Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compo...Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.展开更多
Objective To study the role of sirtuin 1 （SIRT1） in Fas ligand （FasL） expression regulation during vascular lesion formation and to elucidate the potential mechanisms. Methods SIRT1 and FasL protein levels were d...Objective To study the role of sirtuin 1 （SIRT1） in Fas ligand （FasL） expression regulation during vascular lesion formation and to elucidate the potential mechanisms. Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell （VSMC） lysate. Smooth muscle cell （SMC）-specific human SIRT1 transgenic （Tg） C57BL/6 mice and their littermate wild-type （WT） controls underwent complete carotid artery ligation （ligation groups） or the ligation-excluded operation （sham groups）. The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 （SIRT1H363Y）, and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. Results SIRTI was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation （P〈0.001） and VSMCs treated with serum （P〈0.05 at the transcriptional level, P〈0.001 at the protein level）. No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL （P〈0.001）. The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 （P〈0.001）, while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 （P〈0.001）. Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT 1.展开更多
Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain an...Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.展开更多
Background Osteoprotegerin (OPG) is a secreted protein of the tumor necrosis factor receptor family, which regulates bone mass by inhibiting osteoclast differentiation and activation. Although OPG is expressed ubiquit...Background Osteoprotegerin (OPG) is a secreted protein of the tumor necrosis factor receptor family, which regulates bone mass by inhibiting osteoclast differentiation and activation. Although OPG is expressed ubiquitously and abundantly in many tissues and cell types including vascular cells, the role of OPG in other tissues is unknown.Our previous studies demonstrated that OPG was highly expressed in vascular smooth muscle cells (VSMC) and upregulated during vascular lesion formation. Methods and Results We documented, by Northern blot analysis,that the expression of OPG was more prevalent in the aorta and cultured VSMC from spontaneously hypertensive rats (SI-IR) compared to Wistar-Kyoto rats (WKY). In addition, we found that the expression of Angiotensin II (Ang II)type I receptor (AT1R) in SHR VSMC was at significantly increased levels than in WKY VSMC. Furthermore, Ang II potently induced the expression of OPG in VSMC in a time- and dose-dependent manner through the AT1R signaling pathway. Conclusions OPG expression was substantially greater in SHR VSMC, suggesting that OPG may be an important determinant of vascular remodeling in SHR.展开更多
Objective To investigate the mechanism of a novel angiotensin Ⅱ type 1 receptor-associated protein （ATRAP） interfering with angiotensin Ⅱ type 1 （AT1） receptor-mediated vascular smooth muscle cell （VSMC） growt...Objective To investigate the mechanism of a novel angiotensin Ⅱ type 1 receptor-associated protein （ATRAP） interfering with angiotensin Ⅱ type 1 （AT1） receptor-mediated vascular smooth muscle cell （VSMC） growth and neointimal formation. Methods VSMCs isolated from thoracic aorta of adult Sprague-Dawley （SD） rats were used in this study. ATRAP cDNA was subcloned into pcDNA3 vector and then transfected into VSMCs. DNA synthesis and extracellular signal-regulated kinase （ERK） and phospho-ERK expressions in VSMCs were assayed by measurement of ^3H thymidine incorporation and Western blotting, respectively. Morphological changes were observed in the balloon injured artery with or without transfection of ATRAP cDNA using 12-week-old male SD rats. Results ATRAP overexpression in VSMCs inhibited angiotensin Ⅱ （Ang Ⅱ）-induced ^3H thymidine incorporation 48 hours after Ang Ⅱ stimulation （ P 〈 0. 05 ）. In VSMC, Ang Ⅱ stimulation increased the phosphorylation of ERK, which reached the peak around 60 minutes. The activation of phospho-ERK was significantly decreased by ATRAP （ P 〈 0. 05 ）. Neointimal formation was markedly inhibited by ATRAP overexpression in injuried arteries.Conclusions The AT1 receptor-derived activation of ERK plays an essential role in Ang Ⅱ-induced VSMC growth. The growth inhibitory effects of ATRAP might be due to interfering with AT1 receptor-mediated activation of ERK in VSMC growth and neointimal formation.展开更多
