Ing fibers exhibited diffuse Flk-1 and Flt-1 labeling (Figure 2D). In Integrin Associated Protein/CD47 Proteins Purity & Documentation mature fibers, also as in regenerated muscle at 14 days just 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 found 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 develop and divide when cultured in GM and, just after 48 2 in DM, cells fuse to type multinucleated myotubes. In this experimental model, it was investigated whether or not Flk-1, Flt-1, and VEGF expression varied for the duration of differentiation as observed in in vivo during muscle regeneration (Figure two). Western blot evaluation of C2C12 lysates showed that when myoblasts were induced to differentiate by changing from GM to DM each Flk-1 and Flt-1 proteins markedly decreased over a 5-day time period (Figure 5A). Nevertheless, Flt-1 but not Flk-1 was nonetheless detectable at day five of differentiation. These changes in VEGF receptor expression had been paralleled by a progressive raise in myosin heavy chain expression (MyHC), constant using the enhance in differentiation of C2C12 cells (Figure 5A). Additional, right 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. Optimistic cells, Muscle-Specific Kinase (MuSK) Proteins Storage & Stability indicated by arrowheads, were 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 main antibody. Magnification, 40 (inset one hundred); bar, 25 m. Time-course of Flk-1 and Flt-1 expression (C to E). Serial sections from hindlimbs were obtained at three 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 soon after ischemia Flk-1 and Flt-1 have been expressed in regenerating myotubes (D) along with the expression of each receptors decreased at day 14 (E), when the regenerative procedure was almost full. 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 if VEGF levels inside the conditioned medium (CM) varied dur-ing differentiation. CM was collected each and every 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. Just 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 standard skeletal muscle (A). VEGF protein was detected in satellite cells at day three (B) and in regenerating fibers at day 7 (C) right after femoral artery ligation. The immunostaining decreased in regenerating fibers at 14 days right after ischemic in.
