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Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Regulatory T cells (Tregs) have been shown to attenuate the development and progression of atherosclerosis; however, the exact mechanism is still unclear. In our study, Tregs were adoptively transferred into ApoE-/- mice, and type 2 innate lymphoid cells (ILC2s) were expanded by the IL-2/Jes6-1 complex or depleted by anti-CD90.2 mAb in ApoE-/-Rag1-/- mice to study their effects on atherosclerosis. Then, Tregs were cocultured with ILC2s in vitro to analyze ILC2s number and IL-13 production. In vivo, ApoE-/-Rag1-/- mice were treated with activated Tregs with or without anti-CD90.2 mAb to explore whether Tregs reduced atherosclerosis through ILC2s. Finally, neutralizing antibodies and Transwell assay were used to investigate how Tregs regulate ILC2s. Our results show that both Tregs and ILC2s reduce atherosclerosis lesions and macrophage infiltration. Moreover, Tregs effectively expanded the number of ILC2s and increased their production of IL-13 in vivo and in vitro. Furthermore, the reductions in plaque size and macrophage infiltration by Tregs were partly reversed by anti-CD90.2 mAb. Mechanistically, our data reveal that IL-10, TGF-β and cell-cell contacts are required for Tregs-ILC2s regulation. These results show that Tregs may play a partial protective role against atherosclerosis by expanding the number of ILC2s and consequently increasing IL-13 production.

Myocardial infarction results from obstruction of a coronary artery that causes insufficient blood supply to the myocardium and leads to ischemic necrosis. It is one of the most common diseases threatening human health and is characterized by high morbidity and mortality. Atherosclerosis is the pathological basis of myocardial infarction, and its pathogenesis has not been fully elucidated. Innate lymphoid cells (ILCs) are an important part of the human immune system and participate in many processes, including inflammation, metabolism and tissue remodeling, and play an important role in atherosclerosis. However, their specific roles in myocardial infarction are unclear. This review describes the current understanding of the relationship between innate lymphoid cells and myocardial infarction during the acute phase of myocardial infarction, myocardial ischemia-reperfusion injury, and heart repair and regeneration following myocardial infarction. We suggest that this review may provide new potential intervention targets and ideas for treatment and prevention of myocardial infarction.

Bortezomib is a classical proteasome inhibitor and previous researches have reported its roles of anti-oxidation and anti-inflammatory functions in various diseases. However, the role of Bortezomib in myocardial ischemia reperfusion injury (MIRI) is unclear. Thus, our research seeks to reveal the protective effects of Bortezomib pretreatment in the mice model of MIRI. First, by the optimization of Bortezomib concentration and pretreatment timepoints, we found that 0.5 mg/kg Bortezomib pretreatment 2 h before MIRI significantly attenuated pathological damage and neutrophil infiltration. Then we found that pretreatment with Bortezomib obviously increased myocardial systolic function ((left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)) and decreased infarct size, as well as serum Troponin T levels. Meanwhile, Bortezomib pretreatment also remarkably augmented oxidative stress related protein levels of Superoxide dismutase [Cu–Zn] (SOD1), Catalase (CAT) and Glutathione (GSH), while reactive oxygen species (ROS) contents and Malonaldehyde (MDA) protein level were significantly reduced. Mechanistically, Bortezomib pretreatment significantly promoted nuclear translocation of transcriptional factor nuclear factor erythroid 2-related factor 2(Nrf2) and Heme Oxygenase 1(HO-1) expression. Interestingly, co-treatment with ML-385, a new type and selective Nrf2 inhibitor, counteracted antioxidative effects induced by Bortezomib pretreatment. In conclusion, Bortezomib pretreatment mitigates MIRI by inhibiting oxidative damage which is regulated by Nrf2/HO-1 signaling pathway.

