Upregulated LncRNA H19 Sponges MiR-106a-5p and Contributes to Aldosterone-Induced Vascular Calcification via Activating the Runx2-Dependent Pathway.

BACKGROUND: Excess aldosterone is implicated in vascular calcification (VC), but the mechanism by which aldosterone-MR (mineralocorticoid receptor) complex promotes VC is unclear. Emerging evidence indicates that long-noncoding RNA H19 (H19) plays a critical role in VC. We examined whether aldosterone-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) through H19 epigenetic modification of Runx2 (runt-related transcription factor-2) in a MR-dependent manner. METHODS: We induced in vivo rat model of chronic kidney disease using a high adenine and phosphate diet to explore the relationship among aldosterone, MR, H19, and VC. We also cultured human aortic VSMCs to explore the roles of H19 in aldosterone-MR complex-induced osteogenic differentiation and calcification of VSMCs. RESULTS: H19 and Runx2 were significantly increased in aldosterone-induced VSMC osteogenic differentiation and VC, both in vitro and in vivo, which were significantly blocked by the MR antagonist spironolactone. Mechanistically, our findings reveal that the aldosterone-activated MR bound to H19 promoter and increased its transcriptional activity, as determined by chromatin immunoprecipitation, electrophoretic mobility shift assay, and luciferase reporter assay. Silencing H19 increased microRNA-106a-5p (miR-106a-5p) expression, which subsequently inhibited aldosterone-induced Runx2 expression at the posttranscriptional level. Importantly, we observed a direct interaction between H19 and miR-106a-5p, and downregulation of miR-106a-5p efficiently reversed the suppression of Runx2 induced by H19 silencing. CONCLUSIONS: Our study clarifies a novel mechanism by which upregulation of H19 contributes to aldosterone-MR complex-promoted Runx2-dependent VSMC osteogenic differentiation and VC through sponging miR-106a-5p. These findings highlight a potential therapeutic target for aldosterone-induced VC.

Potential ferroptosis key genes in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a highly prevalent condition that comprises a disease continuum, ranging from microscopic changes to profound fibro-calcific leaflet remodeling, culminating in aortic stenosis, heart failure, and ultimately premature death. Ferroptosis has been hypothesized to contribute to the pathogenesis of CAVD. We aimed to study the association between ferroptosis genes and CAVD and reveal the potential roles of ferroptosis in CAVD. CAVD-related differentially expressed genes (DEGs) were identified via bioinformatic analysis of Datasets GSE51472 and GSE12644 obtained from Gene Expression Omnibus. A ferroptosis dataset containing 259 genes was obtained from the Ferroptosis Database. We then intersected with CAVD-related DEGs to identify the ferroptosis DEGs. Subsequently, protein-protein interaction networks and functional enrichment analyses were performed for ferroptosis DEGs. Then, we used miRWalk3.0 to predict the target pivotal microRNAs. An in vitro model of CAVD was constructed using human aortic valve interstitial cells. The qRT-PCR and western blotting methods were used to validate the ferroptosis DEGs identified by the microarray data. A total of 21 ferroptosis DEGs in CAVD containing 12 upregulated and nine downregulated genes were identified. The results of the Gene Set Enrichment Analysis (GSEA) and analysis of the KEGG pathway by WebGestalt indicated that the ferroptosis DEGs were enriched in six signaling pathways among which NAFLD (including IL-6, BID, and PRKAA2 genes) and HIF-1 (including IL-6, HIF-1, and HMOX1 genes) signaling pathways were also verified by DAVID and/or Metascape. Finally, the in vitro results showed that the mRNA and protein expression levels of IL-6, HIF-1α, HMOX1, and BID were higher, while the levels of PRKAA2 were lower in the Pi-treated group than those in the control group. However, the addition of ferrostatin-1 (a selective ferroptosis inhibitor) significantly reversed the above changes. Therefore, IL-6, HIF-1α, HMOX1, BID, and PRKAA2 are potential key genes closely associated with ferroptosis in CAVD. Further work is required to explore the underlying ferroptosis-related molecular mechanisms and provide possible therapeutic targets for CAVD.

