The antiplatelet agent revacept prevents the increase of systemic thromboxane A2 biosynthesis and neointima hyperplasia

Figures

• 

  Figure 1. Effects of platelets on phenotypic modulation of CASMC. Human CASMC (0.8 × 105 cells) were cultured alone or cocultured with human platelets (0.5 × 108). COX-1 (green) (A,B) or COX-2 (green) and α-SMA (red) (C,D) expression was assessed by confocal microscopy. Image magnification was 10X.

• 

  Figure 2. Effects of platelets on α-SMA, COX-2, and COX-1 protein levels in CASMC cultured alone or cocultured with platelets. (A) α-SMA levels were evaluated by Western blot in CASMC cultured alone or cocultured with platelets for 24 h; (B) the protein bands were quantified, and the optical density (OD) was calculated using laser densitometry and normalized to the OD of β-actin; data are reported as % of control (CASMC cultured alone), mean ± SEM, n = 5 (separate experiments); *P < 0.05 versus CASMC cultured alone (using Student’s t-test). (C–E) COX-2 and COX-1 levels were evaluated by Western blot in platelets (Plt) or CASMC cultured alone or in CASMC cocultured with platelets (CASMC + Plt) for 20 h; (D,E) the protein bands were quantified and OD was calculated and normalized to the OD of GAPDH, data are reported as % of control (CASMC cultured alone), mean ± SEM, n = 5 and n = 3 (separate experiments), respectively; (D) **P < 0.01 versus CASMC cultured alone (using Student’s t-test).

• 

  Figure 3. Effects of platelets on CASMC migration. (A) Human CASMC (0.8 × 105 cells) were cultured alone or cocultured with human platelets (0.5 × 108) (pretreated or not with Aspirin, ASA, 100 µM) for 20 h and cell migration of confluent cells was evaluated at different time-points (up to 24 h) by scrape wounding assay, using an inverted microscope and a digital camera (original magnification × 10), the dashed lines indicate the starting position of the cell migration occurring toward the center of the wound; % of covered area values are reported as mean ± SEM, n = 3 (separate experiments), the scale bar is of 100 µm. In each experimental condition, % covered area was significantly different (P < 0.01) at each time-point versus time 0 (using two-way ANOVA, Dunnett's multiple comparisons test), not shown. At 4 h, **P < 0.01, CASMC alone versus CASMC + PLT and CASMC + PLT-ASA; at 8 h, **P < 0.01, CASMC alone versus CASMC + PLT and CASMC + PLT-ASA; at 12 h, **P < 0.01, CASMC alone versus CASMC + PLT and CASMC + PLT-ASA, §P < 0.01, CASMC + PLT versus CASMC + PLT-ASA; at 24 h, **P < 0.01, CASMC alone versus CASMC + PLT and CASMC + PLT-ASA, §P < 0.01, CASMC + PLT versus CASMC + PLT-ASA (using…

• 

  Figure 4. Effects of platelets on prostanoid generation and COX-2 expression of CASMC. (A) Levels of PGE2 and TXB2 in the conditioned medium of CASMC and platelets cultured alone, or cocultured for 20 h, assessed by immunoassays; the effect of Rofecoxib (Rofe, 0.3 µM) or aspirinated platelets (ASA, 100 µM was added during platelet isolation and then washed away) was assessed. **P < 0.01, *P < 0.05; n = 3–4 (separate experiments) (using one-way ANOVA, Tukey's multiple comparisons test). (B,E) The effect of ASA (100 µM) or of the TP antagonist SQ 29,548 (SQ, 10 µM) on COX-2 expression in cocultures of CASMC and platelets, was assessed by Western Blot. (F,G) CASMC were cultured alone or cocultured with platelets in the presence of Revacept (Reva, 40 µg/ml) or vehicle (PBS/4% mannitol/1% sucrose) and COX-2 levels were analyzed by Western blot; interleukin (IL)-1β was used as positive control. (C,E,G) COX-2 protein bands were quantified, and the optical density (OD) was calculated using laser densitometry and normalized to the OD of GAPDH or β-actin; data are reported as % of control (CASMC cultured alone), mean ± SEM. (C) **P < 0.01 versus CASMC and §P < 0.05 versus CASMC + PLT(ASA), n = 3…

• 

  Figure 5. Effects of Revacept administration on urinary levels of TXM in mice in response to injury. (A) Study design of Revacept or Fc-control (2 mg/kg/day) administration to mice using a model of neointima formation caused by the damage of femoral arteries via transluminal wire injury. (B) Urinary levels of TXM were assessed at different time-points, before (0) and after vascular injury (3 and 28 days) in mice treated with Fc control or Revacept (2 mg/kg/day); data are expressed as mean ± SEM, n = 9–10; **P < 0.01 versus the other Fc conditions, #P < 0.05 versus the conditions of Revacept after injury (using mixed-effects analysis for repeated measures data and Tukey's multiple comparisons test).

• 

  Figure 6. Effects of Revacept administration to mice on CD68 and Ki67 expression in femoral artery sections. (A,B) Immunohistochemistry of femoral artery sections to assess CD68 and Ki67 at 28 days after surgery in mice treated with Revacept or Fc. (C,D) CD 68+ and Ki67+ cells per field are presented as mean ± SEM, n = 4 mice; *P < 0.05 versus Fc-control, (using Student’s t-test). Scale bar = 50 μm.

• 

  Figure 7. Effects of Revacept administration neointima formation following vascular injury in mice. (A) Hematoxylin and eosin staining of cross-sections of injured femoral arteries from mice treated with Fc-control or Revacept (2 mg/kg/day), harvested at 4 weeks after transluminal wire injury. (B) Quantification of intima-to-media (I/M) ratio, data are reported as mean ± SEM, n = 6–10; *P < 0.05 versus Fc-control (using Student’s t-test).