The M s7.0 Jiuzhaigou earthquake with the hypocenter depth of 20 km occurred at the NNW extension line of the Huya fault located next to the eastern boundary of the Bayan Har block. The focal mechanism solution of the mainshock based on the W-phase source inversion showed a sinistral slip and the moment magnitude was M w 6.5. Over 3000 aftershocks were recorded within six days, among which the largest was M 4.8 and three others were larger than M 4.0. The value of the aftershock frequency attenuation coefficient P was 1.03, and the b value of the magnitude-frequency relationship was 0.68. The aftershock activities were relatively inactive but the attenuation was normal. Relocation results of 603 aftershocks were obtained by using the double-difference earthquake location algorithm and the location errors of the EW, NS and the vertical directions were 0.81, 0.84 and 1.59 km respectively. The mainshock occurred in the center of the aftershock region and ruptured on both sides along the strike direction. The aftershocks were distributed linearly along a NNW direction, and the fault plane was nearly vertical with a length over 30 km and a width of about 15 km. Aftershocks were more concentrated in the southeast than in the northwest. The focal mechanism solutions of most aftershocks in the central and southeastern regions were consistent with the mainshock, while those in the northwest were thrust-types, which might be related to the Minjiang fault. Meanwhile, the regional stress was released more in the central and southeastern regions than in the northwest. The maximum intensity investigated in the damage zone was up to IX. The maximum intensity region was located in scenic areas, therefore few people were present when the earthquake occurred at night. The peak acceleration was highest in the NS records, followed by the EW and vertical records. The design spectra of intensity for Jiuzhaigou was VIII, which is higher than the acceleration response spectra obtained from the Jiuzhaibaihe station, the nearest station which had strong motion recordings. These aforementioned factors may have contributed to the minor casualties resulting from this earthquake. The aftershock regions of the Jiuzhaigou earthquake, the 1973 M s6.5 Songpan northeast earthquake, and the 1976 M s7.2 Songpan earthquake are connected along the Huya fault which may extend to the northwest in the deep earth. Considering the rupture displacement distribution of the mainshock, the focal mechanism of aftershocks, and the stress drop, strong aftershocks are unlikely to occur in the central and southeastern Jiuzhaigou aftershock regions; however, the development of seismicity in the northwest and the Minjiang River fault should be noted. Static Coulomb stress triggering of other 4 earthquakes larger than M 6.5 occurred near the Ganqingchuan border were calculated using the Coulomb 3.3 software. The results indicated that the Jiuzhaigou earthquake was triggered by the Wenchuan earthquake and related to the stress adjustment in the study area.

Based on the results of multipurpose regional geochemical surveys of the Guizhou Province, geochemical characteristics of soil Se and Se-rich land resources in the central area of Guiyang City were studied and evaluated. Major conclusions are as follows: (1) the Se content in surface soil of the central area of Guiyang City was 0.17–2.89 mg kg−1, and the average was 0.78 mg kg−1, which were respectively 2.6 and 3.9 times of the national background value of soil and the world background value of soil. The Se content in deep soil was 0.11–1.48 mg kg−1, and the average was 0.44 mg kg−1, which were respectively 1.5 and 2.2 times of the national background value of soil and the world background value of soil. The soil Se content decreased with the increase in the soil depth on the vertical profile, and the surface soil had a higher Se content. (2) Distribution of Se content was mainly affected by parent material, physicochemical properties of soil and other components, soil type, and land use type. Parent material played a key role, as the soil Se content was mainly originated from parent rock and increased with the background value of Se in parent rock, physicochemical properties of soil and other components had certain influences upon the Se content. Se was shown to have a significant linear positive correlation with S and organic carbon but no significant correlation with pH value. Se content varied with different types of soil as follows: skeleton soil > yellow soil > paddy soil > limestone soil > purple soil. Land use type also had certain influences upon the soil Se content as follows: dry land > construction land > garden plot > grassland = garden plot > forest land. (3) Taking 0.4 mg kg−1 ≤ ω (Se)

There are different interferences in the satellite communication. A example is given in the paper, it is shown from the simulation that when using smart antenna and PASTd algorithm in the satellite communication, the radiation patterns formed by smart antenna points a main beam in the direction of the desired signal but the ability to cancel inferences.