Background and objectives Proliferation of human vascular smooth muscle cells(VSMCs)induced by hyperinsulinemia is a very common clinical pathology.Extensive research has focused on PKC(Protein kinase C)-MAPK(mitogen-...Background and objectives Proliferation of human vascular smooth muscle cells(VSMCs)induced by hyperinsulinemia is a very common clinical pathology.Extensive research has focused on PKC(Protein kinase C)-MAPK(mitogen-activated protein kinase)intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs.Methods DNA synthesis,signaling of ERK1/2 MAPKs,and changes inα-smooth muscle(SM)actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor,GF109203X.Results Differences among cell types in MAPK signaling,α-SM actin,and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension(EH)and normotension(NT)were identified in response to insulin treatment.Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs.Insulin exposure decreased expression ofα-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs,especially in the EH group.These effects were reversed by treatment with the PKC inhibitor.Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation,MAPK signaling,and degree of changes inα-SM actin and F-actin isoforms immediately following insulin exposure in vitro.展开更多
Ojective: To investigate the effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on the proliferation of vascular smooth muscle cells (VSMCs ). Methods: Based on the computer analysis of the secondary s...Ojective: To investigate the effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on the proliferation of vascular smooth muscle cells (VSMCs ). Methods: Based on the computer analysis of the secondary structure of c-myc mRNA, nt 2029 in rat c-myc oncogene was selected as a cleaving site for hammerhead ribozyme and the ribozyme was designed. With automatic DNA synthesizer, the two complementary DNA strands of the ribozyme were synthesized. The ribozyme gene was cloned into pGEM3Zf ( + ) vector and subcloned into eukaryotic expression pcD-NA3 vector. The recombinant pcDNA-Rz was transfected into the cultured rat VSMCs by lipofectAMINE mediated DNA transfection protocol and individual cell clones were selected by G418. Results: The sequence of ribozyme gene inserted in pGEMSZf ( + ) vector was proved to be perfectly correct. In VSMCs transfected with recombinant pcDNA-Rz, flow cytometry analysis showed that the S phase and G2/M fractions were decreased significantly and cell proliferation stagnated in the G0/G1 phase. Conclusion: The results suggest that hammerhead ribozyme that specifically cleaves c-myc mRNA can significantly inhibit the proliferation of VSMCs.展开更多
Objective： To determine whether balloon catheter denudation can induce vascular smooth muscle cells （VSMCs） to senescence, and whether this senescence can result in inflammation activity. Methods： Twelve male Chin...Objective： To determine whether balloon catheter denudation can induce vascular smooth muscle cells （VSMCs） to senescence, and whether this senescence can result in inflammation activity. Methods： Twelve male Chinese white rabbits were denuded of the carotid arteries or VSMCs. Acidic β-galactosidase activity of carotid arteries or VSMCs was detected. Transfection and chloramphenicol acetyltransferase （CAT） assay for iNOS gene and nitrite （NO2^-） assay were undertaken. Reverse transcription-polymerase chain reaction （RT-PCR） was used to evaluate inflammation cytokines mRNA expression. Measurement of NF-kB activity was detected by electrophoretic mobility shift assay （EMSA）. MMP-9, ICAM-1, P-p65, and IkBα expressions were analyzed by Western blotting. Results： After denudation VSMCs from denuded arteries showed an accumulation of significantly more senescence-associated β-galactosidase （SA-β-Gal） positive ceils and greater iNOS activity. Transcriptional activity of iNOS was highly expressed. The mRNA expressions of IL-1β, ICAM-1,MMP-9, TNF-α and the iNOS enzyme were significantly increased in injuring-induced senescence SMCs. However, the TNF-α or IL-1β-induced the protein production （ICAM-1 and MMP-9） was prevented by PDTC and MG132, which are inhibitors of NF-kB activation. Also, activation of NF-kB and cytokine-induced degradation of IKBα in the denuded VSMC were significantly affected. Conclusion： Intraluminal injury to the artery may lead to the emergence of senescent VSMC. Inflammation activity in SMCs is closely related to the senescence and the activation of NF-kB is involved.展开更多
