Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPβ enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC), respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role of PI3K/PKB/GSK-3β signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the β-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3β, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the β-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and β-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene.

Increased local tumor tissue expression of versican in breast cancer patients is predictive of relapse and has a negative impact on survival rates. It is recognized that bone is a common anatomic site of breast cancer metastasis. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. We exogenously expressed a G3 construct in mouse breast cancer cell line 66c14, and found that G3 expression enhanced breast cancer cell proliferation and migration, and spontaneous metastasis to bone in an orthotopic model by upregulating the EGFR-mediated signaling pathway. Possessing anti-apoptotic and drug resistant properties, overexpression of versican was accompanied by selective sensitization to several chemotherapeutic agents. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may, in part, explain breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. New knowledge gained by our experiments includes the understanding that GSK-3β (S9P) appears to function as a key check-point in this balance. In addition, versican G3 enhanced breast cancer cell self-renewal in vitro and in vivo. Versican was expressed at high levels in breast cancer mammosphere cells, which contained a high percentage of SP cells. Reduction of versican’s functionality through anti-versican shRNA or knocking out the EGF-like motifs using G3ΔEGF reduced the effect of versican on enhancing mammosphere and colony formation. Versican promoted breast cancer cell self-renew appears to play a role in enhanced chemotherapeutic drug resistance (including Docetaxel, Doxorubicin, and Epirubicin), which relates partly to its upregulated expression of EGFR signaling. Versican enhances breast cancer bone metastasis not only by enhancing tumor cell mobility, invasion, and survival in bone tissues, but also through mechanisms inhibiting osteoblast cell growth and differentiation, affording favorable microenvironments for tumor metastasis.

Increased local tumor tissue expression of versican in breast cancer patients is predictive of relapse and has a negative impact on survival rates. It is recognized that bone is a common anatomic site of breast cancer metastasis. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. We exogenously expressed a G3 construct in mouse breast cancer cell line 66c14, and found that G3 expression enhanced breast cancer cell proliferation and migration, and spontaneous metastasis to bone in an orthotopic model by upregulating the EGFR-mediated signaling pathway. Possessing anti-apoptotic and drug resistant properties, overexpression of versican was accompanied by selective sensitization to several chemotherapeutic agents. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may, in part, explain breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. New knowledge gained by our experiments includes the understanding that GSK-3β (S9P) appears to function as a key check-point in this balance. In addition, versican G3 enhanced breast cancer cell self-renewal in vitro and in vivo. Versican was expressed at high levels in breast cancer mammosphere cells, which contained a high percentage of SP cells. Reduction of versican’s functionality through anti-versican shRNA or knocking out the EGF-like motifs using G3ΔEGF reduced the effect of versican on enhancing mammosphere and colony formation. Versican promoted breast cancer cell self-renew appears to play a role in enhanced chemotherapeutic drug resistance (including Docetaxel, Doxorubicin, and Epirubicin), which relates partly to its upregulated expression of EGFR signaling. Versican enhances breast cancer bone metastasis not only by enhancing tumor cell mobility, invasion, and survival in bone tissues, but also through mechanisms inhibiting osteoblast cell growth and differentiation, affording favorable microenvironments for tumor metastasis.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPβ enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC), respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role of PI3K/PKB/GSK-3β signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the β-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3β, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the β-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and β-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPβ enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC), respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role of PI3K/PKB/GSK-3β signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the β-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3β, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the β-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and β-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements,and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPf3 enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC),respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role ofPI3K/PKB/GSK-30 signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the 0-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3[3, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine the irrepressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the P-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and 13-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene.

