| Objective: The adhesion, proliferation and migration of vascular smooth muscle cell (VSMC) are the important pathological bases of vascular remodeling diseases, such as hypertension, atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Adhesion of VSMC plays a key role in VSMC proliferation and migration, and it is vital for cell survival and normal blood vessel structure. All-trans retinoic acid (ATRA) can induce differentiation and apoptosis of many cancer cells. The aim of this study is to explore the mechanisms of ATRA inhibiting proliferation and inducing apoptosis in VSMC by analysis of VSMC adhesion and the expression of the adhesion molecules.Methods:1. VSMC culture and ATRA treatment5-week-old male SD rats were selected. The VSMCs from rat aorta were isolated and were cultured with DMEM medium containing 10% FBS. The cells used for the experiment were passage 5 ~ 6. The cells grown into 70% ~ 80% confluences were serum starved for 24 hours. Then the VSMCs were treated with 10μM ATRA for different times ( 0, 24, 48 h ) or at different doses ( 0, 5, 10μM ) for 48 h.2. VSMC adhesion assayThe cultured VSMCs were digested with 0.25% trypsin after pretreatment by ATRA, and then they were planted in the 96-well plate to incubate for 120 min. The VSMCs were washed by PBS and fixed by 4% paraformaldehyde. The adhesive cells were counted under microscope, and then stained with toluidine blue. The absorbance of cell lystates atλ595 nm was analyzed, which presents adhesive activity.3. Expression of adhesion molecules in VSMCImmunohistochemical staining and Western blot were used to detect the changes of ICAM-1 and VCAM-1 expression. Trizol was used to extract the total RNA of VSMC, and semiquantitative RT-PCR was performed to detect the mRNA of ICAM-1 and VCAM-1.Results:1. Effects of ATRA on VSMC adhesionCell counting results showed that ATRA could inhibit VSMC adhesion. Compared to control group (0 h), VSMC treated by 10μM ATRA for 24 h and 48 h, decreased in adhesion activity to 85.8% (P>0.05) and 35.3% (P<0.05), respectively. Compared to control group (0μM), for VSMC treated by 5, 10μM ATRA for 48 h, the number of adhesion cells was decreased to 59.1% and 34.7% (P<0.05), respectively. Under the same conditions, results of toluidine blue staining were consistent with the results of cell counting.2. Effects of ATRA on expression of adhesion molecules in VSMCResults of immunohistochemical staining indicated that, the positive staining particles of ICAM-1 and VCAM-1 were reduced in ATRA-treated VSMCs. Compared to control group, the positive particles of ICAM-1 and VCAM-1 were dramatically decreased in VSMCs treated with ATRA at different doses.Semiquantitative RT-PCR results showed that ATRA could inhibit the transcriptional activities of ICAM-1 and VCAM-1 genes. After pretreatment by 10μM ATRA for 24 and 48 h, ICAM-1 mRNA was decreased by 16% and 47%, VCAM-1 mRNA was decreased by 32% and 48%, respectively.ICAM-1 and VCAM-1 mRNA was reduced by ATRA in a dose-dependent manner. ICAM-1 mRNA were decreased by 50% and 54% following treatment with ATRA at 5μM or10μM, and VCAM-1 mRNA by 29% and 51%, respectively.Western blot indicated that ATRA inhibited the expression of ICAM-1 and VCAM-1. After pretreatment by 10μM ATRA for 24, 48 h, ICAM-1 protein was reduced to 85.8% and 57.6%, VCAM-1 was reduced to 80.1% and 63.6%, respectively.Similar to the results of RT-PCR, after treated with different doses of ATRA, ICAM-1 and VCAM-1 was reduced in a dose-dependent manner. Conclusion:1. ATRA inhibits VSMC adhesion in a time- and dose-dependent manner.2. ATRA inhibits the gene transcriptional activities and protein expressions of ICAM-1 and VCAM-1 in a time- and dose-dependent manner.
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