Long noncoding RNA ZFAS1/miR-150/ROCK1 axis regulates proliferation and migration of vascular smooth muscle cells

AIM: To investigate whether long noncoding RNA ZNFX1 (zinc finger NFX1-type containing 1) antisense RNA 1 (ZFAS1) promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) by regulating microRNA-150 (miR-150)/ROCK1, and the involving mechanism of atherosclerosis. METHODS: Platelet-derived growth factor-BB (PDGF-BB) was used to induce proliferation and migration of VSMCs. Real-time PCR was used to detect the content of ZFAS1 in the VSMCs. After further down-regulating the expression of ZFAS1 by siRNA, the viability of VSMCs was detected by MTT assay, and the proliferation was measured by EdU staining. The migration ability of VSMCs was detected by Transwell method. The expression levels of miR-150 and ROCK1 were detected by RT-qPCR, and the protein level of ROCK1 was determined by Western blot. Luciferase reporter assay was used to confirm that ROCK1 was the target gene of miR-150. Finally, miR-150 expression was inhibited, and the proliferation and migration ability of VSMCs and expression of ROCK1 after down-regulation of ZFAS1 expression were examined. RESULTS: PDGF-BB up-regulated the expression of ZFAS1 in the VSMCs. After down-regulating the expression of ZFAS1, the proliferation and migration abilities of VSMCs were inhibited (P<0.05), the expression level of miR-150 was increased (P<0.05), and the expression level of ROCK1 was decreased (P<0.05). The results of luciferase reporter assay showed that miR-150 directly targeted ROCK1. Inhibition of miR-150 expression attenuated the inhibition of proliferation and migration of VSMCs by ZFAS1 expression knock-down (P<0.05) and up-regulated the expression level of ROCK1 (P<0.05). CONCLUSION: ZFAS1 promotes the proliferation and migration of VSMCs induced by PDGF-BB by regulating miR-150/ROCK1.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

Molecular mechanism of nontypeable Haemophilus influenzae stimulated MUC5AC production in NCI-H292 cells

AIM: To investigate the molecular mechanism of nontypeable Haemophilus influenzae(NTHi)-induced MUC5AC expression in the human airway NCI-H292 cells.METHODS: Bronchial epithelial NCI-H292 cells were cultured in vitro. The production of MUC5AC and matrix metalloproteinase 9(MMP-9) after stimulation with NTHi was analyzed by enzyme-linked immunosorbent assay(ELISA). The enzymatic activity of MMP-9 was detected by gelatin zymography. In addition, the cells were pretreated with different inhibitors such as AG1478, LY294002, DPI, NAC and GM6001 before stimulation, which specifically inhibit epidermal growth factor receptor(EGFR), phosphatiadylinositol 3-kinase(PI3K), NADPH oxidase, reactive oxygen species(ROS) and MMP-9, respectively, and then the production of MUC5AC or MMP-9 was determined.RESULTS: NTHi-stimulated NCI-H292 cells exhibited a time-dependent increase in MMP-9 secretion and activity. NTHi also increased the activity of Rac1 and the production of ROS. Pretreatment of AG1478 and LY294002 decreased the Rac1 activity, and preincubation of DPI or Rac1 inhibitor significantly abrogated ROS production. In addition, secretion of MMP-9 and the enzymatic activity was decreased by treatment of NAC and NSC23766. Furthermore, inhibition of the MMP-9 activity by GM6001 significantly inhibited MUC5AC production.CONCLUSION: EGFR/PI3K/Rac1/NADPH oxidase/ROS/MMP-9 regulates MUC5AC production in NTHi-challenged NCI-H292 cells.     

Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

Inducing vascular smooth muscle cell proliferation by insulin involves in phosphatidylinositol 3-kinase and ERK1/2

AIM: To investigate the effect of insulin on vascular smooth muscle cells (VSMCs) proliferation and to evaluate the intracellular signaling pathways involved.METHODS: VSMCs separated from Sprague-Dawley rats were used in this study. The proliferation of VSMCs induced by insulin was assayed by ［3H］-thymidin incorporation. The protein expression and activity of p-ERK1/2 were determined by immunblot and ［γ-32P］ATP incorporation.RESULTS: Insulin induced cell proliferation in a concentration-dependent manner. The proliferative effect of insulin on VSMCs was inhibited partly by LY294002 (48.8%), an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059 (43.6%), respectively. Moreover, phosphorylation of ERK1/2 and activity of ERK1/2 induced by insulin were also inhibited partly by LY294002.CONCLUSION: PI-3 kinase and ERK1/2 are involved in insulin induced VSMCs proliferation.     

