lncRNA JPX-Enriched Chromatin Microenvironment Mediates Vascular Smooth Muscle Cell Senescence and Promotes Atherosclerosis.

BACKGROUND: Senescence is a series of degenerative changes in the structure and physiological function of an organism. Whether JPX (just proximal to XIST)-a newly identified age-related noncoding RNA by us-is associated with atherosclerosis is still unknown. Our study was to investigate the role of JPX and provide insights into potential therapies targeting atherosclerosis. METHODS: We analyzed clinical data from multiple tissues including meniscus tissue, leukemia cells, and peripheral blood monocytes to identify age-related noncoding RNAs in senescent vascular smooth muscle cells (VSMCs). The molecular mechanism of JPX was investigated by capture hybridization analysis of RNA targets and chromatin immunoprecipitation. IGVTools and real-time quantitative polymerase chain reaction were used to evaluate the JPX expression during phenotype regulation in age-related disease models. The therapeutic potential of JPX was evaluated after establishing an atherosclerosis model in smooth muscle-specific Jpx knockout mice. RESULTS: JPX expression was upregulated in activated ras allele (H-rasV12)-induced senescent VSMCs and atherosclerotic arteries. JPX knockdown substantially reduced the elevation of senescence-associated secretory phenotype (SASP) genes in senescent VSMCs. Cytoplasmic DNA leaked from mitochondria via mitochondrial permeability transition pore formed by VDAC1 (voltage-dependent anion channel 1) oligomer activates the STING (stimulator of interferon gene) pathway. JPX could act as an enhancer for the SASP genes and functions as a scaffold molecule through interacting with phosphorylated p65/RelA and BRD4 (bromodomain-containing protein 4) in chromatin remodeling complex, promoting the transcription of SASP genes via epigenetic regulation. Smooth muscle knockout of Jpx in ApoeKO mice resulted in a decrease in plaque area, a reduction in SASP gene expression, and a decrease in senescence compared with controls. CONCLUSIONS: As an enhancer RNA, JPX can integrate p65 and BRD4 to form a chromatin remodeling complex, activating SASP gene transcription and promoting cellular senescence. These findings suggest that JPX is a potential therapeutic target for the treatment of age-related atherosclerosis.

Divergent total synthesis of the revised structures of marine anti-cancer meroterpenoids (+)-dysiherbols A-E.

We report here a concise and divergent enantioselective total synthesis of the revised structures of marine anti-cancer sesquiterpene hydroquinone meroterpenoids (+)-dysiherbols A-E (6-10) using dimethyl predysiherbol 14 as a key common intermediate. Two different improved syntheses of dimethyl predysiherbol 14 were elaborated, one starting from Wieland-Miescher ketone derivative 21, which is regio- and diastereoselectively α-benzylated prior to establishing the 6/6/5/6-fused tetracyclic core structure through intramolecular Heck reaction. The second approach exploits an enantioselective 1,4-addition and a Au-catalyzed double cyclization to build-up the core ring system. (+)-Dysiherbol A (6) was prepared from dimethyl predysiherbol 14via direct cyclization, while (+)-dysiherbol E (10) was synthesized through allylic oxidation and subsequent cyclization of 14. Epoxidation of 14 afforded allylic alcohol 45 or unexpectedly rearranged homoallylic alcohol 44. By inverting the configuration of the hydroxy groups, exploiting a reversible 1,2-methyl shift and selectively trapping one of the intermediate carbenium ions through oxy-cyclization, we succeeded to complete the total synthesis of (+)-dysiherbols B-D (7-9). The total synthesis of (+)-dysiherbols A-E (6-10) was accomplished in a divergent manner starting from dimethyl predysiherbol 14, which led to the revision of their originally proposed structures.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB-P300-BRD4 axis.

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)-P300-bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid-liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

Melatonin antagonizes ovarian aging via YTHDF2-MAPK-NF-κB pathway.

