Dazhu Li

Systemic Delivery of MicroRNA-181b Inhibits Nuclear Factor-κB Activation, Vascular Inflammation, and Atherosclerosis in Apolipoprotein E–Deficient Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

Systemic Delivery of MicroRNA-181b Inhibits NF-κB Activation, Vascular Inflammation, and Atherosclerosis in Apoe-/- Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

Thymic Stromal Lymphopoietin Over-Expressed in Human Atherosclerosis: Potential Role in Th17 Differentiation

Background: Adaptive immunity plays a critical role in atherosclerosis and T helper 17 (Th17) cells, a new T-cell lineage, are recently reported to be involved in atherosclerosis. However, the mechanism underlying Th17 inflammation in atherosclerosis remains largely unknown. Thymic stromal lymphopoietin (TSLP) is a novel IL-7-like cytokine and mainly responsible for Th2 inflammation in many inflammatory diseases. Methods and Results: Immunohistochemistry showed that TSLP over-expressed in human atherosclerotic lesion and could be induced by ox-LDL in human vascular smooth muscle cells (HVSMCs) and human umbilical vein endothelial cells (HUVECs). TSLP, in turn, could activate dendritic cells (DCs) to differentiate Th17 inflammation in naive CD4+ T cells. Conclusion: TSLP induced by ox-LDL could promote Th17 immune response in vitro, which may be implicated in Th17 inflammation in atherosclerosis.

Inflammatory Cytokine TSLP Stimulates Platelet Secretion and Potentiates Platelet Aggregation via a TSLPR-Dependent PI3K/Akt Signaling Pathway

Aims: Thymic stromal lymphopoietin (TSLP) plays an important role in inflammatory diseases and is over-expressed in human atherosclerotic artery specimens. The present study investigated the role of TSLP in platelet activation and thrombosis models in vitro and in vivo, as well as the underlying mechanism and signaling pathway. Methods and Results: Western blotting and flow cytometry demonstrated that the TSLP receptor was expressed on murine platelets. According to flow cytometry, platelet stimulation with TSLP induced platelet degranulation and integrin αIIbβ3 activation. A TSLPR deficiency caused defective platelet aggregation, defective platelet secretion and markedly blunted thrombus growth in perfusion chambers at both low and high shear rates. TSLPR KO mice exhibited defective carotid artery thrombus formation after exposure to FeCl3. TSLP increased Akt phosphorylation, an effect that was abrogated by the PI3K inhibitors wortmannin and LY294002. The PI3K inhibitors further diminished TSLP-induced platelet activation. TSLP-mediated platelet degranulation, integrin αIIbβ3 activation and Akt phosphorylation were blunted in platelets that lacked the TSLP receptor. Conclusion: This study demonstrated that the functional TSLPR was surface-expressed on murine platelets. The inflammatory cytokine TSLP triggered platelet activation and thrombus formation via TSLP-dependent PI3K/Akt signaling, which suggests an important role for TSLP in linking vascular inflammation and thrombo-occlusive diseases.

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10

Thrombogenic and inflammatory mediators, such as thrombin, induce NF‐kB–mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti‐inflammatory microRNA‐181b (miR‐181b) on thrombosis remains unknown. Our previous study demonstrated that miR‐181b inhibits downstream NF‐kB signaling in response to TNF‐a. Here, we demonstrate that miR‐181b uniquely inhibits upstream NF‐kB signaling in response to thrombin. Overexpression of miR‐181b inhibited thrombin‐induced activation of NF‐kB signaling, demonstratedbyreductionofphospho‐IKK‐b,‐IkB‐a, and p65 nuclear translocation in ECs. MiR‐181b also reduced expression of NF‐kB target genes VCAM‐1, intercellular adhesion molecule‐1, E‐selectin, and tissue factor. Mechanistically, miR‐181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease‐activated receptor‐1. miR‐181b reduced expression of Card10 mRNA and protein, but not protease‐activated receptor‐1. 39‐Untranslated region reporter assays, argonaute‐2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR‐181b target. Small interfering RNA–mediated knockdown of Card10 expression phenocopied effects of miR‐181b on NF‐kB signaling and targets. Card10 deficiency did not affect TNF‐a–induced activation of NF‐kB signaling, which suggested stimulus‐specific regulation of NF‐kB signaling and endothelial responses by miR‐181b in ECs. Finally, in response to photochemical injury‐induced arterial thrombosis, systemic delivery of miR‐181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6‐fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR‐181b and Card10 are important regulators of thrombin‐induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA‐dependent targets in response to ligand‐specific signaling in ECs.—Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA‐181b inhibits thrombin‐mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10. FASEB J. 30, 3216–3226 (2016). www.fasebj.org

Increased IL-37 concentrations in patients with arterial calcification

BACKGROUND Our previous study indicates that IL-37 plays a critical role in both atherosclerosis and arterial calcification. However, whether IL-37 concentrations are significantly changed in patients with arterial calcification has not yet been investigated. METHODS Anterior tibial arterial wall specimens were obtained from 8 patients with type 2 diabetes mellitus and 8 patients who experienced a traffic accident. IL-37 expression was measured by immunohistochemistry in the calcified and the normal samples. In addition, plasma IL-37 concentrations were measured in 75 patients with coronary artery calcification (CAC) and 50 patients without coronary artery calcification (NCAC). RESULTS High concentrations of IL-37 were detected in calcified samples, whereas low concentrations of IL-37 were detected in the normal arteries. Macrophages and vascular smooth muscle cells were the main source of IL-37. Plasma IL-37 concentrations were significantly increased in CAC patients compared with NCAC patients. A correlation analysis showed that IL-37 was positively correlated with age, fasting glucose, alkaline phosphatase, IL-6, TNF-α, C-reactive protein and Agatston scores. Binary logistic regression analyses demonstrated that fasting glucose and IL-37 were independently associated with the presence of CAC. CONCLUSIONS Increased IL-37 concentrations are associated with the onset of arterial calcification.

