An Effective Isolation of the Vascular Endothelial and Smooth Muscle Cells from the Mouse Aorta / 대한해부학회지

The mechanism of the disease such as artherosclerosis is easily elucidated by the comparison among cells isolated from each aorta of knockout mouse and wild type mouse, respectively. This study was aimed at effectively harvesting the endothelial and smooth muscle cells from 4~6 weeks old wild type C57BL/6J mouse aorta. The tunica adventitia was completely removed to get the aortic tissues only consisting of the tunica intima and the tunica media under the stereoscope. These aortic tissues were treated with type I collagenase or type II collagenase solution, respectively, and then the endothelial or smooth muscle cell was isolated. CD31 marker of the endothelial cell and alphasmooth muscle actin marker of the smooth muscle cell were identified with confocal microscope. The percentages of the labelled cells by each marker represented the extent of purification of endothelial or smooth muscle cells, respectively, for harvested cells according to the collagenase solutions. 70~80% of culture vessel was covered with the endothelial cells 10 days after the treatment of the type I collagenase solution, while 40~50% of culture vessel covering with the cells after the treatment of the type II collagenase solution. 70~80% of culture vessel was covered with the smooth muscle cell regardless of the type of the collagenase solution on the 13th day. Percentages of the CD31 positive cells after the treatment with the type I or the type II collagenase solution was 91.1+/-.865%** and 86.4+/-.641%, respectively (**p <0.05, n=5). Percentages of the alphasmooth muscle actin labelled cells after the treatment with the type I or the type II collagenase solution were 87.9+/-.713% and 86.6+/-.778%, respectively, and these values were not significantly different. Taken together, the aortic tissues using the type I collagenase solution comparing with using the type II collagenase solution were much more effective in the isolation of the endothelial cells

Zinc deficiency decreased cell viability both in endothelial EA.hy926 cells and mouse aortic culture ex vivo and its implication for anti-atherosclerosis

Zinc plays a protective role in anti-atherosclerosis but the clear mechanism has not been proposed yet. In the present study, we evaluated whether zinc modulates atherosclerotic markers, VACM-1 and ICAM-1 and cell viability both in endothelial cells in vitro and mouse aortic cell viability ex vivo. In study 1, as in vitro model, endothelial EA.hy926 cells were treated with TNFalpha for 5 hours for inducing oxidative stress, and then treated with Zn-adequacy (15 micrometer Zn) or Zn-deficiency (0 micrometer Zn) for 6 hours. Pro-atherosclerosis factors, VCAM-1 and ICAM-1 mRNA expression and cell viability was measured. In study 2, as ex vivo model, mouse aorta ring was used. Mourse aorta was removed and cut in ring then, cultured in a 96-well plate. Aortic ring was treated with various TNFalpha (0-30 mg/ml) and intracellular zinc chelator, N, N, N', N', -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 0-30 microM) for cellular zinc depletion for 2 days and then cell viability was measured. The results showed that in in vitro study, Zn-adequate group induced more VCAM-1 & ICAM-1 mRNA expression than Zn-deficient group during 6-hour zinc treatment post-5 hour TNF-alpha treatment, unexpectedly. These results might be cautiously interpreted that zinc would biologically induce the early expression of anti-oxidative stress through the increased adhesion molecule expression for reducing atherosclerotic action, particularly under the present 6-hour zinc treatment. In ex vivo, mouse aortic ring cell viability was decreased as TNF-alpha and TPEN levels increased, which suggests that mouse aortic blood vessel cell viability was decreased, when oxidative stress increases and cellular zinc level decreases. Taken together, it can be suggested that zinc may have a protective role in anti-atherosclerosis by cell viability in endothelial cells and aorta tissue. Further study is needed to clarify how pro-atherosclerosis molecule expression is modulated by zinc.