Cellular senescence is closely associated with age-related diseases. Ovarian aging, a special type of organ senescence, is the pathophysiological foundation of the diseases of the reproductive system. It is characterized by the loss of integrity of the surface epithelium and a gradual decrease in the number of human ovarian surface epithelial cells (HOSEpiCs). To contribute to the research on delaying ovarian aging, we aimed to investigate the novel epigenetic mechanism of melatonin in protecting HOSEpiCs. We discovered that melatonin has antagonistic effects against the oncogene-induced senescence (OIS) of HOSEpiCs. Mechanistically, the oncogene Ras decreased the expression of YTHDF2, which is the reader of RNA-m6A, by stimulating the generation of reactive oxygen species (ROS). Moreover, we found that the suppression of YTHDF2 increased the expression of MAP2K4 and MAP4K4 by enhancing the stability of the transcription of their mRNAs, thereby upregulating the expression of the senescence-associated secretory phenotype (SASP) through the activation of the MAP2K4 and MAP4K4-dependent nuclear factor-κB (NF-κB) signaling pathways. We further determined that melatonin has antagonistic effects against the OIS of HOSEpiCs by inhibiting the ROS-YTHDF2-MAPK-NF-κB pathway. These findings provide key insights into the potential avenues for preventing and treating ovarian aging.

Total Synthesis of Anti-Cancer Meroterpenoids Dysideanone†B and Dysiherbol†A and Structural Reassignment of Dysiherbol†A.

The first total synthesis of marine anti-cancer meroterpenoids dysideanone B and dysiherbol A have been accomplished in a divergent way. The synthetic route features: 1) a site and stereoselective α-position alkylation of a Wieland-Miescher ketone derivative with a bulky benzyl bromide to join the terpene and aromatic moieties together and set the stage for subsequent cyclization reactions; 2) an intramolecular radical cyclization to construct the 6/6/6/6-tetracycle of dysideanone B and an intramolecular Heck reaction to forge the 6/6/5/6-fused core structure of dysiherbol A. A late-stage introduction of the ethoxy group in dysideanone B reveals that this group might come from the solvent ethanol. The structure of dysiherbol A has been revised based on our chemical total synthesis.

PM2.5 promotes plaque vulnerability at different stages of atherosclerosis and the formation of foam cells via TLR4/MyD88/NFκB pathway.

Clinical evidence has shown an elevated myocardial infarction (MI) risk after PM2.5 (particulate matter < 2.5 µm) exposure. Incident MI may result from rupture of vulnerable plaques. To test whether PM2.5 could promote plaque vulnerability, we exposed PM2.5 to apoe-/- mice by intranasal instillation. We detected the lipid, collagen, macrophage and smooth muscle cells (SMCs) content, and fibrous cap thickness to evaluate the plaque vulnerability. Plaques in HFD-fed mice with PM2.5 treatment for 24 weeks had increased lipid content and macrophage recruitment, and reduced collagen content, fibrous cap thickness and SMCs infiltration. Besides, 4-week exposure to PM2.5 could reduce the fibrous cap thickness, collagen content, but increase the macrophage infiltration and SMCs loss in a rapid atherosclerosis model. In existing plaques, PM2.5 could also decrease the fibrous cap thickness, collagen content. In RAW264.7, PM2.5 could promote the transformation of macrophage into foam cells. The expression of TLR4/MyD88/NFκB and CD36 were upregulated by PM2.5 treatment. Besides, the expression of CD36 promoted by PM2.5 was downregulated by the TLR4 inhibitor or MyD88/NFκB SiRNA. In conclusion, our data indicated that short- and long-term PM2.5 exposure increased plaque vulnerability. The underlying mechanism might be the PM2.5-enhanced formation of foam cells via TLR4/MyD88/NFκB pathway.

lncRNA HOTAIR Protects Myocardial Infarction Rat by Sponging miR-519d-3p.

Myocardial infarction (MI) is a cardiovascular disease with high morbidity and mortality. In this study, we focused on exploring the roles and underlying regulatory mechanisms of Hox transcript antisense intergenic RNA (HOTAIR) and miR-519d-3p in myocardial infarction. To comprehensively understand the role of microRNA in MI rat, we construct MI rat model by permanent ligation of the left anterior descending (LAD) coronary artery. Cardiac troponin I and creatine kinase-MB concentration measured by ELISA and infract size of heart section analyzed by TTC staining were served as evaluation indicators to confirmed the established model. Based on the bioinformatics assay and qRT-PCR, we found that the expression of miR-519d-3p was upregulated remarkably. Dual-luciferase reporter assays were performed to investigate the interaction of lncRNA HOTAIR and miR-519d-3p. In order to investigate the potential mechanism of lncRNA HOTAIR and miR-519d-3p, flow cytometry was applied to measure apoptotic cardiomyocytes and western blot was used to detect expressions of apoptotic related protein Bax, Bcl-2, and caspase-3 in cardiomyocytes in vitro and myocardial infraction in vivo. Downregulating miR-519d-3p or overexpressing HOTAIR alleviated MI or hypoxia-induced cardiomyocytes apoptosis. Taken together, our results showed that the interaction of miR-519d-3p and HOTAIR can protect MI and hypoxia-induced cardiomyocytes apoptosis, providing the potential therapeutic target for MI treatment.

