In contrast, compound C had no effect on the expression of cyclin E, p27, and cyclin-dependent kinase 2, 4, and 6. Open in a separate window Fig. (QImaging QICAM; Hitschfel Devices, Inc., St. Louis, MO). The wound area was then measured to determine cell migration. Protein Analysis. Vascular SMCs were lysed in electrophoresis Ethoxzolamide buffer [125 mM Tris (pH 6.8), 12.5% glycerol, 2% SDS, and trace bromphenol blue] and proteins were separated by SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes, membranes were clogged with PBS and nonfat milk (5%) and then incubated Ethoxzolamide with antibodies against cyclin D1 (1:500), cyclin E (1:500), cyclin A (1:500), p27 (1:300), p21 (1:500), cyclin-dependent kinase (1:500), phospho-retinoblastoma protein (1:200), AMPK1/2 (1:500), phospho-AMPK 1/2 (1:100), phospho-ACC (1:300), or -actin (1:200). Membranes were washed in PBS, incubated with horseradish peroxidase-conjugated goat anti-rabbit or rabbit anti-goat antibodies, and developed with commercial chemoluminescence reagents. Protein manifestation was quantified by scanning densitometry and normalized with respect to -actin. AMPK Activation. AMPK activity was determined by Western blotting using phospho-specific antibodies directed against AMPK or ACC (Liu et al., 2011). Carotid Artery Injury. Male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7.5 mg/kg), and experimental balloon injury was performed within the remaining common carotid artery, as described previously (Tulis et al., 2001; Granada et al., 2005; Peyton et al., 2009). Depth of anesthesia was monitored by the absence of a withdrawal reflex to feet and tail pinch and the absence of a blink reflex. In brief, a Fogarty 2F embolectomy catheter (Baxter Healthcare Corporation, Deerfield, IL) was launched through an external carotid arteriotomy site and advanced through the remaining common carotid artery to the level of the aortic arch. The balloon catheter was then inflated and withdrawn with rotation to the level of the FzE3 carotid bifurcation. This was repeated three times, and then the catheter was eliminated and the incision was closed. Immediately after injury, a local polymer-based delivery system was used to administer compound C to the hurt vessel wall. The delivery system consisted of 200 l of a 25% copolymer gel answer (Pluronic F-127; BASF, Chicago, IL) comprising compound C (1 mg) that was applied inside a circumferential manner to the revealed adventitia Ethoxzolamide of the carotid arteries. A separate cohort of animals received an empty gel, which has previously been demonstrated to have no effect on vascular redesigning (Hu et al., 1999; Tulis et al., 2001). After 2 weeks, rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7.5 mg/kg) and euthanized by pneumothorax and exsanguination, and carotid arteries were collected for analysis. All methods conformed to the National Institutes of Health (Institute of Laboratory Animal Resources, 1996) and were authorized by the institutional animal care and use committee. Histology. Carotid arteries were perfusion-fixed, excised, and inlayed in paraffin. Sections (5 m) were stained in Verhoeff-Van Gieson for measurement of vessel sizes. Microscopic dedication of Ethoxzolamide vessel sizes was performed using Image-Pro Plus (Press Cybernetics, Inc., Bethesda, MD) and Adobe Photoshop software (Adobe Systems, Mountain View, CA) linked through a digital video camera (QICAM Fast 1394; Hitschfel Devices, Inc.) to an Olympus model BX41TF light microscope (Olympus America Inc., Center Valley, PA), mainly because explained previously (Tulis et al., 2001; Granada et al., 2005; Peyton et al., 2009). Statistics. Results are indicated as means S.E.M. Statistical analyses were performed with the use of a Student’s two-tailed test and an analysis of variance with the Bonferroni post hoc test when more than two treatment regimens were compared. < 0.05 was considered statistically significant. Results Compound C Inhibits Vascular SMC Proliferation and Migration in an AMPK-Independent Manner. Treatment of vascular SMCs with serum stimulated a time-dependent increase in cell number that was clogged by compound C (10 M) (Fig. 1A). The antiproliferative effect of compound C (0.02C10 M) was concentration-dependent (Fig. 1B). A significant inhibition of cell growth by compound C was mentioned at a concentration of 0.1 M and near-total abolition of proliferation was observed with 10 M. Compound C also inhibited the migration of SMCs after scrape wounding (Fig. 1C). Treatment of SMCs with compound C (0.02C10 M) resulted in a concentration-dependent inhibition of SMC migration beginning at a concentration of 0.2 M. In contrast, compound C experienced no significant effect on cell viability, as determined by trypan blue exclusion [control 96.3 3.3% versus compound C (10 M) 95.2 3.6%, = 4]. Open in a separate windows Fig. 1. Compound C inhibits the proliferation and migration of vascular SMCs in an AMPK-independent manner. A, serum (5%) stimulates a time-dependent increase in Ethoxzolamide cell number that is clogged by compound C (CC; 10 M). B, compound C inhibits the proliferation of SMCs inside a concentration-dependent manner. Cells were treated with serum (5%) in the absence and presence of compound C (0.02C10 M) for.