Mechanism Of Human Amniotic Mesenchymal Stem Cells Inhibiting The Development Of Atherosclerosis In Mice

Objective:Atherosclerosis is a chronic inflammatory disease,and its pathogenesis is complicated.Available evidences have suggested that the dysfunction of arterial endothelial cells might be the first step in atherosclerosis.Endothelium injury would lead to monocytes gathered in the subcutaneous,which would develop into macrophage foam cells by phagocytosis of the modified low density lipoprotein,especially oxidized low density lipoprotein cholesterol.Then the foam cells undergo necrosis or apoptosis,and further promote the development of atherosclerotic plaques,which leads to serious cardiovascular disease^[1].Inflammation,as an important factor,runs through each stage of atherosclerosis,and is associated with a variety of risk factors,such as diabetes,hypertension,and smoking.Inflammation can not only change the inherent function of artery endothelial cells and smooth muscle cells,but can oxidize lipid to oxidized low density lipoprotein,which has the chemotaxis to monocyte and can make monocyte differentiate into macrophage foam cells and secret active molecule,eventually involves in the formation of atherosclerosis.The modified oxidized low-density lipoprotein,as an antigen,will aggravate the inflammatory response of the arterial vessels,ultimately affecting the stability of the plaque^[2-4].Therefore,the reduction of atherosclerosis through anti-inflammatory action has been recognized by more and more studies.Currently,drug therapy for atherosclerosis,especially the application of statins,has had an obvious effect on reducing the risk of cardiovascular disease.Statins have anti-inflammatory effects while lowering lipid,which would benefit patients with cardiovascular disease.However,many patients cannot tolerate statin drugs or cannot follow long-term treatment,as a result they cannot meet the expected lipid reduction target.And some large clinical trials have shown that the reduction of cardiovascular risk,including statins,is still poor.Some patients continue to suffer from the expected cardiovascular events while using statins.Therefore,it is necessary to find new methods for the treatment of atherosclerosis,and to reduce the development of atherosclerosis by reducing the inflammation or inhibiting the formation of foam cells.More and more studies have shown that stem cells can play an important role in tissue repair and anti-inflammation.In particular,mesenchymal stem cells have shown anti-inflammatory and immunological functions,which have been confirmed by many experiments.MSCs are one type of stem cells with self-renewal and multidirectional differentiation potential.The results show that MSCs are easy to be isolated and cultured in vitro,and have the characteristics of low immunogenicity,which make it widely applied in many experimental directions.At present,the role of MSCs has been applied to the area of atherosclerosis.It can be observed that mice bone marrow mesenchymal stem cells can reduce the atherosclerosis plaque area by caudal vein injection to the atherosclerosis model mouse,the mechanism has been considered that MSCs can reduce oxidized low density lipoprotein on vascular endothelial damage.Some studies suggest that MSCs can regulate the role of various inflammatory cells such as macrophages and inhibit the formation of plaques by inhibiting inflammatory responses.Research has showed that the application of mice skin source MSCs in the treatment of atherosclerosis can also reduce plaque area,the mechanisms involved in increasing the role of regulatory T cells and inhibition of macrophage foam cells formation.And in vitro experiments have established that MSCs can be induced to polarize to M2 macrophages,can modulate inflammatory and immune response by secrete a variety of cytokines,which inhibit atherosclerosis development from several aspects.MSCs were first found in the bone marrow and subsequently found in various tissues such as the fatty muscle periosteum and the synovial membrane.Human amniotic mesenchymal stem cells?hAMSCs?are mesenchymal stem cell isolated from human amniotic membrane.It not only has significant self-renewal ability and differentiation potential,but can be more readily available compared to embryonic stem cells and other adult stem cells,and without the ethical controversy,without risk of carcinoma,without expression of MHC class II antigen,expressing a small amount of MHC class I antigen,with low immunogenicity,and has paracrine or autocrine function to secret immunosuppressive factors and neurotrophic factors.These characteristics make hAMSCs have incalculable clinical value in the field of tissue engineering,cell therapy,gene therapy and other related regenerative medicine.The study showed that the conditioned medium of hAMSCs can significantly promote the proliferation and migration of endothelial cells and the capacity of tube formation.The conditioned medium of hAMSCs cannot cause toxic damage to cells,and inhibit macrophages to produce inflammatory cytokines by LPS stimulating.So we transferred hAMSCs by means