Role And Mechanism Of CREG In Vascular Calcification

Objective Vascular calcification（VC）is characterized by different mineral deposits accumulating in blood vessels and valves.The presence of any arterial wall calcification increases the risk of mortality and cardiovascular events by 3-4fold.According to the location of vascular lesions,vascular calcificationcan be divided into intimal calcification,middle model calcification and valve calcification.As an independent risk factor of cardiovascular disease Vascular calcification is the common pathological basis of vascular disease.Similar to bone remodeling,VC is an actively regulated process in which many cells and molecules play a key role.The severity of vascular calcification is commonly accompanied by a heightened risk of cardiovascular events and all-cause mortality;however,no targeted therapeutic methods are currently available.Cellular repressor of E1A-stimulated genes（CREG）is a small molecule secreted glycoprotein and is consisted by 220 amino acids.CREG is mainly located in perinuclear endoplasmic reticulum and lysosome.Studies have shown that CREG is involved in regulating cell growth and differentiation.A series of previous studies of our laboratory have proved that the exogenous CREG recombinant protein can protect the vascular remodeling caused by angiotensin Ⅱ in a dose-dependent manner.In addition,CREG plays an important role in maintaining the mature phenotype of vascular smooth muscle cells and inhibiting the migration and proliferation of vascular smooth muscle cells.Based on the above results,our study proposed a hypothesis that CREG may be involved in the occurrence and development of vascular calcification,by regulating the differentiation of vascular smooth muscle cells.To verify the role of CREG in vascular calcification at animal level,vascular calcification model was established by CREG transgenic mice and CREG heterozygous mice.Then,primary mouse vascular smooth muscle cells were stimulated with phosphate to induce cell calcification model,the role of CREG in vascular calcification and explore its mechanism at the cellular level was clarified.This study provides new experimental basis and theoretical clues for further clarifying the regulatory mechanism of vascular calcification.It may provide a new intervention target for the prevention and treatment of vascular calcification and other related cardiovascular diseases.Methods1.The role of CREG in VitD3 induced vascular calcification.（1）The relationship between CREG expression and VitD3 induced vascular calcificationVitD3 was injected by subcutaneous to establish vascular calcification,the control group was injected subcutaneously with the same dose of PBS.The Alizarin red staining,Von Kossa staining and western blot were used to verify the model.The expression of osteopontin（OPN）、runt related transcription factor 2（RUNX2）and CREG was determined by real-time PCR、western blot and immunohistochemistry in the vascular tissues of VitD3 group and control group.（2）The role of CREG in VitD3 induced vascular calcification calcification.①Using CREG transgenic mice(CREG^tg)and its littermate control mice were used to establish VitD3 induced vascular calcification.After 14 days,Alizarin red staining,Von Kossa staining and immunohistochemistry were used to assess the severity of vascular calcification.Real-time PCR and western blot were used to detect the expression of OPN,RUNX2 and CREG.②VitD3 was injected by subcutaneous in CREG^+/-mice and its littermate control mice,vascular calcification was detected by Alizarin red staining,Von Kossa staining and immunohistochemistry.Real-time PCR and western blot were used to detect the expression of OPN,RUNX2 and CREG.2.Study on the mechanism of CREG in VitD3 induced vascular calcification.（1）In this study,mouse primary vascular smooth muscle cells（VSMCs）were used.The VSMCs of CREG overexpression and low expression were established by adenovirus infection and si RNA gene silencing,respectively.Pi stimulation was used to induce cell calcification.Real-time PCR and western blot were used to detect the expression of OPN,RUNX2 and CREG.（2）Preparation of isolated vascular rings: 8-week-old male C57BL/6J mice,CREG^+/-mice and cregtg mice were killed under anesthesia,and then the aortas were removed,and the adventitia was stripped.Cut into arterial rings with a length of about 4 mm.After 3 days of pi stimulation,Alizarin red staining,Von Kossa staining was used to assess the severity of vascular calcification.（3）Screening differential proteins by mass spectrometry: The calcified vascular tissue of cregtg mice and their control mice were delivered for mass spectrometry,the differential proteins were screened and analyzed.The overexpressed-adenovirus and small interfering RNA of the differential protein were constructed,and CREG or the differential protein were overexpressed or knockdown in mouse primary vascular smooth muscle cells,respectively.To clarify the upstream and downstream regulation relationship between CREG and the differential protein,real-time PCR and western blot were used to detect the expression of the differential protein and CREG.