Effect Of Pg-LPS On Calcification Of Human Umbilical Artery Smooth Muscle Cells Co-cultured With Human Periodontal Ligament Cells

Objective:Periodontitis is an independent risk factor for vascular calcification and significantly enhance the risk of vascular calcification.During periodontitis,porphyromonas gingivalis(Pg)is one of leading pathogenic bacteria,and lipopolysaccharide(LPS),as its main virulence factor,can promote the abnormal proliferation of vascular smooth muscle cells(VSMCs)in rats and activity of Alkaline Phosphatase(ALP),and accelerate the expressions of ALP of related calcification genes,core binding factor al(Cbfal),bone sialoprotein(BSP)and osteopontin(OPN)as well.However,the effect of Pg-LPS on VSMCs remains to further defined in vivo in the future studies due to its complexity of internal environment.Therefore,a co-cultured system using transwell inserts of human periodontal ligament cells(HPDLCs)and human umbilical artery smooth muscle cells(HUASMCs)was established to simulate the micro-environment in vivo to further verify its effects.The aim of this study was to preliminarily explore the effects of stimulated Pg-LPS on HUASMCs proliferation,ALP activity,and expressions of mRNA in involved calcification genes(ALP,Cbfal,BSP).Methods:1.HPDLCs were obtained from the primary culture of the tissues and HUASMCs were bought from the ScienceCell company.HPDLCs and HUASMCs were cultured in a contact co-culture system by the mean of transwell chamber.2.Experimental groups were divided into four groups:Using Pg-LPS(1?g/ml)stimulated the co-culture system(Group CO-P)and the separate HUASMCs culture group(Group C-P),at the same time,negative control groups(Group CO,Group C)were set up.All groups were cultured 48 hours and detected the proliferation of HUASMCs and the activity of ALP with CCK-8 method and ALP kit.3.Mineralization-inducing medium was added on confluent monolayers by addition of DMEM medium containing 5%FBS,with osteogenic supplements such as L-ascorbate,sodium glycerophosphate-? and dexamethasone.The experiment consisted eight groups,including the four groups(Group C,Group CO,Group C-P,Group CO-P)in experiment two and four groups(Group C-C,Group CO-C,Group C-CP,Group CO-CP)mineralization-inducing.Alizarin Red S staining,which detects calcium deposition,was used as an indicator of mineralization.After incubation for 21 days,the culture was terminated,and the cells were analyzed.4.Experimental groups was the same as in experiment three.All groups were cultured 48 hours.Reverse transcription-polymerase chain reaction(RT-PCR)was performed on HUASMCs treated or untreated with Pg-LPS in different groups.The messenger RNA(mRNA)expression of several osteoblastic markers,including ALP,Cbfal,and BSP,was quantified by real-time RT-PCR using the real-time RT-PCR detection system.Results:1.Primary cultures of HPDLCs were obtained by the tissue block method after I collagenase I egestion and co-culture system of HPDLCs and HUASMCs was established in vitro.2.The proliferation and ALP activity of HUASMCs in group C-P and group CO-P were higher than that in group C and group CO.The proliferation and ALP activity of HUASMCs in co-culture system(Group CO-P)was higher than that in the separate HUASMCs culture group(Group C-P).3.Mineralized nodule formation and the mineralization capacity of the HUASMCs were assessed with Alizarin Red S staining to stain the deposited calcium in red.After 21 days,HUASMCs grown on mineralization-inducing medium all displayed more obvious calcification nodules than that on normal culture.With Pg-LPS treatment,HUASMCs displayed more obvious calcification nodules.Moreover,HUASMCs displayed more obvious calcification nodules in co-culture Group CO-C and Group CO-CP than those in separate HUASMCs culture Group C-C and Group C-CP.4.Relative Quantification of HUASMCs Osteogenic:When cultured by mineralization-inducing medium,the expression of specific osteogenic genes(e.g.ALP,Cbf?l,and BSP)was significantly promoted in the presence or absence of Pg-LPS.mineralization-inducing medium.When stimulated with Pg-LPS,the expression of specific osteogenic genes was significantly promoted in the presence or absence of mineralization-inducing medium.In the presence of Pg-LPS or mineralization-inducing medium or the both,the calcification of HUASMCs in co-culture system is higher than that in separate culture.Conclusion:Pg-LPS may involve in the underlying mechanism that is to promote calcification by increased HUASMCs proliferation and ALP activity,and up-regulated expression of calcification genes involved(ALP,Cbf?l,BSP).Meanwhile,its promotion effect is better in HUASMCs than HPDLCs in the co-culture system,which reveals that the periodontitis can better accelerate calcification of blood vessels than simple inflammatory reaction,and plays an important role in occurrence and development of cardiovascular diseases.