Investigation On Endothelial To Mesenchymal Transition In Atherosclerotic Plaque And The Regulation Mechanisms Of Hydrogen Sulfide

Cardiovascular and cerebrovascular diseases caused by atherosclerosis have become the leading cause of death in the world,and vascular endothelial dysfunction is the initial link of atherosclerosis.More and more researches have shown that endothelial to mesenchymal transition（EndMT）plays an vital role in the occurrence and development of atherosclerosis.Hydrogen sulfide（H₂S）is another gas signal molecule after nitric oxide and carbon monoxide,involved in many kinds of physiological and pathological processes of the cardiovascular system.Many studies have shown that H₂S can protect vascular endothelial function and inhibit the occurrence and development of atherosclerosis,but its regulatory mechanism has not been clarified.Therefore,to explore the regulatory mechanism of H₂S antagonizing atherosclerosis is of great significance to protect vascular endothelium and inhibit atherosclerotic lesions.Part 1:The role of hydrogen sulfide on endothelial-to-mesenchymaltransition in atherosclerotic plaques of ApoE^-/-miceAims:To investigate the effect of hydrogen sulfide on the endothelial-to-mesenchymal transition of vascular atherosclerotic plaque in ApoE^-/-mice.Methods:High-fat fed ApoE^-/-mice were divided into three groups:control group,the NaHS treatment group,and the PPG treatment group.Animals were sacrificed at 12 weeks of feeding.Automatic biochemical analyzer was used to detect the blood lipid level of each group of mice.Oil red O staining was used to discover the aortic atherosclerotic plaque area.HE staining,oil red O staining and Masson staining were used to detect the pathological morphology of As plaque in aortic sinus.Improved methylene blue method was used to detect the hydrogen sulfide content in plasma;Enzyme-linked immunosorbent assay was used to measure the concentration of TGF-β1,IL-10,IL-1β,TNF-αin plasma;immunohistochemical method and Western blotting were used to observe the protein expression of CD31,VE-Cadherin,α-SMA,FAPα,N-Cadherin,PTEN,p-PTEN,Akt,p-Akt,FoxO3a,p-FoxO3a in aorta.Results:Compared with the control group and the PPG treatment group,the plasma levels of TC and LDL-C in the NaHS treatment group was significantly reduced,the atherosclerotic lesion area and the aortic sinus plaque area in the aorta of the mice were significantly reduced.Moreover,the lipid accumulation in the plaques was significantly reduced.We also found that the production of H₂S in plasma and the CSE protein expression of the aorta was increased.There were less CD31 andα-SMA,CD31 and FAPα,VE-Cadherin and N-Cadherin double staining positive cells in As plaque of aorta.The increased expression of endothelial cell marker CD31,VE-Cadherin were obvious.The expression of mesenchymal cell markerα-SMA,FAPα,N-Cadherin,p-PTEN,p-Akt,and p-FoxO3a were reduced.Compared with the control group,TGF-β1 and TNFαincreased in the PPG treatment group,and IL-1βdecreased.Compared with the PPG treatment group,the TGF-β1,IL-1β,and TNFαin the NaHS treatment group were significantly reduced.Conclusions:（1）The treatment of NaHS reduced the blood lipid level,the aortic plaque area of ApoE^-/-mice.It also up-regulated the CSE/H₂S system in mouse aorta.（2）Endothelial to mesenchymal transition at aortic plaques were inhibited by the NaHS.