Figure 2 |. Comparison of content of matrix vesicles (MVs) isolated from cells or media.
MVs isolated from the same 4 sources as in Figure 1 were analyzed for content of annexin II(36 kDa), annexin V (36 kDa), annexin VI (47–51 kDa), fetuin-A (59 kDa), CD63 (core protein, MV 26 kDa), CD81 (22–26 kDA), and CD9 (24 kDa) by Western blot (a) with quantification of band intensity normalized by Ponceau S (b). MVs isolated from cells had increased expression of annexins and CD63, but neglible fetuin-A compared to that from MVs isolated from media. Isolation from cells in high-phosphorus (calcifying) media in general increased expression. In contrast, in the MVs isolated from the media, there was high fetuin-A content, high levels of CD81 and CD9, and no differences when isolated from VSMC with and without calcifying (high-phosphorus) media. Transmission electron microscopy revealed that both cellular (c, left) and media MV (c, right) show uniform size of 100 mm–diameter, membrane-bound vesicles. Data are shown as mean ± SD (n = 3 separate experiments). Cal, calcified (MV isolated from CKD VSMC in high-phosphorus media); Ct, control (MV isolated from CKD VSMC in normal-phosphorus media); *P < 0.05, Ct MV versus Cal MV same source (cellular MV or media MV); #P < 0.05, cellular MV versus media MV, same condition (control or calcifying/high-phosphorus). Black bar = 200 nm (c). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.