναι η αθηροσκλήρωση, η υπέρταση, η επαναστένωση των αρτηριών μετά από αγγειοπλαστική, η ίνωση οργάνων όπως οι πνεύμονες, το ήπαρ και οι νεφροί, και η ανάπτυξη μεταστάσεων από συμπαγείς όγκους. Επομένως, κατανόηση των κυτταρικών και μοριακών μηχανισμών που οδηγούν σε τροποποίηση του ΛΜΦ είναι βασικής σημασίας για την αναγνώριση στρατηγικών περιορισμού της εξέλιξης των νόσων αυτών και της εκδήλωσης των κλινικών συνεπειών τους. Αρχικό στόχο αποτέλεσε η ανάπτυξη και καθιέρωση ενός in vitro προτύπου συστήματος για την διαφοροποίηση μη διαφοροποιημένων κυττάρων προς φαινότυπο που προσομοιάζει με αυτό των Λείων Μυικών Κυττάρων (ΛΜΚ), ώστε να χρησιμεύσει στη μελέτη του μοριακού καθορισμού και ελέγχου του φαινότυπου των κυττάρων αυτών. Πρώτα-πρώτα, χαρακτηρίσαμε βασικά, σημαντικά «μοριακά εργαλεία» για την διαπίστωση και μοριακή διερεύνηση του ΛΜ-φαινοτύπου. Χρησιμοποιώντας τα, αναπτύξαμε και χαρακτηρίσαμε πρωτογενώς ένα πρότυπο σύστημα διαφοροποίησης σε ΛΜΚ, βασιζόμενο σε Μεσεγχυματικά Βλαστικά Κύτταρα (ΜΒΚ) προερχόμενα από γέλη Wharton ομφάλιου λώρου. Στα κύτταρα αυτά, η έκφραση γονιδίων και πρωτεϊνών που χαρακτηρίζουν τον ΛΜΦ εξαρτάται από την επαρκή έκφραση της πρωτεΐνης Serum Response Factor (SRF), από την ύπαρξη αλληλουχιών Serum Response Element (SRE) στον υποκινητή των εξεταζόμενων ΛΜΚ-ειδικών γονιδίων, και επάγεται από εξωγενή έκφραση της Μυοκαρδίνης. Επομένως, όπως έχει περιγραφεί και για άλλα πρότυπα συστήματα, η διαφοροποίηση των κυττάρων αυτών σε κύτταρα που προσομοιάζουν ΛΜΚ στηρίζεται στην συνέργεια δύο μεταγραφικών παραγόντων, του SRF και της Μυοκαρδίνης. Το πρότυπο αυτό θα είναι χρήσιμο για να διερευνήσουμε τους μοριακούς μηχανισμούς δράσης φυσιολογικών και φαρμακολογικών παραγόντων στον έλεγχο του ΛΜΦ. Επί πλέον, το πρότυπο σύστημα αυτό δύναται να αποβεί χρήσιμο για την κατανόηση εν γένει διεργασιών που οδηγούν στην βασική κυτταρική αλλαγή γνωστή ως Επιθηλιακή-Μεσεγχυματική Μετάβαση (ΕΜΤ) και κατ’ επέκταση για την κατανόηση μηχανισμών παθογένειας πλείστων νόσων που χαρακτηρίζονται από ΕΜΤ. Παράλληλα, έγινε προσπάθεια διερεύνησης αν η κυτταρική σειρά A7r5 αγγειακών ΛΜΚ αποτελεί βιώσιμο φαρμακολογικό σύστημα για την διερεύνηση των μηχανισμών μέσω των οποίων η έκφραση του ΛΜΦ ελέγχεται σε μοριακό επίπεδο από τους αδρενεργικούς υποδοχείς, μία οικογένεια υποδοχέων που διαδραματίζουν σημαντικό ρόλο στην ομοιόσταση του αγγειακού τοιχώματος και στην αρτηριακή παθοφυσιολογία. Δείξαμε ότι ο κυτταρικός πληθυσμός A7r5 δεν απαντά σε α1-αδρενεργική διέγερση διότι στερείται α1-αδρενεργικών υποδοχέων. Διέγερση αποκτάται με εισαγωγή μέσω πλασμιδίου α1-αδρενεργικών υποδοχέων, άρα το ενδογενές σηματοδοτικό σύστημα είναι παρόν και λειτουργικό. Επιπρόσθετα, ανακαλύψαμε ότι τα κύτταρα A7r5 εκφράζουν ενδογενώς λειτουργικούς β-αδρενεργικούς υποδοχείς. Θέτουμε έτσι τα θεμέλια για μία σε βάθος διερεύνηση του τυχόν ρόλου των β-αδρενεργικών υποδοχέων στον έλεγχο του φαινοτύπου των αγγειακών ΛΜΚ, ο οποίος είναι καθοριστικός για την γένεση και πορεία των καρδιαγγειακών νοσημάτων εν γένει. Συμπερασματικά λοιπόν α) τα μεσεγχυματικά βλαστικά κύτταρα προερχόμενα από τη γέλη Wharton ανθρώπινου ομφάλιου λώρου αποτελούν κατάλληλο πρότυπο σύστημα διερεύνησης της ρύθμισης των μοριακών μηχανισμών που εμπλέκονται στη διαφοροποίηση προς ΛΜΚ από μόρια φαρμακολογικής