（4）Rescue experiment: FHL₂ small interfering RNA was given into CREG-overexpressed cells,western blot were used to detecte the expression of OPN,RUNX2 and CREG.Results1.The role of CREG in VitD3 induced vascular calcification.（1）The transcription and protein levels of CREG were significantly decreased in VitD3 induced vascular calcification.The results of von Kossa and Alizarin red staining showed that the model of vascular calcification VC in mice could be successfully established by subcutaneous injection of VitD3.Immunohistochemical staining showed that the expression of OPN and RUNX2 were increased significantly in VitD3 injection group,while CREG expression was significantly decreased.The results of real-time PCR and western blot showed that the transcription and protein levels of calcification indexes OPN,RUNX2 transcription and protein levels were significantly increased in VitD3 group,accompanied by the decreased expression of α-SMA and CREG.The above results suggested that there was a significant negative correlation between the decrease of CREG expression and vascular calcification.2.CREG played a protective role in VitD3 induced vascular calcification（1）Compared with CREG^tg group and littermate control group,the positive areas of von Kossa and Alizarin red staining in CREG^tg + VitD3 group and VitD3 group were significantly increased.Immunohistochemical staining,real-time PCR and western blot showed that the transcription and protein expression of OPN and RUNX2 were increased significantly.Compared with VitD3 group,CREG^tg + VitD3 group significantly reduced calcification,and the expression of OPN and RUNX2 were significantly decreased.（2）Compared with CREG^+/-group and littermate control group,the positive areas of von Kossa and Alizarin red staining in CREG +/-+ VitD3 group and VitD3 group were significantly increased.CREG^+/-+ VitD3 group increased more significantly than VitD3 group.Compared with CREG +/-group and control group,the expressions of OPN and RUNX2 in CREG^+/-+VitD3 group and VitD3 group were significantly increased,and the increase in CREG^+/-+VitD3 group was more obvious than that in VitD3 group.3.Study on the mechanism of CREG in VitD3 induced vascular calcification.（1）At the cellular level,the cell model of calcification can be successfully established by pi.Compared with the control group,Alizarin red staining showed the formation of orange granular calcium nodules in pi group=.Compared with the control group,the protein and transcription levels of OPN and RUNX2 were significantly higher.The protein and transcription levels of CREG were significantly decreased in pi group.（2）CREG overexpressed VSMCs can be successfully established by adenovirus infection.Western blot showed that compared with Ad Control+pi group,the expressions of OPN and RUNX2 in Ad CREG+pi group were significantly decreased and the expression of α-SMA was increased.In addition,vascular rings cultured were stimulated by pi,the positive areas of von Kossa and Alizarin red staining in CREG^tg group were significantly reduced compared with the littermate control group.（3）CREG low expression（siCREG）VSMCs model was established by transfection with small interfering RNA and then stimulated by pi.Compared with the si Control+pi group,the expression of OPN and RUNX2 was increased and the expression ofα-SMA was decreased in si CREG+pi group.In addition,vascular rings cultured were stimulated by pi,the positive areas of Von Kossa and Alizarin red staining in CREG^+/-group were significantly increased compared with the littermate control group.These results suggested that overexpression of CREG could inhibit VC,while CREG knockdown can aggravate vascular calcification.（4）CREG participated in the regulation of vascular calcification by affecting the expression of FHL₂.In order to further clarify the mechanism of CREG in vascular calcification,the vessels of CREG^tg mice and their littermate control treated with VitD3 were analyzed by Quantitative Proteomics Analysis.After screening,four and a half LIM domain 2（FHL₂）was found.The results of western blot showed that the expression of FHL₂ in vessels of CREG^tg +VitD3 group was significantly higher than that of their littermate control+VitD3 group.At the cell level,it was also found that the change of FHL₂ protein level was positively correlated with the change of CREG expression,and pi stimulation could significantly reduce the expression of FHL₂ protein.After overexpression and low expression of FHL₂ in VSMCs,CREG protein and transcription levels did not change.It is suggested that CREG may play a role as a upstream of FHL₂.Western blot showed that the expression of OPN,RUNX2 in Ad CREG+si FHL₂ group was increased compared with Ad CREG+si Control group.Conclusions1.After VitD3 induced vascular calcification,the transcription level and protein level of CREG was significantly decreased.2.CREG played a protective role in vascular calcification.CREG transgenic mice could significantly alleviated VitD3-induced vascular calcification VC,while CREG knockout mice could aggravate VitD3-induced vascular calcification.3.As the upstream molecule of FHL₂,CREG could regulate the vascular calcification by regulating FHL₂ protein expression.