The vascular endothelium, as the interface between the blood and the surrounding tissues, plays a pivotal role in inflammation. Neferine, which was isolated from Lotus Plumule, has many biological roles, such as antifibrotic, antioxidative, anti-inflammatory, and antineoplastic activities. We demonstrated the role of neferine in the inhibition of pro-adhesion and pro-inflammatory responses of endothelial cells in vitro. We found that neferine could significantly inhibit the adhesion of Tohoku Hospital Pediatrics-1 (THP-1) cells to primary human umbilical vein endothelial cells (HUVECs). At the molecular level, neferine could significantly alleviate the interleukin 1β (IL-1β)-induced mRNA and protein expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1). Our data showed that neferine suppressed nuclear factor-κB (NF-κB) nuclear translocation and inhibited the NF-κB-p65-induced transcriptional activity of ICAM1 and VCAM1. Therefore, we concluded that neferine suppressed the inflammatory response in endothelial cells in vitro, which could be mainly due to inhibition of NF-κB signaling activation. Moreover, we found that neferine alleviated LPS-induced acute inflammation injury in vivo. Thus, neferine may serve as an effective regulator during the pathogenesis of vascular inflammatory diseases.

Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB–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-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB 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. 3′-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-κB signaling and targets. Card10 deficiency did not affect TNF-α–induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB 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.

In this study, we analyze available weak‐ and strong‐motion seismograms to discern the main source characteristics of the 12 August 2015 Tianjin explosions. The disaster involved two large explosions, which can be clearly identified in the near‐field seismic recordings. The first explosion (event 1) took place at 15:34:04.68 (UTC) and the second at 15:34:36.94 (UTC). The average time interval between the two events was about 32.3 s, which is consistent with the result determined by cross correlation of the whispering gallery wave ( WG phase). The two events did not occur at the same site, and the second explosion was located 65±10 m northwest (N352°W) of the first one. The local magnitudes M L of the two explosions were further measured as 2.4 and 2.9, respectively. The Fourier spectra from the two closest stations, DAG and BET, show that the predominant frequency of the waveforms ranged from 0.7 to 0.9 Hz after the long‐period wavetrains had arrived. According to the distribution of the three‐component peak ground acceleration, the area with a shaking intensity of modified Mercalli intensity VI spanned up to 131 km2. However, the peak intensity of this explosion did not reach an intensity of VII, based on the ground‐motion attenuation relation.

Endothelial cells (ECs) and macrophages engage in tight and specific interactions that play critical roles in cardiovascular homeostasis and the pathogenesis of atherosclerosis. Extracellular vesicles (EVs) are circular membrane fragments released from the endosomal compartment as exosomes or shed from the surfaces of the membranes of most cell types. Increasing evidence indicates that EVs play a pivotal role in cell-to-cell communication. However, the contribution of EVs, as determine by oxidized low-density lipoprotein (ox-LDL)-exposed and/or Kruppel-like factor 2 (KLF2)-transduced ECs in the interaction between vascular ECs and monocytes/macrophages, which is a key event in atherosclerotic plaque development, has remained elusive. This study demonstrates the characteristic impact of EVs from ox-LDL-treated and/or KLF2-transduced ECs on the monocyte/macrophage phenotype in vitro and in vivo.Q-PCR showed that both the atherosclerosis inducer ox-LDL and atheroprotective factor KLF2 regulated inflammation-associated microRNA-155 (miR-155) expression in human umbilical vein endothelial cells (HUVECs). Moreover, coculture, immunofluorescence and flow cytometry revealed that miR-155 was enriched in ox-LDL-induced ECs-EVs and subsequently transferred to human monocytic THP1 cells, in which these vesicles enhance monocyte activation by shifting the monocytes/macrophages balance from anti-inflammatory M2 macrophages towards proinflammatory M1 macrophages; EVs from KLF2-expressing ECs suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses, which indicate the potent anti-inflammatory activities of these cells. Furthermore, oil red staining showed that atherosclerotic lesions were reduced in mice that received EVs from KLF2-transduced ECs with decreased proinflammatory M1 macrophages and increased anti-inflammatory M2 macrophages, and this effect is at least partly due to the decreased expression of inflammation-associated miR-155, confirming our in vitro findings. In summary, this study provides novel insights into the pathophysiological effects of altered EV secretion and/or microRNA content and their influence on modulating monocyte activation depending on the environment surrounding EVs-releasing ECs.