This study aims at the theoretical and practical evidence for prevention of restenosis in vitro.Vascular smooth muscle cell(VSMC)model was established using adherent cell culture methods.The proliferation of VSMC was ...This study aims at the theoretical and practical evidence for prevention of restenosis in vitro.Vascular smooth muscle cell(VSMC)model was established using adherent cell culture methods.The proliferation of VSMC was investigated by the cell counting method and 3H-TDR implementation test.The results are as follows.(1) For ^125I-seeds,the inhibition rate was 29.3% at 74B1(P<0.05),35.2% at 148Bq(P<0.05)and 42.4% at 370Bq(P<0.05),For ^103Pd-implanted stents,the inhibition rate was 14.7% at 4.44MBq(P<0.05),24.0% at 5.92MBq(P<0.05) and 38.0%at 7.4MBq(p<0.05),There was no significant difference between the blank tests and non-radioactive tests.(2) 48 hours exposure from ^125I-seeds at 148Bq or ^103Pd-implanted stents at 7.4MBq did not result in VSMC's morphological alteration,but that from ^125I-seeds at 370Bq caused morphological changes,Both ^125I-seeds and ^103Pd0-implanted stents inhibit the VSMC DNA synthesis in vitro.The inhibition effects are significantly related to their exposure duration and doses.展开更多
Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kap...Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-κB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells (SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL+high density lipoprotein (HDL) originated from rats of 2 and 10 months old respectively. Fat stain was used to identify the lipid intake in SMCs. Results The optimal stimulation time of ox-LDL to SMCs was 12 hours. NF-κB intensity increased in most nuclei of SMCs that originated from rats of either 2 or 10 months old co-cultured with ox-LDL. The intensity of NF-κB and the amount of intracellular lipid taken in SMCs were more obvious in cells from 10-month-old rats than from the younger ones. Change of PDGF-B expression in SMCs was not remarkable in each group of rats. Conclusions The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear transloca- tion of NF-κB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-κB.展开更多
Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WH...Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.展开更多
Objective To investigate the effects of insulin-like growth factor-1 （IGF-1） and oxidized low density lipoprotein （oxLDL） on expression ofphosphatase PHLPP 1 in vascular smooth muscle cells （VSMCs）. Methods Rabb...Objective To investigate the effects of insulin-like growth factor-1 （IGF-1） and oxidized low density lipoprotein （oxLDL） on expression ofphosphatase PHLPP 1 in vascular smooth muscle cells （VSMCs）. Methods Rabbit aortic VSMCs were cultured. VSMCs proliferation ability was determined by measuring cell number and mitochondrial dehydrogenase （MD） activity with MTT assay. Western blot was used to detect the protein expression ofphosphatase PHLPP1. Results IGF-1 （100ug/L） increased cell number and MD activity to 3.02 and 3.59 times of that in control group, oxLDL（501xg/ml） elevated the above two parameters to 2.03 and 2.91 times respectively. Western blot showed that IGF-1 and oxLDL inhibited the expression of PHLPPI to 39.27% and 40.26% of the control group （P〈0.01 ）. Conclusion IGF- 1 and oxLDL may enhance the proliferation of VSMCs by decreasing the expression ofphosphatase PHLPP 1.展开更多
Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxi...Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxia group and 8--week hypoxia group. After the isolation of pulmonary artery smoothmuscles, pHi was determined by fluorescence measurement of the pH--sensitive dye BCECF and theexpression of NHE--1 mRNA was detected with reverse transcription--polymerase chain reaction. Results: ThepHi and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell in the hypoxia groups weresignificantly increased than those in the normal group (P < 0. 01 ). There was no remarkable differencebetween the hypoxia groups. Conclusion: With the function of regulation pHi., NHE--1 may play an importantrole in the pulmonary vascular remodeling of pulmonary hypertension. The result provides a new therapeuticmethod with NHE--1 inhibitors and/or gene therapy for the hypoxic pulmonary hypertension.展开更多
Objective To investigate whether Tumor Necrosis Factor-alpha （TNFα） is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and cla...Objective To investigate whether Tumor Necrosis Factor-alpha （TNFα） is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. Methods Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium （MTT）. The expression of Rho kinase genes after the stimulation of TNFα was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen （PCNA） after TNFα stimulation and Rosuvastatin intervention in smooth muscle cell. Results The TNFα stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells （P 〈 0.05）. These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects （P 〈 0.05）. Conclusions The activation of TNFα mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.展开更多
Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We...Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.展开更多
The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the po...The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the potential role of A.cinnamomea in cardiovascular diseases remains unexplored.Herein,using carotid arterial ligation models,we found that ethanol extract from A.cinnamomea(EEAC)signifi cantly inhibited neointimal hyperplasia in a dose-dependent manner,accompanied with the reduced expression of activated p65 and infl ammatory cytokines.We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-infl ammatory cytokine expression in both vascular smooth muscle cells(VSMCs)and macrophages in vitro.Mechanistically,EEAC suppressed expression levels of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in VSMCs,which attenuates the ability of monocytes/macrophages adhesion to VSMCs.Furthermore,the expression level of these adhesion molecules and infi ltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC.Altogether,our results reveal a novel function of A.cinnamomea in suppressing vascular infl ammation upon ligation injury during neointimal formation,likely through inhibition of infl ammatory cell infi ltration via downregulating the adhesion molecules in VSMCs.Thus,A.cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.展开更多
Objective:To determine the effects of a recombinant replication-deficient adenovirus encoding human tissue inhibitor of metalloproteinase-4(Ad.TIMP-4) on vascular smooth muscle cell(VSMC) function in vitro and neointi...Objective:To determine the effects of a recombinant replication-deficient adenovirus encoding human tissue inhibitor of metalloproteinase-4(Ad.TIMP-4) on vascular smooth muscle cell(VSMC) function in vitro and neointimal development in the injured rat carotid artery.Methods:Western blotting,gelatin zymography and reverse zymography were used to characterize the expression and functional activity of the TIMP-4 secreted by Ad.TIMP-4-infected VSMCs.The migration and proliferation of VSMCs in vitro were separately detected by using Millicell-PCF invasion chambers and [3H]-thymidine incorporation assay.Immunohistochemistry and morphometric analysis were used to determine the local expression of TIMP-4 and its effect on neointima development in a rat carotid artery balloon injury model.Results:VSMCs infected with Ad.TIMP-4 expressed functionally active human TIMP-4 which increased with the duration of infection.TIMP-4 expression inhibited VSMC migration,but not significantly affect VSMC proliferation.In a balloon-injured rat carotid artery model,a significant 62% reduction in neointimal area was found in Ad.TIMP-4-infected vessels at 14 days after injury.Ad.TIMP-4 infection had no effect on medial area.Conclusion:Our results indicated TIMP-4 over expression can significantly inhibit the migration of cultured VSMCs and prevent neointimal formation after vascular injury.Our findings provide additional evidence that TIMP-4 could play an important role in vascular pathophysiology,and may be an important therapeutic target for future drug development.展开更多
Objective: To investigate changes of Ca2+ activated potassium channels (KCa) in autogenous vein grafts. Methods: Contraction of venous ring was measured by means of perfusion in vitro. The intimal rabbits proliferatio...Objective: To investigate changes of Ca2+ activated potassium channels (KCa) in autogenous vein grafts. Methods: Contraction of venous ring was measured by means of perfusion in vitro. The intimal rabbits proliferation of vascular and proliferation of cultured smooth muscle cells(vascular smooth muscle cells, VSMCs)were observed by the means of computerised image analysis and MTT method respectively. Furthermore, whole cell mode of patch clamp was used to record KCa of VSMCs isolated from autogenous vein grafts. Results: One week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P<0.05, n=8 vs control), and they was more enhanced 4 weeks after vein transplantation (P<0.01, n=8 vs control).TEA(blocker of Ca2+ activated potassium channels)increased MTT A490 nm value of VSMCs from femoral vein in a dose dependent manner(P<0.05, n=8). KCa current density was significantly attenuated in VSMCs from autogenous vein grafts (1-4) week after transplantation(P<0.05, n=5).Conclusion: KCa is inhibited in autogenous vein graft, which account for vasospasm and intimal proliferation.展开更多
基金This project was supported by Grant 31200884 from the National Natural Science Foundation of China Grant 2016D016, 2016-ZQN-92, and 2016-2-75 from the Natural Science Foundation of Fujian and Grant 3502Z20154048, 3502Z20144061, and 3502Z20154047 from the Natural Scien- ce Foundation of Xiamen.
