PSGL-1 Is Involved In High Salt-induced Vascular Endothelial Injury And Target Organ Damage&Mir-122 Regulates EndMT Via TGF?1/TGF?R1 Pathway

Objective:To investigate the role and mechanism of PSGL-1 in the development of salt-sensitive hypertension in mice via inflammation.Methods:In vivo:PSGL-1 knockout(PSGL-1-/-)and wild type(PSGL-1+/+)mice were fed with high salt(6%NaCl)and normal salt(0.4%NaCl)diets for three months respectively.After mice blood pressures were measured under anesthesia via carotid artery,the status of tissue inflammation and kidney injury was tested by flow cytometry,immunohistochemistry,immunofluorescence,elisa and western blotting.In vitro experiment ?:Peripheral blood mononuclear cells(PBMC)of PSGL-1-/-and PSGL-1+/+ mice were extracted respectively and stained with BCECF-AM,followed by co-incubation with Bend.3 monolayer pretreated with 130 mM or 170 mM NaCl medium.The fluorescence intensity in each group was observed under inverted fluorescence microscope,which represents the number of adherent PBMC.In vitro experiment ?:PSGL-1+/+-PBMC were co-incubated with recombinant mouse P-selectin protein at 130 mM and 170 mM NaCl medium respectively,and the number of PBMCs binding with recombinant P-selectin protein was detected by flow cytometry.In vitro experiment ?:Mouse brain microvascular endothelial cells(Bend.3)were treated with 130 mM NaCl,170 mM NaCl,170 mM NaCl + NF-?B inhibitor(PDTC)for 48 hours.The expression of P-selectin,E-selectin,VCAM-1 VWF and ET-1 were detected by Western Blot,to determine whether high salt can affect the expression of adhesion molecules of endothelial cells through NF-?B signaling pathway.Results:In vivo:Compared with normal salt diet mice,PSGL-1+/+ mice with high salt had a higher level of blood pressure,accompanied by T-lymphocytes and macrophages infiltration in skin,aortic tissue,and kidney tissue,abnormal expression of eNOS and ET-1 in the vascular endothelium,impaired vasodilatation,as well as elevated expression of kidney injury marker Connective Tissue Growth Factor(CTGF)and oxidative stress marker NADPH oxidase 4(NOX4)(P<0.05);However,the inflammation,vasodilatation impairment and kidney injury were not found in PSGL-1-/-mice with high-salt diet(P>0.05).In vitro:The adhesion of PSGL-1+/+-PBMC to Bend.3 cells or the binding of PBMC and P-selectin was significantly greater at 170 mM NaCl group than that at 130 mM NaCl group,but PSGL-1-/--PBMC did not show this effect.Compared with low salt(130 mM NaCl)group,high salt(170 mM NaCl)significantly increased the expression of adhesion factors(E-selectin,VCAM-1,ET-1,VWF)in Bend.3 cells,while NF-?B inhibitor(PDTC)blocked the upregulation of adhesion molecules induced by high salt(P<0.05).Conclusion:PSGL-1 is involved in high salt-induced infiltration of T-lymphocytes and macrophages,accompanied by imbalance of vascular contractile factor ET-1 and vasodilating factor eNOS/NO,abnormal secretion of kidney injury marker CTGF and oxidative stress marker NOX4;PSGL-1 knockout has a protective effect on vasodilation and renal damage,improving high salt-induced hypertension and other vascular diseases,which provide new ideas for for the prevention and treatment of hypertension.Objective:Endothelial cell injury is an initial step in the development of atherosclerosis.Endothelium-to-mesenchymal transition(EndMT)is involved in the occurrence and development of atherosclerosis.The purpose of our study is to investigate whether liver-specific miR-122 is involved in Endothelial-to-mesenchymal transition and atherosclerosis via TGF?1/TGF?R1 Pathway.Method:In vivo:ApoE-/-mice were fed with normal or high-fat diet for 12 weeks respectively.The plaque formation and lipid deposition in the aortic roots of mice were detected by hematoxylin-eosin staining(HE)and oil red 0,and the expression of miR-122 in serum,arteries and liver was detected by real-time PCR.In vitro experiment I:MiR-122 mimic and ASO-122 and the corresponding negative control(NC)were transfected into HUVEC and Bend.3 cells respectively.The transfection efficiency was analyzed by detecting the intracellular level of miR-122,and the species-specific regulatory role of miR-122 on TGF?1 or TGF?R1 was confirmed by detecting the protein level of TGF?1 and TGF?R1 in mouse endothelial cells and human endothelial cells.The effect of miR-122 on EndMT was determined by detecting the expression and colocalization of endothelial cell marker(CD31,VE-cadherin)and mesenchymal cell marker(Vimentin,a-SMA,FSP1)through western blot and immunofluorescence.In vitro experiment II:Human umbilical vein endothelial cells(HUVEC)were transfected with miR-122 prior to incubating with H2O2,thus the cells were divided into three groups respectively:Control group,H2O2 group,and H2O2+miR-122 group.The degree of EndMT was determined by endothelial cells morphological changes,the expression and colocolization of endothelial cell markers(CD31,VE-cadherin)and mesenchymal cell markers(Vimentin,?-SMA,FSP1)through Western Blot,real-time PCR and immunofluorescence assay.Result:miR-122 was down-regulated in hepatic tissue of High-fat diet-induced atherosclerosis of ApoE-/-mice,while it was up-regulated in serum and arteries,and showed the same phenomenon in H2O2 induced-EndMT model of human and mouse endothelial cells.Effect inhibition of miR-122 can induce the development of EndMT,and overexpression of miR-122 blocked H2O2-induced of EndMT,which might be related to its regulation on TGF?1/TGF?R1 Pathway.Conclusion:miR-122 secreted by liver enters into the systemic circulation,acts on the arterial endothelium and arterial tissue,and inhibits the occurrence of atherosclerosis through the TGF?1/TGF?R1-EndMT pathway,which provides a innovative direction for the prevention and treatment of atherosclerosis.