Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters.

BACKGROUND: Advanced glycation end product-modified low-density lipoprotein (AGE-LDL) is related to inflammation and the development of atherosclerosis. Additionally, it has been demonstrated that receptor for advanced glycation end products (RAGE) has a role in the condition known as calcific aortic valve disease (CAVD). Here, we hypothesized that the AGE-LDL/RAGE axis could also be involved in the pathophysiological mechanism of CAVD. METHODS: Human aortic valve interstitial cells (HAVICs) were stimulated with AGE-LDL following pre-treatment with or without interleukin 37 (IL-37). Low-density lipoprotein receptor deletion (Ldlr-/-) hamsters were randomly allocated to chow diet (CD) group and high carbohydrate and high fat diet (HCHFD) group. RESULTS: AGE-LDL levels were significantly elevated in patients with CAVD and in a hamster model of aortic valve calcification. Our in vitro data further demonstrated that AGE-LDL augmented the expression of intercellular cell adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6) and alkaline phosphatase (ALP) in a dose-dependent manner through NF-κB activation, which was attenuated by nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082. The expression of RAGE was augmented in calcified aortic valves, and knockdown of RAGE in HAVICs attenuated the AGE-LDL-induced inflammatory and osteogenic responses as well as NF-κB activation. IL-37 suppressed inflammatory and osteogenic responses and NF-κB activation in HAVICs. The vivo experiment also demonstrate that supplementation with IL-37 inhibited valvular inflammatory response and thereby suppressed valvular osteogenic activities. CONCLUSIONS: AGE-LDL promoted inflammatory responses and osteogenic differentiation through RAGE/NF-κB pathway in vitro and aortic valve lesions in vivo. IL-37 suppressed the AGE-LDL-induced inflammatory and osteogenic responses in vitro and attenuated aortic valve lesions in a hamster model of CAVD.

Abnormally elevated EZH2-mediated H3K27me3 enhances osteogenesis in aortic valve interstitial cells by inhibiting SOCS3 expression.

BACKGROUND AND AIMS: The osteogenic transition of aortic valve interstitial cells (AVICs) plays a critical role for the progression of calcific aortic valve disease (CAVD). Enhancer of zeste homolog 2 (EZH2) is an important methyltransferase for histone H3 Lys27 (H3K27) that has been found to be involved in osteogenesis. Here, we investigated the effect and mechanism of EZH2 in CAVD progression. METHODS: High throughout mRNA sequencing, qRT-PCR and immunoblot were performed to screen differentially expressed genes in non-CAVD and CAVD aortic valves. To investigate the role of EZH2 and SOCS3 in osteogenesis, AVICs were treated with siRNA, adenovirus and specific inhibitors, then osteogenic markers and mineralized deposits were examined. In vivo, the morphology and function of aortic valves were investigated by HE stain and echocardiography in ApoE-/- mice fed a long-term western diet (WD). RESULTS: We discovered that EZH2 was upregulated and SOCS3 was downregulated in calcified aortic valves. In AVICs, inhibition or silencing of EZH2 attenuated the osteogenic responses. On the other hand, demethylases inhibitor (GSK-J4) enhanced osteogenic transition of AVICs. Moreover, SOCS3 knockdown enhanced the expression of osteogenic markers, while SOCS3 overexpression suppressed osteogenesis and calcification. The chromatin immunoprecipitation and restored experiments indicated that EZH2 directly targeted SOCS3 to promote osteogenic responses of AVICs. In vivo, treatment with EZH2 inhibitor through intraperitoneal injection attenuated aortic valve thickening, calcification and dysfunction induced by WD. CONCLUSIONS: Collectively, we found that EZH2-mediated H3K27me3 enhanced osteogenesis and microcalcification of AVICs via inhibiting SOCS3 expression, which provides potential targets for future therapeutic interventions of CAVD.

Machine learning models in heart failure with mildly reduced ejection fraction patients.

