The Soluble VEGF Receptor sFlt-1 Contributes to Impaired Neovascularization in Aged Mice Zhao Guangxian 1 ;W. Cheng Xian 1, 7, 8 ;Piao Limei 1, 2 ;Hu Lina 3 ;Lei Yanna 1 ;Yang Guang 1 ;Inoue Aiko 2 ;Ogasawara Shinyu 2 ;Wu Hongxian 4 ;Hao Chang-Ning 5 ;Okumura Kenji 6 ;Kuzuya Masafumi 3, 7 ; 1 Department of Cardiology, Yanbian University Hospital, Yanji, Jilin 133000, China ; 2 Department of Health Care #cod#x00026; Geriatrics, Nagoya University Graduate School of Medicine, Aichiken 4668550, Japan ; 3 Department of Public Health, Guilin Medical College, Guilin, Guangxi 541004, China ; 4 Department of Cardiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20160527, China ; 5 Department of vascular surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200126, China ; 6 Department of Cardiology, Tohno Kosei Hospital, Mizunai, Japan ; 7 Institute for Future Society, NAGOYA STREAM, Nagoya University, Nagoya, Aichiken 4668550, Japan ; 8 Division of Cardiology, Department of Internal Medicine, Kyung Hee University, Seoul 130701, Republic of Korea ; Figure 5. Effects of aging on BM EPC mobilization and cellular functions. A The flow cytometry analysis showed that the numbers of circulating c-Kit + CD31 + cells were decreased in the aged mice compared to the young mice n=6. B Progenitor cell surface makers of c-Kit and CD31 were expressed in BM-derived c-Kit + cells cultured in EGM-2 for 4 days after isolation with magnetic beads. C and D Representative images and combined quantitative data showing that aging impaired c-Kit + cell migration, invasion, orand proliferation n=5-6. E and F Yc-Kit + Cs or YCD11b + Cs and Ac-Kit + Cs or ACD11b + Cs were cultured six-well-plates in serum-free EBM-2 for the former or RPMI medium 1640 for the later under hypoxic condition plates in hypoxic cambers for 36 hr for c-Kit+ cells or 48 hr CD11b+ cells, respectively. Following collection of the culture medium Yc-Kit + CM, Ac-Kit + CM; YCD11b + CM, ACD11b + CM, the special fraction containing an approx. #cod#x0003E;70 kDa protein was isolated with 150 K and then 50 K AmiconUltra contricons, and we adjusted the protein concentration to 1.5 mgml for the cellular experiments. For the proliferation assays, 5 #cod#x000D7; 10 3 HUVECs were incubated in EBM-2 supplemented with either heated or unheated Yc-Kit + CM, Ac-Kit + CM E, YCD11b + CM, or ACD11b + CM F 20 #cod#x003BC;l100 #cod#x003BC;l EBM-2 for each, respectively, for 48 hr, and then were subjected to MTS assays. G HUVECs were incubated with VEGF-A 50 ngmL, YCD11b + CM, or ACD11b + CM 20 #cod#x003BC;l100 #cod#x003BC;l EBM-2 for both respectively for 48 hr, and then were subjected to an MTS assay. Data are mean #cod#x000B1; SEM n=5-6. #cod#x0002A; P#cod#x0003C; 0.05, #cod#x02020; P#cod#x0003C; 0.05, # P#cod#x0003C; 0.05 by one-way ANOVA and Tukey#cod#x02019;s post hoc tests. Scale bar, 50 #cod#x003BC;m. null,null,0(0),null-null. Doi:10.14336/AD.2016.09120