（3）The inhibition of EndMT at As plaque by NaHS may be related to PTEN/AKT/FOXO3a signaling pathway.Part 2:Hydrogen Sulfide regulates endothelial-to-mesenchymal transition of human umbilical vein Endothelial cells and itsmechanism involvedAims:HUVECs were co-treated by the TGF-βand oxLDL to induce EndMT,and the effects and molecular mechanisms of H₂S treatment on them were observed.Methods:1.Co-treatment of HUVECs with TGF-βand different concentrations of oxLDL,or co-treatment of HUVECs with oxLDL and different concentrations of TGF-β.Western blotting was used to observe the protein expressions of CD31,α-SMA,FAPα,and Vimentin.2.The cells were divided into three groups:control group（TGF-β+oxLDL）,PPG group（PPG+TGF-β+oxLDL）,and GYY4137（H₂S sustained release agent）group（GYY+TGF-β+oxLDL）.The production rate of H₂S was detected by membrane adsorption methylene blue method.Scratch test and Transwell test were used to observe the function of cell migration.The function of tube formation was detected by matrigel tube formation test.Immunofluorescence,real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of CSE,CD31,α-SMA,Vimentin,VE-Cadherin,N-Cadherin,PTEN,p-PTEN,AKT,p-AKT,FoxO3a,p-FoxO3a.3.The cells were divided into three groups:control group（TGF-β+oxLDL）,CSE interference group（CSE interference plasmid+TGF-β+oxLDL）,and CSE overexpression group（CSE overexpression plasmid+TGF-β+oxLDL）.Scratch test was used to detect the function of cell migration.matrigel tube formation test was used to observe the tube formation.The expression of CD31,α-SMA,Vimentin,VE-Cadherin,N-Cadherin,PTEN,P-PTEN,Akt,p-Akt,FoxO3a,p-FoxO3a were detected by immunofluorescence,real-time fluorescence quantitative PCR and Western blotting.4.TGF-βand oxLDL were used to treat HUVECs at the same time.GYY4137 and PTEN,AKT,FoxO3a interfered with or over-expressed plasmids to treat cells.Scratch assay was used to detect cell migration.The expression of CD31,α-SMA,VE-Cadherin,N-Cadherin,PTEN,P-PTEN,Akt,p-Akt,FoxO3a,p-FoxO3a were detected by immunofluorescence,real-time fluorescence quantitative PCR and Western blotting.Results:1.HUVECs treated with 30ng/ml TGF-β+75μg/mL oxLDL reduced the expression of CD31 protein,but the expression ofαSMA,Vimentin,and FAPαincreased most significantly.2.GYY4137 treatment up-regulated the CSE protein expression,the H₂S production rate in HUVECs after co-treated with TGF-βand ox-LDL,and the opposite of PPG treatment.Compared with the control group,the GYY4137 treatment group had the reduced cell migration rate,the increased tube formation,the increased expression of CD31,VE-Cadherin mRNA and protein,the decreased expression ofα-SMA,N-Cadherin mRNA and protein,the decreased protein expression of Vimentin,p-PTEN,p-AKT,p-FoxO3a.The PPG treatment group showed the increased cell migration,the decreased CD31 mRNA and protein expression,the reduced VE-Cadherin mRNA expression,and the increased expression ofα-SMA and N-Cadherin mRNA and Vimentin protein.3.Compared with the control group,the CSE overexpression group had an increased tube formation,expression of CD31 mRNA and protein,VE-Cadherin protein.But the cell migration rate,the expression ofα-SMA mRNA and protein,N-Cadherin,Vimentin,P-PTEN,p-AKT,p-FoxO3a decreased.While the cell migration,tube formation and expression of CD31,VE-Cadherin,Vimentin in the CSE interference group got the opposite.4.Compared with the control group,the cell migration rate of GYY group,GYY+LvPTEN group,GYY+siAKT group,GYY+LvFOXO3a group reduced,the expression of VE-Cadherin,CD31 increased,the N-Cadherin,α-SMA protein expression decreased.The expression of p-AKT and p-FoxO3a proteins in the GYY+LvPTEN group were reduced,and the expression of p-FoxO3a protein in the GYY+siAKT group was reduced.Compared with the GYY group,the cell migration rates of the GYY+siPTEN group,GYY+LvAKT group,and GYY+siFOXO3a group were reduced.The expression of VE-Cadherin,CD31 decreased,the expression ofα-SMA,N-Cadherin increased.Increased expression of p-Akt,p-FoxO3a in GYY+siPTEN group,and increased expression of p-FoxO3a in GYY+LvAKT group.Conclusion:（1）Co-treatment with TGF-βand oxLDL induced endothelial to mesenchymal transition in HUVECs,which is more effective than TGF-βalone;（2）hydrogen sulfide improved the function of TGF-βand oxLDL co-treated HUVECs cells and inhibit their endothelial to mesenchymal transition;（3）hydrogen sulfide inhibits the endothelial to mesenchymal transition after TGF-βand oxLDL co-treatment through regulating the pathway of PTEN/AKT/FOXO3a.