Intracranial aneurysms are pathological dilatations of the cerebral artery, while rupture of intracranial aneurysms causes life-threatening subarachnoid hemorrhage. The molecular mechanisms of pathogenesis of intracranial aneurysms are poorly understood. MicroRNAs have fundamental roles in modulating vascular biology and disease. In the present study, we carried out a genome-wide characterization on expressions of microRNAs, and performed integrative analyses in conjunction with changes of the transcriptome in human intracranial aneurysms.

Atherosclerosis is a typical complex multi-factorial disease and many molecules at different levels and pathways were involved in its development. Some studies have investigated the dysregulation in atherosclerosis at mRNA, miRNA or DNA methylation level, respectively. However, to our knowledge, the studies that integrated these data and revealed the abnormal networks of atherosclerosis have not been reported. Using microarray technology, we analyzed the omics data in atherosclerosis at mRNA, miRNA and DNA methylation levels. Our results demonstrated that the global DNA methylation and expression of miRNA/mRNA were significantly decreased in atherosclerotic plaque than in normal vascular tissue. The interaction network constructed using the integrative data revealed many genes, cellular processes and signaling pathways which were widely considered to play crucial roles in atherosclerosis and also revealed some genes, miRNAs or signaling pathways which have not been investigated in atherosclerosis until now (e.g. miR-519d and SNTB2). Moreover, the overall protein ubiquitination in atherosclerotic plaque was significantly increased. The proteasome activity was increased early but decreased in advanced atherosclerosis. Our study revealed many classic and novel genes and miRNAs involved in atherosclerosis and indicated the effects of ubiquitin-proteasome system on atherosclerosis might be closely related to the course of atherosclerosis. However, the efficacy of proteasome inhibitors in the treatment of atherosclerosis still needs more research.

Apolipoprotein E-knockout (ApoE(-/-)) mice, atherosclerosis-prone mice, show an autoimmune response, but the pathogenesis is not fully understood. We investigated the pathogenesis in female and male ApoE(-/-) mice. The spleens of all ApoE(-/-) and C57BL/6 (B6) mice were weighed. The serum IgG level and titers of anti-nuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA) antibody were assayed by ELISA. Apoptosis of spleen tissue was evaluated by TUNEL. TLR4 level in spleen tissue was tested by immunohistochemistry and Western blot analysis. Levels of MyD88, p38, phosphorylated p38 (pp38), interferon regulatory factor 3 (IRF3) and Bcl-2-associated X protein (Bax) in spleen tissue were detected by Western blot analysis. We also survey the changes of serum autoantibodies, spleen weight, splenocyte apoptosis and the expressions of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue in male ApoE(-/-) mice after 4weeks of lipopolysaccharide (LPS), Toll-like receptor 4 ligand, administration. ApoE(-/-) mice showed splenomegaly and significantly increased serum level of IgG and titers of ANA and anti-dsDNA antibody as compared with B6 mice. Splenocyte apoptosis and the expression of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue were significantly lower in ApoE(-/-) than B6 mice. The expression of TLR4, MyD88, IRF3, pp38, and Bax differed by sex in ApoE(-/-) spleen tissue. The down-regulation of TLR4 signal molecules induced by LPS led to decreased expression of Bax and increased serum titers of ANA and anti-dsDNA antibody. Therefore, the TLR4 signal pathway may participate in maintaining the balance of splenocyte apoptosis and autoantibody production in ApoE(-/-) mice.

Oxidized low-density lipoprotein (ox-LDL) can impair endothelial function and lead to the atherosclerosis development. Protein S-nitrosylation is sensitive to cellular redox state and acts as a crucial regulator and executor of nitric oxide (NO) signaling pathways. Aberrant S-nitrosylation contributes to the pathogenesis of cardiovascular and cerebrovascular diseases. However, the effect of ox-LDL on S-nitrosylation and its significance for endothelial dysfunction have not been studied at proteome level. Herein, the combined quantitative analysis of proteome and S-nitrosoproteome was performed using an integrated biotin switch and iTRAQ labeling approach in EA.hy926 cell line derived from human umbilical vein endothelial cell (HUVEC) treated with ox-LDL. A total of 2204 S-nitrosylated (SNO) peptides of 1318 SNO-proteins were quantified. Notably, 352 SNO-peptides of 262 SNO-proteins were significantly regulated after excluding S-nitrosylation changes caused by protein expression alterations. Many of them belonged to mRNA splicing, ribosomal structure and translational regulatory proteins, covering the entire translation process. The results indicated that S-nitrosylation of the splicing and translational machinery in vascular endothelial cells was susceptible to ox-LDL. Abnormal protein S-nitrosylation may be one pivotal mechanism underlying endothelial dysfunction induced by ox-LDL. This study potentially enriches the present understanding of pro-atherogenic effect of ox-LDL from the perspective of S-nitrosylation. SIGNIFICANCE: The role of ox-LDL in endothelial dysfunction and atherosclerosis development has been recognized from the aspect of impaired NO production. However, its effect on S-nitrosylation, which is directly related to NO signaling pathway, still remains largely unexplored. Our work initially provided a systematic characterization of S-nitrosoproteome in ox-LDL-treated endothelial cells after ruling out the changes of S-nitrosylation modification caused by protein expression alone. MS-based approach coupled with iTRAQ technique indicated 262 SNO-proteins were significantly regulated. Functional enrichment and interaction network analysis revealed that proteins involved in mRNA splicing and translational machinery were susceptible to abnormal S-nitrosylation under ox-LDL treatment. This achievement suggested one potential mechanism underlying endothelial dysfunction induced by ox-LDL from the perspective of S-nitrosoproteome.