Οι πρωτεογλυκάνες και οι αλυσίδες γλυκοζαμινογλυκανών τους αλληλεπιδρούν με αυξητικούς παράγοντες, διαμεμβρανικούς υποδοχείς όπως οι ιντεγκρίνες, ένζυμα, αναστολείς πρωτεασών και με άλλα μόρια της εξωκυττάριας ύλης όπως η ινονεκτίνη, η λαμινίνη και η tenascin. Στην παρούσα διατριβή μελετήσαμε τη χωροχρονική κατανομή των πρωτεογλυκανών versican και CD44 με τη μέθοδο της RT-PCR και του ανοσοφθορισμού από το στάδιο ΧΙ-ΧΙΙ (μορίδιο) έως το στάδιο ΗΗ16+ (28-29 ζεύγη σωμιτών) και τον ρόλο της versican και της CD44 με τη χρήση μονοκλωνικών αντισωμάτων έναντι αυτών κατά την ανάπτυξη του πρώιμου εμβρύου. Η versican είναι πρωτεογλυκάνη θειικής χονδροϊτίνης και αλληλεπιδρά με αυξητικούς παράγοντες, με διάφορες πρωτεΐνες της εξωκυττάριας ύλης και με διαμεμβρανικούς υποδοχείς όπως η CD44. Τα αποτελέσματα της RT-PCR έδειξαν ότι το mRNA της versican εκφράζεται σε όλα τα αναπτυξιακά στάδια που μελετήσαμε. Ενδιαφέρον παρουσιάζουν τα προϊόντα εναλλακτικής ωρίμανσης της versican που ανιχνεύσαμε ακόμα και στο στάδιο του μοριδίου και που η έκφρασή τους ρυθμίζεται αναπτυξιακά. Η παρουσία του mRNA της versican σε υψηλά επίπεδα στο στάδιο του μοριδίου (ΧΙ-ΧΙΙ) υποδεικνύει ότι το mRNA της versican είναι ωογενετικής προέλευσης στο στάδιο αυτό. Τα πειράματα μας του ανοσοφθορισμού έδειξαν ότι η versican πρωτεΐνη ανιχνεύεται στο στάδιο του μοριδίου και εκφράζεται έντονα στην επιβλάστη και στην υποβλάστη στο στάδιο του προχωρημένου βλαστιδίου (στάδιο ΧΙΙΙ). Στο στάδιο ΗΗ3+ (ενδιάμεσο γαστρίδιο / intermediate streak) παρατηρήσαμε μεγάλη ένταση φθορισμού στα κύτταρα που μεταναστεύουν μέσα από την πρωτογενή αύλακα και στα μεσεγχυματικά κύτταρα που θα σχηματίσουν το μεσόδερμα και το ενδόδερμα. Στο στάδιο ΗΗ4 (προχωρημένο γαστρίδιο / definitive streak) ένταση φθορισμού της versican ανιχνεύθηκε κύτταρα που μεταναστεύουν μέσα από την πρωτογενή αύλακα καθώς και στα κύτταρα που έχουν αρχίσει να σχηματίζουν το μεσόδερμα και το ενδόδερμα. Στο στάδιο που το έμβρυο έχει σχηματίσει 4 ζεύγη σωμιτών (στάδιο ΗΗ8), ανιχνεύσαμε υψηλή ένταση φθορισμού της versican στη νευρική πλάκα και στις νευρικές πτυχές καθώς ανασηκώνονται να σχηματίσουν το νευρικό σωλήνα. Στο στάδιο ΗΗ12 (16 ζεύγη σωμιτών), ισχυρή ένταση φθορισμού της versican ανιχνεύσαμε στο νευρικό σωλήνα, στο γειτονικό του εξώδερμα, στα κύτταρα της νευρικής ακρολοφίας (neural crest), στα κύτταρα του σωμίτη, στο μεσονέφρο και στο γειτονικό του πλάγιο μεσόδερμα που θα σχηματίσει τους μεσονεφρικούς σωληνίσκους. Αργότερα στην ανάπτυξη, ισχυρή ένταση φθορισμού της versican ανιχνεύσαμε επίσης στο διεγκέφαλο, στον οπτικό μίσχο, στο μεσεγκέφαλο, στο μυελεγκέφαλο, στα τοιχώματα του φάρυγγα και της ραχιαίας αορτής, στο ραχιαίο μεσοκάρδιο, στο μυοκάρδιο και στο ενδοκάρδιο, στο μυοτόμο και σκληροτόμο στους σωμίτες, στα τοιχώματα του εντέρου, καθώς και στην εξωκυττάρια ύλη των εμβρυϊκών κοιλοτήτων. Πειράματα σε έμβρυα που εκτέθηκαν στο αντίσωμα έναντι της versican σε διαφορετικά στάδια ανάπτυξης από το μορίδιο ως το προχωρημένο γαστρίδιο έδειξαν ότι η versican πιθανόν να συμμετέχει στα μονοπάτια σηματοδότησης που καθοδηγούν τη μετανάστευση των κυττάρων της νευρικής ακρολοφίας, στο μοριακό δίκτυο για το σχηματισμό του νευρικού σωλήνα, στον καθορισμό ή / και στη διαμερισματοποίηση των προκαρδιακών κυττάρων κατά το σχηματισμό της καρδιάς και στη διατήρηση της αρχιτεκτονικής των εμβρυϊκών κοιλοτήτων. Η CD44 είναι πρωτεογλυκάνη της κυτταρικής επιφάνειας και είναι ο κύριος υποδοχέας του υαλουρονικού. Ανιχνεύσαμε τη CD44 πρωτεΐνη ακόμα και στο στάδιο του μοριδίου. Η παρουσία του mRNA της CD44 σε υψηλά επίπεδα στο στάδιο του μοριδίου μπορεί να δείχνει ότι το mRNA είναι ωογενετικής προέλευσης. Με τη μέθοδο του ανοσοφθορισμού ανιχνεύσαμε έντονο φθορισμό της CD44 στα κύτταρα της επιβλάστης και ειδικότερα σε αυτά που γειτονεύουν με το βλαστόκοιλο και στην υποβλάστη στο στάδιο του προχωρημένου βλαστίδιου (ΧΙΙΙ), ενώ στο στάδιο ΗΗ3+ καθώς συνεχίζονται οι μορφογενετικές κινήσεις της γαστριδίωσης, η CD44 παρουσιάζει ισχυρή ένταση φθορισμού στα κύτταρα της επιβλάστης ,στα μεσεγχυματικά κύτταρα και στα κύτταρα του ενδοδέρματος. Στο στάδιο ΗΗ8 (4 ζεύγη σωμίτες), ανιχνεύσαμε