Genistein inhibits proliferation of human oral cancer TCA8113 cells through suppression of VEGF expression

AIM: To investigate the effect of genistein on the proliferation of human oral cancer TCA8113 cells and to explore the underlying mechanisms.METHODS: The cell proliferation was examined by MTT assay, cell counting and colony formation assay. Western blotting was employed to examine the protein levels of vascular endothelial growth factor(VEGF), extracellular signal-regulated kinase(ERK) and p-ERK. RESULTS: Genistein significantly inhibited the proliferation of TCA8113 cells in a concentration-dependent fashion. Moreover, genistein dose-dependently decreased the protein levels of VEGF, ERK and p-ERK. The expression of VEGF was also blunted by U0126, a specific inhibitor of ERK. U0126 and axitinib, a VEGF receptor antagonist, both significantly inhibited the proliferation of TCA8113 cells. CONCLUSION: Genistein inhibits the proliferation of TCA8113 cells, which may be related to its inhibitory effect on ERK expression and activation, thus subsequently decreasing the expression of VEGF.     

Akt and ERK1/2 are involved in brain derived neurotrophic factor-induced angiogenesis of endothelial cells

AIM: To investigate the role of PI3K/Akt and MEK1/ERK pathway in the brain derived neurotrophic factor(BDNF)-induced angiogenesis in vitro and to explore the further molecular mechanisms. METHODS: The phosphorylations of Akt and ERK1/2 were detected in human umbilical vein endothelial cells(HUVECs) by Western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and tubule formation assay. Cell proliferation was determined by crystal violet staining. Cell apoptosis was analysed by FITC-Annexin-V/PI double staining and flow cytometry. RESULTS: BDNF activated the PI3K/Akt and MEK1/ERK pathway in a time-dependent manner. Ly294002 and PD98059 blocked the activation of Akt and ERK1/2 in response to BDNF. BDNF at concentration of 100 μg/L significantly increased HUVECs tube formation, migration and proliferation in vitro to a degree similar to those induced by 25 μg/L VEGF. Furthermore, tube formation and migration of HUVECs toward BDNF were significantly inhibited by treatment with 20 μmol/L Ly294002 and 20 μmol/L PD98059. BDNF-induced survival was only blocked by Ly294002 and BDNF-induced proliferation was only inhibited by PD98059. CONCLUSION: BDNF promotes angiogenesis of HUVECs in vitro. The ERK and Akt are two crucial events in BDNF-mediated signal transduction leading to HUVECs angiogenesis by different mechanisms. Moreover, the latter is more important.     

CREG inhibits proliferation of VSMCs by modulating internalization of IGF2R/IGFII

AIM: To study the molecular mechanisms of cellular repressor of E1A-stimulated genes (CREG) on the proliferation of human vascular smooth muscle cells (VSMCs) in vitro.METHODS: The pLNCX₂-CREG plasmid and the pSM₂-siCREG plasmid were transfected into VSMCs to produce the cell clones of over-expression and down-expression of CREG, respectively.BrdU assay and FACS cell cycle analysis were performed to detect the proliferation of the cells.The expression and localization of insulin-like growth factor Ⅱ receptor(IGF2R) in the hVSMCs were detected by Western blotting and immunocytochemistry.The expression and secretion of insulin-like growth factor Ⅱ(IGFII) were measured by RT-PCR and ELISA.Alexa 488-labeled rhIGFII was used to investigate the endocysis of the cells.The blockade of IGFII internalization was conducted by treating the cells with both neutralized antibody of IGF2R and recombinant IGF2R peptide to detect the effect of IGFII on HVSMCs growth.Furthermore, Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/Akt and ERK on VSMCs proliferation.RESULTS: Compared with the control cells, Western blotting identified that the expression of CREG was increased in VSMCs-CREG cells and was decreased in VSMCs-siCREG cells.Meanwhile, the over-expression of CREG in the cells inhibited the proliferation of VSMCs and enhanced the distribution of IGF2R in cellular membrane.Furthermore, over-expression of CREG also accelerated the endocytosis of IGFII in VSMCs-CREG cells, and attenuated the secretion of IGFII into cell medium.Blockade experiments showed that enhancement of IGFII secretion promoted the proliferation of HVSMCs.PI3K/Akt and ERK signal pathways mediated the effect of IGFII on the VSMCs.CONCLUSION: CREG inhibits the proliferation of VSMCs through interfering with the internalization of IGF2R-IGFII.     