Analysis of high-throughput omics data is one of the most important approaches for obtaining information regarding interactions between proteins/genes. Time-series omics data are a series of omics data points indexed in time order and normally contain more abundant information about the interactions between biological macromolecules than static omics data. In addition, phosphorylation is a key posttranslational modification (PTM) that is indicative of possible protein function changes in cellular processes. Analysis of time-series phosphoproteomic data should provide more meaningful information about protein interactions. However, although many algorithms, databases, and websites have been developed to analyze omics data, the tools dedicated to discovering molecular interactions from time-series omics data, especially from time-series phosphoproteomic data, are still scarce. Moreover, most reported tools ignore the lag between functional alterations and the corresponding changes in protein synthesis/PTM and are highly dependent on previous knowledge, resulting in high false-positive rates and difficulties in finding newly discovered protein-protein interactions (PPIs). Therefore, in the present study, we developed a new method to discover protein-protein interactions with the delayed comparison and Apriori algorithm (DCAA) to address the aforementioned problems. DCAA is based on the idea that there is a lag between functional alterations and the corresponding changes in protein synthesis/PTM. The Apriori algorithm was used to mine association rules from the relationships between items in a dataset and find PPIs based on time-series phosphoproteomic data. The advantage of DCAA is that it does not rely on previous knowledge and the PPI database. The analysis of actual time-series phosphoproteomic data showed that more than 68% of the protein interactions/regulatory relationships predicted by DCAA were accurate. As an analytical tool for PPIs that does not rely on a priori knowledge, DCAA should be useful to predict PPIs from time-series omics data, and this approach is not limited to phosphoproteomic data.

The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein-protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress-induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1-mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.

The NAD+-dependent deacetylases Sirt1 and Sirt2 mediate cellular stress responses and are highly expressed in vascular endothelial cells. In contrast to the well-documented protective actions of Sirt1, the role of endothelial Sirt2 remains unknown. Using cDNA microarray and PCR validation, we examined global gene expression changes in response to Sirt2 knock down in primary human umbilical vein endothelial cells under oxidative stress. We found that Sirt2 knock down changed expression of 340 genes, which are mainly involved in cellular processes including actin binding, cellular amino acid metabolic process, transmembrane receptor protein serine/threonine kinase signaling, ferrous iron transport, protein transport and localization, cell morphogenesis, and functions associated with endosome membrane and the trans-Golgi network. These genes and associated functions were largely non-overlapping with those altered by Sirt1 knock down. Moreover, we showed that pharmacological inhibition of Sirt2 attenuated oxidant-induced cell toxicity in endothelial cells. These suggest that Sirt2 is functionally important in endothelial cells under oxidative stress, and may have a primarily distinct role as compared to Sirt1. Our results may provide a basis for future studies aiming to dissect the specific signaling pathway(s) that mediates specific Sirt2 functions in endothelial cells.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.

Abdominal aortic aneurysm (AAA) is an insidious and lethal vascular disease that lacks effective nonsurgical interventions. Gut microbiota dysbiosis plays key roles in many diseases, but its relationship with AAA has not been fully elucidated. Herein, we reveal significant abnormalities in the gut microbe composition of AAA patients and confirm that gut microbiota dysbiosis is an important cause of AAA. Specifically, R. intestinalis was significantly reduced in AAA patients. Using AAA mice, we show that R. intestinalis and its metabolite butyrate significantly reduce neutrophil infiltration and NOX2-dependent neutrophil extracellular trap formation, inflammation, and abnormal phenotypic switching of vascular smooth muscle cells in the aortic wall, thereby markedly alleviating AAA development. Our research uncovers the role and mechanism of the gut microbiota in AAA development and provides insights into AAA prophylaxis from a microecological perspective.