ένταση φθορισμού της CD44 στη νευρική πλάκα και στις νευρικές πτυχές με πρότυπο έκφρασης παρόμοιο με αυτό της έκφρασης της versican. Αργότερα στην ανάπτυξη, στο στάδιο ΗΗ16+ (28-29 ζεύγη σωμιτών) ανιχνεύσαμε ισχυρή ένταση φθορισμού της CD44 ανιχνεύθηκε στα τοιχώματα του διεγκεφάλου, του μεσεγκεφάλου και του νευρικού σωλήνα, στα τοιχώματα του φάρυγγα, στις ραχιαίες και κοιλιακές αορτές, στα αορτικά τόξα, στη νωτοχορδή, στην εξωκυττάρια ύλη στην κοιλότητα του μεσεγκεφάλου και στην κοιλότητα του φάρυγγα, στο ακουστικό κυστίδιο και στην κοιλότητα του ακουστικού κυστιδίου και στα κύτταρα της νευρικής ακρολοφίας που μεταναστεύουν προς το ακουστικό κυστίδιο. Υψηλή ένταση φθορισμού της CD44 ανιχνεύσαμε επίσης στο σκληροτόμο και στο μυοτόμο στους σωμίτες, στα κύτταρα της νευρικής ακρολοφίας, στο ήπαρ, στο μυοκάρδιο, στο ενδοκάρδιο, στον αγωγό φλέβας και στο μεσονέφρο. Έμβρυα που εκτέθηκαν στο αντίσωμα έναντι της CD44 σε διαφορετικά στάδια ανάπτυξης από το μορίδιο ως το πρώιμο νευρίδιο έδειξαν το σημαντικό ρόλο της CD44 στην μορφογένεση του εγκεφάλου, στη διατήρηση της αρχιτεκτονικής της κοιλότητας του εγκεφάλου και των άλλων εμβρυϊκών κοιλοτήτων, στην μετανάστευση των κυττάρων της νευρικής ακρολοφίας, στον σχηματισμό της καρδιάς και του αγγειακού συστήματος και στη μορφογένεση των σωμιτών. Τα αποτελέσματα μας έδειξαν τη συνεργιστική δράση των CD44 και versican κατά την ανάπτυξη του πρώιμου εμβρύου. / Proteoglycans and their associated glycosaminoglycans can bind growth factors, integrin and non-integrin cell surface molecules, enzymes, protease inhibitors and other extracellular matrix components including fibronectin, laminin and tenascin. We studied the expression and spatiotemporal distribution of versican and CD44 by RT-PCR and immunofluorescence in the chick embryo from the morula stage (stage XI-XII) to early organogenesis (stage HH16+, 28-29 somites). We also studied the versican and CD44 role by using blocking antibodies in the early chick embryo. Versican is a chondroitin sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules including the CD44. Combined RT-PCR and immunohistochemistry demonstrated the expression of versican as early as the morula stage. Interestingly, we detected splice variants of versican at the morula stage, and their expression was developmentally regulated. The presence of versican mRNA at the morula stage may indicate that it is of oogenetic origin. Versican fluorescence was strong in the epiblast and the hypoblast at the late blastula stage (XIII). At stage HH3+ (intermediate streak), versican expression was intense in the cells ingressing through the primitive streak and the migrating mesenchymal cells which will form the mesoderm and endoderm. By the definitive streak stage (HH4), versican fluorescence was intense in the cells ingressing through the primitive streak and in the mesenchymal cells that have already started to form the mesoderm and endoderm. At stage HH8 (4 somite pairs), versican expression was strong in the neural plate, the elevated neural folds and the ectoderm neighboring the neural folds. At stage HH12 (16 somite pairs), versican fluorescence was intense in the neural tube and its adjacent ectoderm, the neural crest cells, the somite and in the mesonephros and in the adjacent lateral mesoderm that will form the mesonephric tubules. Later in development, versican fluorescence was intense in the diencephalon, the optic stalk, mesencephalon, myelencephalon. Versican fluorescence was also intense in the dorsal mesocardium, myocardium and endocardium, dorsal aorta and aortic arches, in the myotome and sclerotome in somites, gut and in the extracellular matrix of embryonic cavities. Inhibition of the function of versican by blocking antibodies showed that versican is crucial for the neural tube closure, neural crest migration, formation of the heart tube, for the architecture of embryonic cavities and consequently tissue and organ morphogenesis. CD44 is a transmembrane part-time proteoglycan and the main receptor for hyaluronan. We detected CD44 protein even at the morula stage. The presence of high levels of