Resumo Fundamento: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. Objetivos: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. Métodos: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. Resultados: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. Conclusões: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.

Abstract Background: Cardiovascular disease is the major cause of death worldwide. Hypoxia-mediated apoptosis in cardiomyocytes is a major cause of cardiovascular disorders. Treatment with vascular endothelial growth factor (VEGF) protein has been tested but operational difficulties have limited its use. However, with the advancements of gene therapy, interest has risen in VEGF-based gene therapy in cardiovascular disorders. However, the precise mechanism by which VEGF replenishment rescues post-hypoxia damage in cardiomyocytes is not known. Objectives: To investigate the effect of post-hypoxia VEGF121 expression using neonatal rat cardiomyocytes. Methods: Cardiomyocytes isolated from neonatal rats were used to establish an in vitro model of hypoxia-induced cardiac injury. The effect of VEGF overexpression, alone or in combination with small-molecule inhibitors targeting calcium channel, calcium sensitive receptors (CaSR), and calpain on cell growth and proliferation on hypoxia-induced cardiomyocyte injury were determined using an MTT assay, TUNEL staining, Annexin V/PI staining, lactate dehydrogenase and caspase activity. For statistical analysis, a value of P<0.05 was considered to be significant. Results: The effect of VEGF121 was found to be mediated by CaSR and calpain but was not dependent on calcium channels. Conclusions: Our findings, even though using an in vitro setting, lay the foundation for future validation and pre-clinical testing of VEGF-based gene therapy in cardiovascular diseases.

OBJETIVO: Revelar a expressão e os possíveis papéis da aquaporina 9 (AQP9) no cérebro de ratos após lesão cerebral traumática (LCT) grave. MÉTODOS: Foram utilizados: determinação do conteúdo cerebral de água, corante cloreto de tetrazólio, corante azul de Evans, imunoistoquímica (IHQ), imunofluorescência (IF), western blot e PCR em tempo real. RESULTADOS: O conteúdo cerebral de água alcançou o primeiro e o segundo (o mais alto) picos após 6 e 72 horas. A função da barreira hematoencefálica se mostrou muito prejudicada após 6 horas da LCT. A pior isquemia cerebral ocorreu após 72 horas da LCT. Astrócitos AQP9 positivos e neurônios no hipotálamo foram detectados difusamente pela IHQ e IF após LCT. A abundância de AQP9 e de sua mRNA aumentou após LCT e alcançou dois picos após 6 e 72 horas, respectivamente, da LCT. CONCLUSÕES: AQP9 aumentada pode contribuir para a eliminação de água e lactato em excesso na fase precoce da LCT. Astrócitos difusamente localizados AQP9 positivos podem ajudar a entrada do lactato nos neurônios, promovendo edema cerebral celular na fase tardia da LCT. Neurônios AQP9 positivos sugerem que AQP9 tem um papel no equilíbrio energético após LCT.

OBJECTIVE: To reveal the expression and possible roles of aquaporin 9 (AQP9) in rat brain, after severe traumatic brain injury (TBI). METHODS: Brain water content (BWC), tetrazolium chloride staining, Evans blue staining, immunohistochemistry (IHC), immunofluorescence (IF), western blot, and real-time polymerase chain reaction were used. RESULTS: The BWC reached the first and second (highest) peaks at 6 and 72 hours, and the blood brain barrier (BBB) was severely destroyed at six hours after the TBI. The worst brain ischemia occurred at 72 hours after TBI. Widespread AQP9-positive astrocytes and neurons in the hypothalamus were detected by means of IHC and IF after TBI. The abundance of AQP9 and its mRNA increased after TBI and reached two peaks at 6 and 72 hours, respectively, after TBI. CONCLUSIONS: Increased AQP9 might contribute to clearance of excess water and lactate in the early stage of TBI. Widespread AQP9-positive astrocytes might help lactate move into neurons and result in cellular brain edema in the later stage of TBI. AQP9-positive neurons suggest that AQP9 plays a role in energy balance after TBI.

The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH) gene was investigated in 59 children with phenylketonuria (PKU) and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%), followed by Ivs7 +2T>A (5.1%) and T278I (2.5%). G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.