Effect of interferon-inducible protein p204 on proliferation of vascular smooth muscle cells in rats

AIM: To study the effect of interferon-inducible protein p204 on the proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Cultured VSMCs were treated with interferon alpha (IFN-α) and p204 gene (Ifi204) small interfering RNA (siRNA) in vitro instantaneously. The cell vitality was detected by MTT me-thod,and the cell cycle was analyzed by flow cytometry. The expression of mRNA and proteins was determined by real-time qRT-PCR and Western blotting,respectively.RESULTS: IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality, inhibited the cell cycle of G₁/S transition, and down-regulated the expression of Ras protein in VSMCs. Meanwhile, the phosphorylation levels of Raf and ERK were decreased. Transfection of Ifi204 siRNA restrained the expression of p204, increased the cell vitality and promoted the cell cycle of G₁/S transition in VSMCs. The up-regulation of Ras protein expression and the increased phosphorylation levels of Raf and ERK were also observed.CONCLUSION: The expression of p204 restrains the proliferation of VSMCs in rats by inhibiting the activation of Ras/Raf/MEK/ERK signal pathway.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.     

Effects of β3 integrin on adhesion and migration of vascular smooth muscle cells induced by growth factor

AIM: To investigate the effect of platelet-derived growth factor (PDGF) on β₃ integrin gene expression and the role of β₃ integrin on adhesion, migration and proliferation of vascular smooth muscle cells (VSMC) induced by PDGF. METHODS: β₃ integxin gene expression was detected by RT-PCr. After β₃ integrin extracellular do-main was blocks, VSMC adhesio, migration and proliferation were measured by adhesion assay awound-culture model an [³H]-TSR incorporation respectively.RESULTS: After the interaction between β₃ integrin and extracellular matrix was blocked, VSMC proliferation was inhibited in some degree and the rate of [³H]-TdR incorporation into VSMC decreased 39%. The cell adhesion and migration were significantly inhibited when 10 mg/L anti-β₃ integrin antibody was added (P＜0.05). When VSMC were treated by PDGF for 6 hours, the expression of β₃ integrin gene was 87% higher than that of control. CONCLUSION: PDGF significantly induces expression of β₃ integrin gene in VSMC, and the interaction between β₃ integrin and ECM protein may play an important role in VSMC adhesion and migration.     

STAT3 mediates PDGF-BB-induced Pim-1 expression in rat vascular smooth muscle cells

AIM: To study the expression of Pim-1 in vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: VSMCs isolated from rats were treated with different concentrations of PDGF-BB for different time. The proliferation of VSMCs was detected by cell counting. The mRNA expression of Pim-1 was measured by real-time RT-PCR. The STAT3 activity was determined by Western blotting. Actinomycin D, AG490, and small interfering RNA (siRNA) for Pim-1 or STAT3 were used to investigate the underlying mechanisms. RESULTS: Pim-1 gene silencing attenuated the proliferation of VSMCs in response to PDGF-BB. The mRNA expression of Pim-1 was up-regulated by PDGF-BB at concentrations of 10 μg/L~50 μg/L for 1 h, and was maximally induced at the concentration of 20 μg/L. The time of Pim-1 mRNA expression maximally occurred 30 min after PDGF-BB exposure. Incubation of VSMCs with PDGF-BB resulted in a significant activation of STAT3. VSMCs pretreated with actinomycin D showed a significant decrease in the mRNA expression of Pim-1. Treatment with AG490 or knockdown of STAT3 in VSMCs resulted in inactivation of STAT3, and significantly suppressed the mRNA expression of Pim-1. CONCLUSION: PDGF-BB-induced VSMC proliferation is partly attributed to Pim-1. VSMCs strongly increase Pim-1 mRNA upon stimulation with PDGF-BB, and STAT3 signaling pathway appears to be efficient for regulation of Pim-1 expression. This process may play a critical role in development of vascular remodeling.     

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.     

Transforming growth factor α promotes the proliferation,migration and adhesion of human endothelial progenitor cells

AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.     

Role of angiotensin II and angiotensin II receptors in vascular smooth muscle cells migration

AIM:To determine the effects of Angiotensin II(AngII) on migration of rat smooth muscle cells and to investigate the mechanisms underlying Ang II action in the development of injured vascular disease. METHODS:VSMCs isolated from aortic media of Wistar rats and cultured by the modified explant method were adopted. In prersence and absence of AngII, the expression of AngII receptor and reorganization of the actin cytoskeleton of VSMCs were studied by immunocytochemistry technique, fluorocytochemistry technique. The migration assays were performed by a modified Boyden's chamber. And the effects of AT₁R antagonist (CV-11974), AT₂R antagonist (PD123319) on aforementioned target were studied.RESULTS:VSMCs migration was stimulated by addition of AngII. The dynamic reorganization of actin cytoskeleton may be an important mechanism by which AngII facilitates VSMC motility. The expression of AT₁R in VSMCs can be upregulated after treatment with AngII initially, then decreased gradually. The expression of AT₁R was downregulated by AT₁R antagonist. The effect of AngII on VSMCs migration was mediated by AT₁R, while AT₂R had no significant effect.CONCLUSION:The dynamic reorganization of actin cytoskeleton is required for AngII-induced VSMC migration, and this effect is mediated by AT₁R .