Lei Lv

Essential role of Pin1 via STAT3 signalling and mitochondria-dependent pathways in restenosis in type 2 diabetes

Type 2 diabetes (T2D) is associated with accelerated restenosis rates after angioplasty. We have previously proved that Pin1 played an important role in vascular smooth muscle cell (VSMC) cycle and apoptosis. But neither the role of Pin1 in restenosis by T2D, nor the molecular mechanism of Pin1 in these processes has been elucidated. A mouse model of T2D was generated by the combination of high‐fat diet (HFD) and streptozotocin (STZ) injections. Both Immunohistochemistry and Western blot revealed that Pin1 expression was up‐regulated in the arterial wall in T2D mice and in VSMCs in culture conditions mimicking T2D. Next, increased activity of Pin1 was observed in neointimal hyperplasia after arterial injury in T2D mice. Further analysis confirmed that 10% serum of T2D mice and Pin1‐forced expression stimulated proliferation, inhibited apoptosis, enhanced cell cycle progression and migration of VSMCs, whereas Pin1 knockdown resulted in the converse effects. We demonstrated that STAT3 signalling and mitochondria‐dependent pathways played critical roles in the involvement of Pin1 in cell cycle regulation and apoptosis of VSMCs in T2D. In addition, VEGF expression was stimulated by Pin1, which unveiled part of the mechanism of Pin1 in regulating VSMC migration in T2D. Finally, the administration of juglone via pluronic gel onto injured common femoral artery resulted in a significant inhibition of the neointima/media ratio. Our findings demonstrated the vital effect of Pin1 on the VSMC proliferation, cell cycle progression, apoptosis and migration that underlie neointima formation in T2D and implicated Pin1 as a potential therapeutic target to prevent restenosis in T2D.

Lentivirus-mediated RNA Interference Targeting STAT4 Inhibits the Proliferation of Vascular Smooth Muscle Cells

BACKGROUND STAT4 is a key transcription factor regulating Th1 development. However, its presence and role in vascular smooth muscle cells (VSMCs) has not been well studied. In the current study, we have utilized lentivirus-mediated shRNA for functional gene knockdown in human umbilical artery smooth muscle cells in order to access the potential role of STAT4 in VSMC growth. METHODS Cells were isolated from the umbilical arteries of newborns and used at passage 3-5. Recombinant lentivirus producing STAT4 siRNA was prepared. Protein and mRNA expression of STAT4 and relevant genes were examined by Western blot, ELISA, and quantitative RT-PCR analysis, and the effects of the lentivirus on cell growth and apoptosis were determined using MTT assay and flow cytometry, respectively. RESULTS Lentivirus-mediated RNAi effectively reduced endogenous STAT4 expression and downregulation of STAT4 in VSMCs and significantly reduced VSMC growth rate in vitro. We found that STAT4 knockdown led to impaired pSTAT4 protein expression. SOCS-3 as well as MCP-1 production were also markedly decreased, consistent with the suppression of STAT4 expression. CONCLUSIONS Results from our study suggest that STAT4 may play a role in VSMC proliferation, and thus is a novel therapeutic target for neointima formation following vascular injury, e.g., post-angioplasty restenosis.

Fine-tuning the expression of microRNA-155 controls acetaminophen-induced liver inflammation.

Treatment of acetaminophen (APAP) in overdose can cause a potentially serious and fatal liver injury. MicroRNA-155 (miR-155), a multifunctional microRNA, is known to mediate inflammatory responses via regulating various target genes. In this study, we aimed to study the role of miR-155 in APAP-induced liver injury, using miR-155-/- mice and miR-155 in vivo intervention. We noted that miR-155 expression was significantly increased in liver and blood after APAP treatment. Knockout of miR-155 deteriorated APAP-induced liver damage, with the elevated serum levels of AST and ALT. The levels of various inflammatory mediators, such as TNF-α and IL-6, were markedly augmented in livers in the absence of miR-155. Moreover, miR-155 deficiency aberrantly activated NF-kappa-B signaling via enhancing p65 and IKKε expression. Finally, in vivo administration of miR-155 agomir attenuated APAP-induced liver damage, reduced the serum levels of AST and ALT, and dampened the NF-kB signaling. In conclusion, our data demonstrated that miR-155 protects the mice against APAP-induced liver damage via mediating NF-KB signaling pathway, suggesting that miR-155 might be a potential pharmaceutic target for treatment of APAP-induced liver inflammation.

Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial injury

OBJECTIVE Restenosis engenders surgical vascular intervention failure. Zinc finger protein 191 (ZFP191) is a novel member of the SCAN domain family of Krüppel-like zinc finger transcription factors. Previous work reveals that ZFP191 is a pleiotropic factor that plays important roles in hematopoiesis, brain development, and tumor growth. Here, we sought to determine whether intimal hyperplasia was affected by the activity of ZFP191 and to investigate the molecular mechanisms that may underpin the process. METHODS Intimal hyperplasia was induced by guidewire injury in mouse femoral arteries. The arteries were harvested for morphometric assessment and determination of ZFP191 expression. Next, ZFP191 knockdown in cultured mouse aortic vascular smooth muscle cells (VSMCs) was achieved by lentiviral transduction of short-hairpin RNA. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, [(3)H]thymidine incorporation assay, scratch assay, and transwell migration assay were used to evaluate the effects of ZFP191 knockdown on VSMC growth and migration. In addition, β-catenin, c-myc, cyclin D1, matrix metalloproteinase (MMP) 9, MMP2, and MMP7 were measured by Western blotting in the absence of ZFP191 in vitro and in vivo. Zymography was used to evaluate MMP activity in cell culture-conditioned media. Lastly, artery injury was performed in wild-type (WT) and heterozygous ZFP191 knockout (KO) mice, and morphometric analysis of the arteries was determined. RESULTS Guidewire injury was associated with development of intimal hyperplasia, and ZFP191 expression was enhanced by 51% in the injured arteries. Cultured primary VSMCs transfected with lentiviral shZFP191 displayed reduced proliferation and migration compared with controls. Mechanically, ZFP191 knockdown potently decreased the level of β-catenin and its downstream targets c-myc and cyclin D1. ZFP191 knockdown downregulated the expression of MMP9, MMP2, and MMP7, and zymography confirmed that ZFP191 knockdown reduced the activity of MMPs. Consistent with the in vitro data, elevated expression of β-catenin, c-myc, cyclin D1, MMP9, MMP2, and MMP7 accompanied upregulation of ZFP191 after injury in the femoral arteries of mice, and these levels were downregulated in ZFP191 KO vessels. Finally, intimal hyperplasia was greatly blocked in heterozygous ZFP191 KO mice compared with WT mice (intima/media ratio, 0.124 vs 0.412; P < .05). CONCLUSIONS ZFP191 played an essential role in aggressive proliferation and migration of VSMCs, which in turn facilitated intimal hyperplasia. Our findings offer the first genetic evidence of ZFP191 as a potential therapeutic target to prevent restenosis.

Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescence

Objective Pin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. Methods Immunohistochemistry and Western blotting were used to assess Pin1 protein level in human normal and atherosclerotic tissues. &bgr;‐galactosidase staining, cumulative population doubling level, telomerase activity, and relative telomere length measurement were used to confirm VSMC senescence. The expressions of Pin1 and other genes involved in this research were analyzed by quantitative reverse‐transcription polymerase chain reaction and Western blotting in VSMCs. Apolipoprotein E gene‐deleted mice (ApoE−/−) fed a high‐fat diet were treated with juglone or 10% ethanol, respectively, for 3 weeks. The extent of atherosclerosis was evaluated by Oil Red O, Masson trichrome staining, and immunohistology. Results Pin1 protein level decreased in human atherosclerotic tissues and VSMCs, synchronously with increased VSMC senescence. Adenoviral‐mediated Pin1 overexpression rescued cellular senescence in atherosclerotic VSMCs, with concurrent down‐regulation of P53, p21, growth arrest and DNA‐damage‐inducible protein 45‐alpha (Gadd45a), phosphorylated retinoblastoma (p‐pRb), p65 and upregulation of cyclin subfamilies (cyclin B, D, and E), and cyclin‐dependent kinase subfamilies (2, 4, and 6), whereas Pin1 knockdown resulted in the converse effects, indicating that VSMC senescence mediated by Pin1 is an integrated response to diverse signals. In vivo data from ApoE−/− mice showed that treatment of juglone led to accelerated atherosclerosis development. Conclusions Altogether this work supports a role for Pin1 as a vital modulator of VSMC senescence, thereby providing a novel target for regulation and control of atherosclerosis. Clinical Relevance We found that decreased Pin1 protein level in human atherosclerotic tissues and vascular smooth muscle cells (VSMCs) was related to increased VSMC senescence, and in vivo data from apolipoprotein E−/−mice showed that treatment of a Pin1 inhibitor led to accelerated atherosclerosis development. This research indicated that interventions targeted at Pin1, such as cell‐specific drugs, are potential novel approaches to the retardation of atherosclerosis, in which VSMC senescence has a prominent role. Patients with atherosclerosis may benefit from pharmacologic interference with Pin1.

Thrombospondin-4 ablation reduces macrophage recruitment in adipose tissue and neointima and suppresses injury-induced restenosis in mice.

OBJECTIVE Thrombospondin-4 (Thbs4) is a member of the extracellular calcium-binding protein family and is linked to cell adhesion and migration. Given the involvement of Thbs4 in vascular inflammation, we hypothesized that Thbs4 plays a role in restenosis. METHODS AND RESULTS Here we show evidence that Thbs4 is upregulated in wire-injured mouse arteries and correlated with CD68 expression. Macrophage infiltration is reduced in both adipose tissue (AT) and neointima of Thbs4/ApoE double knockout (DKO) mice after injury. Moreover, Thbs4 deficiency prevents restenosis in ApoE KO mice fed a Western-type diet (WTD). Lethally irradiated DKO mice that receive bone marrow from ApoE KO or DKO mice have reduced neointima development. While considering related mechanisms, we note decreased chemokine production in both AT and neointima of DKO mice. In addition, vascular smooth muscle cells (VSMCs) derived from DKO mice display suppressed proliferation and migration in comparison with controls. Thioglycollate (TG)-induced macrophages from DKO mice show retarded adhesion to VSMCs. Recombinant Thbs4 promoted macrophage adhesion to VSMCs, and enhanced VSMC proliferation and migration. CONCLUSION Collectively, these data highlight the significance of Thbs4 in regulating macrophage accumulation and treating restenosis.

STAT4 deficiency protects against neointima formation following arterial injury in mice

Signal transducer and activator of transcription 4 (STAT4) has been associated with susceptibility to autoimmune diseases. Intriguingly, we previously reported that STAT4 might play a critical role in vascular smooth muscle cell (VSMC) proliferation. The present study therefore investigated the impact of STAT4 on VSMC migration, apoptosis and neointimal hyperplasia postinjury, as well as the underlying mechanisms. Guide-wire injury was associated with development of intimal neointima, STAT4 and phosphorylated STAT4 (p-STAT4) expressions were apparently up-regulated in the injured arteries. Neointima was greatly blocked in STAT4 knockout (KO) mice compared with wild type (WT) mice. A marked loss of inflammatory cells was identified in the vasculature postinjury in STAT4 KO mice. VSMC apoptosis was enhanced in the vasculature postinjury in STAT4 KO mice compared with WT mice. Cultured primary STAT4 KO VSMCs displayed reduced migration in comparison with WT controls. Mechanically, the deletion of STAT4 potently decreased the level of MCP-1, and its downstream targets MMP1 and MMP2. The effect of STAT4 on VSMC apoptosis was mainly mediated by the activation of the mitochondrial apoptotic pathway, as manifested by increased cytochrome c release and the activation of caspase-3. STAT4 therefore represents a promising molecular target to limit restenosis after artery intervention.