Kruppel-Like Factor 2-Mediated Suppression of MicroRNA-155 Reduces the Proinflammatory Activation of Macrophages.

Objective Recent evidence indicates that significant interactions exist between Kruppel-like factor 2 (KLF2) and microRNAs (miRNAs) in endothelial cells. Because KLF2 is known to exert anti-inflammatory effects and inhibit the pro-inflammatory activation of monocytes, we sought to identify how inflammation-associated miR-155 is regulated by KLF2 in macrophages. Approach and Results Peritoneal macrophages from wild-type (WT) C57Bl/6 mice were transfected with either recombinant adenovirus vector expressing KLF2 (Ad-KLF2) or siRNA targeting KLF2 (KLF2-siRNA) for 24 h–48 h, then stimulated with oxidized low-density lipoproteins (ox-LDL, 50 μg/mL) for 24 h. Quantitative real-time polymerase chain reaction showed that KLF2 markedly reduced the expression of miR-155 in quiescent/ox-LDL-stimulated macrophages. We also found that the increased expression of miR-155, monocyte chemoattractant protein (MCP-1) and interleukin (IL)-6 and the decreased expression of the suppressor of cytokine signaling (SOCS)-1 and IL-10 in ox-LDL-treated macrophages were significantly suppressed by KLF2. Most importantly, over-expression of miR-155 could partly reverse the suppressive effects of KLF2 on the inflammatory response of macrophages. Conversely, the suppression of miR-155 in KLF2 knockdown macrophages significantly overcame the pro-inflammatory properties associated with KLF2 knockdown. Finally, Ad-KLF2 significantly attenuated the diet-induced formation of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/-) mice, which was associated with a significantly reduced expression of miR-155 and its relative inflammatory cytokine genes in the aortic arch and in macrophages. Conclusion KLF2-mediated suppression of miR-155 reduced the inflammatory response of macrophages.

TSLPR deficiency attenuates atherosclerotic lesion development associated with the inhibition of TH17 cells and the promotion of regulator T cells in ApoE-deficient mice.

AIMS We generated thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice to directly explore the role of thymic stromal lymphopoietin (TSLP) in atherogenesis. METHODS AND RESULTS Both thymic stromal lymphopoietin (TSLP) and its receptor are expressed in atherosclerotic aortas of apolipoprotein E knockout (ApoE KO) mice. Serum thymic stromal lymphopoietin (TSLP) is markedly increased in apolipoprotein E knockout (ApoE KO) mice fed with a high fat diet (HFD). Arterial lesion formation was significantly decreased in thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice compared with apolipoprotein E knockout (ApoE KO) mice. Bone marrow chimera studies indicated reduced lesions in apolipoprotein E knockout (ApoE KO) mice which received the bone marrow of thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice as well as in TSLPR KO mice which received bone marrow of ApoE-TSLPR DKO mice. Compared with apolipoprotein E knockout (ApoE KO) mice, IFN-γ secretion by activated T cells was increased but IL-4 expression was reduced in thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice. Consisted with these results, the mRNA of IFN-γ was increased but IL-4 was reduced in root. These findings suggest that a reduction in atherosclerotic lesions in thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice may not be due to a Th1/Th2 imbalance. On the other hand, the number of Th17 cells, the secretion of IL-17A by activated CD4(+) T cells and the mRNA expression of IL-17A in root were decreased in thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice. Notably, the number of regulatory T cell expression of IL-10 was increased in thymic stromal lymphopoietin R-chain deficient apolipoprotein E-double knockout (ApoE-TSLPR DKO) mice. CONCLUSIONS Collectively, our data suggest that activating thymic stromal lymphopoietin (TSLP) promotes atherosclerosis by inducing Th17/Treg imbalance through thymic stromal lymphopoietin/thymic stromal lymphopoietin R-receptor (TSLP/TSLPR) signal way in apolipoprotein E-deficient mice fed with HFD model.

Kruppel-Like Factor 2 Regulates Dendritic Cell Activation in Patients with Acute Coronary Syndrome

Objective: Dendritic cells (DCs) activation is important in atherosclerosis and coronary heart disease, but the mechanisms regulating activation of dendritic cells remain largely unclear. The aim of this study was to evaluate the effect of transcription factor Kruppel-like factor 2 (KLF2) in the proinflammatory activation of DCs in acute coronary syndrome. Methods and Results: In this study, the expression of CD80 and KLF2 was detected in DCs in normal health controls, patients with stable angina (SA), and acute coronary syndrome (ACS). Our study found that compared with normal control and SA, KLF2 expression in DCs is reduced in patients with ACS. Moreover, the surface expression of CD80 was increased in ACS. In vitro experiment, we found that ox-LDL could increase CD80 expression and decrease KLF2 expression. Furthermore, down-regulated KLF2 could in turn increase CD80 expression via NF-κB pathway. Conclusions: These observations identify KLF2 as a novel negative regulator of DC function and it may play an essential role in DC activation in ACS.

A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor

Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway.