Study On The Mechanism Of Glutathione S-transferase Pi On Inhibiting Phenotypic Transformation Of Vascular Smooth Cells

Vascular smooth muscle cells(VSMC)are the main components of the media of the blood vessel wall.VSMC exhibits a contractile phenotype in normal blood vessels,with a high degree of differentiation,showing a typical spindle or ribbon shape,and expressing VSMC contractile marker genes such as vascular actin-related protein(SM22 alpha,SM22α),smooth muscle α-actin gene(smooth muscle alpha-actin,α-SMA)and SM-MHC,etc.When blood vessels are damaged,VSMC can transform into a synthetic phenotype under the stimulation of various factors such as growth factors,with low differentiation,decreased cell shrinkage,enhanced proliferation and migration capabilities,and can synthesize and secrete a large amount of extracellular matrix.Morphologically similar to fibroblasts,they are flat,large in cell size,lack of muscle fibers,rich in rough endoplasmic reticulum,Golgi,ribosomes,etc.,and express VSMC synthetic marker genes such as osteopontin(OPN).More and more evidences indicate that VSMC phenotypic transformation is widespread in cardiovascular diseases such as atherosclerosis,intimal hyperplasia,hypertension,and restenosis after angioplasty.Therefore,inhibiting the phenotypic transformation of VSMC is one of the fundamental measures to treat vascular disease and prevent cardiovascular disease.Platelet-derived growth factor-B(PDGF-BB)is a strong mitogen factor,which can induce the transformation of VSMC phenotype and plays a vital role in the formation of new intima effect.In normal mature blood vessels,the expression level of PDGF and its receptors is low.When blood vessels are damaged,the expression of PDGF increases abnormally.Studies have shown that PDGF can promote the transformation of VSMC from a resting contractile phenotype to a synthetic phenotype by inhibiting the expression of α-SMA and calmodulin.Glutathione S-transferases-pi(Glutathione S-transferases-pi,GSTpi)is ubiquitously present in cells.It was originally discovered that it can catalyze the combination of glutathione(Glutathione,GSH)and electrophilic substrates to form a complex to perform Detoxification.GSTpi can also regulate various kinases through protein-protein binding.There is evidence that GSTpi has an inhibitory effect on the proliferation and migration of VSMC induced by Angiotensin II.However,there is no clear mechanism for how GSTpi regulates the phenotypic transformation-related proteins of VSMC.We aim to illustrate the effect of GSTpi on inhibiting the phenotypic transformation of vascular smooth muscle cells.Our initial study showed that the expression of endogenous GSTpi protein increased in PDGF-BB-stimulated VSMCs.It is speculated that the upregulated GSTpi protein level may be involved in the PDGF-BB-induced intimal proliferative disease-related vascular remodeling.The pc DNA3-Xpress-GSTpi(1.5μg/m L)eukaryotic expression plasmid was transfected into VSMC with a transfection reagent.We found that overexpression of GSTpi can increase the level of contractile phenotype protein(SMA and SM22α)reduced by PDGF-BB,and decrease the level of synthetic phenotypic protein(OPN),thereby blocking the cell cycle from G0/G1 phase to S phase Process.Further laser confocal and nucleocytoplasmic separation results showed that GSTpi serine 184 was phosphorylated and translocated from the cytoplasm into the nucleus.The results of qRTPCR showed that after PDGF-BB stimulated VSMC,the mRNA levels of synthetic phenotypic marker OPN increased significantly,while the mRNA levels of contractile phenotypic markers SMA and SM22α decreased significantly.The overexpression of GSTpi(S184D)significantly reversed the regulation of OPN,SMA and SM22α mRNA levels induced by PDGF-BB.Therefore,we speculate that GSTpi may regulate the phenotypic transformation of VSMC by influencing related transcription processes in the nucleus.CREB binding protein(CREB binding protein,CBP)is a transcriptional cofactor that can bind to sequence-specific DNA transcription factors;it is also an acetyltransferase that can acetylate histones near the target promoter to participate in DNA Repair,cell growth,apoptosis and inflammation,etc.We found that overexpression of GSTpi can promote CBP to enter the nucleus and maintain the nuclear activity of CBP.When VSMC are co-treated with GSTpi and CBP bromine domain inhibitor SGC-CBP30,the inhibitory effect of GSTpi on the phenotypic transformation of VSMC can be reversed.In summary,GSTpi is highly phosphorylated into the nucleus,and enters the nucleus.By regulating the protein level and activity of CBP,it has a negative regulatory effect on PDGF-BB-triggered VSMC phenotypic transformation.This study provides new ideas for the subsequent research on intimal proliferative diseases and atherosclerosis.