Triglyceride-glucose index in the development of peripheral artery disease: findings from the Atherosclerosis Risk in Communities (ARIC) Study.

BACKGROUND: It remains unclear whether triglyceride-glucose (TyG) index, a surrogate marker of insulin resistance, is prospectively associated with incident peripheral arterial disease (PAD). METHODS: We included 12,320 Atherosclerosis Risk in Communities Study participants (aged 54.3 ± 5.7 years) free of a history of PAD at baseline (visit 1: 1987-1989). The TyG index was determined using ln (fasting triglycerides [mg/dL] × fasting glucose [mg/dL]/2), and measured at 5 visits between 1987 and 2013. Incident PAD was defined as the first hospitalization with PAD diagnosis or a new onset of measured ABI < 0.90 during follow-up visits. We quantified the association of both baseline and trajectories of TyG index with incident PAD using Cox regression and logistic regression analysis, respectively. RESULTS: Over a median follow-up of 23 years, 1300 participants developed PAD. After adjustment for traditional PAD risk factors, each 1-SD (0.58) increase in TyG index was associated with an 11.9% higher risk of incident PAD [hazard ratio, 1.119 (95% CI, 1.049-1.195)]. Results were similar when individuals were categorized by TyG index quartiles [hazard ratio, 1.239 (95% CI, 1.028-1.492); comparing extreme quartiles]. Four distinct trajectories of stable TyG indexes at various levels along the follow-up duration were identified [low (22.2%), moderate (43.2%), high (27.5%), and very high (7.1%) trajectory groups]. Compared with those with a TyG index trajectory at a low level, those participants with TyG index trajectories at high and very high levels had an even greater risk of future incident PAD [odds ratio (95%CI): 1.404 (1.132-1.740) and 1.742 (1.294-2.344), respectively] after multivariate adjustments for traditional PAD risk factors. CONCLUSIONS: Higher TyG index is independently associated with an increased risk of incident PAD. Long-term trajectories of TyG index help identify individuals at a higher risk of PAD who deserve specific preventive and therapeutic approaches. TRIAL REGISTRATION: Clinical trial registration number: The ARIC trial was registered at clinicaltrials.gov as NCT00005131.

Astragaloside IV Prevents Cardiac Remodeling in the Apolipoprotein E-Deficient Mice by Regulating Cardiac Homeostasis and Oxidative Stress.

BACKGROUND: Hypercholesterolemia is a risk factor for the development of cardiac hypertrophy. Astragaloside IV (AST-IV) possesses cardiovascular protective properties. We hypothesize that AST-IV prevents cardiac remodeling with hypercholesterolemia via modulating tissue homeostasis and alleviating oxidative stress. METHODS: The ApoE-/- mice were treated with AST-IV at 1 or 10 mg/kg for 8 weeks. The blood lipids tests, echocardiography, and TUNEL were performed. The mRNA expression profile was detected by real-time PCR. The myocytes size and number, and the expressions of proliferation (ki67), senescence (p16INK4a), oxidant (NADPH oxidase 4, NOX4) and antioxidant (superoxide dismutase, SOD) were observed by immunofluorescence staining. RESULTS: Neither 1 mg/kg nor 10 mg/kg AST-IV treatment could decrease blood lipids in ApoE-/- mice. However, the decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS) in ApoE-/- mice were significantly improved after AST-IV treatment. The cardiac collagen volume fraction declined nearly in half after AST-IV treatment. The enlarged myocyte size was suppressed, and myocyte number was recovered, and the alterations of genes expressions linked to cell cycle, proliferation, senescence, p53-apoptosis pathway and oxidant-antioxidants in the hearts of ApoE-/- mice were reversed after AST-IV treatment. The decreased ki67 and increased p16INK4a in the hearts of ApoE-/- mice were recovered after AST-IV treatment. The percentages of apoptotic myocytes and NOX4-positive cells in AST-IV treated mice were decreased, which were consistent with the gene expressions. CONCLUSION: AST-IV treatment could prevent cardiac remodeling and recover the impaired ventricular function induced by hypercholesterolemia. The beneficial effect of AST-IV might partly be through regulating cardiac homeostasis and anti-oxidative stress.

Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.