Impact of age-related gut microbiota dysbiosis and reduced short-chain fatty acids on the autonomic nervous system and atrial fibrillation in rats.

Objective: Aging is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). Dysbiosis of gut microbiota has been implicated in age-related diseases, but its role in AF development remains unclear. This study aimed to investigate the correlations between changes in the autonomic nervous system, short-chain fatty acids (SCFAs), and alterations in gut microbiota in aged rats with AF. Methods: Electrophysiological experiments were conducted to assess AF induction rates and heart rate variability in rats. 16S rRNA gene sequences extracted from fecal samples were used to assess the gut microbial composition. Gas and liquid chromatography-mass spectroscopy was used to identify SCFAs in fecal samples. Results: The study found that aged rats exhibited a higher incidence of AF and reduced heart rate variability compared to young rats. Omics research revealed disrupted gut microbiota in aged rats, specifically a decreased Firmicutes to Bacteroidetes ratio. Additionally, fecal SCFA levels were significantly lower in aged rats. Importantly, correlation analysis indicated a significant association between decreased SCFAs and declining heart rate variability in aged rats. Conclusions: These findings suggest that SCFAs, as metabolites of gut microbiota, may play a regulatory role in autonomic nervous function and potentially influence the onset and progression of AF in aged rats. These results provide novel insights into the involvement of SCFAs and autonomic nervous system function in the pathogenesis of AF. These results provide novel insights into the involvement of SCFAs and autonomic nervous system function in the pathogenesis of AF.

Identification and validation of pyroptosis-related genes as potential biomarkers for hypertrophic cardiomyopathy: A comprehensive bioinformatics analysis.

Pyroptosis plays a key role in the death of cells including cardiomyocytes, and it is associated with a variety of cardiovascular diseases. However, the role of pyroptosis-related genes (PRGs) in hypertrophic cardiomyopathy (HCM) is not well characterized. This study aimed to identify key biomarkers and explore the molecular mechanisms underlying the functions of the PRGs in HCM. The differentially expressed genes were identified by GEO2R, and the differentially expressed pyroptosis-related genes (DEPRGs) of HCM were identified by combining with PRGs. Enrichment analysis was performed using the "clusterProfiler" package of the R software. Protein-protein interactions (PPI) network analysis was performed using the STRING database, and hub genes were screened using cytoHubba. TF-miRNA coregulatory networks and protein-chemical interactions were analyzed using NetworkAnalyst. RT-PCR/WB was used for expression validation of HCM diagnostic markers. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western Blot (WB) were used to measure and compare the expression of the identified genes in the cardiac hypertrophy model and the control group. A total of 20 DEPRGs were identified, which primarily showed enrichment for the positive regulation of cytokine production, regulation of response to biotic stimulus, tumor necrosis factor production, and other biological processes. These processes primarily involved pathways related to Renin-angiotensin system, Adipocytokine signaling pathway and NF-kappa B signaling pathway. Then, a PPI network was constructed, and 8 hub genes were identified. After verification analysis, the finally identified HCM-related diagnostic markers were upregulated gene protein tyrosine phosphatase non-receptor type 11 (PTPN11), downregulated genes interleukin-1 receptor-associated kinase 3 (IRAK3), and annexin A2 (ANXA2). Further GSEA analysis revealed these 3 biomarkers primarily related to cardiac muscle contraction, hypertrophic cardiomyopathy, fatty acid degradation and ECM - receptor interaction. Moreover, we also elucidated the interaction network of these biomarkers with the miRNA network and known compounds, respectively. RT-PCR/WB results indicated that PTPN11 expression was significantly increased, and IRAK3 and ANXA2 expressions were significantly decreased in HCM. This study identified PTPN11, IRAK3, and ANXA2 as pyroptosis-associated biomarkers of HCM, with the potential to reveal the development and pathogenesis of HCM and could be potential therapeutic targets.

MiR-431 promotes cardiomyocyte proliferation by targeting FBXO32 expression.