文摘Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.
基金Supported by the National Natural Science Foundation of China(81102444)Special Fund of the National Laboratory of China(2060204)
文摘Objective To study the role of sirtuin 1 （SIRT1） in Fas ligand （FasL） expression regulation during vascular lesion formation and to elucidate the potential mechanisms. Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell （VSMC） lysate. Smooth muscle cell （SMC）-specific human SIRT1 transgenic （Tg） C57BL/6 mice and their littermate wild-type （WT） controls underwent complete carotid artery ligation （ligation groups） or the ligation-excluded operation （sham groups）. The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 （SIRT1H363Y）, and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. Results SIRTI was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation （P〈0.001） and VSMCs treated with serum （P〈0.05 at the transcriptional level, P〈0.001 at the protein level）. No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL （P〈0.001）. The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 （P〈0.001）, while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 （P〈0.001）. Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT 1.
基金The work was supported by grant from The National Natural Sciences Foundation of China (No.30070280)
文摘Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.
文摘Background Osteoprotegerin (OPG) is a secreted protein of the tumor necrosis factor receptor family, which regulates bone mass by inhibiting osteoclast differentiation and activation. Although OPG is expressed ubiquitously and abundantly in many tissues and cell types including vascular cells, the role of OPG in other tissues is unknown.Our previous studies demonstrated that OPG was highly expressed in vascular smooth muscle cells (VSMC) and upregulated during vascular lesion formation. Methods and Results We documented, by Northern blot analysis,that the expression of OPG was more prevalent in the aorta and cultured VSMC from spontaneously hypertensive rats (SI-IR) compared to Wistar-Kyoto rats (WKY). In addition, we found that the expression of Angiotensin II (Ang II)type I receptor (AT1R) in SHR VSMC was at significantly increased levels than in WKY VSMC. Furthermore, Ang II potently induced the expression of OPG in VSMC in a time- and dose-dependent manner through the AT1R signaling pathway. Conclusions OPG expression was substantially greater in SHR VSMC, suggesting that OPG may be an important determinant of vascular remodeling in SHR.
文摘Objective To investigate the mechanism of a novel angiotensin Ⅱ type 1 receptor-associated protein （ATRAP） interfering with angiotensin Ⅱ type 1 （AT1） receptor-mediated vascular smooth muscle cell （VSMC） growth and neointimal formation. Methods VSMCs isolated from thoracic aorta of adult Sprague-Dawley （SD） rats were used in this study. ATRAP cDNA was subcloned into pcDNA3 vector and then transfected into VSMCs. DNA synthesis and extracellular signal-regulated kinase （ERK） and phospho-ERK expressions in VSMCs were assayed by measurement of ^3H thymidine incorporation and Western blotting, respectively. Morphological changes were observed in the balloon injured artery with or without transfection of ATRAP cDNA using 12-week-old male SD rats. Results ATRAP overexpression in VSMCs inhibited angiotensin Ⅱ （Ang Ⅱ）-induced ^3H thymidine incorporation 48 hours after Ang Ⅱ stimulation （ P 〈 0. 05 ）. In VSMC, Ang Ⅱ stimulation increased the phosphorylation of ERK, which reached the peak around 60 minutes. The activation of phospho-ERK was significantly decreased by ATRAP （ P 〈 0. 05 ）. Neointimal formation was markedly inhibited by ATRAP overexpression in injuried arteries.Conclusions The AT1 receptor-derived activation of ERK plays an essential role in Ang Ⅱ-induced VSMC growth. The growth inhibitory effects of ATRAP might be due to interfering with AT1 receptor-mediated activation of ERK in VSMC growth and neointimal formation.
基金This work was supported by grants from the National Science Foundation of China(No.30170384 and No.30570764)
文摘Background and objectives Proliferation of human vascular smooth muscle cells(VSMCs)induced by hyperinsulinemia is a very common clinical pathology.Extensive research has focused on PKC(Protein kinase C)-MAPK(mitogen-activated protein kinase)intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs.Methods DNA synthesis,signaling of ERK1/2 MAPKs,and changes inα-smooth muscle(SM)actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor,GF109203X.Results Differences among cell types in MAPK signaling,α-SM actin,and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension(EH)and normotension(NT)were identified in response to insulin treatment.Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs.Insulin exposure decreased expression ofα-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs,especially in the EH group.These effects were reversed by treatment with the PKC inhibitor.Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation,MAPK signaling,and degree of changes inα-SM actin and F-actin isoforms immediately following insulin exposure in vitro.
基金Supported by the National Natural Science Foundation of China(No.39600064)
文摘Ojective: To investigate the effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on the proliferation of vascular smooth muscle cells (VSMCs ). Methods: Based on the computer analysis of the secondary structure of c-myc mRNA, nt 2029 in rat c-myc oncogene was selected as a cleaving site for hammerhead ribozyme and the ribozyme was designed. With automatic DNA synthesizer, the two complementary DNA strands of the ribozyme were synthesized. The ribozyme gene was cloned into pGEM3Zf ( + ) vector and subcloned into eukaryotic expression pcD-NA3 vector. The recombinant pcDNA-Rz was transfected into the cultured rat VSMCs by lipofectAMINE mediated DNA transfection protocol and individual cell clones were selected by G418. Results: The sequence of ribozyme gene inserted in pGEMSZf ( + ) vector was proved to be perfectly correct. In VSMCs transfected with recombinant pcDNA-Rz, flow cytometry analysis showed that the S phase and G2/M fractions were decreased significantly and cell proliferation stagnated in the G0/G1 phase. Conclusion: The results suggest that hammerhead ribozyme that specifically cleaves c-myc mRNA can significantly inhibit the proliferation of VSMCs.
文摘Objective： To determine whether balloon catheter denudation can induce vascular smooth muscle cells （VSMCs） to senescence, and whether this senescence can result in inflammation activity. Methods： Twelve male Chinese white rabbits were denuded of the carotid arteries or VSMCs. Acidic β-galactosidase activity of carotid arteries or VSMCs was detected. Transfection and chloramphenicol acetyltransferase （CAT） assay for iNOS gene and nitrite （NO2^-） assay were undertaken. Reverse transcription-polymerase chain reaction （RT-PCR） was used to evaluate inflammation cytokines mRNA expression. Measurement of NF-kB activity was detected by electrophoretic mobility shift assay （EMSA）. MMP-9, ICAM-1, P-p65, and IkBα expressions were analyzed by Western blotting. Results： After denudation VSMCs from denuded arteries showed an accumulation of significantly more senescence-associated β-galactosidase （SA-β-Gal） positive ceils and greater iNOS activity. Transcriptional activity of iNOS was highly expressed. The mRNA expressions of IL-1β, ICAM-1,MMP-9, TNF-α and the iNOS enzyme were significantly increased in injuring-induced senescence SMCs. However, the TNF-α or IL-1β-induced the protein production （ICAM-1 and MMP-9） was prevented by PDTC and MG132, which are inhibitors of NF-kB activation. Also, activation of NF-kB and cytokine-induced degradation of IKBα in the denuded VSMC were significantly affected. Conclusion： Intraluminal injury to the artery may lead to the emergence of senescent VSMC. Inflammation activity in SMCs is closely related to the senescence and the activation of NF-kB is involved.