Polymorphism in the promoter region of lncRNA GAS5 is functionally associated with the risk of gastric cancer.

BACKGROUND AND AIMS: Previous studies showed that down-regulation of GAS5 was involved in the development of gastric cancer (GC). However, the regulatory mechanism of down-expressed GAS5 in GC remains obscure. We aimed to investigate the role of rs145204276 of GAS5 in the development and metastasis process of GC. METHODS: 853 GC patients and 954 healthy controls were recruited. The variant rs145204276 was genotyped and the Chi2 test was used to compare the frequency of the genotype and the allele between the patients and the controls. Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated to estimate the association of rs145204276 with the risk of development and metastasis of GC. RESULTS: Patients were found to have significantly lower rate of genotype del/del than the controls (7.2% vs. 8.9%, P=0.016). The allele del was significantly associated with a decreased risk of GC (26.4% vs. 30.7%, P=0.005) with an OR of 0.81 (95% CI=0.70-0.94). Patients with allele del were less likely to develop lymph node metastasis (P=0.01), with an OR of 0.75 (95% CI=0.60-0.93). Comparably, rs145204276 was also significantly associated with a decreased risk of distant metastasis of GC (P=0.007; OR=0.55). CONCLUSION: We confirmed that rs145204276 of GAS5 is a functional variant associated with the susceptibility and metastasis of GC. It plays a protective role in the development of GC possibly through the regulation of GAS5.

Restoration of motor function after operative reconstruction of the acutely transected spinal cord in the canine model.

BACKGROUND: Cephalosomatic anastomosis or what has been called a "head transplantation" requires full reconnection of the respective transected ends of the spinal cords. The GEMINI spinal cord fusion protocol has been developed for this reason. Here, we report the first randomized, controlled study of the GEMINI protocol in large animals. METHODS: We conducted a randomized, controlled study of a complete transection of the spinal cord at the level of T10 in dogs at Harbin Medical University, Harbin, China. These dogs were followed for up to 8 weeks postoperatively by assessments of recovery of motor function, somato-sensory evoked potentials, and diffusion tensor imaging using magnetic resonance imaging. RESULTS: A total of 12 dogs were subjected to operative exposure of the dorsal aspect of the spinal cord after laminectomy and longitudinal durotomy followed by a very sharp, controlled, full-thickness, complete transection of the spinal cord at T10. The fusogen, polyethylene glycol, was applied topically to the site of the spinal cord transection in 7 of 12 dogs; 0.9% NaCl saline was applied to the site of transection in the remaining 5 control dogs. Dogs were selected randomly to receive polyethylene glycol or saline. All polyethylene glycol-treated dogs reacquired a substantial amount of motor function versus none in controls over these first 2 months as assessed on the 20-point (0-19), canine, Basso-Beattie-Bresnahan rating scale (P<.006). Somatosensory evoked potentials confirmed restoration of electrical conduction cranially across the site of spinal cord transection which improved over time. Diffusion tensor imaging, a magnetic resonance permutation that assesses the integrity of nerve fibers and cells, showed restitution of the transected spinal cord with polyethylene glycol treatment (at-injury level difference: P<.02). CONCLUSION: A sharply and fully transected spinal cord at the level of T10 can be reconstructed with restoration of many aspects of electrical continuity in large animals following the GEMINI spinal cord fusion protocol, with objective evidence of motor recovery and of electrical continuity across the site of transection, opening the way to the first cephalosomatic anastomosis. (Surgery 2017;160:XXX-XXX.).

Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway.

