H neurocan και η phosphacan είναι πρωτεογλυκάνες θειικής χονδροϊτίνης. Η neurocan θεωρείτο οτι είναι μια πρωτεογλυκάνη αποκλειστικά του εγκεφάλου. Η phosphacan έχει μελετηθεί σε προχωρημένα στάδια ανάπτυξης εμβρύων ποντικού και αρουραίου. Δεν ήταν γνωστό πότε αρχίζουν να εκφράζονται και πως κατανέμονται χωρο-χρονικά οι neurocan και phosphacan καθώς το έμβρυο αρχίζει την ανάπτυξή του. Μελετήσαμε την έκφραση της neurocan και της phosphacan με RΤ-PCR και ανοσοφθορισμό στο έμβρυο όρνιθας από το στάδιο Χ (μορίδιο) έως το στάδιο HH17 (29 σωμίτες/πρώιμη οργανογένεση). Επίσης μελετήσαμε το ρόλο της neurocan και phosphacan με τη χρήση μονοκλωνικών αντισωμάτων έναντι αυτών κατά την ανάπτυξη του πρώιμου εμβρύου. Το mRNA της neurocan πρωτοανιχνεύτηκε στο στάδιο του προχωρημένου γαστριδίου (HH4) και η έκφραση του ρυθμίζεται αναπτυξιακά. Τα πειράματα μας του ανοσοφθορισμού επίσης έδειξαν ότι η neurocan άρχισε να ανιχνεύεται στη νευρική πλάκα και στην εξωκυττάρια ουσία στο στάδιο του προχωρημένου γαστριδίου (HH4). Στα έμβρυα στο στάδιο των 19 σωμιτών (ΗΗ13), ο φθορισμός ήταν έντονος στον μυελεγκέφαλο, τη νωτοχορδή, στα κύτταρα των νευρικών κρηπίδων, στο κάτω τοίχωμα του φάρυγγα, στο ραχιαίο μεσοκάρδιο και μυοκάρδιο και λιγότερο στο ενδοκάρδιο και στα φαρυγγικά τόξα όπου έχουν μεταναστεύσει κύτταρα των νευρικών κρηπίδων. Στο στάδιο των 29 σωμιτών (HH17), η έκφραση της neurocan ήταν ισχυρή στον τελεγκέφαλο, μυελεγκέφαλο και στο νευρικό σωλήνα. Η έκφραση της neurocan ήταν ισχυρή στον αμφιβληστροειδή, λιγότερη στο φακό και έδειχνε μεγάλη ένταση στον κερατοειδή χιτώνα στον οφθαλμό, έντονη στο ραχιαίο μεσοκάρδιο, στο μυοκάρδιο και αχνή στο ενδοκάρδιο στην καρδιά και έντονη στους σωμίτες, στο μεσονέφρο και στις νησίδες αίματος. Σε μια σειρά λειτουργικών πειραμάτων με τη χρήση μονοκλωνικών αντισωμάτων έναντι της neurocan προκάλεσε τα νευροεξωδερμικά κύτταρα να επιδεικνύουν μεσεγχυματικά χαρακτηριστικά και ο εγκέφαλος να μην έχει αναπτυχθεί φυσιολογικά. Επίσης ανεστάλη η μορφογένεση της καρδιάς και των σωμιτών στα έμβρυα αυτά. / Neurocan, a chondroitin sulfate proteoglycan, interacts with other molecules of the extracellular matrix and the cell surface and participates in signalling pathways. Phosphacan is a chondroitin/ keratan sulfate proteoglycan that has been studied extensively in the late embryonic and postnatal brain and in the spinal cord. Relatively little is known about the neurocan tissue-specific distribution or function during the development of the embryo. We studied the neurocan and phosphacan spatio-temporal expression pattern by RT-PCR and immunofluorescene in the early chick embryo from the morula stage (stage XI) to early organogenesis (stage HH17, 29 somites). We also studied the role of neurocan and phosphacan by using blocking antibodies directed against neurocan or phosphacan in a set of functional studies. Neurocan mRNA was first detectable at the late gastrula stage (HH4) and its expression was developmentally regulated. Neurocan protein was first detectable in cells of the inchoate neural plate and in the extracellular matrix in embryos at stage HH4. At stage HH13 (19 somites), neurocan fluorescence was intense in the myelencephalon, notochord, neural crest cells, the foregut lower wall, pharyngeal arches, dorsal mesocardium, myocardium and in the endocardium. At stage HH17 (29 somites), neurocan expression was intense in the telencephalon, myelecenphalon, diencephalon and in the neural tube. Expression of neurocan was strong in the retina, lens and intense in the cornea in the eye, intense in the myocardium and lower in endocardium in the heart. In a set of functional studies using monoclonal antibodies directed against neurocan, the inhibition of neurocan function resulted in neural plate duplications, the neurepithelial cells exhibited mesenchymal characteristics and the somite, heart and gut formation were inhibited. The observed neural plate duplications point to an important role of neurocan to modulate the activity and availability of growth factor(s) during the induction of neurectoderm. Phosphacan mRNA was first