BACKGROUND: The induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRNAs) have been reported to regulate CM proliferation. In particular, miR-431 expression decreases during cardiac development, according to Gene Expression Omnibus (GEO) microarray data. However, whether miR-431 regulates CM proliferation has not been thoroughly investigated. METHODS: We used integrated bioinformatics analysis of GEO datasets to identify the most significantly differentially expressed miRNAs. Real-time quantitative PCR and fluorescence in situ hybridization were performed to determine the miRNA expression patterns in hearts. Gain- and loss-of-function assays were conducted to detect the role of miRNA in CM proliferation. Additionally, we detected whether miR-431 affected CM proliferation in a myocardial infarction model. The TargetScan, miRDB and miRWalk online databases were used to predict the potential target genes of miRNAs. Luciferase reporter assays were used to study miRNA interactions with the targeting mRNA. RESULTS: First, we found a significant reduction in miR-431 levels during cardiac development. Then, by overexpression and inhibition of miR-431, we demonstrated that miR-431 promotes CM proliferation in vitro and in vivo, as determined by immunofluorescence assays of 5-ethynyl-2'-deoxyuridine (EdU), pH3, Aurora B and CM count, whereas miR-431 inhibition suppresses CM proliferation. Then, we found that miR-431 improved cardiac function post-myocardial infarction. In addition, we identified FBXO32 as a direct target gene of miR-431, with FBXO32 mRNA and protein expression being suppressed by miR-431. FBXO32 inhibited CM proliferation. Overexpression of FBXO32 blocks the enhanced effect of miR-431 on CM proliferation, suggesting that FBXO32 is a functional target of miR-431 during CM proliferation. CONCLUSION: In summary, miR-431 promotes CM proliferation by targeting FBXO32, providing a potential molecular target for preventing myocardial injury.

Trimethylamine N-oxide promotes abdominal aortic aneurysm by inducing vascular inflammation and vascular smooth muscle cell phenotypic switching.

OBJECTIVE: Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). Trimethylamine N-oxide (TMAO) has emerged as a crucial risk factor in cardiovascular diseases, inducing vascular inflammation and calcification. We aimed to evaluate the effect of TMAO on the formation of AAA. APPROACH AND RESULTS: Here, we showed that TMAO was elevated in plasma from AAA patients compared with nonaneurysmal subjects by liquid chromatography‒mass spectrometry (LC‒MS) detection. Functional studies revealed that increased TMAO induced by feeding a choline-supplemented diet promoted Ang II-induced AAA formation. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analyses revealed that TMAO induced macrophage infiltration and inflammatory factor release. Conversely, inhibition of TMAO by supplementation with DMB suppressed AAA formation and the inflammatory response. Molecular studies revealed that TMAO regulated VSMC phenotypic switching. Flow cytometry analyses showed that TMAO induces macrophage M1-type polarization. Furthermore, pharmacological intervention experiments suggested that the nuclear factor-κB (NF-κB) signaling pathway was critical for TMAO to trigger AAA formation. CONCLUSIONS: TMAO promotes AAA formation by inducing vascular inflammation and VSMC phenotypic switching through activation of the NF-κB signaling pathway. Thus, TMAO is a prospective therapeutic AAA target.

Predictive value of serum lncRNA MALAT1 for the recurrence of persistent atrial fibrillation after radiofrequency ablation.

Objective: The authors investigated the predictive value of MALAT1 for persistent atrial fibrillation (PAF) recurrence after radiofrequency ablation. Methods: Serum MALAT1 level was determined. The correlation between MALAT1 and high-sensitivity C-reactive protein/left atrial diameter (LAD) was analyzed. The predictive value of MALAT1 was evaluated. The postoperative recurrence rate in patients with high/low MALAT1 was compared. Independent risk factors for postoperative recurrence were analyzed. Results: MALAT1 was elevated in PAF patients and positively correlated with high-sensitivity C-reactive protein/LAD. MALAT1/high-sensitivity C-reactive protein/LAD were enhanced in patients with recurrent PAF. Patients with high MALAT1 had a higher recurrence rate. Upregulated MALAT1 was an independent risk factor for postoperative PAF recurrence. Conclusion: Serum MALAT1 level >2.03 predicts postoperative recurrence of PAF, and PAF patients with high MALAT1 have a higher risk of postoperative recurrence.

Atrial fibrillation (AF) is a common cardiac arrhythmia (abnormal heartbeat), which usually manifests as an irregular and rapid rhythm. In severe cases, AF can lead to cardiovascular diseases such as thromboembolism (narrowing and blockage of blood vessels) and heart failure (impaired pumping function of the heart). Cardiac radiofrequency ablation is a common method used to treat AF. However, patients with PAF still have a high rate of late recurrence after the operation, so there is an urgent need to identify suitable biochemical markers for predicting the postoperative recurrence of PAF. lncRNAs are a type of noncoding nucleic acid; they do not encode proteins, have various biological functions and are widely distributed in living organisms. The lncRNA MALAT1 has been considered a potential therapeutic target and biomarker in several cardiovascular diseases. This study demonstrated that the serum level of MALAT1 in PAF patients was significantly higher than that in normal subjects and MALAT1 level was elevated in patients with recurrent PAF compared with patients without recurrence. The authors also found that serum MALAT1 could predict whether PAF will recur after operation, with a high accuracy. In addition, PAF patients with high expression of serum MALAT1 had a higher risk of postoperative recurrence. In summary, serum level of lncRNA MALAT1 can help predict postoperative recurrence of PAF and a high level of MALAT1 is indicative of a higher risk of postoperative recurrence. Analysis of serum lncRNA MALAT1 level in PAF patients before surgery can predict postoperative recurrence, and intervention programs that lower the MALAT1 level can be implemented to reduce the risk of postoperative recurrence of PAF and increase the success rate of the operation. This study has important implications for reducing the recurrence rate after radiofrequency ablation in PAF patients.

Erratum: Therapeutic ultrasound combined with microbubbles improves atherosclerotic plaque stability by selectively destroying the intraplaque neovasculature: Erratum.

[This corrects the article DOI: 10.7150/thno.39553.].

Protective activity of Malus doumeri leaf extract on H2O2-induced oxidative injury in H9C2 rat cardiomyocytes.

In this study, Malus doumeri leaf extract (MDLE) was used to test its anti-oxidation capacity in vitro, it has been preliminarily analyzed for H2O2-induced oxidative damage in H9C2 cells and its main active components. The antioxidant capacity through DPPH (1, 1-Diphenyl-2-Picrylhydrazyl), ABTS+• [2,2,2'-azino-BIS-(3-ethylbenzo-thiazoline-6-sulfonic acid)] radical ion, •OH (hydroxyl radical), and • O 2 - (superoxide anion) were determined in vitro. The proliferation of H9C2 cells was examined by MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-Tetrazolium bromide]. MDA (malondialdehyde), SOD (superoxide dismutase), CAT (catalase), GSH (glutathione), and GSH-Px (glutathione peroxidase) were determined by colorimetry. Apoptosis induced by oxidative damage was detected by flow cytometry. The mRNA expression of antioxidant related genes of SOD, CAT, GSH, and GSH-Px were checked by qRT-PCR (quantitative real-time polymerase chain reaction). The MDLE main active components were analyzed by HPLC (high-performance liquid chromatography). MDLE had significant scavenging effects on DPPH, ABTS+•, •OH, and superoxide anion radicals in a concentration-dependent manner. H2O2 treatment could significantly lead to oxidative stress injury of H9C2 cells, and MDLE treatment significantly improved the degree of H9C2 cell damage, and showed a positive correlation with concentration. MDLE can also reduce apoptosis caused by oxidative damage. MDLE treatment could significantly reduce MDA content and increase CAT, SOD, GSH, and GSH-Px contents and expression. In addition, by HPLC analysis, the following six bioactive components were detected from MDLE: chlorogenic acid, isoquercitrin, quercetin, baicalin, and phloretin. Therefore, MDLE has a good protective effect on myocardial cells.

CircRNA Samd4 induces cardiac repair after myocardial infarction by blocking mitochondria-derived ROS output.

Reactive oxygen species (ROS) derived from oxygen-dependent mitochondrial metabolism are the essential drivers of cardiomyocyte (CM) cell-cycle arrest in adulthood. Mitochondria-localized circular RNAs (circRNAs) play important roles in regulating mitochondria-derived ROS production, but their functions in cardiac regeneration are still unknown. Herein, we investigated the functions and underlying mechanism of mitochondria-localized circSamd4 in cardiac regeneration. We found that circSamd4 was selectively expressed in fetal and neonatal CMs. The transcription factor Nrf2 controlled circSamd4 expression by binding to the promoter of circSamd4 host gene. CircSamd4 overexpression reduced while circSamd4 silenced increased mitochondrial oxidative stress and subsequent oxidative DNA damage. Moreover, circSamd4 overexpression induced CM proliferation and prevented CM apoptosis, which reduced the size of the fibrotic area and improved cardiac function after myocardial infarction (MI). Mechanistically, circSamd4 reduced oxidative stress generation and maintained mitochondrial dynamics by inducing the mitochondrial translocation of the Vcp protein, which downregulated Vdac1 expression and prevented the mitochondrial permeability transition pore (mPTP) from opening. Our findings suggest that circSamd4 is a novel therapeutic target for heart failure after MI.

3-iodothyronamine inhibits apoptosis induced by myocardial ischemia reperfusion via the Akt/FoxO1 signaling pathway.

Background: This study investigated the potential effects of 3-iodothyronamine (T1AM) on myocardial ischemia reperfusion injury (MIRI) and the underlying molecular mechanisms. Methods: A total of 16 adult male Sprague-Dawley rats were randomly divided into 4 groups and administered the following: control [60% dimethyl sulfoxide (DMSO) and 40% saline, pH 7.4], T1AM (25 mg/kg), T1AM (50 mg/kg), or T1AM (100 mg/kg). The rectal temperatures of the rats were measured at different time points. A further 30 adult male Sprague-Dawley rats were randomized and divided into the following 3 groups (n=10 in each group): sham operation, ischemia/reperfusion (I/R), and I/R + T1AM. In the I/R and I/R + T1AM groups, the left anterior descending (LAD) coronary artery of the rats were occluded for 0.5 hour to induce myocardial ischemia, followed by reperfusion for 3 hours in the I/R group. The electrocardiography (ECG), cardiac function, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were examined in rats to evaluate the myocardial injury. The differences in the expression of apoptosis-related and Akt-FoxO1 signaling-related proteins were determined via Western blot. Results: This work verified that T1AM reduced the body temperature of rats in a dose-dependent manner. Additionally, T1AM improved cardiac function and decreased the infarction size caused by MIRI. T1AM reduced the expression of biochemical parameters and apoptosis of myocardial cells. In addition, after treatment with T1AM, the expression of Glut1, pFoxO1 and Akt were reduced, while the expression of FoxO1 and PPARα were increased significantly. Conclusions: Pretreatment of cardiomyocytes with T1AM inhibited apoptosis and protected against ischemia reperfusion injury via the Akt/FoxO1 signaling pathway.

Vitamin D receptor gene polymorphism predicts left ventricular hypertrophy in maintenance hemodialysis.

BACKGROUND: To verify that the single nucleotide polymorphisms (SNP) of vitamin D receptor (VDR) may lead to genetic susceptibility to left ventricular hypertrophy (LVH), the present study was designed to study four SNPs of VDR associated with LVH in maintenance hemodialysis (MHD) patients of Han nationality. METHODS: 120 MHD patients were recruited at Department of Nephrology, Zhongnan Hospital of Wuhan University to analyze the expression of genotype, allele and haplotype of Fok I, Bsm I, Apa I and Taq I in blood samples, and to explore their correlation with blood biochemical indexes and ventricular remodeling. RESULTS: The results showed that the risks of CVD included gender, dialysis time, heart rate, SBP, glycated hemoglobin, calcium, iPTH and CRP concentration. Moreover, LAD, LVDd, LVDs, IVST and LVMI in B allele of Bsm I increased significantly. Fok I, Apa I and Taq I polymorphisms have no significant difference between MHD with LVH and without LVH. Further study showed that VDR expression level decreased significantly in MHD patients with LVH, and the B allele was positively correlated with VDR Expression. CONCLUSION: VDR Bsm I gene polymorphism may predict cardiovascular disease risk of MDH patients, and provided theoretical basis for early detection and prevention of cardiovascular complications.

MicroRNA-23b prevents aortic aneurysm formation by inhibiting smooth muscle cell phenotypic switching via FoxO4 suppression.

AIMS: Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for the formation of abdominal aortic aneurysms (AAAs). MicroRNA-23b (miR-23b) has recently been shown to play a vital role in maintaining the VSMC contractile phenotype; however, little is known about the role of miR-23b in the formation of AAAs. Here, we investigated whether miR-23b prevents AAA formation by inhibiting VSMC phenotypic switching. MATERIALS AND METHODS: We administered angiotensin II (Ang II, 1000 ng/kg/min) or vehicle to 10-12-week-old male apolipoprotein E knockout (ApoE-/-) or C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks. KEY FINDINGS: The expression of miR-23b was significantly reduced in the aorta during the early onset of AAA in angiotensin II-treated ApoE-/- mice and in human AAA samples. In vitro experiments showed that the suppression of SMC contractile marker gene expression induced by Ang II was accelerated by miR-23b inhibitors but inhibited by mimics. In vivo studies revealed that miR-23b deficiency in Ang II-treated C57BL/6J mice aggravated the formation of AAAs in these mice compared with control mice; the opposite results were observed in miR-23b-overexpressing mice. Mechanistically, miR-23b knockdown significantly increased the expression of the transcription factor forkhead box O4 (FoxO4) during VSMC phenotypic switching induced by Ang II. In addition, a luciferase reporter assay showed that FoxO4 is a target of miR-23b in VSMCs. SIGNIFICANCE: Our study revealed a pivotal role for miR-23b in protecting against aortic aneurysm formation by maintaining the VSMC contractile phenotype.

Hyperuricemia and hypertriglyceridemia indicate tubular atrophy/interstitial fibrosis in patients with IgA nephropathy and membranous nephropathy.

PURPOSE: Hyperuricemia (HUA) and hypertriglyceridemia (HTG) were very common in chronic kidney disease (CKD) and associated with accelerated progression of CKD. This was a retrospective, cross-sectional study which aimed to explore the relationship between serum uric acid levels or triglyceride levels and tubular atrophy/interstitial fibrosis (proven by renal biopsy). METHODS: The present study enrolled 229 CKD individuals who included 127 biopsy-proven primary IgA nephrology (IgAN) patients and 102 biopsy-proven primary membranous nephropathy (MN) patients. The baseline characteristics at the time of the kidney biopsy were collected. According to the serum uric acid (UA) or triglyceride (TG) whether it exceeds the normal reference range, patients were divided into non-HUA (n = 127), HUA (n = 102), non-HTG (n = 119), and HTG group (n = 110). Based on the extent of tubular atrophy/interstitial fibrosis, patients were divided into no/mild injury (T0, n = 127), moderate injury (T1, n = 102). Multivariable logistic regression for factors predicting moderate tubular atrophy/interstitial fibrosis was performed. RESULTS: There were 127 IgAN and 102 MN cases among 229 patients in the present study. The prevalence of HUA was 44.5% (n = 102), 40.9% (n = 52), and 49.0% (n = 50) in all patients, IgAN patients and MN patients, respectively (P = 1.49). The prevalence of HTG was 48.0% (n = 110), 29.9% (n = 38), and 70.6% (n = 72) (P < 0.001), respectively, as well. Multivariate logistic regression analysis showed that HUA and HTG were independent risk factors for moderate tubular atrophy/interstitial fibrosis (HUA OR = 2.335, 95% CI = 1.147-4.755, P = 0.019; HTG OR = 2.646, 95% CI = 1.289-5.432, P = 0.008). The area under curve (AUC) of model 1 (HUA + eGFR + HTG + age + serum globulin + 24 h urinary protein, AUC = 0.876) was larger than the other two models; however, there was no significant difference among these models (all P > 0.05). CONCLUSIONS: Hyperuricemia and hypertriglyceridemia, which were prevalent in CKD patients, were the independent risk factors for moderate tubular atrophy/interstitial fibrosis. HUA together with HTG could improve the value of diagnosis for moderate tubular atrophy/interstitial fibrosis to some extent.

