The Role Of Estrogen Mediated PI(3)K Pathway In The Proliferation Of Endothelial Progenitor Cells (EPCs) In Response To Estrogen

Section OneThe Isolation and Culture of Endothelial Progenitor Cells in Peripheral Blood Abstract Objective: To investigate how to isolate and culture endothelial progenitor cells and how to make it differentiate into endothelial cells. Methods: mononuclear cells were isolated from peripheral blood of younge volunteers with Ficoll-paque by density gradient centrifugation. MNCs were cultured in M199 supplemented with VEGF. Every 3-4 days, culture media was changed. After 5 days of culture, attached cells were detached and stained with DiI-LDL and FITC-UEA, and then observed under fluromicroscope. Results:Attached cells were observed after 3 or 4 days of culture and cord-like structure were noticed after 16 days or so. According to immunofluorescence, more than 90% attached cells were double positive fluorescence. Conclusions: Endothelial progenitor cellsenriched in peripheral blood and can differentiate into endothelial cells under certain conditions. Section TwoThe effect of estrogen on proliferation and migration endothelial progenitor cells(EPCs)Abstract Aim is to study the effect of estrogen(E₂) on proliferation and migration of EPCs and its' relation with the change of culture time and concentration of estrogen. We also want to know changes of cell cycle and expression of specific endothelial surface antigen, Flt-1,KDR and VE-cadherin at mRNA and protein level, in response to estrogen. Method MNCs were isolated from peripheral blood of younge volunteers. Cells were cultured under endothelial cultre condition to differentiate into endothelial cells. Proliferation test(MTS analysis) and migration (transwell assay) test was performed with 7d EPCs in response to different concentrations of 17β —estradiol (10⁶ mol/L, 10⁷mol/L, 10^-8mol/L, 10^-9mol/Lor 10l0mol/L). To investigate the relation of culture time and proliferative activity and to confirm increase in proliferative activity of EPCs in response to estrogen, manual counting of EPCs was conducted with 4X 10D 5d, 7d, 9d and 13d EPCs in response to 17 3 —estradiol at a dose of 10"smol/L for 24 hours. To further understand the proliferative effect of estrogen on EPCs, cell cycle analysis was performed after 5d, 7d, 9d and 13d EPCs respectively was exposed to l(T8mol/L estradiol for 24 hours. Real-time PCR technique and flowcytometry was employed to detect expression of specific endothelial antigens Flt-1, KDR and VE-cadherin at both mRNA and protein levels after 5d, 7d, 9d, 13d and 20d EPCs respectively was exposed to l(T8mol/L estradiol for 24 hours.. Results MTS assay showed that estrogen increased EPC proliferative activity and the peak impact occurred with the stimulation of 10⁸mol/L 17 {3 —estradiol. The increase in proliferative activity was confirmed by manual counting of EPCs. 10d-14d EPCs exhibited most powerful proliferative activity. Transwell assay revealed that estrogen profoundly enhanced EPCs migration and the peak impact occurred with the stimulation of 10⁸mol/L 17 0 — estradiol. Cell cycle analysis disclosed that S phase, G2^M phase EPCs increased significantly (/XO. 05) in response to estrogen, however , Go phase cells decreased significantly. Estrogen enhances the expression of Flt-1, KDR and VE-cadherin at both mRNA and protein levels. Their expression at mRNA level aarived at highest level in 8d and lOd EPCs, in consistent with change of cell cycle. Expression at protein level was highest in 14d and 21d. Conclusion Estrogen can enhance the proliferation and migration of EPCs and increase the numbers of EPCs in S and G2^M phase concomitant with the increased expression of endothelial specific antigens, Flt-1, KDR and VE-cadherin at mRNA and protein level.Section ThreeThe role of estrogen receptor (ER) mediated activation of PI(3)K pathway in the proliferation and migration of endothelial progenitor cells (EPCs)in response to estrogenAbstract Our aim is to investigate the effect of estrogen receptor (ER) mediated PI(3)K pathway on proliferation and migration of EPCs in response toE2. Method To determine the effect of ER blocker, ICI182, 780, andPI(3)K inhibitor on proliferative activity of EPCs in response to estrogen: Four groups of 7d EPCs were used as subjects. There are 4X105EPCs in every group. In two groups, EPCs were incubated respectively with ER blocker, ICI182, 780 ,and PI(3)K inhibitor, LY294002 at 37°C for 1 hour ,then 10 ⁸mol/L E2BSA was added into culture medium for 24h. In the other two groups, after EPCs were incubated respectively with the same dose of control vehicle for lh, 10⁸mol/L E2BSA and the same dose of control vehicle was added into culture medium respectively for 24 hours. Manual counting the numbers of EPCs was performed. Four groups of EPCs, containing 4X 105cells in every group, were incubated with ER blocker, ICI182, 780 , and PI(3)K inhibitor, LY294002 and two same doses of control vehicle for 1 hour and put into upper layer of transwell chamber respectively . Three doses of 600 M-L cultur medium containing 10⁸mol/L 17 P -estradiol and 600 ML culture medium without 17P-estradiol were added into the lower layer of transwell chamber for 24 hours in turn. Mannual counting of cell numbers in the lower layer of every transwell chamber was performed. Four groups of EPCs, containing 4X105 cells in every group, were incubated with ER blocker, ICI182, 780 , and PI(3)K inhibitor, LY294002 and two same doses of control vehicle for 1 hour respectively and then three doses of 10" 8mol/L 17 3-estradiol and control vehicle was added into into culture medium. Expression of Flt-1, KDR and VE-cadherin at mRNA and protein level was determined with Real-time and RT-PCR technique respectively. Results Compared with control group, the increase of EPCs numbers in response to estrogen was abolished after incubation with ICI182, 780 and LY294002. When compared with control group, no significant difference in the increase of EPCs numbers in response to estrogen was found after EPCs treated with ICI182, 780 and LY294002. Expression of Flt-1, KDR and VE-cadherin at mRNA level and protein level in response to estrogen was abolished after EPCs were incubated with ICI182, 780 and LY294002. Conclusion: ER mediated PI(3)K pathway plays an important role in proliferation and migration of EPCs in response to estrogen.