of tail intravenous infusion to apolipoprotein E knockout mice from in vitro to observe whether infusion hAMSCs would have an impact on development of atherosclerosis in mice,and discuss the role of hAMSCs in regulating blood vessel inflammation in mice,and whether the paracrine function of hAMSCs was involved and played a role.Methods:hAMSCs were extracted from the original generation,and the third and sixth generation cells were selected for the experiment after the cultivation and identification.In the animal experiment section,eight weeks old apolipoprotein E gene knockout mice were given a high-fat diet to build atherosclerosis model.The first 18 mice were studied for atherosclerotic formation,which were begun to intervene while giving a high-fat diet.Six of them were injected with hAMSCs?5x10⁵ cells suspended in 150ul PBS?,and 6were injected with the same dose of 150ul PBS by the tail vein,and the other 6 did not give injection intervention as the positive control group.They were injected every two weeks,injected five times,and the mice were executed after 10 weeks of intervention.The remaining 18 mice,as the second batch,began to intervene after 8 weeks of high-fat diet,to study the effect on the formation of plaques.Also 6 of them were injected with hAMSCs?5x10⁵ cells suspended in 150ul PBS?,and 6 were injected with the same dose of 150ul PBS by the tail vein,and the other 6 did not give injection intervention as the positive control group.Once every two weeks,and totally they were injected 5 times and executed after 10 weeks of intervention.Before the mice were executed,the weight was measured,and the blood was collected from the heart to compare the differences in blood lipid indicators.A paraffin section was performed on the aortic root vessels,and HE staining was used to evaluate the differences in plaque area.Total RNA and total protein were extracted from the whole artery?from aortic arch to bilateral iliac artery?,and the influence of hAMSCs intervention on the expression of vascular inflammatory factors in mice was observed through qRT-PCR and Western Blot.In the cell experiment section,the conditioned medium of hAMSCs was collected to explore the paracrine effects of hAMSCs on the proliferation and inflammatory response of macrophages?thp-1 cells stimulated by oxLDL?.The multiplication was detected by MTS experiment.Total RNA and total protein were extracted from macrophages,which were cultured by the conditioned medium of hAMSCs or co-cultured with hAMSCs,and the effects of the paracrine secretion of hAMSCs on the expression of inflammatory cytokines in macrophages induced by oxLDL were analyzed through qRT-PCR and Western Blot analysis.The effect on NF-?B pathway of macrophages was discussed by Western Blot.Results:The original generation of extracted hAMSCs presented long spindle angle and adherent growth.The cells changed into a fibrous sample and fish shape distribution after the third generation.Flow cytometry showed that hAMSCs were positive in CD90,CD44,SSEA-4,and negative in CD31,CD45,HLA-DR.In the animal experiment,there was no statistical difference between the two groups of mice in weight.The HE staining results showed that the area of the aortic root of the hAMSC group in the first group of mice was significantly lower than that of the PBS group and the Control group.The second group of mice showed the same result.There was no difference in the hAMSC of the second group between the Control of the first group,which suggested that the injection of hAMSCs after the formation of the plaques had no reversal effect on the plaques that had been formed.The results showed that the expression of TNF?was lower in the hAMSC group than the other two groups,and the anti-inflammatory factor IL10 was higher than the other two groups.In the Cell experiment part,we selected the experimental conditions after MTS tests:100 ng/ml PMA 24h and 50 mg/L oxLDL 24h,and then the cells were collected for experimental analysis.The result showed that oxLDL could stimulate the proliferation of macrophages.After cultured with hAMSCs conditioned medium,the proliferation of macrophages was significantly inhibited.The results showed that with the increase of oxLDL concentration,the stimulation of macrophages produced a significant increase in TNF?,and no significant change in the anti-inflammatory factor IL10.It could reduce the inflammatory stimulation of oxLDL on macrophages whether cultured macrophages with the conditioned medium or co-cultured with hAMSCs.The culture of macrophages with conditioned medium had inhibiting effects on NF-?B pathway,through inhibiting the phosphorylation of p65 and I?B?.Conclusion:hAMSCs could inhibit the formation of atherosclerotic plaques of the apolipoprotein E gene knockout mice and delay the progression of atherosclerotic plaques,but could not reverse the plaque formation.It might be based on the regulation of NF-?B signaling pathway to suppress the inflammatory stimulation of oxLDL on macrophage,the inhibition of macrophage proliferation,to implement the inhibition of vascular nflammation in mice,so as to reduce the formation of plaque.