σημασίας, και β) τα κύτταρα A7r5 αποτελούν καλό πρότυπο σύστημα για την διερεύνηση του τυχόν ρόλου των β-αδρενεργικών υποδοχέων στον έλεγχο του φαινοτύπου των ΛΜΚ των αγγείων. / The control of the genes that specify the Smooth Muscle Cell Phenotype is of great importance for our understanding, at a molecular level, of the processes central in a number of human pathologies. Among the diseases whose onset and progress is influenced by alterations in Smooth Muscle-Like (SM-L) phenotype are atherosclerosis, organ fibrosis (lung, liver and kidney), and metastasis associated with solid tumors. For these reasons, the understanding of the cell and molecular mechanisms that lead to changes in the SM phenotype expression are of central importance in our efforts to identify new approaches in limiting the progress of these diseases and the manifestation of the associated clinical symptoms. The first Aim of this work was the development and initial characterization of an in vitro model of differentiation towards a Smooth-Muscle-Like phenotype, to serve for the study of its molecular control. Initially, we characterized basic important molecular tools useful in determining the SM-L phenotype. With their aid, we developed and characterized a model system based on Wharton’s Jelly-derived Mesenchymal stem Cells (MSCs). In these cells, the expression of genes and proteins characteristic of the SM Phenotype depends on the protein levels of Serum Response Factor (SRF) and on the existence of SRF-binding elements on the promoters of the SM-specific genes; it is also potently induced by the exogenous expression of the transcription factor Myocardin. Therefore, this population of MSCs behaves as other characterized model systems, in that their differentiation to a SM-L phenotype is supported by the synergistic action of SRF and Myocardin. This novel model system based on Wharton’s Jelly MSCs will be useful to study the role of specific physiological and pharmacological agents in the control of the SM phenotype. In addition, such a system can offer insights in the basic cellular process of Epithelial-to-Mesenchymal Transition (EMT) and by extent, in the pathological mechanisms of diseases characterized by EMT. In parallel, we investigated whether the differentiated SMC line A7r5 is a viable pharmacological model system to investigate the control of the SMC phenotype by adrenergic receptors, a family of receptors that plays a crucial role in the homeostasis of the vessel wall. We showed that A7r5 cells do not express functional α1-adrenergic receptors; however, the intracellular signaling system linked to α-adrenergic receptors is present and functional. In contrast, A7r5 cells endogenously express functional β-adrenergic receptors, and A7r5 cells are therefore an attractive model to study the role of these receptors in the control of the SMC-phenotype. In conclusion, a) Mesenchymal Stem Cells from Wharton’s Jelly surrounding the human umbilical cord are a suitable in vitro model for the study of the molecular mechanisms that modulating Smooth Muscle Cell differentiation, and b) A7r5 cells are a good in vitro model system to investigate the role of the β-adrenergic receptor in controlling the phenotype of Vascular Smooth Muscle cells.

No description available.