MiR-155-dependent Regulation Of MST2 Coordinates Inflammation,Oxidative Stress And Promotes Vascular Remodeling

In response to vascular injury, vascular smooth muscle cells(VSMCs) undergo a series of characteristic changes including phenotypic modulation, abnormal proliferation, migration, matrix synthesis, and inflammation; these events are crucial for the development and progression of vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. VSMCs are the principal effector cells in this process; therefore VSMCs coordinate and synchronize extremely complex inflammatory, proliferative, differentiation and oxidative stress programs. Although a few pathophysiological mechanisms associated with VSMC proliferation, inflammation and oxidative stress are known, the molecular mechanisms by which the VSMC responses to injury are coordinated remain unclear.Micro RNAs(mi RNAs) regulate gene expression at the post-transcriptional level by promoting m RNA degradation or by inhibiting translation and play a critical role in vascular inflammation and remodeling. mi R-155 is a target of several inflammatory mediators. Recently, we found that mi R-155 is involved in neointimal formation in carotid arteries after angioplasty. mi R-155 is a pleiotropic regulator of inflammation-related diseases and is critical for various physiological and pathological processes including inflammation, differentiation, carcinogenesis, oxidative stress, and cardiovascular remodeling. However, the role of mi R-155 in coordinating inflammation, oxidative stress and vascular remodeling has not been elucidated.As a core component of the Hippo pathway in mammalian cells, the mammalian sterile 20–like kinase 2(MST2) regulates cell proliferation, growth and apoptosis. MST2 binds to Raf-1 at two distinct sites that partially overlap with the mitogen-activated protein kinase kinase(MEK)-binding domain on Raf-1, suggesting that MST2 competes with MEK to bind to Raf-1 and affects the MAPK signaling cascade. Furthermore, the interaction between MST2 and Raf-1 regulates the ERK1/2 pathway and inhibits the pro-apoptotic activation of MST2. Although MST1/2 activates Ser/Thr protein kinases and regulates the Raf-1/ERK pathway activity, the role of MST2 in regulation of inflammation and oxidative stress in VSMCs has not been studied. In this study, we investigated whether and how MST2 mediates mi R-155-promoted inflammation and oxidative stress, which lead to VSMC proliferation and vascular remodeling, through integrating inflammatory and oxidative stress signaling.PartⅠ mi R-155 is involved in vascular remodeling by promoting the proliferation of VSMCsObjective: To investigate the role of mi R-155 in proliferation and apoptosis of VSMCs as well as in neointimal hyperplasia after vascular injury.Methods: 1 Western blot, cell-counting and MTS assays were performed to examine the proliferation of cell after overexpression and knockdown of mi R-155 in VSMCs. 2 Femoral artery wire injury was performed in mi R-155-/-and wild-type(WT) mice, and morphology of neointima hyperplasia was assessed by hematoxylin/eosin staining and images were acquired using a Leica microscope and digitized with LAS V.4.4 software. 3 Expression of mi R-155 in injured vessels of mi R-155-/- and WT mice was determined by q RT-PCR. 4 mi R-155 was overexpressed by using adenovirus(Ad)-mediated gene transfer and up-regulated or down-regulated by local transfecting with an agomir or antagomir after vascular injury, and morphology of neointima hyperplasia was assessed by hematoxylin/eosin staining. 5 Proliferation of VSMCs and infiltration of macrophages were detected by immunofluorescence staining with SM22α and MAC2 antibodies. 6 In situ hybridization was performed to locate the expression of mi R-155. 7 TUNEL staining and Western blot were performed to measure apoptosis after overexpression mi R-155 in H2O2-treated VSMCs.Results:1 Loss and gain of mi R-155 significantly decrease and increase proliferation of cultured VSMCs, respectivelyOverexpression of mi R-155 significantly strengthened the expression of proliferating cell nuclear antigen(PCNA) compared with control group, and increased cell number and A490 absorption values. In contrast, transfection of VSMCs with anti-mi R-155 but not anti-mi R-ctl inhibited PDGF-BB-induced expression of PCNA, and cell-counting and MTS assays yielded similar results.2 Genetic deletion of mi R-155 significantly inhibits neointimal hyperplasia induced by femoral artery wire injuryMorphometric analysis showed that neointimal formation significantly increased after femoral artery wire injury. The injured vessels in mi R-155-/-mice showed decreased neointimal hyperplasia, intima-to-media