Ing fibers exhibited diffuse Flk-1 and Flt-1 labeling (Figure 2D). In mature fibers, at the same time as in regenerated muscle at 14 days right after ischemia, immunostaining for Flk-1 and Flt-1 returned for 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 For the duration of 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, soon after 48 2 in DM, cells fuse to type multinucleated myotubes. In this experimental model, it was investigated no matter whether Flk-1, Flt-1, and VEGF expression varied throughout differentiation as observed in in vivo for the duration of muscle regeneration (Figure 2). Western blot evaluation of C2C12 lysates showed that when myoblasts have been induced to differentiate by altering from GM to DM each Flk-1 and Flt-1 proteins markedly decreased over a 5-day time period (Figure 5A). Nonetheless, Flt-1 but not Flk-1 was nonetheless detectable at day five of differentiation. These alterations in VEGF receptor expression were paralleled by a progressive increase in myosin heavy chain expression (MyHC), constant together with the boost in differentiation of C2C12 cells (Figure 5A). Additional, just after 5 days in DM, a big numberVEGF GITR/CD357 Proteins site receptors Expression in Skeletal Muscle 1421 AJP October 2003, Vol. 163, No.Figure 2. 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 had been immunostained for Flk-1 and Flt-1. Good cells, indicated by arrowheads, had been identified as satellite cells by their immunoreactivity with M-cadherin antibody. Insets show higher-power photomicrographs of satellite cell. Manage immunostaining was performed by omitting the primary antibody. 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 three days (C), 7 days (D), and 14 days (E) following the induction of ischemia. Flk-1 and Flt-1 were expressed in activated satellite cells as identified by desmin labeling (C); 7 days following Gastrin Proteins Biological Activity ischemia Flk-1 and Flt-1 had been expressed in regenerating myotubes (D) and also the expression of both receptors decreased at day 14 (E), when the regenerative procedure was practically total. Magnification, 40; bar, 25 m.of myotubes was observed inside the culture dishes (not shown). In more experiments it was determined regardless of whether VEGF was secreted from C2C12 cells and, if so, no matter whether VEGF levels within the conditioned medium (CM) varied dur-ing differentiation. CM was collected each 24 hours from developing 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. Immediately after 1 day of culture in DM, VEGF level decreased to 270 pg/mg of1422 Germani et al AJP October 2003, Vol. 163, No.Figure 3. 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 regular skeletal muscle (A). VEGF protein was detected in satellite cells at day 3 (B) and in regenerating fibers at day 7 (C) following femoral artery ligation. The immunostaining decreased in regenerating fibers at 14 days immediately after ischemic in.
