Study On BMSCs Migration Induced By TGF-β1 In Collaboration With MCP-1 In Vascular Calcification

Objective:This study aims to investigate the role of TGF-β1 and MCP-1 in the migration of BMSCs during vascular calcification.Methods:Thirty LDLR knockout C57 mice(LDLR^-/-mice)and thirty ordinary C57 mice,one month old,were divided into four groups according to high fat diet and ordinary diet:LDLR^-/-mice high-fat diet group（Group LH）,C57 mice high-fat diet group（Group CH）LDLR^-/-mice ordinary diet group（Group LL）and C57 mice ordinary diet group（Group CL）,with 15 mice in each group.Five rats were killed at2W,6W and 12W after feeding,and their aorta and BMSCs were extracted.The study was carried out in three parts.The first part consisted of four groups:LH,CH,LL and CL.RNA was extracted from aorta,and the expression levels of osteogenic gene Collagen Type I Alpha 1（COL1A1）,Osteocalcin（OCN）,and Runt-related transcription factor 2（RUNX2）were analyzed.The fixed paraffin-embedded sections of the aorta were performed by hematoxylin-eosin（HE）,alizarin red and MOVAT staining to evaluate the degree of atherosclerosis and calcification of the aorta.In the second part,divided into seven groups:TGF-β1,TGF-β1Ab,MCP-1,MCP-1Ab,TGF-β1-MCP-1,TGF-β1Ab-MCP-1Ab,NC were used to evaluate the mode and degree of regulation of BMSCs migration by TGF-β1 and MCP-1.In the third part,the Group LH was fed for 12W to the aorta that had successfully formed aortic calcification to make culture solution and added to the lower petri dish to be divided into four groups:Calcified aorta,Calcified aorta-TGF-β1Ab,Calcified aorta-MCP-1Ab,Calcified aorta-TGF-β1Ab-MCP-1Ab,to evaluate the mode and extent of BMSCs migration regulated by TGF-β1 and MCP-1 in the aorta.Results:1.Expressions of osteogenic genes:COL1A1,OCN,RUNX2,there was no significant difference in the early 2W Group LH compared with the other three control groups（P>0.05）;Intermediately in 6W COL1A1,Group LH was up-regulated compared with Group CH and Group CL,the difference was significant（P<0.05）;In 6W RUNX2,Group LH was up-regulated compared with Group CH,Group LL and Group CL,the difference was statistically significant（P<0.05）.There was no statistical significance in 6W OCN and Group LH compared with the other three control groups（P>0.05）.Lately in 12W COL1A1,Group LH was significantly up-regulated compared with the other three control groups,the difference was statistically significant（P<0.05）;Lately in 12W OCN,RUNX2,Group LH and the other three control groups were significantly up-regulated,the difference was statistically significant（P<0.01）.2.Aortic atherosclerosis and calcification:HE staining,alizarin red staining and MOVAT staining showed that the aorta structure was complete in the early 2W groups,and no obvious abnormal cells,plaques and calcification foci were observed.A small number of foam cells in the intima of Group LH and Group LL were observed by 6W HE staining in the middle stage,while thickening of intima and destruction of elastic fibers in Group LH and Group CL were observed by MOVAT staining.The 12W HE staining showed a large number of foam cells in the intima of the Group LH,obvious atherosclerotic plaques,and calcification in the plaques;alizarin red staining showed obvious calcification foci;MOVAT staining showed aortic plaques in the Group LH,and acidic mucopolysaccharide appeared in the plaques,indicating that the high-fat LDLR^-/-mice in the Group LH were successfully constructed as a model of aortic calcification.3.The migration of BMSCs was regulated by TGF-β1 and MCP-1:There was statistical difference between TGF-β1 group and TGF-β1Ab group（P<0.01）.The difference between MCP-1 group and MCP-1Ab group was statistically significant（P<0.05）.There was statistical difference between TGF-β1-MCP-1 group and TGF-β1Ab-MCP-1Ab group（P<0.01）.There was statistical difference between TGF-β1-MCP-1 group and TGF-β1 group（P<0.05）.TGF-β1Ab-MCP-1Ab group was significantly different from NC group（P<0.01）.There was no significant difference between MCP-1Ab group and NC group（P>0.05）.These results indicated that both TGF-β1 and MCP-1 could independently induce BMSCs migration,and the independent induction of TGF-β1 was stronger.TGF-β1 and MCP-1 co-induced BMSCs migration ability exceeded TGF-β1independent induction.Dual inhibition of TGF-β1 and MCP-1 was more potent than single inhibition of MCP-1.4.The regulation mode and degree of TGF-β1 and MCP-1 in aorta:There was statistical difference between the aortic calcification group and the aortic calcification-TGF-β1Ab group（P<0.01）.There was statistical difference between the aortic calcification group and the aortic calcification-TGF-β1Ab-MCP-1Ab group（P<0.01）.There was statistical difference between aortic calcification group and aortic calcification MCP-1Ab group（P<0.01）.This indicated that TGF-β1 and MCP-1 in calcified aortic culture medium had a significant ability to jointly induce BMSCs migration,and the induction ability of single inhibition of TGF-β1 or single inhibition of MCP-1 was lower than that of the two factors combined induction.There was no significant difference between aortic calcification-TGF-β1Ab group and aortic calcification-TGF-β1AB-MCP-1AB group（P>0.05）.There was statistical difference between aortic calcification-MCP-1Ab group and aortic calcification-TGF-β1Ab-MCP-1Ab group（P<0.05）.These results showed that MCP-1 inhibited TGF-β1 only,and the migration ability of BMSCs decreased.The ability of TGF-β1 to induce BMSCs migration was almost unaffected by single inhibition of MCP-1.Conclusion:Before aortic calcification in LDLR^-/-mice,the expression of osteogenic genes began to increase in the middle stage and increased significantly in the late stage.In vascular calcification,both TGF-β1 and MCP-1 can independently induce BMSCs migration,and they have a synergistic effect.Conversely,inhibition of TGF-β1 or MCP-1 decreased the migration capacity of BMSCs.