Ing fibers exhibited diffuse Flk-1 and Flt-1 labeling (Figure 2D). In mature fibers, too as in regenerated muscle at 14 days following 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 located 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 two in DM, cells fuse to form multinucleated myotubes. Within this experimental model, it was investigated whether Flk-1, Flt-1, and VEGF expression varied through differentiation as observed in in vivo through muscle regeneration (Figure 2). Western blot analysis of C2C12 lysates showed that when myoblasts were induced to differentiate by changing from GM to DM both Flk-1 and Flt-1 proteins markedly decreased over a 5-day time CD200R Proteins Recombinant Proteins period (Figure 5A). On the other hand, Flt-1 but not Flk-1 was still detectable at day five of differentiation. These alterations in VEGF receptor expression have been paralleled by a progressive raise in myosin heavy chain expression (MyHC), consistent using the enhance in differentiation of C2C12 cells (Figure 5A). Additional, just after 5 days in DM, a large 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 have been immunostained for Flk-1 and Flt-1. Positive 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. Handle immunostaining was performed by omitting the key 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 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 right after 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 complete. Magnification, 40; bar, 25 m.of myotubes was observed in the culture dishes (not shown). In extra experiments it was determined regardless of whether VEGF was secreted from C2C12 cells and, if that’s the case, regardless of whether VEGF levels inside the conditioned medium (CM) varied dur-ing differentiation. CM was collected every 24 hours from increasing 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 Gastrin Proteins Formulation 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 three (B) and in regenerating fibers at day 7 (C) following femoral artery ligation. The immunostaining decreased in regenerating fibers at 14 days right after ischemic in.