文摘This study aims at the theoretical and practical evidence for prevention of restenosis in vitro.Vascular smooth muscle cell(VSMC)model was established using adherent cell culture methods.The proliferation of VSMC was investigated by the cell counting method and 3H-TDR implementation test.The results are as follows.(1) For ^125I-seeds,the inhibition rate was 29.3% at 74B1(P<0.05),35.2% at 148Bq(P<0.05)and 42.4% at 370Bq(P<0.05),For ^103Pd-implanted stents,the inhibition rate was 14.7% at 4.44MBq(P<0.05),24.0% at 5.92MBq(P<0.05) and 38.0%at 7.4MBq(p<0.05),There was no significant difference between the blank tests and non-radioactive tests.(2) 48 hours exposure from ^125I-seeds at 148Bq or ^103Pd-implanted stents at 7.4MBq did not result in VSMC's morphological alteration,but that from ^125I-seeds at 370Bq caused morphological changes,Both ^125I-seeds and ^103Pd0-implanted stents inhibit the VSMC DNA synthesis in vitro.The inhibition effects are significantly related to their exposure duration and doses.
文摘Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-κB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells (SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL+high density lipoprotein (HDL) originated from rats of 2 and 10 months old respectively. Fat stain was used to identify the lipid intake in SMCs. Results The optimal stimulation time of ox-LDL to SMCs was 12 hours. NF-κB intensity increased in most nuclei of SMCs that originated from rats of either 2 or 10 months old co-cultured with ox-LDL. The intensity of NF-κB and the amount of intracellular lipid taken in SMCs were more obvious in cells from 10-month-old rats than from the younger ones. Change of PDGF-B expression in SMCs was not remarkable in each group of rats. Conclusions The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear transloca- tion of NF-κB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-κB.
文摘Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.
文摘Objective To investigate the effects of insulin-like growth factor-1 （IGF-1） and oxidized low density lipoprotein （oxLDL） on expression ofphosphatase PHLPP 1 in vascular smooth muscle cells （VSMCs）. Methods Rabbit aortic VSMCs were cultured. VSMCs proliferation ability was determined by measuring cell number and mitochondrial dehydrogenase （MD） activity with MTT assay. Western blot was used to detect the protein expression ofphosphatase PHLPP1. Results IGF-1 （100ug/L） increased cell number and MD activity to 3.02 and 3.59 times of that in control group, oxLDL（501xg/ml） elevated the above two parameters to 2.03 and 2.91 times respectively. Western blot showed that IGF-1 and oxLDL inhibited the expression of PHLPPI to 39.27% and 40.26% of the control group （P〈0.01 ）. Conclusion IGF- 1 and oxLDL may enhance the proliferation of VSMCs by decreasing the expression ofphosphatase PHLPP 1.
文摘Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxia group and 8--week hypoxia group. After the isolation of pulmonary artery smoothmuscles, pHi was determined by fluorescence measurement of the pH--sensitive dye BCECF and theexpression of NHE--1 mRNA was detected with reverse transcription--polymerase chain reaction. Results: ThepHi and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell in the hypoxia groups weresignificantly increased than those in the normal group (P < 0. 01 ). There was no remarkable differencebetween the hypoxia groups. Conclusion: With the function of regulation pHi., NHE--1 may play an importantrole in the pulmonary vascular remodeling of pulmonary hypertension. The result provides a new therapeuticmethod with NHE--1 inhibitors and/or gene therapy for the hypoxic pulmonary hypertension.
文摘Objective To investigate whether Tumor Necrosis Factor-alpha （TNFα） is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. Methods Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium （MTT）. The expression of Rho kinase genes after the stimulation of TNFα was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen （PCNA） after TNFα stimulation and Rosuvastatin intervention in smooth muscle cell. Results The TNFα stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells （P 〈 0.05）. These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects （P 〈 0.05）. Conclusions The activation of TNFα mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.
基金supported by the National Key Research Project of China(2022YFF1100300)National Natural Science Foundation of China(32272328)+5 种基金Natural Science Foundation of Hebei Province(B2022321001)Major Public Welfare Projects in Henan Province(201300110200)National Key Research Project of Hebei Province(20375502D)National Key Research Project of Hebei Province(H2021206427)University Science and Technology Research Project of Hebei Province(QN2017107)Postdoctoral Research Funds of Hebei Medical University(307050100163759).