ratio and stenosis compared with WT mice. q RT-PCR analysis revealed that mi R-155 expression was elevated by 3.5-fold in the injured arteries of WT mice compared to uninjured arteries and was barely detectable in mi R-155-/- mice.3 Overexpression of mi R-155 facilitates neointimal formation after wire injurymi R-155 was overexpressed using adenovirus(Ad)-mediated gene transfer; Ad-mi R-155 was administered intraluminally after wire-induced injury in WT mice. q RT-PCR analysis revealed increased mi R-155 expression in injured vessels 14 days after Ad-mi R-155 infection. As expected, mi R-155 overexpression significantly increased neointimal formation, the intima/media ratio, and stenosis.4 Transfection with mi R-155 agomir promotes and with antagomir inhibits wire-injury-induced neointimal formationTo further confirm the role of mi R-155 in vascular injury, wire-injured femoral arteries were treated locally with an agomir or antagomir of mi R-155 to up-regulate or down-regulate mi R-155, respectively. The results showed that treatment with the mi R-155 agomir increased neointimal hyperplasia, intima-to-media ratio and stenosis compared with the control agomir. Conversely, mi R-155 antagomir decreased neointimal formation, the intima/media ratio, and stenosis compared with control antagomir. These results suggest that mi R-155 plays a vital role in neointimal formation induced by vascular endothelial injury.5 VSMCs are the primary cellular source of mi R-155 in injured vesselsBecause VSMC proliferation and macrophage infiltration underlie the pathogenesis of neointimal formation, we examined the VSMC and macrophage contents in injured vessels. SM22α-positive cells comprised 73.9 % of the lesion area within the neointima in WT mice but only 23.7 % of the lesion area in mi R-155-/- mice. However, the macrophage numbers in the lesion area(MAC2-positive cells) were not significantly different in WT and mi R-155-/- mice. These observations suggest that VSMCs are likely the primary cellular source of mi R-155 in injured vessels. To test this hypothesis, we performed in situ hybridization of mi R-155 probe combined with SM22α immunostaining and observed that mi R-155 was expressed mainly in the VSMCs but not the macrophages in the neointima. Together, these results indicate that mi R-155 mediates VSMC proliferation, leading to neointimal formation after wire-induced vascular injury.6 Overexpression of mi R-155 inhibits apoptosis in cultured VSMCsThe number of TUNEL positive cells was markedly increased in VSMCs after VSMCs were treated with H2O2. The H2O2–induced apoptosis cells were significantly reduced in mi R-155-overexpressed VSMCs compared with control. TUNEL staining and caspase-3 cleavage assay showed that mi R-155 overexpression significantly inhibited VSMC apoptosis and the activation of caspase-3.Summary:mi R-155 promotes VSMC proliferation in vitro and in vivo. mi R-155 is involved in the wire-injury-induced intimal hyperplasia and vascular remodeling.and oxidative stress and is involved in the vascular smooth muscle cell proliferation PartⅡ mi R-155-targeted wuppression of MST2 regulates inflammationObjective: To elucidate whether mi R-155-dependent regulation of MST2 integrates inflammation and oxidative stress and participates in vascular remodeling.Method: 1 Potential targets of proliferation and migration-related genes were predicted in the mi Randa and RNAhybrid sites. 2 Wild-type and mutant 3’ UTR was inserted into pmir-GLO dual luciferase reporter plasmid and transfected into 293 A cells, and its activity was measured by dual luciferase reporter assay. 3 Western blot analyses examined the expression of MST2 and TRIM39 in VSMCs after mi R-155 was overexpressed and knock downed. 4 Immunofluorescence staining of MST2 and SM22α was performed in injured vessels of WT and mi R-155-/- mice. 5 Inflammatory and oxidative stress-related gene expression was detected by q RT-PCR in the injured vessels. 6 Wound-scratch healing assay detected VSMC proliferation and migration.Results:1 MST2 is a direct target of mi R-155 in VSMCsEight potential targets related to VSMC proliferation or inflammation were tested using luciferase reporter assays. MST2, TRIM39 and Itch reporters were repressed by a mi R-155 mimic. We mutated the mi R-155-binding site in the 3’UTR of the MST2 and TRIM39 m RNAs and performed luciferase reporter assays using a mi R-155 mimic and a mi R-155 inhibitor. We observed that in cells containing wild-type(WT) MST2 3’UTR, luciferase activity was significantly reduced after treatment with the mi R-155 mimic compared with control micro RNA; mutation of the mi R-155-binding site in the MST2 3’UTR almost