Atherosclerosis is a chronic and multifactorial inflammatory disease and is closely associated with cardiovascular and cerebrovascular diseases. circRNAs can act as competing endogenous RNAs to mRNAs and function in various diseases. However, there is little known about the function of circRNAs in atherosclerosis. In this study, three rabbits in the case group were fed a high-fat diet to induce atherosclerosis and another three rabbits were fed a normal diet. To explore the biological functions of circRNAs in atherosclerosis, we analyzed the circRNA, miRNA and mRNA expression profiles using RNA-seq. Many miRNAs, mRNAs and circRNAs were identified as significantly changed in atherosclerosis. We next predicted miRNA-target interactions with the miRanda tool and constructed a differentially expressed circRNA-miRNA-mRNA triple network. A gene ontology enrichment analysis showed that genes in the network were involved in cell adhesion, cell activation and the immune response. Furthermore, we generated a dysregulated circRNA-related ceRNAs network and found seven circRNAs (ocu-cirR-novel-18038, -18298, -15993, -17934, -17879, -18036 and -14389) were related to atherosclerosis. We found these circRNAs also functioned in cell adhesion, cell activation and the immune response. These results show that the crosstalk between circRNAs and their competing mRNAs might play crucial roles in the development of atherosclerosis.

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of KATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration approximately 10 nM. The channel activation was voltage-independent and could be blocked by KATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the KATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of KATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.

Sepsis is a severe medical condition causing a large number of deaths worldwide. Recent studies indicate that the septic susceptibility is attributable to the vascular ATP-sensitive K ؉ (K ATP ) channel. However, the mechanisms underlying the channel modulation in sepsis are still unclear. Here we show evidence for the modulation of vascular K ATP channel by septic pathogen lipopolysaccharides (LPS). In isolated mesenteric arterial rings, phenylephrine (PE) produced concentration-dependent vasoconstriction that was relaxed by pinacidil, a selective K ATP channel opener. The PE response was disrupted with a LPS treatment. In acutely dissociated aortic smooth myocytes the LPS treatment augmented K ATP channel activity, and hyperpolarized the cells. Quantitative PCR analysis showed that LPS raised Kir6.1 and SUR2B transcripts in a concentration-dependent manner, which was suppressed by transcriptional inhibition. Consistently, the same LPS treatment did not affect Kir6.1/ SUR2B channels in a heterologous expression system. The LPS effect on Kir6.1 and SUR2B expression was abolished in the presence of NF-B inhibitors. Several other Toll-like receptor ligands also stimulated Kir6.1 and SUR2B expression to a similar degree as LPS. Thus, the effect of LPS on vasodilation involves up-regulation of K ATP channel expression, in which the NF-B-dependent signaling plays an important role.

BackgroundBmi1 has been identified as an important regulator in breast cancer, but its relationship with other signaling molecules such as ERα and HER2 is undetermined.MethodsThe expression of Bmi1 and its correlation with ERα, PR, Ki-67, HER2, p16INK4a, cyclin D1 and pRB was evaluated by immunohistochemistry in a collection of 92 cases of breast cancer and statistically analyzed. Stimulation of Bmi1 expression by ERα or 17β-estradiol (E2) was analyzed in cell lines including MCF-7, MDA-MB-231, ERα-restored MDA-MB-231 and ERα-knockdown MCF-7 cells. Luciferase reporter and chromatin immunoprecipitation assays were also performed.ResultsImmunostaining revealed strong correlation of Bmi1 and ERα expression status in breast cancer. Expression of Bmi1 was stimulated by 17β-estradiol in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells, while the expression of Bmi1 did not alter expression of ERα. As expected, stimulation of Bmi1 expression could also be achieved in ERα-restored MDA-MB-231 cells, and at the same time depletion of ERα decreased expression of Bmi1. The proximal promoter region of Bmi1 was transcriptionally activated with co-transfection of ERα in luciferase assays, and the interaction of the Bmi1 promoter with ERα was confirmed by chromatin immunoprecipitation. Moreover, in breast cancer tissues activation of the ERα-coupled Bmi1 pathway generally correlated with high levels of cyclin D1, while loss of its activity resulted in aberrant expression of p16INK4a and a high Ki-67 index, which implied a more aggressive phenotype of breast cancer.ConclusionsExpression of Bmi1 is influenced by ERα, and the activity of the ERα-coupled Bmi1 signature impacts p16INK4a and cyclin D1 status and thus correlates with the tumor molecular subtype and biologic behavior. This demonstrates the important role which is played by ERα-coupled Bmi1 in human breast cancer.

Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K(+) (K(ATP)) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3'-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3'-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular K(ATP) channels, and impairment of arterial function.

Aims/hypothesis Alcohol consumption levels frequently fluctuate over the life course, but studies examining the association between alcohol consumption trajectories and type 2 diabetes are limited. This study aims to investigate the association of alcohol consumption trajectories with the risk of type 2 diabetes and its related factors. Methods Weighted longitudinal data were obtained for 12,186 adults who completed a questionnaire about alcohol consumption and diabetes status as part of the China Health and Nutrition Survey (1993-2011). Participants were designated into subgroups based on alcohol consumption trajectory, and subgroup analyses included 5436 individuals who were tested for specified diabetes-related factors. Light alcohol consumption was defined as fewer than seven standard drinks per week; moderate as 7-21 drinks per week; and heavy as more than 21 drinks per week. Latent class trajectory modelling was used to identify different alcohol consumption trajectories by sex. Multivariate Cox regression models and general linear regression models were used to assess association of trajectories with type 2 diabetes and its related factors. Results Compared with stable abstainers (individuals who never drank alcohol), two trajectories in men showing reduction to moderate or light levels after heavy alcohol consumption during early adulthood were significantly associated with increased risk of type 2 diabetes (HR 1.66 [95% CI 1.18, 2.33]; HR 1.93 [95% CI 1.01, 3.70]), while no significant association between trajectories and risk of type 2 diabetes was observed in women (p for trend = 0.404). Triacylglycerol, HDL-cholesterol (HDL-C), uric acid and high sensitivity C-reactive protein were significantly higher in these two trajectories than other trajectories in men (all p < 0.05), while only HDL-C showed significant increasing trends in women. Trajectories showing light-stable, or increase to moderate, levels were not associated with reduced risk of type 2 diabetes. Conclusions This study indicated that heavy alcohol consumption in early adulthood is significantly associated with increased risk of type 2 diabetes and higher levels of its biomarkers throughout adulthood in men. Gradually reducing alcohol consumption to moderate levels may not make a difference, which demonstrates the importance of alcohol intervention strategies in early adulthood. Although association between alcohol consumption and increased HDL-C levels has been observed, the results of this study did not support the hypothesis regarding the protective effect of moderate alcohol consumption on risk of type 2 diabetes in the Asian population. Data availability Data from China Health and Nutrition Survey was used in this study, which can be downloaded at www.cpc. unc.edu/projects/china.

Vasoactive intestinal polypeptide (VIP), an endogenous neuropeptide normally present in lungs and other organs, relaxes pulmonary arteries (PAs) in different species, whereas the underlying mechanisms are still not fully understood. The aim of this study, therefore, is to investigate the signal transduction of VIP in the relaxation of isolated rat PA rings. The isometric tension of the rings was studied in vitro with force-electricity transducers. In endothelium-intact (EI) rings, VIP elicited concentration-dependent relaxation after the rings were pre-contracted by phenylephrine. A similar effect, though smaller, was observed in endothelium-denuded (ED) rings. Inhibition of the endothelial nitric oxide synthase (eNOS) by NG-nitro-L-arginine methyl ester diminished the VIP-induced vasodilatation of PA rings. The VIP-induced vasorelaxation was markedly reduced by the inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway with wortmannin and LY294002, respectively, which was seen in EI rings, but not in ED rings. Western blot analysis revealed that VIP increased the phosphorylation of eNOS at Ser 1177, but did not affect the overall expression of eNOS. In ED rings, the PKA inhibitor H-89 and K(ATP) channel inhibitor glibenclamide almost totally abolished the vasodilatation effect of VIP. The results suggested that the vasodilatation effect of VIP on rat PAs is mediated by both vascular endothelium and smooth muscle, involving respectively the PI3K/Akt-eNOS pathway and the PKA-K(ATP) channel pathway.

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