Effects Of Statins And Antioxidants On The Biological Behaviors Of Vascular Smooth Muscle Cells

Atherosclerosis is a systematic pathophysiologic process which induces arterial stenosis or even occlusion. It is the principal cause of myocardial infarction, stroke, and peripheral vascular diseases. Percutaneous transluminal angioplasty and stenting represents a well-established technique for revascularization of stenotic/occluded arteries. However, the long-term efficacy of the procedure remains limited by progressive arterial restenosis and reocclusion within the following few months after operation.The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is thought to play an important role in the pathogenesis of both atherosclerosis and restenosis. The ability of activated VSMCs to proliferate and migrate often associates with their phenotypic switching. Inhibition of abnormal proliferation, migration and phenotypic switching of VSMCs, therefore, is of great value in the prevention and treatment of atherosclerosis and restenosis.Statins and antioxidants are often prescribed to patients with atherosclerosis or restenosis. Statins not only inhibit the proliferation but also induce the apoptosis of VSMCs, thus suppress intimal hyperplasia after angioplasty and stenting. However, the effects of antioxidants on the biological behaviors of VSMCs remain obscure. Further exploration, therefore, is needed to demonstrate the relationship between antioxidants and the biological behaviors of VSMCs and to compare the effects on VSMCs between statins and antioxidants.In this study, we induce the proliferation, migration and phenotypic switching of VSMCs with oxidized low density lipoprotein (oxLDL), after which VSMCs are treated with Atorvastatin and Probucol, alone or combined. Differences of the effects on VSMCs between Atorvastatin and Probucol are compared, and the effects of the two drugs combined are also observed.Part Ⅰ In vitro isolation and primary culture of vascular smooth muscle cells ObjectiveTo isolate, culture and identify VSMCs derived from the thoracoabdominal aortas of male SD rats with simple and effective method.MethodsThoracoabdominal aortas were resected from healthy male SD rats in sterilized condition. Adventitia and intima were removed gently. VSMCs were obtained from the tunica media with the explant method, and were cultured subsequently. VSMCs were timely subcultured, frozen and thawed. The growth of VSMCs was observed continuously with inverted microscope. The cell viability was assessed with trypan blue staining, while the purity of VSMC cultures was identified with immunocytochemical staining.ResultsThe cultivation period of VSMCs with explant technique was 2 weeks to 3 weeks. The majority of VSMCs displayed a spindle-or triangle-like shape and appeared as the "hills and valleys" arrangement when cells reached confluence. After subcultured, passaged VSMCs still showed an elongated shape and a parallel appearance similar to the primary cultures. The cell viability was 96.3%±1.8%, while the immunopositive proportion of VSMC cultures for SMa-Actin was 95.4%±2.7%.ConclusionsVSMC primary cultures could be acquired from aortic media of rats with explant technique, and could be subcultured stably without contamination. The VSMC cultures were characterized by high viability and high purity, which could be used in the following researches on their biological behaviors.Part Ⅱ Effects of statins and antioxidants on proliferation of vascular smooth muscle cellsObjectiveTo assess the effects of statins and antioxidants on proliferation of vascular smooth muscle cells, to compare the effects among Atorvastatin, Probucol and the two drugs combined, and to find out the optimal concentration of Atorvastatin and Probucol, respectively.MethodsCytotoxicity of Atorvastatin at a concentration between 0.1μmol/L to 10μmol/L, Probucol between 1μmol/L to 100μmol/L, alone respectively or in combination, were assessed with MTT-colorimetric method. VSMCs were stimulated by oxLDL and subsequently treated with Atorvastatin and/or Probucol at concentration above. Optical density (OD) values were compared among different groups.ResultsNeither alone respectively nor in combination did Atorvastatin and Probucol showed cytotoxicity on VSMCs at concentration above (P>0.05 for all). OxLDL induced VSMCs to proliferate, thus increased the OD value, which could be reversed by Probucol alone (P<0.001) or combined with Atorvastatin (P=0.001). The optimal concentration of Atorvastatin and Probucol were 10μmol/L and 100μmol/L, respectively.ConclusionsProbucol alone or combined with Atorvastatin dose-dependently inhibits proliferation of VSMCs. It needs research of larger sample size to testify whether Atorvastatin alone can reverse the proliferation induced by oxLDL.Part Ⅲ Effects of Statins and Antioxidants on migration of Vascular Smooth Muscle CellsObjectiveTo evaluate the effects of statins and antioxidants on migration of vascular smooth muscle cells, and to compare the effects among Atorvastatin, Probucol and the two drugs combined.MethodsVSMCs were stimulated by oxLDL and then treated with Atorvastatin (10μmol/L) and/or Probucol (100μmol/L). Fluorescence microscope was used to observe the migrated VSMCs. Migration of VSMCs were evaluated by the percentage of the area occupied by migrated VSMCs in the linear wound, and were compared among control group, oxLDL group, Atorvastatin group, Probucol group and Atorvastatin plus Probucol group.ResultsThe percentage of the area occupied by migrated VSMCs in the linear wound was 5.80%±2.85% in control group,17.42%±9.97% in oxLDL group,9.68%±2.65% in Atorvastatin group,11.84%±5.14% in Probucol group and 5.28%±2.24% in Atorvastatin plus Probucol group. Significant differences were observed both between control group and oxLDL group (P=0.004) and between Atorvastatin plus Probucol group and oxLDL group (P=0.002).ConclusionsAtorvastatin combined with Probucol significantly inhibits migration of VSMCs. Further exploration is needed to demonstrate whether Atorvastatin or Probucol alone can inhibit migration of VSMCs.Part IV Effects of Statins and Antioxidants on phenotypic switching of Vascular Smooth Muscle CellsObjectiveTo assess the effects of statins and antioxidants on phenotypic switching of VSMCs, and to compare the effects among Atorvastatin, Probucol and the two drugs combined.MethodsVSMCs were stimulated by oxLDL and were subsequently treated with Atorvastatin (lOμmol/L) and Probucol (100μmol/L), alone respectively or in combination. Ultrastructures of VSMCs were observed with transmission electron microscope (TEM). The mRNA and protein levels of both the SMC phenotypic markers and collagen synthesized by VSMCs were analyzed by immunofluorescent staining, real-time reverse transcriptional polymerase chain reaction (RT-PCR), and Western blot.ResultsIn the cytoplasm of VSMCs in oxLDL group, when compared with control group, myofilament disappeared, while the synthetic organelles such as endoplasmic reticulum increased and dilated, and mitochondria also increased. In the cytoplasm of VSMCs in Atorvastatin group, Probucol group and Atorvastatin plus Probucol group, myofilament were more than that in oxLDL group but less than that in control group, while both endoplasmic reticulum and mitochondria were less than those in oxLDL group. Compared with control group, both mRNA and protein levels of SMα-Actin, SM-MHC and Smoothelin were significantly reduced in oxLDL group, while those of OPN were markedly elevated, and the amounts of the newly synthesized collagens I and III were also remarkably increased. The levels of the contractile markers were higher, while the amounts of OPN and collagens were smaller in Atorvastatin group and Probucol group compared with oxLDL group. The levels of the contractile markers were further elevated in Atorvastatin plus Probucol group, and those of OPN and collagens were further decreased.ConclusionsEither Atorvastatin or Probucol alone can inhibit the phenotypic switching of VSMCs, and this inhibitory effect is enhanced when these two drugs are used in combination.