ILC populations elaborate a similar cytokine expression pattern with helper T cell subsets Th1, Th2 and Th17. Recent studies indicate that CD25+ILC2 could alleviate atherosclerosis by altering lipid metabolism, whereas the depletion of CD90-expressing ILCs had no influence on atherosclerosis. Thus, these findings raise the question of whether ILC1 cells react on atherosclerosis. Hence, our group attempted to explore the role of ILC1 cells in atherosclerosis. We found that ILC1 cells have a high Th1-like gene expression of T-bet and IFN-γ, which is distinct from ILC2, ILC3 or conventional NK (cNK) cells. Moreover, atherosclerotic lesions were greatly reduced in ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs for depleting ILC1 cells (ILC1+cNK cells), compared to ApoE-/-Rag1-/- mice treated with anti-IL-15R mAbs for depleting cNK cells, and these effects could be fully rescued through the adoptive transfer of ILC1 cells sorted from the spleen of ApoE-/-TLR4+/+ mice into ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs. However, the adoptive transfer of ILC1 cells sorted from the spleen of ApoE-/-TLR4-/- mice into ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs blocked the progression of atherosclerosis, indicating that the pro-atherosclerotic role of ILC1 cells is dependent on TLR4. Furthermore, oxLDL-induced increase in IFN-γ expression from ApoE-/- ILC1 cells was correlated with the decrease in BACH2 expression. Taken together, ILC1 cells exist in atherosclerosis and aggravate atherosclerosis via increasing pro-inflammatory cytokine expression in a TLR4/BACH2-dependent manner.

Background: CD4+CD25+FoxP3+ regulatory T cells (Treg cells) play a protective role against the development and progression of the inflammatory disease atherosclerosis (AS). MicroRNA-21 (miR-21) is expressed in Treg cells and is up-regulated in the context of AS and other inflammatory diseases. Aims: This study aimed to determine the role of miR-21 in Treg cell regulation and gene expression during the development of AS in patients with coronary heart disease (CHD). Methods and Results: MiR-21 expression in peripheral blood mononuclear cells (PBMCs) was significantly up-regulated in patients with CHD (acute myocardial infarction (AMI) group, n=24; unstable angina (UA) group, n=21; stable angina (SA) group, n=24) compared with patients with chest pain syndrome (CPS, n=27), and miR-21 expression showed an increasing trend from SA to UA to AMI patients. Moreover, flow cytometry analysis indicated that the frequencies of circulating Treg cells decreased in a manner proportionate opposite with the level of miR-21. Quantitative real-time PCR (qRT-PCR) revealed a decrease in mRNA expression of forkhead box P3 (foxp3), transforming cell growth factor beta 1(TGF-β1) and smad7 (a known target gene of miR-21). ELISA analysis revealed a decrease in TGF-β1 secreted into the plasma. In addition, we transfected PBMCs with a miRNA negative control (NS-m), a miR-21 mimic (miR-21-m), a miRNA inhibitor negative control (NS-i), or a miR-21 inhibitor(miR-21-i). Up-regulation of miR-21 decreased the frequency of circulating Treg cells, decreased the expression levels of foxp3, TGF-β1 and smad7, and decreased the amount of TGF-β1 secreted into the plasma. Consistent with these observations, miR-21 down-regulation increased the frequency of circulating Treg cells, increased the expression of foxp3, TGF-β1 and smad7, and increased the amount of TGF-β1 secreted into the plasma. Conclusions: Because the smad7 expression pattern was similar to that of TGF-β, our study suggests that miR-21 can negatively regulate the frequency of circulating Treg cells through a TGF-β1/smad-independent signaling pathway in PBMCs.

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.

Background/Aims: Recently, studies have shown that interleukin-37 (IL-37) is involved in atherosclerosis-related diseases. However, the regulatory mechanisms of IL-37 in atherosclerosis remain unknown. This study aims to determine the role of IL-37 in atherosclerosis and to investigate the underlying mechanisms involved. Methods: IL-37 expression in human atherosclerotic plaques was detected by immunohistochemical staining and real-time reverse transcription polymerase chain reaction (RT-PCR). Oil Red O staining was used to measure the size of plaques. Cell apoptosis in vitro and in vivo was tested by flow cytometric analysis and terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling (TUNEL) staining, respectively. Protein expression levels of IL-37, IL-18Rα and p-Smad3 were measured by Weston blotting. Results: Immunohistochemical staining revealed that IL-37 was highly expressed in human atherosclerotic plaques. Intracellular cytokine staining revealed that infiltrated CD4+ T lymphocytes and vascular smooth muscle cells (VSMCs), but not macrophages, were the major sources of IL-37. Mice that overexpressed IL-37 exhibited significant improvements in their atherosclerotic burden, as demonstrated by reduced plaque size, increased collagen levels, and reduced numbers of apoptotic cells in vivo. Subsequently, mechanistic studies showed that IL-37 played an anti-atherosclerotic role, at least partially, through reducing inflammation by promoting the differentiation of the T helper cell anti-inflammatory phenotype, and through increasing plaque stability by decreasing matrix metalloproteinase (MMP)-2/13-mediated degradation of collagen and inhibiting VSMCs apoptosis. Conclusion: IL-37 may be a novel potential therapeutic target in patients with atherosclerotic heart disease.

Myocardial infarction triggers an intense inflammatory response that is essential for dead tissue clearance but also detrimental to cardiac repair. Macrophages are active and critical players in the inflammatory response after myocardial infarction. Understanding the molecular mechanisms by which macrophage-mediated inflammatory response is regulated is important for designing new therapeutic interventions for myocardial infarction. In current study, we examined the role of Sestrin2, which is a stress-inducible protein that regulate metabolic homeostasis, in the regulation of inflammatory response of cardiac macrophages after myocardial infarction. We found that cardiac macrophages up-regulated Sestrin2 expression in a mouse myocardial infarction model. Using a lentiviral transduction system to overexpress Sestrin2 in polarized M1 and M2 macrophages, we revealed that Sestrin2 predominantly functioned on M1 rather than M2 macrophages. Sestrin2 overexpression suppressed inflammatory response of M1 macrophages both in vitro and in vivo. Furthermore, in the mouse myocardial infarction model with selective depletion of endogenous macrophages and adoptive transfer of exogenous Sestrin2-overexpressing macrophages, the anti-inflammatory and repair-promoting effect of Sestrin2-overexpressing macrophages was demonstrated. Furthermore, Sestrin2 significantly inhibited mTORC1 signaling in M1 macrophages. Taken together, our study indicates the importance of Sestrin2 for suppression of M1 macrophage-mediated cardiac inflammation after myocardial infarction.

Objective: Thrombogenic and inflammatory mediators such as thrombin induce NF-kB-mediated EC activation and dysfunction, which contribute to the pathogenesis of arterial thrombosis. The role of the anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Methods and Results: It has been demonstrated that miR-181b inhibits downstream NF-kB signaling in response to TNF-a. Herein, we reveal that miR-181b can inhibit upstream NF-kB signaling uniquely in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-kB signaling demonstrated by the reduction of phospho-IKK-b, -IkB-a, and p65 nuclear translocation in ECs. MiR-181b also reduced the expression of NF-kB target genes VCAM-1, ICAM-1, E-selectin, PAI-1, and tissue factor. Mechanistically, miR-181b targets Card10, an adaptor protein that participates in the activation of the IKK complex in response to signals transduced from G-protein coupled receptors such as PAR-1. MiR-181b reduced the expression of Card10 mRNA and protein, but not PAR-1. In addition, miR-181b inhibited Card10 3’-UTR luciferase reporter activity, and Argonaute-2 miRNP-IP studies revealed 4-fold enrichment of Card10 mRNA in the RNA-induced silencing complex in the presence of miR-181b, indicating that Card10 is a bona fide direct target. SiRNA-mediated knockdown of Card10 expression phenocopied miR-181b’s effects on NF-kB signaling and targets. Interestingly, knockdown of Card10 does not affect TNF-a-induced activation of NF-kB signaling and target gene expression, suggesting 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 the time to occlusion by 1.6-fold. Conclusions: MiR-181b is an important regulator of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of miRNA-dependent targets in response to ligand-specific signaling in ECs. Delivery of miR-181b or Card10 inhibition may represent a new therapeutic approach to reduce arterial thrombosis by improving vascular EC function.

Objective: To investigate the regulation of CD4+CD25+ Regulatory T cells (Tregs) on pro-inflammatory adhesion molecules, Kruppel-Like Factor-2 (KLF-2) and its downstream transcriptional targets in human umbilical vein endothelial cells (HUVECs) impaired by ox-LDL and the mechanisms of it. Methods and results: HUVECs were cultured in the continuous presence of ox-LDL(0 mg/L,25 mg/L,50 mg/L,100 mg/L) for 4, 6, 12 and 24 hours to allow identification of early-and late-induced genes, respectively, whereas non-stimulated controls were taken at 0 hours. The expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, KLF-2 and its target genes eNOS, PAI-1 were determined by real time RT-PCR and/or western-blot analysis. Expression of pro-inflammatory adhesion molecules, KLF-2, eNOS and PAI-1 in HUVEC cultured alone or with anti-CD3 mAbs activated Tregs, followed by addition of ox-LDL (50 mg/L) for 6 hours, are compared to expression levels in control cultures. Ox-LDL treated HUVECs increased pro-inflammatory adhesion molecules expression, as well as increased PAI-1 but decreased eNOS expression accompanied with significant downregulating of KLF-2 at a dose and time dependent manner. Furthermore, ox-LDL increased pro-inflammatory adhesion molecules but inhibited KLF2 expression was reversed by addition of Tregs. Small interfering RNA reduced endogenous KLF-2 expression and partly reversed the suppressive effect of Tregs on HUVECs activation, which strongly implicate KLF-2 as a transcriptional regulator of the Tregs-mediated effects in endothelial cells. Mechanism studies reveal that Treg-mediated KLF2 expression in HUVECs impaired by ox-LDL requires cell contact as well as soluble factors. Conclusions: Tregs could protect endothelial function that is largely dependent on KLF2 and its downstream transcriptional targets regulation involving cell-to-cell contact and soluble factors.

CD4(+)CD25(+) regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect oxLDL-induced proinflammatory response in macrophages. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxLDL for 48 hours to induce proinflammatory response. Our data showed that with oxLDL challenge, the Treg-modulated macrophages have decreased NO production and iNOS expression, decreased HLA-DR and CD86 expression, and down-regulated proinflammatory cytokine/chemokine production. Tregs can inhibit the pro-inflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that Treg-mediated suppression of the monocyte response to oxLDL was reflected by a reduction in the up-regulation of NF-kappaB activity accompanied by a decreased expression of TLR2 but not TLR4 at the transcriptional level. These results suggest that CD4(+)CD25(+)Foxp3(+) regulatory T cells may exert its suppressive functions on pro-inflammatory properties of OxLDL induced-macrophages partly through TLR2-NF-kappaB signaling pathway.

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.

The effect of thymic stromal lymphopoietin (TSLP) on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein (ox-LDL). The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors (CD36 and SRA) as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.

Rationale: Activated nuclear factor (NF)-κB signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-κB may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-κB 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-κB signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-κB-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-α3, an effect that reduced NF-κB nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-κB signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-κB nuclear translocation in leukocytes does not involve importin-α3, but rather importin-α5, which miR-181b does not target, highlighting that inhibition of NF-κB signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-κB 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.

EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB-responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b "mimics" reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB-mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.