CD44 mRNA at the morula stage indicated that this is an oogenetic mRNA. CD44 fluorescence was strong in the epiblast cells, especially those neighboring the blastocoele, and in the hypoblast at the late blastula stage (XIII). At stage HH3+, during gastrulation, CD44 was expressed strongly in the epiblast cells, in mesenchymal cells and in endoderm cells. At stage HH8 (4 somite pairs), strong CD44 expression was detected in the neural plate and neural folds and their adjacent ectoderm and this expression pattern was similar to that of versican. Later in development, CD44 expression was intense in the diencephalon, optic stalks, mesencephalon, myelencephalon, metencephalon, auditory vesicles and the neural crest cells migrating towards the auditory vesicle and the neural tube. CD44 fluorescence was also intense in the dorsal mesocardium, myocardium, endocardium, aortae and aortic arches, sclerotome and myotome in somites, mesonephros, liver, gut and in the migrating neural crest cells that will form the sympathetic and enteric ganglia. Inhibition of CD44 function by blocking antibodies showed that CD44 is crucial for the architecture of the embryonic cavities such as the brain lumen, neural tube closure, neural crest cell migration, cardiac and cardiovascular formation and somite morphogenesis. Our results showed a synergistic role of CD44 and versican during the development of the early embryo.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年4月5日 西田佳弘氏の博士論文として提出された

La calpaïne 1 est une protéase à cystéine activée par le calcium, qui peut être partiellement externalisée. Les calpaines extracellulaires favorisent la résolution de l'inflammation et la réparation des tissus, à travers la prolifération et la migration cellulaire. Le récepteur Toll like (TLR) 2 a été identifié comme une cible des calpaïnes extracellulaires dans les lymphocytes. L'objectif est d'étudier le rôle de la calpaïne extracellulaire 1 dans la progression tumorale de l'adénocarcinome pulmonaire lepidique (ADL). La calpaïne extracellulaire, le fragment soluble de TLR2, le versican et les cytokines étaient analysés par ELISA dans des surnageants de lavage bronchoalvéolaire (LBA) de patients atteints d'ADL (n = 68). La source de calpaïne était analysée par immunohistochimie. TLR2, cible de la calpaïne extracellulaire était étudiée par cytométrie de flux sur les polynucléaires neutrophiles (PNN) et des lignées humaines de cancer bronchiques. Calpain 1 extracellulaire, sécrété par les cellules tumorales, était associée à la progression tumorale, l'inflammation à neutrophiles, avec un facteur de mauvais pronostic de survie (p = 0,003). TLR2 était exprimé sur les cellules tumorales ou les PNN avec une diminution d¿expression après traitement par calpaïne. Le fragment soluble de TLR2 était corrélée à la concentration extracellulaire de calpaïne 1 dans les surnageants de LBA (r = 0,624; p <0,001). Le fragment soluble de TLR2 élevé était associé à la progression tumorale et un environnement pro-inflammatoire La calpain extracellulaire sécrétée par la cellule tumorale, favorise un microenvironnement inflammatoire et la progression tumorale médiée par TLR2 dans ADL. / Calpain 1 is pro inflammatory calcium-activated cysteine proteases, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the role of extracellular calpain 1 in tumor progression of lepidic pulmonary adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry. TLR2 as target of extracellular calpain was studied by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular Calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (p=0,003). TLR2 was expressed on PMN or tumor cells and decreased after calpain treatment. The soluble fragment of TLR2 was correlated to the extracellular calpain 1 concentration in the BALF supernatants (r=0.624; p<0.001). High soluble fragment of TLR2 was associated with tumor progression and a pro-inflammatory environment. Extracellular Calpain 1 secreted by tumor cell, promotes inflammatory microenvironment and tumor progression through TLR2 in LPA.

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.