miR­449a­5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR­449a­5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR­449a­5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR­449a­5p modulates CM proliferation and to identify the molecular mechanism via which miR­449a­5p regulates CM proliferation. The current study demonstrated that miR­449a­5p expression levels were significantly increased during heart development. Furthermore, the results suggested that miR­449a­5p mimic inhibited CM proliferation in vitro as determined via immunofluorescence for ki67 and histone H3 phosphorylated at serine 10 (pH3), as well as the numbers of CMs. However, miR­449a­5p knockdown promoted CM proliferation. CDK6 was identified as a direct target gene of miR­449a­5p, and CDK6 mRNA and protein expression was suppressed by miR­449a­5p. Moreover, CDK6 gain­of­function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR­449a­5p on CM proliferation, indicating that CDK6 was a functional target of miR­449a­5p in CM proliferation. In conclusion, miR­449a­5p inhibited CM proliferation by targeting CDK6, which provides a potential molecular target for preventing myocardial injury.

circRNA Hipk3 Induces Cardiac Regeneration after Myocardial Infarction in Mice by Binding to Notch1 and miR-133a.

The synergism between cardiomyogenesis and angiogenesis is essential for cardiac regeneration. Circular RNAs (circRNAs) play pivotal roles in cell growth and angiogenesis, but their functions in cardiac regeneration are not yet known. In this study, we investigated the role and underlying mechanisms of circRNA Hipk3 (circHipk3) in both cardiomyogenesis and angiogenesis during cardiac regeneration. We found that circHipk3 was overexpressed in the fetal or neonatal heart of mice. The transcription factor Gata4 bound to the circHipk3 promoter and increased circHipk3 expression. Cardiomyocyte (CM) proliferation in vitro and in vivo was inhibited by circHipk3 knockdown and increased by circHipk3 overexpression. Moreover, circHipk3 overexpression promoted coronary vessel endothelial cell proliferation, migration, and tube-forming capacity and subsequent angiogenesis. More importantly, circHipk3 overexpression attenuated cardiac dysfunction and decreased fibrotic area after myocardial infarction (MI). Mechanistically, circHipk3 promoted CM proliferation by increasing Notch1 intracellular domain (N1ICD) acetylation, thereby increasing N1ICD stability and preventing its degradation. In addition, circHipk3 acted as a sponge for microRNA (miR)-133a to promote connective tissue growth factor (CTGF) expression, which activated endothelial cells. Our findings suggested that circHipk3 might be a novel therapeutic target for preventing heart failure post-MI.

METTL3 Induces AAA Development and Progression by Modulating N6-Methyladenosine-Dependent Primary miR34a Processing.

Identifying effective drugs to delay the progression of aortic aneurysms is a formidable challenge in vascular medicine. Methyltransferase-like 3 (METTL3) plays a key role in catalyzing the formation of N6-methyladenosine (m6A), but despite the functional importance of METTL3 and m6A in various fundamental biological processes, their roles in abdominal aortic aneurysm (AAA) are unknown. Here, we found that METTL3 knockdown in apolipoprotein E-deficient (ApoE-/-) mice treated with angiotensin II suppressed the formation of AAAs, while METTL3 overexpression exerted the opposite effects. Similar results were obtained in a calcium chloride (CaCl2)-induced mouse AAA model. Mechanistically, METTL3-dependent m6A methylation promoted primary microRNA-34a (miR-34a, pri-miR34a) maturation through DGCR8. Moreover, miR-34a overexpression significantly decreased SIRT1 expression and aggravated AAA formation, while miR-34a deficiency produced the opposite effects. In a rescue experiment, miR-34a knockdown or forced expression of SIRT1 partially attenuated the protective effects of METTL3 deficiency against AAA formation. Our studies reveal an important role for METTL3/m6A-mediated miR-34a maturation in AAA formation and provide a novel therapeutic target and diagnostic biomarker for AAA treatment.

Therapeutic ultrasound combined with microbubbles improves atherosclerotic plaque stability by selectively destroying the intraplaque neovasculature.