The T cell receptor (TCR) β repertoire directly reflects the status of T cell function. Meanwhile, the immune/inflammatory responses regulated by T cells are the critical determinants of atherosclerosis development. However, due to technical limitations, the composition and molecular characteristics of the TCR repertoire in atherosclerotic patients have not been fully elucidated. In the present study, we use powerful immune repertoire sequencing technology to study this issue. Results show that the utilization of V and/or J genes and the diversity of TCRβ repertoire in atherosclerotic plaques are significantly reduced compared to those in the peripheral blood of normal subjects and atherosclerotic patients. The frequencies of the common T cell clones with certain lengths of the complement determining region 3 regions are notably different among all groups. The high-frequency common clones are also increased in the atherosclerotic plaques compared to that in the other two groups. The expansion of several T cell clonotypes (V29-1J2-1, V20-1J1-6, V6-3J2-7 and V11-2J2-2) is validated in atherosclerotic patients. In short, this study reveals that the diversity of TCR β repertoire significantly decreases in atherosclerotic plaques, probably because of the reduced utilization of VJ genes and marked expansion of some T cell subclones. It provides the basis for understanding the roles of T lymphocytes in the pathogenesis of atherosclerosis.

Subclinical hypothyroidism is associated with cardiovascular diseases, yet the underlying mechanism remains largely unknown. Herein, in a common population (n = 1,103), TSH level was found to be independently correlated with both carotid plaque prevalence and intima-media thickness. Consistently, TSH receptor ablation in ApoE -/- mice attenuated atherogenesis, accompanied by decreased vascular inflammation and macrophage burden in atherosclerotic plaques. These results were also observed in myeloid-specific Tshr-deficient ApoE -/- mice, which indicated macrophages to be a critical target of the proinflammatory and atherogenic effects of TSH. In vitro experiments further revealed that TSH activated MAPKs (ERK1/2, p38α, and JNK) and IκB/p65 pathways in macrophages and increased inflammatory cytokine production and their recruitment of monocytes. Thus, the present study has elucidated the new mechanisms by which TSH, as an independent risk factor of atherosclerosis, aggravates vascular inflammation and contributes to atherogenesis.

The inflammatory responses of vascular endothelial cells (VECs) are critical in the development of many cardio-cerebrovascular diseases. Let-7e is an important regulator of endothelial function and inflammation. However, the effects and mechanisms of let-7e on VECs inflammation have not been studied until recently. Thus, we investigated these issues and found that in addition to proliferation, apoptosis and cell adhesion, let-7e was also implicated in the regulation of inflammatory responses through a complex network, including IκBβ and lncRNA lnc-MKI67IP-3. Let-7e promoted NF-κB activation and translocation to the nucleus by inhibiting its target gene (IκBβ) expression and subsequently increased the expression of inflammatory and adhesion molecules. Meanwhile, lnc-MKI67IP-3 acted as a sponge or competing endogenous RNA (ceRNA) for let-7e, suppressing its pro-inflammatory effects, and let-7e decreased lnc-MKI67IP-3 expression, thereby forming a positive feedback loop to aggravate inflammation. Moreover, let-7e, lnc-MKI67IP-3 and IκBβ were also abnormal in oxLDL-treated VECs and atherosclerotic plaques. The present study revealed let-7e as a pro-inflammatory mediator and a novel regulatory mechanism for the NF-κB pathway through ceRNA crosstalk, comprising let-7e and its target IκBβ and the ceRNA lnc-MKI67IP-3. Thus, this molecule might play important roles in the inflammatory responses of VECs and development of atherosclerosis.

Arsenic trioxide exhibits therapeutic effects on certain blood malignancies, at least partly by modulating cell differentiation. Previous in vitro studies in human hematopoietic progenitor cells have suggested that arsenic may inhibit erythroid differentiation. However, these effects were all observed in the presence of arsenic compounds, while the concomitant cytostatic and cytotoxic actions of arsenic might mask a prodifferentiating activity. To eliminate the potential impacts of the cytostatic and cytotoxic actions of arsenic, we adopted a novel protocol by pretreating human bone marrow CD34+ cells with a low, noncytotoxic concentration of arsenic trioxide, followed by assaying the colony forming activities in the absence of the arsenic compound. Bone marrow specimens were obtained from chronic myeloid leukemia patients who achieved complete cytogenetic remission. CD34+ cells were isolated by magnetic-activated cell sorting. We discovered that arsenic trioxide enhanced the erythroid colony forming activity, which was accompanied by a decrease in the granulomonocytic differentiation function. Moreover, in erythroleukemic K562 cells, we showed that arsenic trioxide inhibited erythrocyte maturation, suggesting that arsenic might have biphasic effects on erythropoiesis. In conclusion, our data provided the first evidence showing that arsenic trioxide could prime human hematopoietic progenitor cells for enhanced erythroid differentiation.

