Carbonylation of Runx2 at K176 by 4-Hydroxynonenal Accelerates Vascular Calcification.

BACKGROUND: Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated. METHODS: Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2-/-) mice, smooth muscle cell-specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing ß-glycerophosphate and CaCl2 to investigate cell calcification and the underlying molecular mechanisms. RESULTS: Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell-specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE-induced upregulation of Runx2. CONCLUSIONS: Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

Causal relationship between iron status and preeclampsia-eclampsia: a Mendelian randomization analysis.

BACKGROUND: Preeclampsia/eclampsia is a severe pregnancy-related disorder associated with hypertension and organ damage. While observational studies have suggested a link between maternal iron status and preeclampsia/eclampsia, the causal relationship remains unclear. The aim of this study was to investigate the genetic causality between iron status and preeclampsia/eclampsia using large-scale genome-wide association study (GWAS) summary data and Mendelian randomization (MR) analysis. METHODS: Summary data for the GWAS on preeclampsia/eclampsia and genetic markers related to iron status were obtained from the FinnGen Consortium and the IEU genetic databases. The "TwoSampleMR" software package in R was employed to test the genetic causality between these markers and preeclampsia/eclampsia. The inverse variance weighted (IVW) method was primarily used for MR analysis. Heterogeneity, horizontal pleiotropy, and potential outliers were evaluated for the MR analysis results. RESULTS: The random-effects IVW results showed that ferritin (OR = 1.11, 95% CI: .89-1.38, p = .341), serum iron (OR = .90, 95% CI: .75-1.09, p = .275), TIBC (OR = .98, 95% CI: .89-1.07, p = .613), and TSAT (OR = .94, 95% CI: .83-1.07, p = .354) have no genetic causal relationship with preeclampsia/eclampsia. There was no evidence of heterogeneity, horizontal pleiotropy, or possible outliers in our MR analysis (p > .05). CONCLUSIONS: Our study did not detect a genetic causal relationship between iron status and preeclampsia/eclampsia. Nonetheless, this does not rule out a relationship between the two at other mechanistic levels.

LincRNA-p21 Upregulates Nuclear Orphan Receptor Nr4a2 and Aggravates Myocardial Ischemia/Reperfusion Injury via Targeting MiR-466i-5p.

Myocardial ischemia/reperfusion (I/R) injury can bring about more cardiomyocyte death and aggravate cardiac dysfunction, but its pathogenesis remains unclear. This study aimed to investigate the role of long intergenic noncoding RNA-p21 (LincRNA-p21) in myocardial I/R injury and its underlying mechanism. Mice were subjected to myocardial I/R injury by ligation and release of the left anterior descending artery, and HL-1 cardiomyocytes were treated with hydrogen peroxide. Infarct area, cardiac function, and cardiomyocyte apoptosis were determined. Consequently, LincRNA-p21 was found to significantly be elevated both in the reperfused hearts and H2O2-treated cardiomyocytes. Moreover, genetic inhibition of LincRNA-p21 brought about reduced infarct area and improved cardiac function in mice subjected to myocardial I/R injury. LincRNA-p21 knockdown was also demonstrated to inhibit cardiomyocyte apoptosis both in vivo and in vitro. Notably, LincRNA-p21 silencing increased the expression of microRNA-466i-5p (miR-466i-5p) and suppressed the expression of nuclear receptor subfamily 4 group A member 2 (Nr4a2). Mechanically, LincRNA-p21 downregulated and directly interacted with miR-466i-5p, while application of miR-466i-5p inhibitor promoted cardiomyocyte apoptosis that was improved by LincRNA-p21 inhibition. Furthermore, Nr4a2 upregulation caused by LincRNA-p21 overexpression was partially reversed by miR-466i-5p mimics. Thus, LincRNA-p21 positively regulated the expression of Nr4a2, through sponging miR-466i-5p, promoting cardiomyocyte apoptosis in myocardial I/R injury. The current study revealed a novel LincRNA-p21/miR-466i-5p/Nr4a2 pathway for myocardial I/R injury, indicating that LincRNA-p21 may serve as a potential target for future therapy.

Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury.

Increased adenosine helps limit infarct size in ischaemia/reperfusion-injured hearts. In cardiomyocytes, 90% of adenosine is catalysed by adenosine kinase (ADK) and ADK inhibition leads to higher concentrations of both intracellular adenosine and extracellular adenosine. However, the role of ADK inhibition in myocardial ischaemia/reperfusion (I/R) injury remains less obvious. We explored the role of ADK inhibition in myocardial I/R injury using mouse left anterior ligation model. To inhibit ADK, the inhibitor ABT-702 was intraperitoneally injected or AAV9 (adeno-associated virus)-ADK-shRNA was introduced via tail vein injection. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to elucidate the underlying mechanisms. ADK was transiently increased after myocardial I/R injury. Pharmacological or genetic ADK inhibition reduced infarct size, improved cardiac function and prevented cell apoptosis and necroptosis in I/R-injured mouse hearts. In vitro, ADK inhibition also prevented cell apoptosis and cell necroptosis in H/R-treated H9c2 cells. Cleaved caspase-9, cleaved caspase-8, cleaved caspase-3, MLKL and the phosphorylation of MLKL and CaMKII were decreased by ADK inhibition in reperfusion-injured cardiomyocytes. X-linked inhibitor of apoptosis protein (XIAP), which is phosphorylated and stabilized via the adenosine receptors A2B and A1/Akt pathways, should play a central role in the effects of ADK inhibition on cell apoptosis and necroptosis. These data suggest that ADK plays an important role in myocardial I/R injury by regulating cell apoptosis and necroptosis.