Duffy antigen / receptor for chemokines correlates with inflammatory reaction in rats with venous hypertension: implication for the pathogenesis of primary chronic venous disease.

BACKGROUND Duffy antigen / receptor for chemokines (DARC) possesses high affinity for several chemokine subgroups of CC and CXC. Although DARC has been shown to play a role in many inflammatory diseases, its effect on chronic venous disease (CVD) remains unidentified. We explored whether the expression of DARC in skin tissue was activated under venous hypertension as well as the relationships between DARC and inflammation. MATERIALS AND METHODS The inflammation in a rat model of venous hypertension caused by a femoral arterial-venous fistula (AVF) was studied. At specified intervals the pressure in the femoral veins was recorded within 42 days. Hindlimb skin specimens were harvested at different time points. The expressions of DARC, interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) in skin tissue were examined. Mononuclear cells infiltrated in skin tissue were detected. RESULTS Femoral venous pressures in AVF groups increased significantly at different time points (P < 0.01). DARC was expressed in skin tissue and its expression level increased significantly in AVF groups from the 7nd day on and was enhanced in a time-dependent manner within 42 days (P < 0.05). Meanwhile, both MCP-1 and IL-8 had higher levels, accompanied by increased mononuclear cells infiltrating into skin tissue (P < 0.05). CONCLUSIONS A rat AVF model which can maintain venous hypertension for at least 42 days is competent for researching the pathogenesis of CVD. DARC, which plays a role in the inflammation of skin tissue under venous hypertension, may become a new molecular target for diagnosis and treatment of CVD at a very early stage.

Long Noncoding RNA uc001pwg.1 Is Downregulated in Neointima in Arteriovenous Fistulas and Mediates the Function of Endothelial Cells Derived from Pluripotent Stem Cells

Recent studies indicate important roles for long noncoding RNAs (lncRNAs) as essential regulators of gene expression. However, the specific roles of lncRNAs in stenotic lesions of arteriovenous fistula (AVF) failure are still largely unknown. We first analyzed the expression profiles of lncRNAs in human stenosed and nonstenotic uremic veins using RNA-sequencing methodology. A total of 19 lncRNAs were found to be differentially expressed in stenotic lesions. Among these, uc001pwg.1 was one of the most significantly downregulated lncRNAs and enriched in both control vein segments and human umbilical vein endothelial cells (HUVECs). Further studies revealed that uc001pwg.1 overexpression could increase nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production in endothelial cells (ECs) derived from human-induced pluripotent stem cells (HiPSCs). Mechanistically, uc001pwg.1 improves endothelial function via mediating MCAM expression. This study represents the first effort of identifying a novel candidate lncRNA for modulating the function of iPSC-ECs, which may facilitate the improvement of stem cell-based therapies for AVF failure.

Pyruvate kinase M2 interacts with DNA damage-binding protein 2 and reduces cell survival upon UV irradiation

Pyruvate Kinase M2 (PKM2) is highly expressed in many solid tumors and associated with metabolism reprogramming and proliferation of tumors. Here, we report that PKM2 can bind to DNA Damage-Binding Protein 2 (DDB2), which is necessary for global nucleotide excision repair of UV induced DNA damage. The binding is promoted by UV irradiation and K433 acetylation of PKM2. Over expression of PKM2 facilitates phosphorylation of DDB2 and impairs DDB2-DDB1 binding. Furthermore, knocking down of PKM2 increases cell survival upon UV irradiation, while over expression of PKM2 reduces cell survival and over expression of DDB2-DDB1 reverts this effect. These results reveal a previously unknown regulation of PKM2 on DDB2 and provide a possible mechanism for UV induced tumorigenesis.