Biological markers that can be used to predict the risk of intracranial aneurysms (IAs) are not available.

Myocardial infarction (MI) remains the leading cause of death worldwide. Cardiac fibroblasts (CFs) are abundant in the heart and are responsible for cardiac repair post-MI. NF-κB-repressing factor (NKRF) plays a significant role in the transcriptional inhibition of various specific genes. However, the NKRF action mechanism in CFs remains unclear in cardiac repair post-MI. This study investigates the NKRF mechanism in cardiac remodeling and dysfunction post-MI by establishing a CF-specific NKRF-knockout (NKRF-CKO) mouse model. NKRF expression is downregulated in CFs in response to pathological cardiac remodeling in vivo and TNF-α in vitro. NKRF-CKO mice demonstrate worse cardiac function and survival and increased infarct size, heart weight, and MMP2 and MMP9 expression post-MI compared with littermates. NKRF inhibits CF migration and invasion in vitro by downregulating MMP2 and MMP9 expression. Mechanistically, NKRF inhibits human antigen R (HuR) transcription by binding to the classical negative regulatory element within the HuR promoter via an NF-κB-dependent mechanism. This decreases HuR-targeted Mmp2 and Mmp9 mRNA stability. This study suggests that NKRF is a therapeutic target for pathological cardiac remodeling.

Thyroid-stimulating hormone (TSH) treatment activates inhibitor of NF-κB/nuclear factor κB (IκB/NFκB) and extracellular signal-regulated kinase (ERK)-P38 in macrophages, but how these pathways are activated, and how they contribute to the proinflammatory effect of TSH on macrophages remain unknown. The TSH receptor (TSHR) is coupled to 4 subfamilies of G proteins (Gs, Gi/o, Gq/11, and G12/13) for its downstream signaling. This study investigated the G protein subtypes responsible for the proinflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, and G15 are abundantly expressed by macrophages. The contribution of different G protein pathways to the proinflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, or Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interference of either G13 or G15 could block the proinflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by the G13/ERK-P38/Rho GTPase and G15/phospholipase C (PLC)/protein kinases C (PKCs)/IκB pathways.

Angiogenesis is a critical pathophysiological process involved in organ growth and various diseases. Transcription factors Sp1/Sp3 are necessary for fetal development and tumor growth. Sp1/Sp3 proteins were downregulated in the capillaries of the gastrocnemius in patients with critical limb ischemia samples. Endothelial-specific Sp1/Sp3 knockout reduces angiogenesis in retinal, pathological, and tumor models and induced activation of the Notch1 pathway. Further, the inactivation of VEGFR2 signaling by Notch1 contributes to the delayed angiogenesis phenotype. Mechanistically, endothelial Sp1 binds to the promoter of Notch1 and inhibits its transcription, which is enhanced by Sp3. The proangiogenic effect of ACEI is abolished in Sp1/Sp3-deletion male mice. We identify USP7 as an ACEI-activated deubiquitinating enzyme that translocated into the nucleus binding to Sp1/Sp3, which are deacetylated by HDAC1. Our findings demonstrate a central role for endothelial USP7-Sp1/Sp3-Notch1 signaling in pathophysiological angiogenesis in response to ACEI treatment.

CX-5461, a novel selective RNA polymerase I inhibitor, shows potential anti-inflammatory and immunosuppressive activities. However, the molecular mechanisms underlying the inhibitory effects of CX-5461 on macrophage-mediated inflammation remain to be clarified. In the present study, we attempted to identify the systemic biological processes which were modulated by CX-5461 in inflammatory macrophages. Primary peritoneal macrophages were isolated from normal Sprague Dawley rats, and primed with lipopolysaccharide or interferon-γ. Genome-wide RNA sequencing was performed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used for gene functional annotations. Enrichment analysis was conducted using the ClusterProfiler package of R software. We found that CX-5461 principally induced a molecular signature related to cell cycle inhibition in primed macrophages, featuring downregulation of genes encoding cell cycle mediators and concomitant upregulation of cell cycle inhibitors. At the same concentration, however, CX-5461 did not induce a systemic anti-inflammatory transcriptional program, although some inflammatory genes such as IL-1β and gp91phox NADPH oxidase were downregulated by CX-5461. Our data further highlighted a central role of p53 in orchestrating the molecular networks that were responsive to CX-5461 treatment. In conclusion, our study suggested that limiting cell proliferation predominated in the inhibitory effects of CX-5461 on macrophage-mediated inflammation.

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.