detectable at the morula stage (XI) and it continued to be expressed during development. At the blastula stage (XIII)phosphacan immunofluorescence was strong in the epiblast and hypoblast. At the gastrula stage (HH3-ΗΗ4), immunofluorescence was strong in the cells around and also ingressing through the streak and in mesenchymal cells. At stage HH8 (4 somites), immunofluorescence was intense in the elevated neural plate, especially in its dorsal surface. This implies a role for phosphacan in the fusion of neural folds medially during the formation of the neural tube. At stage HH11 (13 somites), phosphacan immunofluorescence was strong in the neural tube, somites, gut lower wall and in the myocardium, endocardium and dorsal mesocardium in the heart and intense in the blood islands. By stage HH17 (29 somites), phosphacan immunofluorescence was strong in the myelencephalon and diencephalon, in the lens and retina in the eye, the neural crest cells, the myotome but not the dermatome and sclerotome in somites, the myocardium and endocardium in the heart and the dorsal aorta. Inhibition of function of phosphacan by blocking antibodies showed that phosphacan participates in the fusion of neural folds to form the neural tube, in the opticoele determination to form the retina and in somite, heart and gut morphogenesis.

INTRODUÇÃO: As doenças retinianas associadas à neovascularização, tais como a degeneração macular relacionada à idade e as retinopatias diabética e da prematuridade são as principais e mais importantes causas da cegueira em todo o mundo. Nos últimos anos, injeções intravítreas de fármacos com ação antiangiogênica, como o bevacizumabe (BVZ), têm sido de grande valia tanto em pacientes na fase adulta quanto nos recém-natos. Todavia, estudos experimentais in vitro e in vivo sugerem que essas drogas promovam efeitos adversos sobre alguns processos celulares, interferindo diretamente em mecanismos fisiológicos que mantém a homeostase do tecido retiniano, incluindo os mecanismos de proliferação, diferenciação e morte celular. OBJETIVO: investigar o efeito do BVZ nos processos de transcrição e tradução de marcadores da gliose: GFAP e vimentina, de morte celular, caspase-3 e beclina-1, e dos proteoglicanos relacionados à manutenção e desenvolvimento de tecido retiniano: neurocam, fosfacam e sindecam-3. MÉTODOS: Dois modelos experimentais foram usados nesse estudo: 1) linhagem celular de Müller de Glia humana adulta (MIO-M1), cultivada em meio de cultura D-MEM na presença e ausência de BVZ por 12 e 24 horas nas concentrações de 0,25 mg/mL e 0,50 mg/mL e 2) explantes de retinas de ratos 2 dias pós-nascidos submetidos à 0,50 mg/mL da droga por 48 horas. Durante este período foram mantidos a 5% de dióxido de carbono à temperatura de 37°C. A análise de proteínas foi realizada por imunocitohistoquímica e Western Blotting e a expressão de RNAm, pela reação em cadeia da polimerase em tempo real (PCR Real Time). Foi utilizado o Teste de ANOVA - fator único para a comparação entre os grupos controle e tratados com BVZ de um mesmo período (12h ou 24h) e o teste t de Student para a comparação entre as mesmas concentrações de 12h e 24h, e para a comparação entre os grupos controle e tratado com BVZ dos explantes (p < 0,05). RESULTADOS: Nas células MIO-M1, o BVZ, aumentou a expressão gênica e diminui a tradução de VEGF na concentração de 0,50 mg/mL em 24h comparado a 12h. Para o GFAP, houve um aumento da transcrição em 0,50 mg/mL em 24h comparado a 12h e aos outros grupos em 24h. Entretanto, houve diminuição da tradução para estes mesmos períodos e condições. Para a vimentina, houve aumento na transcrição em 0,50 mg/mL após 24h. Os achados de beclina-1 revelaram uma diminuição da transcrição e tradução em 0,25 mg/mL em 24h comparado a 12h. A transcrição entre os grupos do mesmo período aumentou nos grupos tratados com BVZ tanto em 12h quanto em 24h. A tradução da beclina-1 diminuiu em 0,25 mg/mL, mas aumentou em 0,50 mg/mL em 24h em relação à 12h. A comparação entre os grupos de 24h revelou aumento da tradução em 0,50 mg/mL. Para a caspase-3, houve