| Recently, it was proposed that endogenous formaldehyde(FA) may be involved in endothelial damage, and may be a potential factor of vulnerability of atherosclerosis. Endogenous FA is derived from methylamine(MA) deamination in vivo. Researches have showed that MA, in the presence of SSAO, is toxic to human endothelial cells and forms patch-like lesions. But till now, there is no research focusing on the relation between concentration of FA and the extent of endothelial injury. In the present study we investigated the association between the concentration of FA and endothelial injury by detecting DNA-protein cross-links (DPC) levels, cell viability, morphological effects, and cytoskeleton changes. Procedures and methods:1. Isolated and cultured rat aorta endothelial cells by explant technique. Then, the cells were identified by staining with a specific antibody to von-Willebrand Factor by using the immunohistochemistry (S-P method). We modified the explant technique by introducing short-time heat treatment (60 C , 2s), to inhibit the outgrowth of adventitial fibroblasts.2. Compared the purity of ECs between heat treatment culture and those without treatment by immunostaining.3. The cells obtained on passages 3 and 4 were used for the experiment. The cell were treated with either various concentrations of FA (0, 0.01, 0.05, 0.1, 0.5, 1, 2mM) for 1.5h or 0.05mM FA and 0.1mM FA for different duration of time (0, 0.5, 1, 1.5, 2, 4h). Then cells were collected and using a KC1-SDS precipitation assay for DPC determination.4. After treatment (as above), the culture media were collected and determined for the activity of lactate dehydrogenase (LDH) by using cytotoxic detection kit.5. After treatment with various concentrations of FA for 1.5h, the cells were observed under the inverted phase contrast microscope.6. After treatment with various concentrations of FA for 1.5h, the F-actin of cells was labeled by FITC-Phalloidin, and observed for the changes of cytoskeleton withConfocal Laser Scaning Microscope (CLSM). Results:1. Endothelial cells of rat aorta exhibited polygonal, when confluency was reached, they exhibit a cobblestone pattern. After immunostaining, ECs showed brownish yellow positive reaction in cytoplasm.2. After heat treatment, the ratio of endothelial cells/total cells (x s) rose from 0.678 0.154 to0.431 + 0.194 (P <0.05)03. Incubation of RAECs with 0~2mM FA resulted in cocentration-dependent increases in DPC levels. The DNA in the cross-links fraction(x +s ) were 1.43 0.63, 2.17 1.44, 3.27 1.26, 6.23+3.22, 8.12 2.49, 9.57 1.87, 13.34+1.82%, respectively; after treatment with O.OSmM FA for various duration of time, the DNA in the cross-links( x s) were 1.18 0.29, 3.07 0.37, 3.13 0.68, 3.27 1.26, 6.93 0.73, 10.15 2.91%, respectively; after treatment with 0.1 mM FA at various time point the DNA in the cross-link( x s) were 93 0.45, 3.9 1.45, 4.2 1.04, 6.23 3.22, 10.8 0.90, 18.6 4.5%, respectively. At the 1.5h time point, the DPC level increased significantly after treatment with 0.5~2mM FA(P<0.05). At the 2h and 4h time point, the DPC level increased significantly after treatment with 0.05 and 0. ImM FA(PO.Ol).4. After treatment of RAECs with various concentration of FA for 1.5h, the released LDH (x + s) were 3.26 3.62, 2.01 1.36, 2.19 1.10, 3.09+1.48, 4.59 2.13, 5.97 + 4.16, 11.48 + 2.26%, respectively; after treatment with 2mM FA, the released LDH increased significantly(P<0.05). After treatment with 0.05 and O.lmM FA at various time points, the released LDH did not exhibit elevated levels compared with controls.5. The morphology of RAECs didn't change after treatment with 0.0ImM and O.OSmM FA for 1.5h; after 0.1~2mM FA treatment, the morphological changes were characterized by the disruption of cell-cell contacts, cell rounding, and the formation of gaps in the monolayer.6. Normal RAECs display a faint ring of polymerized actin at their periphery. RAECs cytoskeleton was disorganized after treatment with 0. l~2mM FA.Conclusion:1. Successful...
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