To investigate the regulation of CD4(+)CD25(+) Regulatory T cells (Tregs) on pro-inflammatory adhesion molecules, Krüppel-Like Factor-2 (KLF-2) and its downstream transcriptional targets in human umbilical vein endothelial cells (HUVECs) impaired by ox-LDL and the mechanisms of it.HUVECs were cultured in the continuous presence of ox-LDL(0 mg/L,25 mg/L,50 mg/L,100 mg/L) for 4, 6, 12 and 24 hours to allow identification of early-and late-induced genes, respectively, whereas non-stimulated controls were taken at 0 hours. The expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, KLF-2 and its target genes eNOS, PAI-1 were determined by real time RT-PCR and/or western-blot analysis. Expression of pro-inflammatory adhesion molecules, KLF-2, eNOS and PAI-1 in HUVEC cultured alone or with anti-CD3 mAbs activated Tregs, followed by addition of ox-LDL (50 mg/L) for 6 hours, are compared to expression levels in control cultures. Ox-LDL treated HUVECs increased pro-inflammatory adhesion molecules expression, as well as increased PAI-1 but decreased eNOS expression accompanied with significant downregulating of KLF-2 at a dose and time dependent manner. Furthermore, ox-LDL increased pro-inflammatory adhesion molecules but inhibited KLF2 expression was reversed by addition of Tregs. Small interfering RNA reduced endogenous KLF-2 expression and partly reversed the suppressive effect of Tregs on HUVECs activation, which strongly implicate KLF-2 as a transcriptional regulator of the Tregs-mediated effects in endothelial cells. Mechanism studies reveal that Treg-mediated KLF2 expression in HUVECs impaired by ox-LDL requires cell contact as well as soluble factors.Tregs could protect endothelial function that is largely dependent on KLF2 and its downstream transcriptional targets regulation involving cell-to-cell contact and soluble factors.

Objective— To investigate the role of CD4 + CD25 + forkhead box P 3 (Foxp3) + T-regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and the mechanisms of it. Methods and Results— ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4 + CD25 − T cells in the presence of anti–CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule, such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide. Moreover, Tregs downregulate proinflammatory factor nuclear factor-κB activation and induce resistance to suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus. Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth factor [TGF]-β). Conclusion— Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving cytotoxic T-lymphocyte antigen-4–dependent cell-to-cell contact and also requiring soluble factors (mainly interleukin 10 and TGF-β).

CD4(+)CD25(+) regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect oxLDL-induced proinflammatory response in macrophages. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxLDL for 48 hours to induce proinflammatory response. Our data showed that with oxLDL challenge, the Treg-modulated macrophages have decreased NO production and iNOS expression, decreased HLA-DR and CD86 expression, and down-regulated proinflammatory cytokine/chemokine production. Tregs can inhibit the pro-inflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that Treg-mediated suppression of the monocyte response to oxLDL was reflected by a reduction in the up-regulation of NF-kappaB activity accompanied by a decreased expression of TLR2 but not TLR4 at the transcriptional level. These results suggest that CD4(+)CD25(+)Foxp3(+) regulatory T cells may exert its suppressive functions on pro-inflammatory properties of OxLDL induced-macrophages partly through TLR2-NF-kappaB signaling pathway.

Background and Objective Cluster of differentiation (CD)4+ CD25+ regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis, but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect macrophages foam-cell formation. Methods and Results Tregs were isolated by magnetic cell sorting-column and analysed by fl ow cytometry. Macrophages were cultured with or without Tregs in the presence of oxidised LDL (oxLDL) for 48 h to transform foam cells. After co-culture with Tregs, macrophages showed a decrease in lipid accumulation, which was accompanied by a significantly downregulated expression of CD36 and SRA but no obvious difference in ABCA1 expression. Conclusions Tregs can inhibit the proinflammatory properties of macrophages and steer macrophage differentiation toward an anti-infl amatory cytokine producing phenotype. Mechanistic studies reveal that both cell-to-cell contact and soluble factors are required for Treg-mediated suppression on macrophage foam-cell formation. Cytokines, interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta) are the key factors for these suppressive functions.