Aldehyde dehydrogenase 2 activation ameliorates cyclophosphamide-induced acute cardiotoxicity via detoxification of toxic aldehydes and suppression of cardiac cell death.

Cyclophosphamide (CY)-induced acute cardiotoxicity is a common side effect which is dose dependent. It is reported that up to 20% of patients received high dose of CY treatment suffered from acute cardiac dysfunction. However, the effective intervention strategies and related mechanisms are still largely unknown. We aimed to investigate the effects of aldehyde dehydrogenase 2 (ALDH2), an important endogenous cardioprotective enzyme, on CY-induced acute cardiotoxicity and the underlying mechanisms. It was found that ALDH2 knockout (KO) mice were more sensitive to CY-induced acute cardiotoxicity, presenting as higher serum levels of creatine kinase-MB isoform and lactate dehydrogenase, and significantly reduced myocardial contractility compared with C57BL/6 (WT) mice. In addition, cardiac cell death, especially necrosis, was obviously increased in ALDH2 KO mice compared with WT mice after CY treatment. Furthermore, accumulation of toxic aldehydes such as acrolein and 4-HNE and reactive oxygen species (ROS) in the myocardium were significantly elevated after CY in ALDH2 KO mice. Importantly, ALDH2 activation by Alda-1 pretreatment markedly attenuated CY-induced accumulation of toxic aldehydes, cardiac cell death and cardiac dysfunction, without affecting CY's anti-tumor efficacy. In conclusion, the cardioprotective effects of ALDH2 activation against CY-induced acute cardiotoxicity are exerted via reducing toxic aldehydes accumulation and potentially interrupting the acrolein-ROS-aldehydes vicious circles, and thus alleviates myocardial cell death, without antagonizing the anti-tumor efficacy of CY. Therefore, ALDH2 might be a promising prevention and treatment target for CY-induced acute cardiotoxicity.

Protective effect of ALDH2 against cyclophosphamide-induced acute hepatotoxicity via attenuating oxidative stress and reactive aldehydes.

Cyclophosphamide (CY) is a widely used chemotherapeutic agent that is associated with severe side effects, such as hepatotoxicity and nephrotoxicity. However, the extent, mechanisms and potential prevention and treatment strategies of CY-induced acute hepatotoxicity and nephrotoxicity are largely unknown. In this study, we determined the existence and extent of CY-induced acute hepatotoxicity and nephrotoxicity, and demonstrated the effect of ALDH2 on CY-induced acute tissue toxicity and related mechanisms. Adult male C57BL/6J (wide-type, WT) and ALDH2-/- (KO) mice were divided into four groups: WT, WT + CY, KO + CY and WT + CY + Alda-1. Biochemical analysis showed that plasma ALT was increased by 35.8% in KO + CY group and decreased by 21.1% in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). However, there was no significant difference among WT, WT + CY and KO + CY groups regarding plasma renal marker enzymes, including blood urea nitrogen (BUN), creatinine and cystatin C (CysC). Levels of reactive oxygen species (ROS) and toxic aldehydes (acrolein, 4-hydroxynonenol and malondialdehyde) were increased significantly in KO + CY group and decreased significantly in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). These findings demonstrate that CY could induce acute hepatotoxicity without nephrotoxicity, and ALDH2 plays a protective role in CY-induced acute hepatotoxicity. The underlying mechanisms are associated with attenuating oxidative stress and detoxifying reactive aldehydes.

Estimation of invasive coronary perfusion pressure using electrocardiogram and Photoplethysmography in a porcine model of cardiac arrest.

BACKGROUND: Coronary perfusion pressure (CPP) indicates spontaneous return of circulation and is recommended for high-quality cardiopulmonary resuscitation (CPR). This study aimed to investigate a method for non-invasive estimation of CPP using electrocardiography (ECG) and photoplethysmography (PPG) during CPR. METHODS: Nine pigs were used in this study. ECG, PPG, invasive arterial blood pressure (ABP), and right atrial pressure (RAP) signals were simultaneously recorded. The CPPs were estimated using three datasets: (a) ECG, (b) PPG, and (c) ECG and PPG, and were compared with invasively measured CPPs. Four machine-learning algorithms, namely support vector regression, random forest (RF), K-nearest neighbor, and gradient-boosted regression tree, were used for estimation of CPP. RESULTS: The RF model with a combined ECG and PPG dataset achieved better estimation of CPP than the other algorithms. Specifically, the mean absolute error was 4.49 mmHg, the root mean square error was 6.15 mm Hg, and the adjusted R2 was 0.75. A strong correlation was found between the non-invasive estimation and invasive measurement of CPP (r = 0.88), which supported our hypothesis that machine-learning-based analysis of ECG and PPG parameters can provide a non-invasive estimation of CPP for CPR. CONCLUSIONS: This study proposes a novel estimation of CPP using ECG and PPG with machine-learning-based algorithms. Non-invasively estimated CPP showed a high correlation with invasively measured CPP and may serve as an easy-to-use physiological indicator for high-quality CPR treatment.