diminuição da transcrição em 0,25 mg/mL e 0,50 mg/mL em 24h em relação a 12h e entre nos grupos tratados com BVZ em 24h. A tradução revelou um aumento em 0,50 mg/mL em 24h em relação a 12h. No fosfacam, houve diminuição da transcrição em 0,50 mg/mL em 24h comparado a 12h e entre os grupos tratados com BVZ e controles para 12h e 24h. A transcrição de neurocam diminuiu em 0,25 mg/mL e 0,50 mg/mL em 24h comparado a 12h e entre os grupos tratados com BVZ e controles em 12h e 24h. A tradução aumentou em 0,50 mg/mL em 24h em relação a 12h, mas diminuiu entre os grupos em 24h. Nos explantes, a transcrição e tradução de VEGF diminuiram no grupo tratado com BVZ após 48h. CONCLUSÃO: Nossos resultados relacionados às células MIO-M1 e ao explante de ratos, in vitro, nos permitem aventar o possível comprometimento ocasionado pela depleção do VEGF pelo BVZ na homeostase do tecido retiniano, in vivo, interferindo nas moléculas envolvidas na morte e diferenciação celular e na neuroproteção em indivíduos em fase adulta e recém-nato / Backgraound: Vasoproliferative retinal disorders such as age-related macular, degeneration, diabetic retinopathy and retinopathy of prematurity are major causes of blindness in the world. In recent years, intravitreal injections of drugs with antiangiogenic action, as bevacizumab, have been very useful for both patients in adulthood and in newborns. However, experimental studies, in vivo and in vitro, suggest that antiangiogenic drugs may promote side effects in cellular proceedings, interfering directly in physiological mechanisms of cellular proliferation, differentiation and death. POURPOSE: Investigate the bevacizumab effects in transcription and translation processes of gliosis, GFAP and vimentin, cellular death markers, caspase-3 and beclin-1, and proteoglycans involved in retinal tissue maintenance and development, neurocan, phosphacan and syndecan-3. METHODS: Two experimental models were used on this research: cellular lineage of adult and human Müller glial cell(MIO-M1) were cultivated on D-MEM medium with 0,25 and 0,50 mg/mL bevacizumab for 12 and 24 hours, and two days old rat retinal explants submitted to 0,50 mg/mL for 48 hours. During this period were stored in laboratory ovens at 5% carbon dioxide pressure and 37 °C average temperature. Molecular techniques were used to evaluate gene expression and protein content. Protein assessments were performed by immunocytochemistry and western blotting analysis, while Real Time PCR was used to measure mRNA content. ANOVA tests one factor were applied to compare the control and BVZ groups of the same period (12h or 24h) and t test from Student to compare the same conditions of 12h and 24h, and to compare the control and BVZ retinal explants groups (p<0.05). RESULTS: At MIO-M1 cells, BVZ increased the gene expression and reduced the translation of VEGF at concentration of 0.50 mg / mL in 24 hours compared to 12 hours. For GFAP, there was an increase of transcription at 0.50 mg / mL in 24 hours compared to 12 hours and to the other groups at 24 hours. However, there was a decrease in translation for these same periods and conditions. For vimentin, there was an increase in transcription at 0.50 mg / mL after 24 hours. The beclin-1 findings revealed a decrease of transcription and translation at 0.25 mg / ml compared at 24 h compared to 12h. Transcription among groups increased in BVZ treated groups at 12h and 24h. The translation of beclin-1 decreased at 0.25 mg / ml, but increased at 0.50 mg / mL at 24 hours compared to 12 hours. The comparison between the groups at 24h revealed an increased in translation at 0.50 mg / mL. For caspase-3, there was a decrease in transcription at 0.25 mg / ml and 0.50 mg / ml at 24 compared to 12 hours and among BVZ treated groups at 24h. Translation revealed an increase at 0.50 mg / mL at 24 hours compared to 12 hours. For fosfacam, there was a decreased in transcription at 0.50 mg / mL in 24 hours compared to 12 hours and among BVZ treated groups and controls at 12h and 24h. The transcription of neurocam decreased at 0.25 mg / ml and 0.50 mg / ml at 24 hours compared to 12 hours and among BVZ treated groups and controls at 12h and 24h. Translation increased at 0.50 mg / mL at 24 compared to 12 hours, but decreased among the groups at 24 hours. For explants, transcription and translation of VEGF decreased in the BVZ group treated after 48h. CONCLUSION: Our results related to the MIO-M1 cells and explants of rats,in vitro, allow us to suggest the possible impairment caused by depletion of VEGF by BVZ in the homeostasis of retinal tissue, in vivo, interfering in the molecules involved in cell death and cell differentiation and neuroprotection in individuals in adulthood and newborns

INTRODUÇÃO: As doenças retinianas associadas à neovascularização, tais como a degeneração macular relacionada à idade e as retinopatias diabética e da prematuridade são as principais e mais importantes causas da cegueira em todo o mundo. Nos últimos anos, injeções intravítreas de fármacos com ação antiangiogênica, como o bevacizumabe (BVZ), têm sido de grande valia tanto em pacientes na fase adulta quanto nos recém-natos. Todavia, estudos experimentais in vitro e in vivo sugerem que essas drogas promovam efeitos adversos sobre alguns processos celulares, interferindo diretamente em mecanismos fisiológicos que mantém a homeostase do tecido retiniano, incluindo os mecanismos de proliferação, diferenciação e morte celular. OBJETIVO: investigar o efeito do BVZ nos processos de transcrição e tradução de marcadores da gliose: GFAP e vimentina, de morte celular, caspase-3 e beclina-1, e dos proteoglicanos relacionados à manutenção e desenvolvimento de tecido retiniano: neurocam, fosfacam e sindecam-3. MÉTODOS: Dois modelos experimentais foram usados nesse estudo: 1) linhagem celular de Müller de Glia humana adulta (MIO-M1), cultivada em meio de cultura D-MEM na presença e ausência de BVZ por 12 e 24 horas nas concentrações de 0,25 mg/mL e 0,50 mg/mL e 2) explantes de retinas de ratos 2 dias pós-nascidos submetidos à 0,50 mg/mL da droga por 48 horas. Durante este período foram mantidos a 5% de dióxido de carbono à temperatura de 37°C. A análise de proteínas foi realizada por imunocitohistoquímica e Western Blotting e a expressão de RNAm, pela reação em cadeia da polimerase em tempo real (PCR Real Time). Foi utilizado o Teste de ANOVA - fator único para a comparação entre os grupos controle e tratados com BVZ de um mesmo período (12h ou 24h) e o teste t de Student para a comparação entre as mesmas concentrações de 12h e 24h, e para a comparação entre os grupos controle e tratado com BVZ dos explantes (p < 0,05). RESULTADOS: Nas células MIO-M1, o BVZ, aumentou a expressão gênica e diminui a tradução de VEGF na concentração de 0,50 mg/mL em 24h comparado a 12h. Para o GFAP, houve um aumento da transcrição em 0,50 mg/mL em 24h comparado a 12h e aos outros grupos em 24h. Entretanto, houve diminuição da tradução para estes mesmos períodos e condições. Para a vimentina, houve aumento na transcrição em 0,50 mg/mL após 24h. Os achados de beclina-1 revelaram uma diminuição da transcrição e tradução em 0,25 mg/mL em 24h comparado a 12h. A transcrição entre os grupos do mesmo período aumentou nos grupos tratados com BVZ tanto em 12h quanto em 24h. A tradução da beclina-1 diminuiu em 0,25 mg/mL, mas aumentou em 0,50 mg/mL em 24h em relação à 12h. A comparação entre os grupos de 24h revelou aumento da tradução em 0,50 mg/mL. Para a caspase-3, houve diminuição da transcrição em 0,25 mg/mL e 0,50 mg/mL em 24h em relação a 12h e entre nos grupos tratados com BVZ em 24h. A tradução revelou um aumento em 0,50 mg/mL em 24h em relação a 12h. No fosfacam, houve diminuição da transcrição em 0,50 mg/mL em 24h comparado a 12h e entre os grupos tratados com BVZ e controles para 12h e 24h. A transcrição de neurocam diminuiu em 0,25 mg/mL e 0,50 mg/mL em 24h comparado a 12h e entre os grupos tratados com BVZ e controles em 12h e 24h. A tradução aumentou em 0,50 mg/mL em 24h em relação a 12h, mas diminuiu entre os grupos em 24h. Nos explantes, a transcrição e tradução de VEGF diminuiram no grupo tratado com BVZ após 48h. CONCLUSÃO: Nossos resultados relacionados às células MIO-M1 e ao explante de ratos, in vitro, nos permitem aventar o possível comprometimento ocasionado pela depleção do VEGF pelo BVZ na homeostase do tecido retiniano, in vivo, interferindo nas moléculas envolvidas na morte e diferenciação celular e na neuroproteção em indivíduos em fase adulta e recém-nato / Backgraound: Vasoproliferative retinal disorders such as age-related macular, degeneration, diabetic retinopathy and retinopathy of prematurity are major causes of blindness in the world. In recent years, intravitreal injections of drugs with antiangiogenic action, as bevacizumab, have been very useful for both patients in adulthood and in newborns. However, experimental studies, in vivo and in vitro, suggest that antiangiogenic drugs may promote side effects in cellular proceedings, interfering directly in physiological mechanisms of cellular proliferation, differentiation and death. POURPOSE: Investigate the bevacizumab effects in transcription and translation processes of gliosis, GFAP and vimentin, cellular death markers, caspase-3 and beclin-1, and proteoglycans involved in retinal tissue maintenance and development, neurocan, phosphacan and syndecan-3. METHODS: Two experimental models were used on this research: cellular lineage of adult and human Müller glial cell(MIO-M1) were cultivated on D-MEM medium with 0,25 and 0,50 mg/mL bevacizumab for 12 and 24 hours, and two days old rat retinal explants submitted to 0,50 mg/mL for 48 hours. During this period were stored in laboratory ovens at 5% carbon dioxide pressure and 37 °C average temperature. Molecular techniques were used to evaluate gene expression and protein content. Protein assessments were performed by immunocytochemistry and western blotting analysis, while Real Time PCR was used to measure mRNA content. ANOVA tests one factor were applied to compare the control and BVZ groups of the same period (12h or 24h) and t test from Student to compare the same conditions of 12h and 24h, and to compare the control and BVZ retinal explants groups (p<0.05). RESULTS: At MIO-M1 cells, BVZ increased the gene expression and reduced the translation of VEGF at concentration of 0.50 mg / mL in 24 hours compared to 12 hours. For GFAP, there was an increase of transcription at 0.50 mg / mL in 24 hours compared to 12 hours and to the other groups at 24 hours. However, there was a decrease in translation for these same periods and conditions. For vimentin, there was an increase in transcription at 0.50 mg / mL after 24 hours. The beclin-1 findings revealed a decrease of transcription and translation at 0.25 mg / ml compared at 24 h compared to 12h. Transcription among groups increased in BVZ treated groups at 12h and 24h. The translation of beclin-1 decreased at 0.25 mg / ml, but increased at 0.50 mg / mL at 24 hours compared to 12 hours. The comparison between the groups at 24h revealed an increased in translation at 0.50 mg / mL. For caspase-3, there was a decrease in transcription at 0.25 mg / ml and 0.50 mg / ml at 24 compared to 12 hours and among BVZ treated groups at 24h. Translation revealed an increase at 0.50 mg / mL at 24 hours compared to 12 hours. For fosfacam, there was a decreased in transcription at 0.50 mg / mL in 24 hours compared to 12 hours and among BVZ treated groups and controls at 12h and 24h. The transcription of neurocam decreased at 0.25 mg / ml and 0.50 mg / ml at 24 hours compared to 12 hours and among BVZ treated groups and controls at 12h and 24h. Translation increased at 0.50 mg / mL at 24 compared to 12 hours, but decreased among the groups at 24 hours. For explants, transcription and translation of VEGF decreased in the BVZ group treated after 48h. CONCLUSION: Our results related to the MIO-M1 cells and explants of rats,in vitro, allow us to suggest the possible impairment caused by depletion of VEGF by BVZ in the homeostasis of retinal tissue, in vivo, interfering in the molecules involved in cell death and cell differentiation and neuroprotection in individuals in adulthood and newborns