A Chinese herbal formula "Gan-Lu-Yin" suppresses vascular smooth muscle cell migration by inhibiting matrix metalloproteinase-2/9 through the PI3K/AKT and ERK signaling pathways

Abstract

Background: This study was to explore the effects of Gan-Lu-Yin (GLY) on the migration of vascular smooth muscle cells (VSMCs) induced by fetal bovine serum and on neointima formation in a rat model of carotid artery balloon injury.

Methods: VSMCs were treated with different concentrations of GLY, and then analyzed with Flow cytometric analysis, zymography, transwell, and western blotting. SD rats received balloon-injury were analyzed with H&E staining.

Results: Our results showed that GLY significantly decreased the thickness of neointima. The inhibition by non-cytoxic doses of GLY of VSMCs migration was through its negative regulatory effects on phosphorylated ERK1/2, PI3K/AKT, and FAK. The data showed that GLY can inhibit the migration of VSMCs cells, and might block injury-induced neointima hyperplasia via the inhibition of VSMCs migration, without inducing apoptosis.

Conclusions: These observations provide a mechanism of GLY in attenuating cell migration, thus as a potential intervention for restenosis.

Figure 1

Effects of GLY on cell viability and cell cycle analysis. (A). Effects of GLY on cell growth of VSMCs by MTT assay. The cells were incubated for 24 h with 10% fetal bovine serum alone (control) or with different concentrations of GLY (0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL). (B). Flow cytometric analysis of GLY on the cell cycle of VSMCs cells. All the cells were treated with 15% fetal bovine serum with the addition of GLY at 0.0625,0.125,0.25,0.5,1 mg/mL for 18 h. (C). The percentage of subG1 under flow cytometric analysis. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group :* p < 0.05,** p < 0.01, *** p < 0.001.

Figure 2

Effects of GLY on wound healing migration of VSMCs cells. Wound was introduced by scraping confluent cell layers with a pipet tip. VSMCs cells were incubated with GLY (0.0625, 0.125, 0.25 and 0.5 mg/mL) for 18 h, and the migration distances of cells were calculated. (A) Representative photographs of invading cells that received either control (15% FBS) or GLY treatment. (B) Migrated cells across the black lines were counted in six random fields from each treatment. The mean number of cells in the denuded zone is quantified by three independent experiments. (C). Effects of GLY on transwell migration assay vascular smooth muscle cells. VSMCs were incubated with GLY (0.0625, 0.125, 0.25 and 0.5 mg/mL) for 18 h, and (D). The transwell migration cells were calculated. Photos of the migration VSMCs cells were taken under a microscope (100-fold magnification). Mean value was significantly different from that of the control group (15% FBS): * p < 0.05,** p < 0.01.

Figure 3

GLY inhibits the release of MMP-2 and MMP-9 in VSMCs. (A). To examine the possible antimigration mechanisms of GLY, we determined the activity of MMP-2 and MMP-9 in culture media of VSMCs cells by zymographic analysis. (B). GLY inhibited MMP-9 and MMP-2 activities in a concentration-dependent manner. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group :* p < 0.05,** p < 0.01, *** p < 0.001.

Figure 4

Molecular mechanisms of GLY on FBS-induced VSMCs. (A). Effects of GLY on TIMP-1, TIMP-2, MMP-2, MMP-9 proteins expression. VSMCs were treated with 0.125, 0.25 and 0.5 mg/mL for 24 h, and cell lysates were subjected to SDS-PAGE followed by Western blotting. (B). Dose-dependent effects of GLY on the protein expression level of FAK, the phosphorylated FAK, ERK1/2, the phosphorylated ERK1/2, PI3K, MMP-2, MMP-9, AKT and the phosphorylated AKT. VSMCs were treated with 0.125, 0.25 and 0.5 mg/mL of GLY for 24 h. The expression of these proteins were analyzed by Western blotting. β-actin was used as a loading control.

Figure 5

Responses of rat carotid arteries to balloon injury, and the effects of extract of GLY on balloon injury. (A). The left panel represents the low-power (100×) observations from a balloon-injured vessel, a balloon-injured vessel treated with GLY at 0.5 g/ml,and uninjruied vessel (sham control). And the right panel represents the high-power (200×). (L, lumen; N, neointima; M, medium.). (B). The control group shows a significantly higher neointima to media ratio as compared with the groups treated with extract of GLY at 0.5 g/mL. Values are means of two separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group:* p < 0.05.