completely restored luciferase activity in the presence of the mi R-155 mimic. Similar results were obtained in cells containing WT and mutant TRIM39 3’UTR. Western blot analysis showed that anti-mi R-155 increased the MST2 protein level but not the TRIM39 protein level in a dose-dependent manner. Conversely, overexpression of mi R-155 in VSMCs decreased expression of the MST2 protein but not the TRIM39 protein in a dose-dependent manner.2 mi R-155-dependent regulation of MST2 promotes proliferation and migration of VSMCs and mediates vascular remodelingq RT-PCR measured expression of MST2 in wire-injured vessels showed that MST2 expression was increased in mi R-155-/- mice and decreased in WT mice overexpressing mi R-155. Immunofluorescent staining was performed on sections of the injured femoral arteries from WT and mi R-155-/- mice. We observed that MST2 expression was significantly increased in the injured lesions of mi R-155-/- mice compared with WT mice. Wound-scratch assay showed that overexpression of mi R-155 markedly increased the migration of VSMCs. However, the promoting action of mi R-155 on VSMC migration could be abolished by overexpressing MST2. Together, these results suggest that mi R-155 regulates VSMC proliferation and migration by targeting MST2 and mediates vascular remodeling.3 mi R-155 promotes expression of NF-κB, TNF-α, p47 phox and HO-1 by targeting MST2Using q RT-PCR, we examined the expression of iNOS, HO-1, p47, p22 phox, NF-κB, TNF-α, and IL-1β in the injured arteries of WT, mi R-155-/-and mi R-155-overexpressing mice. Notably, the i NOS, HO-1, p47 phox, and NF-κB expression levels were significantly reduced in the injured arteries of mi R-155-/- mice compared with WT mice. Conversely, the expression of these genes was significantly higher in mi R-155-overexpressing mice compared with Ad-GFP-infected mice. However, there was no significant difference in p22 phox, TNF-α, and IL-1β expression in the injured arteries of mi R-155-overexpressing mice compared with Ad-GFP-infected mice. Together, these data suggest that mi R-155-mediated downregulation of MST2 increases inflammation and oxidative stress, as evidenced by upregulation of NF-κB and p47 phox expression.Summary:MST2 is a direct target of mi R-155 in VSMCs. mi R-155 promotes VSMC proliferation by depressing MST2 and that activating ERK1/2 signaling pathway.PartⅢ mi R-155-dependent regulation of MST2 mediates inflammation and oxidative stress crosstalk, and promotes proliferation of VSMCs via Raf-1-MEK-ERK1/2 signaling pathwayObjective: To explore the effect of mi R-155-dependent regulation of MST2 on Raf-1-MEK-ERK signaling pathway.Method: 1 Western blot detected cell proliferation, inflammation, and oxidative stress-related gene expression after mi R-155 was overexpressed and knock downed in VSMCs. 2 VSMCs were transfected with sictl or si MST2 for 24 h. The total protein lysates were then collected and Western blotting was performed to analyze the expression of cell proliferation, inflammation, and oxidative stress-related genes. 3 VSMCs were transfected with si MST2, and Ad-mi R-155 or anti-mi R-155; total protein lysates were collected and analyzed by Western blotting to examine the expression of cell proliferation, inflammation, oxidative stress-related genes. 4 VSMCs were transfected with si MST2 or sictl and then treated with the different inhibitors. Total protein lysates were collected, and the expression of cell proliferation, inflammation, and oxidative stress-related genes was examined by Western blotting. 5 VSMCs were transfected with Ad-GFP, Ad-mi R-155, anti-mi R-ctl, or anti-mi R-155. Anti-Raf-1 immunoprecipitates were analyzed by Western blotting to examine changes in MST2, MEK and Raf-1 levels. 7 PDGF expression in injured arteries from WT, mi R-155-/- and mi R-155-overexpressing mice was detected by q RT-PCR.Results:1 mi R-155 promotes VSMC proliferation by targeting MST2 via ERK1/2 signaling pathwayOverexpression of mi R-155 significantly downregulated MST2 expression and increased PI3K/Akt and ERK1/2 phosphorylation in VSMCs but did not affect the phosphorylation of JNK and p38. In contrast, ERK1/2 activation was significantly repressed upon transfection with anti-mi R-155 but not anti-mi R-ctl; however, there were no significant changes in PI3K/Akt, JNK and p38 phosphorylation. These results suggest that additional mechanisms might regulate the activation of PI3K/Akt in VSMCs overexpressing mi R-155. Western blotting analysis revealed that si MST2 could efficiently knock down MST2 expression compared with control si RNA. Notably, MST2 knockdown also resulted in the activation of ERK1/2 but no significant change in PI3K/Akt