Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes.

PURPOSE. Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS.Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared.RESULTS. No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls.CONCLUSIONS. Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue. (Invest Ophthalmol Vis Sci. 2012;53:7904-7911)

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Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Purpose VEGF is an important in nervous development, promoting neurogenesis and glial cell growth. Bevacizumab (BVZ), an anti‐VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. In this study we investigate BVZ effect on proteoglycans and on cell death, proliferation and differentiation in newborn rat retina. Methods Retinal explants of sixty 2‐day‐old Lister hooded rats were maintained in culture media with or without BVZ for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA), caspase‐3 and beclin‐1 and vimentin and glial fibrillary acidic protein (GFAP). Gene expressions for PCNA, caspase‐3, beclin‐1, vimentin, GFAP and for proteoglycans were quantified by real‐time reverse‐transcription polymerase chain reaction. Results from treatment and control groups were compared Results No significant difference in staining intensity of PCNA, caspase‐3, beclin‐1 and GFAP or in the mRNA levels of PCNA, caspase‐3, beclin‐1, vimentin, neurocan and phosphacan was observed. However, a significant increase in vimentin levels, decrease in GFAP and in syndecan‐3 mRNA expression were observed in BVZ‐treated retinas compared to controls. Conclusion Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation (by interfering with vimentin and GFAP). It can also alter syndecan‐3 gene expression. Thus, we suggest anti‐VEGF agents be used with caution in developing retinal tissue.

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Aos meus pais, Jarbas e Divani, pelo amor incondicional, paciência interminável e palavras certas e carinhosas que sempre estimulam. Vocês são meu espelho, meu orgulho e meus amores. Por toda paciência, plenitude, calma e investimento durante os anos de desenvolvimento deste projeto. Sem vocês nada disso teria sentido. Aos meus irmãos, Frederico e Gláucia, pelas conversas, risadas e cumplicidade. Aos meus sobrinhos, Thaís, Letícia e Frederico, por me mostrarem a simplicidade da vida e a minha importância em suas vidas por um sorriso e um abraço depois de algum tempo sem me ver. AGRADECIMENTOS vi Agradecimentos Ao Prof. Dr. Mário Luiz Ribeiro Monteiro, orientador e chefe do programa de Pós-Graduação do Departamento de Oftalmologia, por ter autorizado meu ingresso no doutorado e pela orientação na publicação deste trabalho. Á minha co-orientadora, Dr a Monique Matsuda, pela ajuda, amizade e convívio diário. Á minha prima, amiga e colaboradora do projeto, Dr a Mônica Valéria Marquezini que, além de ter me ensinado muito do que sei, acolheu-me em sua casa no primeiro ano deste trabalho e me ensinou que a inteligência emocional é tão importante quanto capacidade profissional. Agradeço imensamente a orientação e a escrita desde trabalho. Á Dr a Odaly Toffoletto, pela ajuda na formatação da tese e análise crítica e científica dos resultados e principalmente por agregar novas visões deste trabalho. À Prof a Dr a Nádia Campos de Oliveira Miguel, da Universidade Federal do Rio de Janeiro, por sua colaboração no projeto e convivência durante este período. Aos colegas de laboratório Renato Batista Paceli, Robson Seriani e Bryan Hudson Hossy, que compartilharam comigo os melhores anos profissionais da minha vida, contribuindo para meu crescimento intelectual. Á Maria Paula Elias Martins, Paulinha, pelas conversas em dias difíceis e pela paciência em me escutar muitas vezes sem precisar falar. À Regina Ferreira de Almeida, secretaria da pós-graduação, pela atenção e pontualidade nas informações discentes necessárias para esta tese. Ao Ricardo Issa, por ter me acolhido em sua casa, pela companhia e parceria nos meus momentos difíceis. Aos laboratórios que cederam seus espaços para que pudéssemos realizar parte dos nossos experimentos: ao laboratório de Oncologia, por ter disponibilizado o aparelho de PCR Real Time; ao laboratório de Neurologia que, por muitos anos, nos permitiu o uso do aparelho de nanoespectofotometria, ao laboratório de Nefrologia experimental, pelo uso irrestrito do fotodocumentador para Western Blotting e ao laboratório de Neuro-histologia e vii Ultraestrutura da Universidade Federal do Rio de Janeiro-UFRJ, pela realização integral das reações de imunocitohistoquimica e análise por microscopia confocal a laser. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo suporte financeiro por meio da bolsa de Doutorado demanda social. Às Fundações de Amparo à Pesquisa do Estado de São Paulo (FAPESP, N° 2011/12271-3) e do Estado do Rio de Janeiro (FAPERJ, N° E-26/111.777/2012) e ao Conselho Na...