The Role And Mechanism Of GDF-15 And CNPY2 In Atherosclerosis

Background and objective:Atherosclerosis（AS）is a chronic inflammation of the arteries characterized by the formation of dense lipid plaques in the blood vessels.The main pathological process of AS is the impairment of vascular endothelial cell（VEC）function.Macrophages enter the subendothelial space through damaged vascular endothelial cells,engulf oxidized low-density lipoprotein（ox-LDL）to form foam cells,promote lipid deposition to the vascular wall and form plaques.Therefore,the damage and dysfunction of macrophages and vascular endothelial cells are important factors in the formation of AS,but the mechanism of their damage is not completely clear.Whether growth differentiation factor-15（GDF-15）,as a macrophage inhibitory factor,affects macrophage function and thus plays a protective role in AS remains unclear.Therefore,we explored the role and mechanism of GDF-15 in the formation of AS through cell and animal experiments.In addition,the abnormal expression of Canopy FGF Signaling Regulator 2（CNPY2）gene in the aorta of Apo E^-/-mouse AS model was screened by RNA-seq.At the same time,it was found that the expression of CNPY2 was increased in vascular endothelial cells induced by ox-LDL,so it was speculated that CNPY2 may play a role in AS by affecting vascular endothelial cells.Therefore,we explored the role and mechanism of CNPY2 in the formation of AS through cell and animal experiments.By studying the role of macrophages and vascular endothelial cell in the formation of AS,it lays a foundation for the future study of multi-cell interaction in the pathogenesis of AS.Method:1.The co-localization relationship between GDF-15 and macrophages was determined by immunofluorescence staining,and the downstream signal TLR4 of GDF-15 was screened to explore the effect and mechanism of GDF-15 on lipid accumulation and inflammatory response of macrophages in the formation of AS.In order to verify whether GDF-15 plays a role through TLR4,firstly,in terms of cells,macrophages were treated with GDF-15 or/and TLR4 pathway activator（Neoseptin-3）,respectively.Oil red O staining was used to evaluate lipid accumulation.ELISA and Western blot were used to detect pro-inflammatory factors levels and proteins.Secondly,in animals,AS model mice were treated with GDF-15 or/and Neoseptin-3,and the arterial plaque tissues of mice were taken to evaluate the levels of GDF-15 and TLR4by immunohistochemical staining.Western blot was used to detect lipid accumulation-related proteins.The levels of pro-inflammatory factors were detected by ELISA.HE staining was used to evaluate the pathological condition of blood vessels.2.In order to screen the genes related to AS in vascular endothelial cells,RNA-seq and RT-q PCR were used to establish the research gene-CNPY2.Immunofluorescence staining confirmed that CNPY2 was co-localized with vascular endothelial cells.In order to explore the effect and mechanism of CNPY2 on apoptosis and inflammatory response of vascular endothelial cells in the formation of AS,the downstream signal PERK of CNPY2 was determined.In order to verify whether CNPY2 plays a role through PERK,firstly,vascular endothelial cells were treated with sh RNA interfering CNPY2（sh CNPY2）or/and PERK signaling pathway activator（CCT020312）,respectively.The apoptosis rate was detected by flow cytometry.Western blot was used to detect vascular endothelial cell apoptosis-related proteins.The levels of pro-inflammatory factors were detected by ELISA.Secondly,in vivo,AS model mice were treated with sh CNPY2 or/and CCT020312,and arterial plaque tissues were taken to evaluate the levels of CNPY2 and PERK by immunohistochemical staining.Western blot was used to detect apoptosis-related proteins.The levels of pro-inflammatory factors were detected by ELISA.HE staining was used to evaluate the pathological condition of blood vessels.3.The serum samples of 65 normal healthy people and 101 patients with coronary AS were collected,and the contents of GDF-15 and CNPY2 were detected by ELISA.Result:1.The results of immunofluorescence staining showed that GDF-15 was localized on macrophages and not on vascular endothelial cells.Cells:The expression of GDF-15 in macrophages increased after ox-LDL stimulation,and the addition of GDF-15could reduce ox-LDL-induced lipid accumulation and pro-inflammatory factors in macrophages.In addition,GDF-15 significantly reduced the lipid accumulation-related gene TLR4 m RNA,suggesting that GDF-15 may play a role through TLR4.In order to verify the above speculation,Neoseptin-3 treatment could reverse the inhibition of GDF-15 on ox-LDL-induced macrophage lipid accumulation and pro-inflammatory factors.Clinically,the content of GDF-15 in serum of AS patients was significantly higher than that of normal healthy people.Animals:GDF-15 is highly expressed in AS plaques of AS mouse models.GDF-15 can inhibit the levels of lipid accumulation-related proteins and pro-inflammatory factors in mouse arterial plaque tissues,and reduce the degree of AS.In addition,TLR4 was significantly reduced after GDF-15treatment,while Neoseptin-3 could reverse the above effects of GDF-15.2.The abnormal expression genes screened by RNA-seq were detected by RT-q PCR.It was found that CNPY2 was abnormally highly expressed in plaque tissues of AS model mice and located in vascular endothelial cells.In terms of cells,ox-LDL stimulation can increase the expression of CNPY2 in vascular endothelial cells,while sh CNPY2 can alleviate ox-LDL-induced apoptosis and pro-inflammatory factors in vascular endothelial cells.In addition,sh CNPY2 could inhibit the activation of PERK signaling pathway,while CCT020312 could reverse the inhibitory effect of sh CNPY2on ox-LDL-induced vascular endothelial cell apoptosis and pro-inflammatory factors.Clinical aspects:The content of CNPY2 in serum of AS patients was significantly higher than that of normal healthy people;In animals,CNPY2 is highly expressed in atherosclerotic plaques of AS mouse models,and sh CNPY2 can inhibit the levels of apoptosis-related proteins and pro-inflammatory factors in mouse arterial plaque tissues,and reduce the degree of AS.In addition,PERK was significantly reduced after sh CNPY2 treatment,while CCT020312 reversed the above effects of sh CNPY2.Conclusion:1.GDF-15 is mainly expressed on macrophages.Cell and animal experiments show that GDF-15 can inhibit lipid accumulation and inflammatory response of macrophages induced by ox-LDL by inhibiting TLR4,thus alleviating AS.2.CNPY2 is mainly expressed in vascular endothelial cells.Cell and animal experiments have shown that CNPY2 can promote ox-LDL-induced vascular endothelial cell apoptosis and inflammatory response by activating the PERK signaling pathway,thereby exacerbating AS.