Post-myocardial infarction fibrosis: Pathophysiology, examination, and intervention.

Cardiac fibrosis plays an indispensable role in cardiac tissue homeostasis and repair after myocardial infarction (MI). The cardiac fibroblast-to-myofibroblast differentiation and extracellular matrix collagen deposition are the hallmarks of cardiac fibrosis, which are modulated by multiple signaling pathways and various types of cells in time-dependent manners. Our understanding of the development of cardiac fibrosis after MI has evolved in basic and clinical researches, and the regulation of fibrotic remodeling may facilitate novel diagnostic and therapeutic strategies, and finally improve outcomes. Here, we aim to elaborate pathophysiology, examination and intervention of cardiac fibrosis after MI.

Metabolic reprogramming of pulmonary fibrosis.

Pulmonary fibrosis is a progressive and intractable lung disease with fibrotic features that affects alveoli elasticity, which leading to higher rates of hospitalization and mortality worldwide. Pulmonary fibrosis is initiated by repetitive localized micro-damages of the alveolar epithelium, which subsequently triggers aberrant epithelial-fibroblast communication and myofibroblasts production in the extracellular matrix, resulting in massive extracellular matrix accumulation and interstitial remodeling. The major cell types responsible for pulmonary fibrosis are myofibroblasts, alveolar epithelial cells, macrophages, and endothelial cells. Recent studies have demonstrated that metabolic reprogramming or dysregulation of these cells exerts their profibrotic role via affecting pathological mechanisms such as autophagy, apoptosis, aging, and inflammatory responses, which ultimately contributes to the development of pulmonary fibrosis. This review summarizes recent findings on metabolic reprogramming that occur in the aforementioned cells during pulmonary fibrosis, especially those associated with glucose, lipid, and amino acid metabolism, with the aim of identifying novel treatment targets for pulmonary fibrosis.

Activin receptor-like kinase 7 promotes apoptosis of vascular smooth muscle cells via activating Smad2/3 signaling in diabetic atherosclerosis.

Vascular smooth muscle cells (VSMCs) is a vital accelerator in the late phase of diabetic atherosclerosis, but the underlying mechanism remains unclear. The aim of our study was to investigate whether activin receptor-like kinase 7 (ALK7)-Smad2/3 pathway plays an important role in VSMC apoptosis of diabetic atherosclerosis. It was shown that ALK7 expression was obviously elevated in the aorta of ApoE-/- mice with type 2 diabetes mellitus. Inhibition of ALK7 expression significantly improved the stability of atherosclerotic plaques and reduced cell apoptosis. Further experiments showed that ALK7 knockdown stabilized atherosclerotic plaques by reducing VSMC apoptosis via activating Smad2/3. Our study uncovered the important role of ALK7-Smad2/3 signaling in VSMCs apoptosis, which might be a potential therapeutic target in diabetic atherosclerosis.

4-Hydroxy-2-Nonenal Promotes Cardiomyocyte Necroptosis via Stabilizing Receptor-Interacting Serine/Threonine-Protein Kinase 1.

Background: Necroptosis is a vital regulator of myocardial ischemia/reperfusion (MI/R) injury. Meanwhile, 4-hydroxy-2-nonenal (4-HNE) is abundantly increased during MI/R injury. However, whether 4-HNE induces cardiomyocyte necroptosis during MI/R remains unknown. Methods: To observe the relationship between 4-HNE and necroptosis during MI/R, C57BL/6 mice and aldehyde dehydrogenase 2-transgenic (ALDH2-Tg) mice were both exposed to left anterior descending artery ligation surgery to establish MI/R injury models. For further study, isolated mouse hearts and H9c2 cells were both treated with 4-HNE to elucidate the underlying mechanisms. Results: Necroptosis and 4-HNE were both upregulated in I/R-injured hearts. Cardiomyocyte necroptosis was significantly decreased in I/R-injured hearts from ALDH2-Tg mice as compared with that of wild-type mice. In vitro studies showed that necroptosis was enhanced by 4-HNE perfusion in a time- and concentration-dependent manner. Knockdown of receptor-interacting serine/threonine-protein kinase 1 (RIP1) using small interfering RNA (siRNA) prevented 4-HNE-induced cardiomyocyte necroptosis, manifesting that RIP1 played a key role in the upregulation of cell necroptosis by 4-HNE. Further studies found that 4-HNE reduced the protein degradation of RIP1 by preventing K48-polyubiquitination of RIP1. Conclusion: 4-HNE contributes to cardiomyocyte necroptosis by regulating ubiquitin-mediated proteasome degradation of RIP1.