Ing fibers exhibited diffuse Flk-1 and Flt-1 labeling (Figure 2D). In mature fibers, at the same time as in regenerated CD66e/CEACAM5 Proteins Accession muscle at 14 days right after ischemia, immunostaining for Flk-1 and Flt-1 returned to the basal level observed in normoperfused muscle (Figure 2E). VEGF expression in skeletal muscle was also investigated. In normoperfused hindlimbs VEGF immunostaining was discovered in satelliteVEGF, Flk-1, and Flt-1 Expression Throughout in Vitro Myogenic Differentiation of C2C12 CellsThe sequence of events involved in muscle regeneration was reproduced in an in vitro model of differentiation. C2C12 myoblasts grow and divide when cultured in GM and, after 48 two in DM, cells fuse to form multinucleated myotubes. Within this experimental model, it was investigated whether or not Flk-1, Flt-1, and VEGF expression varied through differentiation as observed in in vivo throughout muscle regeneration (Figure 2). Western blot analysis of C2C12 lysates showed that when myoblasts were induced to differentiate by altering from GM to DM both Flk-1 and Flt-1 proteins markedly decreased more than a 5-day time period (Figure 5A). Even so, Flt-1 but not Flk-1 was nonetheless detectable at day 5 of differentiation. These adjustments in VEGF receptor expression had been paralleled by a progressive improve in myosin heavy chain expression (MyHC), constant with all the improve in differentiation of C2C12 cells (Figure 5A). Further, after five days in DM, a sizable numberVEGF Receptors Expression in Skeletal Muscle 1421 AJP October 2003, Vol. 163, No.Figure two. Expression of VEGF and its receptors in skeletal muscle cells in vivo. Flk-1 and Flt-1 expression in normoperfused mouse skeletal muscle (A) and in vascular structures (B). Serial muscle sections were immunostained for Flk-1 and Flt-1. Good cells, indicated by arrowheads, have been identified as satellite cells by their immunoreactivity with M-cadherin antibody. Insets show higher-power photomicrographs of satellite cell. Control immunostaining was performed by omitting the primary antibody. CD52 Proteins Source Magnification, 40 (inset 100); bar, 25 m. Time-course of Flk-1 and Flt-1 expression (C to E). Serial sections from hindlimbs had been obtained at 3 days (C), 7 days (D), and 14 days (E) following the induction of ischemia. Flk-1 and Flt-1 had been expressed in activated satellite cells as identified by desmin labeling (C); 7 days following ischemia Flk-1 and Flt-1 had been expressed in regenerating myotubes (D) as well as the expression of each receptors decreased at day 14 (E), when the regenerative procedure was practically comprehensive. Magnification, 40; bar, 25 m.of myotubes was observed inside the culture dishes (not shown). In additional experiments it was determined whether or not VEGF was secreted from C2C12 cells and, if that’s the case, irrespective of whether VEGF levels inside the conditioned medium (CM) varied dur-ing differentiation. CM was collected every 24 hours from expanding and differentiating C2C12 cells, and assayed for the presence of VEGF by ELISA. In GM, VEGF concentration was 550 pg/mg of protein/24 hours. Following 1 day of culture in DM, VEGF level decreased to 270 pg/mg of1422 Germani et al AJP October 2003, Vol. 163, No.Figure three. VEGF expression in skeletal muscle cells in vivo. Time-course of VEGF expression in mouse ischemic hindlimb. A: VEGF immunostaining was observed in satellite cells of normal skeletal muscle (A). VEGF protein was detected in satellite cells at day 3 (B) and in regenerating fibers at day 7 (C) immediately after femoral artery ligation. The immunostaining decreased in regenerating fibers at 14 days after ischemic in.
