Reseach Of Indoleamine 2, 3-dioxygenase(IDO) On Regulating The Phenotype Transformation Of Vascular Smooth Muscle Cells

Objective: To study the role of indoleamine 2, 3-dioxygenase(IDO) and its microenvironment(low-tryptophan and tryptophan metabolites accumulation) in the pathological process of arteriosclerosis obliterans(ASO), post-percutaneous angioplasty and stent implantation restenosis(RS), especially in the proliferation and phenotypic transformation of vascular smooth muscular cell.Methods: Through culturing mouse thoracic aorta derived smooth muscle cells(Smooth Muscular Cells, SMCs) in vitro, and simulating the microenvironment of indoleamine 2, 3-dioxygenase(IDO) by low tryptophan and adding its metabolites(Kynurenine), and co-culturing with the EOMA cell line(EOMA, ATCC® CRL-2586 ?) with high expression of m IDO, to study their regulatory role in proliferation and phenotypic transformation of SMCs by MTT test and real-time quantitative PCR(q RTPCR) respectively. Using SPSS 19.0 software package for data analysis.Results: The mouse thoracic aorta derived smooth muscle cells(SMCs) was successfully cultured through the method of aortic ring adherent. Cell eruption for 5-10 days, and about 6×105 second-generation SMCs will be harvested for every two mice. The third to tenth generation of cell will present a typical "hills" and "valleys" growing form, which was identified by immunohistochemistry with SMα-actin(brown), and cell purity was more than 95%. There is significant morphological changes and increased apoptosis when the cells generated for 10-12 and more. Taking the 3-5 generations SMCs for subsequent experiments. Relatively low tryptophan can inhibit the proliferation of smooth muscle cells, but its metabolite, kynurenine, will inhibit smooth muscle cell proliferation only at high concentrations, and this inhibition only work for 96 h and 120 h culture time. RT-PCR showed that whether it is a lowtryptophan or kynurenine has no significant impact on the expression of smooth muscle cell phenotype gene Smoothelin-B and syndecan-1 within the culture time for 120 h. The expression of syndecan-1 was significantly reduced in low tryptophan(10μM) and low tryptophan- kynurenine joint culture group by the culture time of 72 h, 96 h, 120 h. Considerring the impact of the environment for low-tryptophan, and metabolites kynurenine(200μM) did not significantly affect the expression of these two phenotypegene. When co-cultured with high mice IDO-expressed EOMA for 96 h and 120 h, the proliferation of smooth muscle cells was inhibited significantly, and this inhibition disappeared by adding 1-m T, the IDO specific inhibitor, which suggests that IDO can inhibit the proliferation of smooth muscle cells in vitro. RT-PCR result suggests that there was a significantly up-regulated expression for Smoothelin-B and downregulated expression for syndecan-1 when co-cultured 96 h and 120 h with EOMAIDO+, and this effect disappeared or weakened by adding 1-m T.Conclusion: The method of improved vascular ring adherence, which is easy for operation and can effectively avoid contamination with adventitial fibroblasts and endothelial cells, can be used to obtain high purity SMCs and provide cellular material for experiments. Low tryptophan(10μM) can inhibit the proliferation of smooth muscle cells, and its metabolites kynurenine has this inhibition only at high concentrations(200μM). And can not regard that both have a significant effect on the SMCs phenotype conversion in vitro. IDO can inhibit the proliferation and dedifferentiation of SMCs, and may contribute to conversion to the contractile phenotype for SMCs. Currently there is no enough evidence to prove that this effect is caused by tryptophan starvation and its metabolites gathering. There may be other mechanisms to be studied.