Endotélio venoso: estabelecimento e caracterização de culturas primárias e a produção de fatores derivados do endotélio de veia cava e veia porta de ratos

• Ano de defesa: 2017
• Autor(a) principal: Trindade, Marcio Renato [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo
• Palavras-chave em Português: Endothelium; Cell culture; Vein; Prostaglandins; Rats; Endotélio; Cultura celular; Veia; Prostaglandinas; Ratos
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5636120; http://repositorio.unifesp.br/handle/11600/50268

The endothelial cell plays an essential role in vascular control by the release of Endothelium- Derived Relaxing Factors (EDRFs) [nitric oxide (NO), prostacyclin (PGI2) and endotheliumderived hyperpolarizing factor (EDHF)], and Endothelium-Derived Constrictor Factors (EDCFs) [prostaglandin H2 (PGH2) and Thromboxane A2 (TXA2), reactive oxygen species, Angiotensin II (Ang II) and Endothelin-1 (ET-1)]. Most of the studies related to EDRFs and EDCFs refers to arterial territories and little is known about distinct venous territories, such as vena cava (VC) and portal vein (PV). The present study investigated the physiology of the venous endothelial cell using primary cultures of VC and PV of rats. Venous segments were sectioned longitudinally, plated with the endothelial side facing down, covered with culture medium and kept in a 5% CO2 incubator at 37°C for 5 days. After removal of the explants, the cells were subcultured and cultures were studied between 4th and 5th passages. The characterization of the cultures was performed by immunocytochemistry for the specific markers von Willebrand Factor (vWF) and endothelial NO synthase (eNOS), and positive staining was also observed for ET-1 receptor (ETB) and Ang II receptors (AT1 and AT2). Basal NO production was observed in endothelial cultures pre-incubated with the DAF-2DA fluorescent probe and observed in confocal microscopy, and complemented by eNOS expression analysis determined by western blot (n = 4). Prostanoids production was quantified in the culture supernatants by enzyme-immunoassay technique (n = 5 to 6). Endothelial cultures of VC and PV showed similar values in the baseline rates of PGH2 (0.75 ± 0.04 ng/mL vs 0.76 ± 0.02 ng / mL, respectively); these levels were increased after stimulation with Ang II [1μM] (1.00 ± 0.07 * ng/mL for VC vs 1.13 ± 0.07 * ng/mL for PV). Baseline levels of PGI2 were different between cultures, with values of 29.12 ± 1.24 ng/mL for VC vs 35.10 ± 1.27 * ng/mL for PV. Ang II stimulation [1μM] increased PGI2 production in VC cells (36.50 ± 1.89 * ng/mL) but not in PV (37.70 ± 1.38 ng/mL) (* P <.05); however, COX expression (1 and 2) was similar between cultures (determined by western blot, n = 7). Stimulation with ET-1 [1μM] did not modify the release of prostanoids in both cultures. In summary, the venous endothelium can be isolated and studied through primary cultures; these cells, when studied in vitro, produce detectable amounts of NO and prostaglandins. The participation of PGI2 in the venodilation can be pronounced in PV when compared to VC. The consistent increase in PHG2 production by Ang II in VC and PV endothelium indicates the important endothelial modulation in the venoconstriction. The present data reveal new aspects of the vascular physiology, and may contribute to a better understanding of the venous responses to the blocking agents of the Renin Angiotensin System.