BACKGROUND: Many studies have identified trimethylamine N-oxide (TMAO) as a new risk factor of cardiovascular diseases. It has been suggested that TMAO promotes atherosclerosis development. However, the underlying mechanism is still unclear. METHODS: Apoe-/- mice were fed a high-fat diet and given water with or without TMAO for 8-week. Histological and immunohistological analyses were used to evaluate the atherogenic effect of TMAO in vivo. We also employed peritoneal elicited macrophages and RAW264.7 to assess the role of MAPK/JNK pathway in TMAO-induced formation of foam cells. RESULTS: TMAO significantly promoted plaque progression in apoe-/- mice fed with high-fat diet for 8 weeks. Besides, macrophage recruitment, CD36 and proinflammatory cytokine expressions were enhanced by TMAO in plaque lesions. In vitro, TMAO increased the macrophage migration and the expression of TNF-α, IL-6 and ICAM1. In addition, CD36 expression and foam cell formation induced by ox-LDL were also enhanced by TMAO, which could be attenuated by siRNA-mediated knockdown of CD36. We additionally used MAPK inhibitor (SB230580) and JNK inhibitor (SP600125) to assess the MAPK/JNK pathway in TMAO-induced CD36 expression. Western blotting showed that both SB230580 and SP600125 could reduce the expression of CD36 induced by ox-LDL and TMAO. Moreover, SB230580 and SP600125 could also reduce the formation of foam cells. CONCLUSIONS: TMAO promotes the atherosclerosis in vivo and in vitro.CD36/MAPK/JNK pathway may play a crucial role in TMAO-induced formation of foam cells.

Premature senescence of cardiac fibroblasts and atrial fibrosis in patients with atrial fibrillation.

Premature senescence is associated with atrial fibrosis and has an antifibrotic effect in mice. However, the role of senescence in atrial fibrillation (AF) remains unclear. Here, we investigated the association of premature senescence with fibrosis and also determined the role of senescence in the recurrence of AF after surgery ablation. Western blot, Sirius red staining, SA-ß-gal staining and immunohistochemistry were performed to detect the degree of atrial fibrosis ,the expression of TGF-ß and collagens, and also the senescence markers in 72 tissue specimens of left atrial appendage in this study. Then the patients undergoing successful surgical ablation were followed up for 12 months. The expression of collagens and TGF-ß was paralleled by a high level of atrial fibrosis and were increased in AF group, especially in the persistent AF group. Western blotting of P16 and SA-ß-gal staining showed an increased premature senescence in the sinus rhythm, paroxysmal AF and persistent AF groups. In addition, positive area of senescence markers, SA-ß-gal and P16, was correlated positively with fibrotic lesions. We also found a lower ratio of P16/TGF-ß in patients with recurrence of AF than in patients without recurrent AF. In conclusion, premature senescence is associated with atrial fibrosis in AF, and may have an antifibrotic role in AF.

Increased M1 Macrophages Infiltration Is Associated with Thrombogenesis in Rheumatic Mitral Stenosis Patients with Atrial Fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia. In patients with AF, the role of macrophage subsets in thrombogenesis is unclear. In the present study, we analyzed the role of M1 and M2 macrophages and related cytokines in thrombogenesis of AF. Immunohistochemistry, Western blot, and TUNEL assay were used to detect M1/M2 macrophage infiltration, the expression pattern of IL-1ß and inflammasome components, and apoptosis of cardiomyocytes in 71 specimens obtained from the left atrial appendage of patients with rheumatic mitral stenosis (MS) with or without thrombosis. We demonstrated that proinflammatory M1 macrophages were predominant in the atrium of MS patients with AF and thrombus. NLRP3 inflammasomes and IL-1ß, which are primarily functional in macrophages, were activated in those patients. We also showed that increased cell death was associated with thrombogenesis in MS patients. These data indicate that infiltration of M1 macrophages and over-activation of NLRP3 inflammasomes may play a role in progressive atrial inflammation and thrombogenesis in rheumatic mitral stenosis patients with AF.

Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy.

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.

[Overexpression of integrin-linked kinase improves cardiac function in a rat model of doxorubicin-induced chronic heart failure].