Objective: The current antiangiogenic therapy for atherosclerotic plaques was mainly achieved by the use of antiangiogenic drugs, but serious side effects have limited the clinical application. The present study investigated whether therapeutic ultrasound (TUS) treatment with appropriate pressure could selectively deplete the neovasculature in vulnerable plaques to improve its stability with no side effects on the body; the underlying mechanisms were also explored. Methods and Results: A mouse model of advanced atherosclerosis was generated by maintaining apolipoprotein E-deficient (ApoE-/-) mice on a hypercholesterolemic diet (HCD). Plaque, skeletal muscle, mesentery and skin tissue from 114 atheroma-bearing mice were subjected to sham therapy, an ultrasound application combined with microbubbles at four different ultrasound pressures (1.0, 2.0, 3.0, 5.0 MPa), or ultrasound at 5.0 MPa alone. Microvessel density (MVD) was assessed by immunofluorescence and immunohistochemical methods. The plaque necrotic center/fiber cap (NC/FC) ratio and vulnerability index were calculated to evaluate plaque vulnerability. Twenty-four hours after TUS treatment at 3.0 MPa, the MVD in the plaque was substantially decreased by 84% (p < 0.05), while there was almost no change in MVD and neovessel density (NVD) in normal tissues, including skeletal muscle, mesentery and skin. Additionally, a marked reduction in the number of immature vessels was observed in the plaques (reduced by 90%, p < 0.05), whereas the number of mature vessels was not significantly decreased. Furthermore, TUS treatment at 3.0 MPa significantly improved plaque stability, as reflected by the NC/FC ratio and vulnerability index, which may be due to the selective destruction of intraplaque neovascularization by TUS treatment, thereby decreasing the extravasation of erythrocytes and leading to vascular inflammation alleviation and thin-cap fibroatheroma reduction. Conclusions: TUS treatment at 3.0 MPa selectively depleted plaque neovessels and improved the stability of vulnerable plaques through a reduction in erythrocyte extravasation and inflammatory mediator influx, with no significant effect on normal tissue.

Sirt1-inducible deacetylation of p21 promotes cardiomyocyte proliferation.

Inducing cardiomyocyte proliferation is a hopeful approach for cardiac regeneration following myocardial infarction. Previous studies have shown that p21 inhibits the cardiomyocyte proliferation and cardiac regeneration. Deacetylation of p21 by Sirt1 deacetylase may reduce p21 abundance and remove p21-induced cell cycle arrest. However, whether p21 deacetylation and Sirt1 deacetylate control cardiomyocyte proliferation is unclear. Here, we show that acetylation of p21 induces cardiomyocyte proliferation arrest, whereas blocking the acetylation of p21 increases cardiomyocyte proliferation. P21 can be acetylated by Sirt1, and Sirt1 activate p21 ubiquitination through deacetylation. Additionally, overexpression of Sirt1 induces EdU-, pH3-, and Aurora B-positive cardiomyocytes in neonatal and adult mice. In contrast, depletion of Sirt1 reduces cardiomyocyte proliferation in vitro and in vivo. Moreover, Sirt1 protects cardiac function, reduces cardiac remodeling, inhibits cardiomyocyte apoptosis, and attenuates cardiomyocyte hypertrophy post-myocardial infarction. These results suggest that Sirt1-induced p21 deacetylation plays an essential role in cardiomyocyte proliferation and that it could be a novel therapeutic strategy for myocardial infarction.

Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis.

Objective: Long noncoding RNAs (lncRNAs) may serve as specific targets for the treatment of abdominal aortic aneurysms (AAAs). LncRNA GAS5, functionally associated with smooth muscle cell (SMC) apoptosis and proliferation, is likely involved in AAA formation, but the exact role of GAS5 in AAA is unknown. We thus explored the contribution of GAS5 to SMC-regulated AAA formation and its underlying mechanisms. Methods: Human specimens were used to verify the diverse expression of GAS5 in normal and AAA tissues. The angiotensin II (Ang II)-induced AAA model in ApoE-/- mice and the CaCl2-induced AAA model in wild-type C57BL/6 mice were used. RNA pull-down and luciferase reporter gene assays were performed in human aortic SMCs to detect the interaction between GAS5 and its downstream targets of protein or microRNA (miR). Results: GAS5 expression was significantly upregulated in human AAA specimens and two murine AAA models compared to human normal aortas and murine sham-operated controls. GAS5 overexpression induced SMC apoptosis and repressed its proliferation, thereby promoting AAA formation in two murine AAA models. Y-box-binding protein 1 (YBX1) was identified as a direct target of GAS5 while it also formed a positive feedback loop with GAS5 to regulate the downstream target p21. Furthermore, GAS5 acted as a miR-21 sponge to release phosphatase and tensin homolog from repression, which blocked the activation and phosphorylation of Akt to inhibit proliferation and promote apoptosis in SMCs. Conclusion: The LncRNA GAS5 contributes to SMC survival during AAA formation. Thus, GAS5 might serve as a novel target against AAA.