文摘Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.
基金This work was supported by the National Key Research Project of China(2019YFC1606400)Major Public Welfare Projects in Henan Province(201300110200)+4 种基金National Key Research Project of Hebei Province(20375502D)Natural Science Foundation of Hebei Province(H2019206212)High-level Talent Funding Project of Hebei Province(A201905006)Fund of National R&D Center for Edible Fungus Processing Technology,Henan University(20200109)the Open Fund from Beijing Advanced Innovation Center for Food Nutrition and Human Health(20182025).
文摘The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the potential role of A.cinnamomea in cardiovascular diseases remains unexplored.Herein,using carotid arterial ligation models,we found that ethanol extract from A.cinnamomea(EEAC)signifi cantly inhibited neointimal hyperplasia in a dose-dependent manner,accompanied with the reduced expression of activated p65 and infl ammatory cytokines.We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-infl ammatory cytokine expression in both vascular smooth muscle cells(VSMCs)and macrophages in vitro.Mechanistically,EEAC suppressed expression levels of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in VSMCs,which attenuates the ability of monocytes/macrophages adhesion to VSMCs.Furthermore,the expression level of these adhesion molecules and infi ltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC.Altogether,our results reveal a novel function of A.cinnamomea in suppressing vascular infl ammation upon ligation injury during neointimal formation,likely through inhibition of infl ammatory cell infi ltration via downregulating the adhesion molecules in VSMCs.Thus,A.cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.
基金Supported by the National Natural Science Foundation of China (30630056)
文摘Objective:To determine the effects of a recombinant replication-deficient adenovirus encoding human tissue inhibitor of metalloproteinase-4(Ad.TIMP-4) on vascular smooth muscle cell(VSMC) function in vitro and neointimal development in the injured rat carotid artery.Methods:Western blotting,gelatin zymography and reverse zymography were used to characterize the expression and functional activity of the TIMP-4 secreted by Ad.TIMP-4-infected VSMCs.The migration and proliferation of VSMCs in vitro were separately detected by using Millicell-PCF invasion chambers and [3H]-thymidine incorporation assay.Immunohistochemistry and morphometric analysis were used to determine the local expression of TIMP-4 and its effect on neointima development in a rat carotid artery balloon injury model.Results:VSMCs infected with Ad.TIMP-4 expressed functionally active human TIMP-4 which increased with the duration of infection.TIMP-4 expression inhibited VSMC migration,but not significantly affect VSMC proliferation.In a balloon-injured rat carotid artery model,a significant 62% reduction in neointimal area was found in Ad.TIMP-4-infected vessels at 14 days after injury.Ad.TIMP-4 infection had no effect on medial area.Conclusion:Our results indicated TIMP-4 over expression can significantly inhibit the migration of cultured VSMCs and prevent neointimal formation after vascular injury.Our findings provide additional evidence that TIMP-4 could play an important role in vascular pathophysiology,and may be an important therapeutic target for future drug development.
文摘Objective: To investigate changes of Ca2+ activated potassium channels (KCa) in autogenous vein grafts. Methods: Contraction of venous ring was measured by means of perfusion in vitro. The intimal rabbits proliferation of vascular and proliferation of cultured smooth muscle cells(vascular smooth muscle cells, VSMCs)were observed by the means of computerised image analysis and MTT method respectively. Furthermore, whole cell mode of patch clamp was used to record KCa of VSMCs isolated from autogenous vein grafts. Results: One week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P<0.05, n=8 vs control), and they was more enhanced 4 weeks after vein transplantation (P<0.01, n=8 vs control).TEA(blocker of Ca2+ activated potassium channels)increased MTT A490 nm value of VSMCs from femoral vein in a dose dependent manner(P<0.05, n=8). KCa current density was significantly attenuated in VSMCs from autogenous vein grafts (1-4) week after transplantation(P<0.05, n=5).Conclusion: KCa is inhibited in autogenous vein graft, which account for vasospasm and intimal proliferation.