OBJECTIVE: The aim of this study is to investigate the effects of cardiac integrin-linked kinase (ILK) overexpression in a rat model of doxorubicin-induced heart failure and the underlying mechanisms. METHODS: Rat heart failure model was induced by intraperitoneal administration of doxorubicin (6 injections within 2 weeks: total dose = 15 mg/kg). Five weeks after the first injection, rats with heart failure were confirmed by echocardiography and then randomly divided into Ad-ILK group (intra-myocardial injected with adenoviral vector expressing ILK) and Ad-null group (intra-myocardial injected with empty ad-null, n = 20 each). After 4 weeks, ILK expression and activity were detected by Western blot, cardiac function was determined by echocardiographic and hemodynamic examinations. Apoptosis was measured by TUNEL analysis and cardiomyocyte proliferation was estimated by phospho-histone-H3 staining. RESULTS: Western blot analysis revealed higher expression of ILK as well as the phosphorylation levels of Akt in Ad-ILK hearts as compared with ad-null controls. Four weeks after transfection, LVEF and LVFS were significantly higher in Ad-ILK group as compared with control group [LVEF: (60.56 ± 2.61)% vs. (51.94 ± 2.28)%, P < 0.05; LVFS: (28.10 ± 1.83)% vs. (22.82 ± 1.68)%, P < 0.05]. The LVEDD and LVESD, as well as LVEDP were significantly lower in Ad-ILK group compared with control group [LVEDD: (6.22 ± 0.24) mm vs. (7.15 ± 0.21) mm, P < 0.05; LVESD: (4.42 ± 0.23) mm vs. (5.65 ± 0.25) mm, P < 0.05; LVEDP: (12.96 ± 2.10) mmHg vs. (21.45 ± 2.48) mmHg (1 mmHg = 0.133 kPa) , P < 0.05]. Reduced levels of serum BNP was also seen in the Ad-ILK group. TUNEL analysis showed that ILK treatment significantly inhibited the apoptosis of cardiomyocytes [(0.23 ± 0.02)% vs. (0.45 ± 0.04)%, P < 0.05]. Moreover, increased cardiomyocyte proliferation was found in Ad-ILK group through the phospho-histone H3 staining [(0.60 ± 0.07)% vs. (0.24 ± 0.03)%, P < 0.01]. CONCLUSION: ILK gene therapy improves cardiac function in this rat model of heart failure, and is associated with reduced apoptosis and increased cardiomyocyte proliferation.

Properties and coagulation performance of coagulant poly-aluminum-ferric-silicate-chloride in water and wastewater treatment.

An efficient inorganic polymer coagulant, poly-aluminum-ferric-silicate-chloride (PAFSC), was developed using two approaches: (i) hydroxylation of the mixture of AlCl3, FeCl3 and fresh polysilicic acid in different Al/Fe/Si molar ratios to obtain PAFSCc and (ii) hydroxylated poly-aluminum-iron-chloride (PAFC) combined with aged polysilicic acid in different Al/Fe/Si ratios to produce PAFSCm. The properties of PAFSC in comparison with polyaluminum silicate chloride (PASC) and polyferric silicate chloride (PAFC) were characterized by various experimental methods. The effect of Al/Fe/Si molar ratio on the hydrolysis-polymerization process of Al (III) and Fe (III) in PAFSC solutions was examined by pH titration, and the effect of Al/Fe/Si molar ratio on electrokinetic mobility of PAFSC was studied by Zeta potential measurement. The laboratory experiments were performed to evaluate the PAFSC in comparison with polyaluminum chloride (PAC) for the coagulation of synthetic water samples, actual surface water and wastewater. The results show that interactions exist among aluminum species, ferric species and polysilicic acid, and the Al/Fe/Si molar ratio affects the Zeta potential of the hydrolyzate and the coagulating performance. PAFSC achieved a better water treatment result than PAC. At the same basicity (B) value and Al/Fe/Si ratio, PAFSCc has better coagulation performance than PAFSCm. PAFSC is a new type and high efficiency composite inorganic polymer coagulant.

Electrophoretic nature and evaluation of poly-aluminum-chloride-sulfate (PACS) as a coagulant for water and wastewater treatment.

A series of poly-aluminum-chloride-sulfates (PACS), which have different OH/Al (gamma) and Al(3+)/SO4(2-) mole ratios, has been prepared using AlCl3 x 6H2O, A(SO4)3 x 18H2O and Na2CO3 as raw materials. The electrophoretic nature of PACS was investigated by electrophoresis. Laboratory experiments were undertaken to evaluate the PACS in comparison with polyaluminum chloride (PAC) for the coagulation of simulating water and actual wastewaters. The experimental results show that the gamma value and the Al(3+)/SO4(2-) mole ratio affect the electrophoretic nature of PACS. PACS has a maximum zeta potential at about a gamma value of 1.5 and Al(3+)/SO4(2-) mole ratio of 12-16. The zeta potential of PACS varies with pH. The performance of PACS as coagulant is affected by gamma value and Al(3+)/SO4(2-) mole ratio. PACS of gamma = 2.1 and Al(3+)/SO4(2-) = 15 gives best turbidity removal effectiveness. In comparison with PAC of gamma = 2.0, PACS of gamma = 2.0 and Al(3+)/SO4(2-) = 16 gives higher removal efficiency for turbidity and COD, and shows the following advantages in the clarification of waters and wastewaters: rapid aggregation velocity, larger and heavier flocs, and lower required dosage.