Magnetic Targeting Improves the Therapeutic Efficacy of Microbubble-Mediated Obstructive Thrombus Sonothrombolysis.

BACKGROUND: Magnetic targeting may help microbubbles (MBs) reach obstructive thrombi and improve the efficacy of MB-mediated sonothrombolysis, but the role of magnetic targeting in MB-mediated sonothrombolysis remains elusive. OBJECTIVES: We investigate the feasibility and efficacy of magnetically targeted MB-mediated sonothrombolysis for the treatment of obstructive thrombi. MATERIALS AND METHODS: Red and white thromboembolic models were established in vitro and in vivo. The models were randomly assigned to the control, ultrasound plus control MB (US + C-MB), ultrasound plus magnetic MB (US + M-MB), or US + M-MB + recombinant tissue-type plasminogen activator (r-tPA) groups and treated for 30 minutes. The recanalization rate, average blood flow velocity, hindlimb perfusion, and skeletal muscle injury marker levels were recorded. RESULTS: The recanalization rate, average blood flow velocity, and hindlimb perfusion in the red and white thromboembolic models were all significantly higher in the US + M-MB and US + M-MB + r-tPA groups than in the control and US + C-MB groups both in vitro and in vivo. Moreover, the levels of the skeletal muscle injury markers were all significantly lower in the US + M-MB and US + M-MB + r-tPA groups than in the other two groups in vivo for both thromboembolic models. However, the thrombolytic effects of red thrombi performed better than those of white thrombi in the US + M-MB + r-tPA group. CONCLUSION: M-MB-mediated sonothrombolysis improves the efficacy of thrombolysis both in vitro and in vivo, and reduces tissue damage in clogging model; thus, this method may serve as a promising approach for treating thrombus-occlusive diseases.

The pseudogene PTENP1 regulates smooth muscle cells as a competing endogenous RNA.

The long non-coding RNA (lncRNA) PTENP1 is a pseudogene of phosphatase and tensin homologue deleted on chromosome ten (PTEN), has been implicated in smooth muscle cell (SMC) proliferation and apoptosis. PTENP1 is the pseudogene of PTEN. However, it is unclear whether and how PTENP1 functions in the proliferation and apoptosis of human aortic SMCs (HASMCs). Here, we hypothesised that PTENP1 inhibits HASMC proliferation and enhances apoptosis by promoting PTEN expression. PCR analysis and Western blot assays respectively showed that both PTENP1 and PTEN were up-regulated in human aortic dissection (AD) samples. PTENP1 overexpression significantly increased the protein expression of PTEN, promoted apoptosis and inhibited the proliferation of HASMCs. PTENP1 silencing exhibited the opposite effects and mitigated H2O2-induced apoptosis of HASMCs. In an angiotensin II (Ang II)-induced mouse aortic aneurysm (AA) model, PTENP1 overexpression potentiated aortic SMC apoptosis, exacerbated aneurysm formation. Mechanistically, RNA pull-down assay and a series of luciferase reporter assays using miR-21 mimics or inhibitors identified PTENP1 as a molecular sponge for miR-21 to endogenously compete for the binding between miR-21 and the PTEN transcript, releasing PTEN expression. This finding was further supported by in vitro immunofluorescent evidence showing decreased cell apoptosis upon miR-21 mimic administration under baseline PTENP1 overexpression. Ex vivo rescue of PTEN significantly mitigated the SMC apoptosis induced by PTENP1 overexpression. Finally, Western blot assays showed substantially reduced Akt phosphorylation and cyclin D1 and cyclin E levels with up-regulated PTENP1 in HASMCs. Our study identified PTENP1 as a mediator of HASMC homeostasis and suggests that PTENP1 is a potential target in AD or AA intervention.