Effects Of Different FimA Variants Of P.gingivalis-LPS On The Function Of Human Umbilical Artery Smooth Muscle Cells

Objective: To explore the possible effects of lipopolysaccharide from I, IV fim A genotype Porphyromonas gingivalis on the function of human umbilical artery smooth muscle cells, including ability of proliferation, migration and phenotype transformation and the effects on the development of atherosclerosis.Methods: 1. I fim A genotype P.gingivalis(ATCC33277) and IV fim A genotype P.gingivalis(W83) were cultured under anaerobic conditions. 2. P.gingivalis-LPS was extracted by extraction kit, and identified by SDS-PAGE. The purified lipopolysaccharide was tested by Tachypleus Amebocyte Lysatc kit. 3.HUASMCs were cultured in vitro. The 5~6 generations of HUASMCs were seeded into 96-well plates, then different concentrations of P.gingivalis-LPS(0.5μg/ml、1μg/ml、2μg/ml、5μg/ml、10μg/ml) were co-cultured with HUASMCs,after 2h, 8h, 24 h, 48 h, the proliferation of vascular smooth muscle cells was detected by CCK-8, enzyme labeled instrument measure the OD values at 450 nm. 4. HUASMCs were stimulated by 10μg/ml P.gingivalis-LPS in subsequent experiment. Employing cells scratch assay and Transwell cell migration assay to detect the migration of HUASMCs, and observing the ultrastructure of cells through transmission electron microscope. The levels of contraction phenotypic marker protein including α smooth muscle actin(α-SM-actin), smooth muscle myosin heavy chain(SM-MHC) and synthetic symbol cellular retinol binding protein 1(CRBP-1) were detect through flow cytometer.Results: 1. when HUASMCs were stimulated with the I, IV fim A genotype P.gingivalis-LPS at different time, the proliferation of HUASMCs were higher than that of the negative control group, there is time- and concentration- dependence maner in up-proliferating effects on HUASMCs(P<0.05). The up-proliferation effects on HUASMCs by IV fim A genotype P.gingivalis-LPS were stronger than that by I fim A genotype P.gingivalis-LPS, when HUASMCs were stimulated with the same concentration and co-cultured at the same time(P<0.05).2.The migration of HUASMCs was detected by scarification and Transwell. At 2h, 8h, 24 h, 48 h, I, IV fim A genotype P.gingivalis-LPS can promote the migration of HUASMCs. The distance of HUASMCs migration increased with prolongation of stimulation time. To the end of observation(48h), compared with control group, the distance of HUASMCs migration reached a peak, the difference was statistically significant(P<0.05). At 24 h, 48 h, the number of HUASMCs migration stimulated by IV fim A genotype P.gingivalis-LPS was higher than that stimulated by the I fim A genotype P.gingivalis-LPS, the difference was statistically significant(P<0.05). The results of Transwell assay and scratch test are close to each other, but only at 48 h, the number of HUASMCs stimulated by IV fim A genotype P.gingivalis-LPS migration was higher than the number of HUASMCs stimulated by I fim A genotype P.gingivalis-LPS, the difference was statistical significance(P<0.05).3. HUASMCs were stimulated by I, IV fimA genotype P.gingivalis-LPS. After 2h, 8h, 24 h, 48 h, the HUASMCs were observed by transmission electron microscope(TEM). HUASMCs gradually switched from the contraction phenotype to the synthetic phenotype, cells volume turned larger than the negative control group, the number of mitochondria, rough endoplasmic reticulum and Golgi organelles gradually increased, glycogen synthesis increased, the ingredient of myofilament gradually decreased and the number of phagosomes increased. At 2h, 8h, 24 h, 48 h, the HUASMCs which were stimulated by IV fim A genotype P.gingivalis-LPS were observed that lipid droplets gradually appeared and increased in intracellular. There were not lipid droplets appeared in the HUASMCs which were stimulated by I fim A genotype P.gingivalis-LPS, but to some extent the rough endoplasmic reticulum became edema at the end of observation(48h).4. There were up-regulating effects on HUASMCs synthetic symbol molecular CRBP-1 expression and down-regulating effects on contraction phenotypic symbol molecular ɑ-SMA and SM-MHC expression, after HUASMCs were stimulated by I, IV fim A genotype P.gingivalis-LPS. The effects of HUASMCs were stimulated by IV fim A genotype P.gingivalis-LPS up-regulating synthetic symbol molecular CRBP-1 expression were stronger than that by I fim A genotype P.gingivalis-LPS. But there were statistical significant difference between the down-regulating effects on the ɑ-SMA only at 24 h and SM-MHC only at 48 h expression by I, IV fim A genotype P.gingivalis-LPS.Conclusion: Stimulated by I, IV fim A genotype P.gingivalis-LPS, the HUASMCs gradually switched from the contraction phenotype to the more activity synthetic phenotype. And I, IV fim A genotype P.gingivalis-LPS stimulated HUASMCs which can promote the proliferation and migration of HUASMCs, which may participate in and have an influence on the development of atherosclerosis. To some extent, there are different effects on HUASMCs proliferation, migration and phenotypic conversion when stimulated by different fim A genotype P.gingivalis-LPS.