Objective: Heart failure with mildly reduced ejection fraction (HFmrEF) has been recently recognized as a unique phenotype of heart failure (HF) in current practical guideline. However, risk stratification models for mortality and HF re-hospitalization are still lacking. This study aimed to develop and validate a novel machine learning (ML)-derived model to predict the risk of mortality and re-hospitalization for HFmrEF patients. Methods: We assessed the risks of mortality and HF re-hospitalization in HFmrEF (45-49%) patients enrolled in the TOPCAT trial. Eight ML-based models were constructed, including 72 candidate variables. The Harrell concordance index (C-index) and DeLong test were used to assess discrimination and the improvement in discrimination between models, respectively. Calibration of the HF risk prediction model was plotted to obtain bias-corrected estimates of predicted versus observed values. Results: Least absolute shrinkage and selection operator (LASSO) Cox regression was the best-performing model for 1- and 6-year mortality, with a highest C-indices at 0.83 (95% CI: 0.68-0.94) over a maximum of 6 years of follow-up and 0.77 (95% CI: 0.64-0.89) for the 1-year follow-up. The random forest (RF) showed the best discrimination for HF re-hospitalization, scoring 0.80 (95% CI: 0.66-0.94) and 0.85 (95% CI: 0.71-0.99) at the 6- and 1-year follow-ups, respectively. For risk assessment analysis, Kansas City Cardiomyopathy Questionnaire (KCCQ) subscale scores were the most important predictor of readmission outcome in the HFmrEF patients. Conclusion: ML-based models outperformed traditional models at predicting mortality and re-hospitalization in patients with HFmrEF. The results of the risk assessment showed that KCCQ score should be paid increasing attention to in the management of HFmrEF patients.

4-Octyl itaconate suppresses the osteogenic response in aortic valvular interstitial cells via the Nrf2 pathway and alleviates aortic stenosis in mice with direct wire injury.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in older individuals, but there is a lack of drug treatment. The cellular biological mechanisms of CAVD are still unclear. Oxidative stress and endoplasmic reticulum stress (ER stress) have been suggested to be involved in the progression of CAVD. Many studies have demonstrated that 4-octyl itaconate (OI) plays beneficial roles in limiting inflammation and oxidative injury. However, the potential role of OI in CAVD has not been thoroughly explored. Thus, we investigated OI-mediated modulation of ROS generation and endoplasmic reticulum stress to inhibit osteogenic differentiation in aortic valve interstitial cells (VICs). In our study, calcified aortic valves showed increased levels of ER stress and superoxide anion, as well as abnormal expression of Hmox1 and NQO1. In VICs, OI activated the Nrf2 signaling cascade and contributed to Nrf2 stabilization and nuclear translocation, thus augmenting the expression of genes downstream of Nrf2 (Hmox1 and NQO1). Moreover, OI ameliorated osteogenic medium (OM)-induced ROS production, mitochondrial ROS levels and the loss of mitochondrial membrane potential in VICs. Furthermore, OI attenuated the OM-induced upregulation of ER stress markers, osteogenic markers and calcium deposition, which were blocked by the Nrf2-specific inhibitor ML385. Interestingly, we found that OM-induced ER stress and osteogenic differentiation were ROS-dependent and that Hmox1 silencing triggered ROS production, ER stress and elevated osteogenic activity, which were inhibited by NAC. Overexpression of NQO1 mediated by adenovirus vectors significantly suppressed OM-induced ER stress and osteogenic markers. Collectively, these results showed the anti-osteogenic effects of OI on AVICs by regulating the generation of ROS and ER stress by activating the Nrf2 signaling pathway. Furthermore, OI alleviated aortic stenosis in a mouse model with direct wire injury. Due to its antioxidant properties, OI could be a potential drug for the prevention and/or treatment of CAVD.

Marine-Derived Piericidin Diglycoside S18 Alleviates Inflammatory Responses in the Aortic Valve via Interaction with Interleukin 37.

Calcific aortic valve disease (CAVD) is a valvular disease frequently in the elderly individuals that can lead to the valve dysfunction. Osteoblastic differentiation of human aortic valve interstitial cells (HAVICs) induced by inflammation play a crucial role in CAVD pathophysiological processes. To date, no effective drugs for CAVD have been established, and new agents are urgently needed. Piericidin glycosides, obtained from a marine-derived Streptomyces strain, were revealed to have regulatory effects on mitochondria in previous studies. Here, we discovered that 13-hydroxypiericidin A 10-O-α-D-glucose (1→6)-ß-D-glucoside (S18), a specific piericidin diglycoside, suppresses lipopolysaccharide- (LPS) induced inflammatory responses of HAVICs by alleviating mitochondrial stress in an interleukin (IL)-37-dependent manner. Knockdown of IL-37 by siRNA not only exaggerated LPS-induced HAVIC inflammation and mitochondrial stress but also abrogated the anti-inflammatory effect of S18 on HAVICs. Moreover, S18 alleviated aortic valve lesions in IL-37 transgenic mice of CAVD model. Microscale thermophoresis (MST) and docking analysis of five piericidin analogues suggested that diglycosides, but not monoglycosides, exert obvious IL-37-binding activity. These results indicate that S18 directly binds to IL-37 to alleviate inflammatory responses in HAVICs and aortic valve lesions in mice. Piericidin diglycoside S18 is a potential therapeutic agent to prevent the development of CAVD.