phosphorylation. Subsequently, we knocked down MST2 and treated VSMCs with mi R-155 inhibitor anti-mi R-155. Western blotting analysis revealed that knockdown of MST2 in anti-mi R-155-transfected cells rescued ERK1/2 but not PI3K/Akt phosphorylation and increased the PCNA protein level. Conversely, knockdown of MST2 using a specific si RNA further promoted ERK1/2 phosphorylation induced by mi R-155 overexpression. The downregulation of MST2 by mi R-155 and the ability of MST2 to block activation of ERK1/2 by mi R-155 suggest that MST2 is a crucial mediator of mi R-155 in regulating ERK1/2 signaling and promoting proliferation of VSMCs.2 mi R-155 regulates inflammation and oxidative stress response by targeting MST2We knocked down MST2 expression in VSMCs and monitored the expression of inflammation- and oxidative stress-related genes. Notably, downregulation of MST2 caused increased expression of p47 phox and NF-κB. To examine whether mi R-155 was involved in these processes, we tested the effect of mi R-155 overexpression and anti-mi R-155-mediated functional inhibition of mi R-155 activity on the expression and phosphorylation of p47 phox and NF-κB. Results showed that mi R-155 overexpression significantly increased phosphorylation of p47 phox and NF-κB. By contrast, inhibition of mi R-155 by anti-mi R-155 reduced phosphorylation of p47 phox and NF-κB. These results suggest that mi R-155 participates in both inflammatory and oxidative stress responses by downregulating MST2 in VSMCs. Silencing of MST2 in VSMCs transfected with anti-mi R-155 increased the expression and phosphorylation of p47 phox and NF-κB compared with control groups. Conversely, overexpression of MST2 in Ad-mi R-155-infected VSMCs reduced the expression and phosphorylation of p47 phox and NF-κB. Together, these results indicate that mi R-155-dependent regulation of p47 phox and NF-κB by MST2 links inflammation and oxidative stress.3 MST2 mediates mi R-155 regulation of inflammation and oxidative stress through Raf-1-MEK-ERK1/2 signaling pathwayVSMCs were incubated with the PI3K/Akt, ERK1/2, NF-κB, and NAD(P)H inhibitors LY294002, PD98059, CAY10576 and APOCYNIN, respectively, for 2 h before exposure to 10 % FCS. We found that inhibition of ERK1/2 blocked si MST2-induced phosphorylation of p47 phox and NF-κB. To confirm this observation, VSMCs(not transfected with si MST2) were incubated with PD98059, and Western blotting was used to analyze the phosphorylation of p47 phox and NF-κB. Inhibition of the ERK1/2 pathway reduced the phosphorylation of p47 phox and NF-κB. Interestingly, inhibition of NF-κB by CAY10576 decreased the activation of p47 phox in VSMCs transfected with si MST2. Consistent with this observation, inhibition of NAD(P)H oxidase using APOCYNIN also reduced the phosphorylation of NF-κB.Coimmunoprecipitation analysis of the lysates from VSMCs infected with Ad-mi R-155 revealed a significant increase in MEK levels and decrease of MST2 levels in anti-Raf-1-immunoprecipitates compared with Ad-GFP-infected cells. By contrast, transfection with anti-mi R-155 increased MST2 levels and reduced MEK levels in the complexes. As expected, mi R-155 overexpression also increased phosphorylation of MEK and ERK1/2, whereas transfection with anti-mi R-155 reduced their phosphorylation. Together, these observations indicate that MST2 competes with MEK to bind Raf-1; furthermore, downregulation of MST2 by mi R-155 alters the interaction of MEK with Raf-1 and MST2, leading to activation of ERK1/2 signaling.4 mi R-155 and NF-κB forms a positive feedback loop by targeting MST2Luciferase reporter assays showed that overexpression of NF-κB significantly increased the activity of mi R-155/BIC promoter. Similar resultswere obtained in VSMCs by overexpressing NF-κB. The PDGF expression levels were significantly reduced in the injured arteries of mi R-155-/- mice compared with WT mice. Conversely, the expression of PDGF genes was significantly higher in mi R-155-overexpressing mice than that of Ad-GFP-infected mice.Summary:Downregulation of MST2 by mi R-155 alters the interaction of MEK with Raf-1 and MST2, leading to activation of ERK1/2 signaling. MST2 mediates mi R-155 regulation of inflammation and oxidative stress response via Raf-1-MEK-ERK1/2 signaling pathway.Conclusion:1 mi R-155 promotes VSMC proliferation.2 mi R-155 is involved in the wire-injury-induced intimal hyperplasia and vascular remodeling.3 MST2 is a direct target of mi R-155 in VSMCs.4 mi R-155 promotes VSMC proliferation by depressing MST2 and that activating ERK1/2 signaling pathway.5 MST2 mediates mi R-155 regulation of inflammation and oxidative stress response via Raf-1-MEK-ERK1/2 signaling pathway.