A Novel Mechanism Of H₂s Against Atherogenesis: Inhibition Of MEST Expression Induced By Low Shear Stress And Its Mediated Endothelial-to-Mesenchymal Transition

Background:Atherosclerosis（As）is closely related to the inflammation of the blood vessel wall,vascular endothelial cell function,and local hemodynamic environment.It usually occurs in parts where blood flow is slow or disturbed.Endothelial-to-mesenchymal transition（End MT）induced by low shear stress plays an important role in the development of As,but its regulatory mechanism has not been clarified.Based on recent studies,it has been found that the expression of Mesoderm-specific transcripts（MEST）can be up-regulated by abnormal shear stress,and MEST may induce a phenotypic transition of cells to mesenchymal cells.The previous study of our group found that MEST was highly expressed in Apo E^-/-mouse As model induced by low shear stress,but the effect of MEST on the development of As lesion has not been reported.Multiple studies have shown that hydrogen sulfide（H₂S）has vascular protective properties.Our group’s previous experiments found that H₂S negatively regulates End MT induced by low shear stress and inhibits As lesions,and the bioinformatics predictions suggest that H₂S has the potential to inhibit the expression of MEST.Therefore,an in-depth study of the regulatory effect of H₂S on MEST and its effect on End MT is expected to reveal a novel mechanism for H₂S to against As.Aims:To investigate the inhibitory effect of H₂S on End MT induced by low shear stress and its effect and mechanism on the formation of As lesions from the perspective of regulation of MEST.Methods:1.Use the parallel plate flow chamber system to treat HUVECs with physiological laminar shear stress or low shear stress.Immunofluorescence and Western blotting were used to detect cell MEST protein expression.2.HUVECs were pretreated with different concentrations of GYY4137（H₂S sustained-release agent donor）and then subjected to low-shear stress treatment.Western blotting was used to detect cell MEST protein expression.3.MEST overexpression lentivirus（Lv MEST）and lentiviral empty vector（EV）were transfected into HUVECs,and the efficiency of viral transfection was detected by immunofluorescence and Western blotting.4.HUVECs are divided into three groups:Empty Vector group,Lv MEST group,and Lv MEST+GYY4137 group.Western blotting was used to detect the expression of CD31,VE-Cadherin,α-SMA,and N-Cadherin proteins.5.Apo E^-/-mice with local ligation of the left common carotid artery were divided into two groups:the control group and the Na HS group.The mice were fed a high-fat diet for 2 weeks and then euthanized and the tissues were collected.Immunofluorescence was used to detect the protein expression of MEST in the carotid artery of mice.6.High-fat diet Apo E^-/-mice were divided into two groups:the control group and the endothelial cell-specific overexpression group of MEST.At 16 weeks,they were euthanized and the tissues were collected.Immunofluorescence and Western blotting were used to detect the protein expression of MEST in the mouse aorta.Western blotting was used to detect the expression of mouse aortic mesenchymal cell markers N-Cadherin,Vimentin,FAP,and a-SMA,and the expression of endothelial cell markers Tie2,v WF,and CD31.Immunofluorescence and immunohistochemical staining were used to observe the expression of a-SMA,Vimentin,CD31,and Tie2 proteins in the aortic root.Observation under a stereo microscope and oil red O staining to detect the area of As plaque in the aorta.HE staining,oil red O staining,and Masson staining were used to detect the pathological changes of As plaques in the aortic sinus.An automatic biochemical analyzer was used to detect blood lipid levels in mice.Results:1.The parallel plate flow chamber experiment showed that compared with the physiological shear stress group,the MEST protein of HUVECs in the low shear stress group was increased by 123.00%（P<0.05）.2.For HUVECs treated with low shear stress,after treatment with GYY4137 at a concentration of 25,50,100 or 200μmol/L,the expression of MEST protein was down-regulated by 28.72%（P<0.01）45.36%（P<0.01）51.69%（P<0.01）or 60.94%（P<0.01）.3.The lentivirus Lv MEST was successfully transfected into HUVECs,and the expression of MEST protein was increased by 115.40%（P<0.01）.4.Compared with the EV group,the expression of CD31 and VE-Cadherin in the Lv MEST group decreased by 46.55%（P<0.01）and 30.36%（P<0.01）,and the expression ofα-SMA and N-Cadherin proteins increased by 46.09%（P<0.01）and 19.53%（P<0.05）.Compared with the Lv MEST group,the CD31 protein expression in the Lv MEST+GYY group increased by141.40%（P<0.01）,the VE-Cadherin protein expression was not significantly different,and theα-SMA and N-Cadherin protein expression decreased by 17.67%（P<0.05）and 29.04%（P<0.01）.5.Compared with the right common carotid artery of mice in the control group,the expression of MEST protein increased.Compared with the left common carotid artery of mice in the Na HS group,the expression of MEST was reduced.6.Compared with the control group,the expression of MEST protein in the AAV-MEST group increased by 86.22%（P<0.05）.The expression of N-Cadherin,Vimentin,FAP and a-SMA proteins in the aorta increased by 306.10%（P<0.05）,67.01%（P<0.01）,21.12%（P<0.05）and 52.42%（P<0.01）.Tie2 expression decreased by 32.41%（P<0.01）.There was no significant difference in v WF expression.CD31 expression increased by 359.70%（P<0.05）.The number of CD31 andα-SMA double positive cells and Vimentin and Tie2 double positive cells increased in As plaques of aortic sinus.The aortic As lesion area of mice was increased by 180.73%（P<0.05）.The aortic sinus plaque area was increased by45.47%（P<0.05）,and the lipid accumulation in the plaque was increased.There was no significant difference in body weight and blood lipids between the two groups of mice.Conclusion:Inhibition of the expression of MEST and its mediated End MT in vascular endothelial cells induced by low shear stress is a novel mechanism of H₂S antagonism against the occurrence and development of As.