Sodium ferulate inhibits neointimal hyperplasia in rat balloon injury model

Abstract

Background/aim: Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved.

Methods: Cultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed.

Results: In presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group.

Conclusion: Sodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs.

Figure 1. Effect of sodium ferulate on VSMCs proliferation and migration under 1 µmol/L Ang II stimulation.

VSMCs were pre-incubated with sodium ferulate in a range from 50 to 200 µmol/L for 1 hour and stimulated with 1 µmol/L Ang II for the indicated time. (A) Cell proliferation was quantified by CCK-8 kit (n = 6). (B) Cell migration was performed with transwell method (n = 3). ^* P<0.05 vs. the control group, ^{* *} P<0.05 vs. the Ang II group.

Figure 2. Sodium ferulate attenuates Ang II-induced oxidative stress in VSMCs.

After synchronization, VSMCs were stimulated with 1 µmol/L Ang II for 1 hour, with or without sodium ferulate at a dose of 200 µmol/L. (A) Intracellular ROS generation was detected using DCFH-DA probe (× 100 magnifications). NAPDH oxidase (B) and SOD activity (C) were analyzed using ELISA kits. Protein levels of p-p38 and p-ERK1/2 (D) were examined by western blot. N = 3 for each group, ^* P<0.05 vs. the control group, ^{* *} P<0.05 vs. the Ang II group.

Figure 3. Sodium ferulate upregulates contractile markers expression in response to 10% serum.

Cultured VSMCs were pre-treated with serum starvation for 72 hours and then challenged by 10% serum for another 48 hours to induce synthetic VSMCs, with or without 200 µmol/L sodium ferulate. (A) Representative Immunoblots. (B) Relative expression of SM α-actin protein. (C) Relative expression of SM-MHC protein. N = 3 for each group, ^* P<0.05 vs. the non-serum group, ^{* *} P<0.05 vs. the serum group.

Figure 4. Sodium ferulate inhibits Notch pathway transduction.

After serum starvation for 72 hours, VSMCs were exposed to 10% serum for another 48 hours in presence or absence of sodium ferulate at a dose of 200 µmol/L. The nuclear Notch-1 protein (A), the mRNA levels of Jagged-1, Hey-1 and Hey-2 (B) were measured using Western blot and real-time PCR, respectively. N = 3 for each group, ^* P<0.05 vs. the non-serum group, ^{* *} P<0.05 vs. the serum group.

Figure 5. Sodium ferulate blocks activity of Wnt pathway.

After serum starvation for 72 hours, VSMCs were exposed to 10% serum for another 48 hours in presence or absence of sodium ferulate at a dose of 200 µmol/L. (A) Total β-catenin protein was detected by Western blot. (B) Cyclin D1 mRNA was measured by real-time PCR. N = 3 for each group, ^* P<0.05 vs. the non-serum group, ^{* *} P<0.05 vs. the serum group.

Figure 6. Sodium ferulate increases contractile markers expression and reduces collagen generation in injured arteries.

(A) Representative artery sections with immunofluoresence staining for SM α-actin and SM-MHC (n = 12). Scale bar represents 100 µm. (C) Representative Masson trichrome stained vessel sections (n = 12). Collagen displayed as the blue performance and calculated as the percentage of collagen area to intimal and medial area. Scale bar represents 100 µm. *P < 0.05 vs sham group; **P < 0.05 vs saline group.

Figure 7. Sodium ferulate suppresses neointimal hyperplasia at 7 days after artery injury.

(A) Representative hematoxylin-eosin stained carotid artery sections from each group (n = 12). Scale bar represents 100 µm. Neointimal area (B) and the ratio of intimal area to medial area (C) were measured. *P < 0.05 vs sham group; **P < 0.05 vs saline group.