Physcion 8-O-ß-Glucopyranoside Alleviates Oxidized Low-Density Lipoprotein-Induced Human Umbilical Vein Endothelial Cell Injury by Inducing Autophagy Through AMPK/SIRT1 Signaling.

AIM: Vascular endothelial cell dysfunction plays a crucial role in the initiation and development of atherosclerosis. Physcion 8-O-ß-glucopyranoside (PG), an anthraquinone extracted from Polygonum cuspidatum, has a number of pharmacological functions. The aim of this study was to elucidate the protective effects of PG against oxidized low-density lipoprotein (ox-LDL) in VECs. METHODS AND MATERIALS: Human umbilical vein endothelial cells (HUVECs) were used as the in vitro model. Cell viability and apoptosis were, respectively, assessed by CCK-8 assay and Annexin-V/PI staining. Formation of autophagosomes was visualized by acridine orange staining, and the autophagy flux was tracked after infecting the cells with the mRFP-GFP-LC3 adenovirus. The expression levels of various apoptosis and autophagy-associated marker proteins were detected by Western blotting. RESULTS: Pretreatment with PG protected the HUVECs from ox-LDL-induced apoptosis. In addition, PG promoted autophagy in HUVECs, which was responsible for its antiapoptotic effects. Finally, activation of AMPK/SIRT1 signaling was upstream of PG-induced autophagy. CONCLUSIONS: PG has potential pharmacological effects against oxidative damage-induced HUVEC injury through inducing AMPK/SIRT1-mediated autophagy.

GATA1 activated lncRNA (Galont) promotes anoxia/reoxygenation-induced autophagy and cell death in cardiomyocytes by sponging miR-338.

The hypernomic autophagy is associated with various cardiovascular diseases. Long noncoding RNAs (lncRNAs) are emerging as important regulators in gene expression, which have been involved in multiple physiological and pathological processes. However, the function of lncRNAs and how they functioned in the autophagy in cardiomyocytes were rarely reported. In this study, we report that a lncRNA, named GATA1 activated lncRNA (Galont), can directly interact with miR-338 and promote ATG5-mediated autophagic cell death in murine cardiomyocytes. First, we found that Galont was upregulated by anoxia/reoxygenation (A/R) stimulus, and it was able to promote autophagy and cell death in cardiomyocytes exposure to A/R. Then, miR-338 was identified as a novel suppressor in autophagy and autophagic cell death. Our results from bioinformatic analysis and luciferase reporter gene assay indicated that ATG5 is a target gene of miR-338. Furthermore, RNA pull-down assays demonstrated that Galont directly interacted with miR-338, and thus promoted ATG5 expression and autophagic cell death. Our findings reveal a novel regulatory circuit in the autophagy in cardiomyocytes, which consists of Galont, miR-338 and ATG5.

Apolipoprotein A1 regulates coenzyme Q10 absorption, mitochondrial function, and infarct size in a mouse model of myocardial infarction.

HDL and apolipoprotein A1 (apoA1) concentrations inversely correlate with risk of death from ischemic heart disease; however, the role of apoA1 in the myocardial response to ischemia has not been well defined. To test whether apoA1, the primary HDL apolipoprotein, has an acute anti-inflammatory role in ischemic heart disease, we induced myocardial infarction via direct left anterior descending coronary artery ligation in apoA1 null (apoA1(-/-)) and apoA1 heterozygous (apoA1(+/-)) mice. We observed that apoA1(+/-) and apoA1(-/-) mice had a 52% and 125% increase in infarct size as a percentage of area at risk, respectively, compared with wild-type (WT) C57BL/6 mice. Mitochondrial oxidation contributes to tissue damage in ischemia-reperfusion injury. A substantial defect was present at baseline in the electron transport chain of cardiac myocytes from apoA1(-/-) mice localized to the coenzyme Q (CoQ) pool with impaired electron transfer (67% decrease) from complex II to complex III. Administration of coenzyme Q10 (CoQ10) to apoA1 null mice normalized the cardiac mitochondrial CoQ pool and reduced infarct size to that observed in WT mice. CoQ10 administration did not significantly alter infarct size in WT mice. These data identify CoQ pool content leading to impaired mitochondrial function as major contributors to infarct size in the setting of low HDL/apoA1. These data suggest a previously unappreciated mechanism for myocardial stunning, cardiac dysfunction, and muscle pain associated with low HDL and low apoA1 concentrations that can be corrected by CoQ10 supplementation and suggest populations of patients that may benefit particularly from CoQ10 supplementation.

Connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL: an involvement of ERK signaling pathway.

Oxidized low-density lipoprotein (ox-LDL), one of the most important risk factors of atherosclerosis, is a highly antigenic, potent chemoattractant that facilitates the development of atherosclerosis. Gap junctions also play an important in the development of atherosclerosis. In this study, we investigated the effects of ox-LDL on connexin43 and the mechanisms of connexin43 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cell (HUVEC), to clarify the role of connexin43 in atherosclerosis. Our results showed that ox-LDL significantly inhibited the growth and promoted apoptosis of HUVEC in a dose-dependent manner. Also, ox-LDL upregulated the expression of connexin43. Furthermore, knockdown connexin43 by siRNA promoted proliferation and inhibited apoptosis in ox-LDL-stimulated HUVEC. Moreover, the level of phosphor-ERK1/2 and connexin43 was remarkably attenuated by a ERK pathway inhibitor (PD98059). These results suggest that connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL, and ERK signaling pathway appears to be involved in these processes.

Edoxaban for stroke prevention in atrial fibrillation and factors associated with dosing: patient characteristics from the prospective observational ETNA-AF-China registry.

Real-world data on effectiveness and safety of a single non-vitamin K antagonist oral anticoagulant in the Chinese population with atrial fibrillation (AF) are limited. This study reports characteristics of patients treated with edoxaban and factors associated with dosing patterns from routine care in China. ETNA-AF-China (NCT04747496) is a multicentre, prospective, observational study enrolling edoxaban-treated patients from four economic regions with a targeted 2-year follow-up. Of the 4930 patients with AF (mean age: 70.2 ± 9.5 years; male, 57.1%), the mean creatinine clearance (CrCl), CHA2DS2-VASc, and HAS-BLED scores were 71.2 mL/min, 2.9, and 1.6. Overall, 6.4% of patients were perceived as frail by investigators. Available label dose reduction criteria (N = 4232) revealed that 3278 (77.5%) patients received recommended doses and 954 (22.5%) non-recommended doses. Northeast (53.0%) and West (43.1%) regions had the highest prescriptions of 60 mg and 30 mg recommended doses, respectively. Non-recommended 30 mg doses were more frequently prescribed in patients with antiplatelet use and history of heart failure than recommended 60 mg. Multivariate analysis identified advanced age as the strongest associated factor with non-recommended doses. Frailty had the strongest association with 30 mg except for age, and history of TIA was the most relevant factor associated with 60 mg. In conclusion, patients in the ETNA-AF-China study were predominantly aged 65 years and older, had mild-to-moderate renal impairment and good label adherence. Advanced age was associated with non-recommended doses, with frailty most common for non-recommended 30 mg and a history of TIA for the non-recommended 60 mg dose.

Autophagy of umbilical cord mesenchymal stem cells induced by rapamycin conduces to pro-angiogenic function of the conditioned medium.

Angiogenesis is critical for wound healing and tissue repair. Umbilical cord mesenchymal stem cells (UCMSCs)-conditioned medium has certain actions to promote angiogenesis, and is expected for wound healing and tissue repair. However, recent studies showed that the pro-angiogenic efficacy of unprocessed MSCs-conditioned medium is low, and insufficient for tissue repair. Autophagy is a process for protein recycling and a contributor for cell exocrine, which may enhance pro-angiogenic efficacy of the conditioned medium by stimulating cytokine release from UCMSCs. Therefore, in this study we attempted to obtain enhanced autophagy in UCMSCs using different concentrations of rapamycin and compared pro-angiogenic functions of the conditioned media. The in vitro data showed that although 100 nM-10 µM rapamycin all could induce autophagy in UCMSCs, 100 nM was the best dose to optimize the angiogenic effect of the conditioned medium. The in vivo data also showed that pro-angiogenic effect of the optimized conditioned medium was more obvious than that of the control conditioned medium (0 nM group) in the injected matrigel plaques. Further, the expressions of VEGF, FGF-2, MMP-9, PDGF-α and PDGF-ß were markedly increased in UCMSCs treated with 100 nM rapamycin. In conclusion, appropriately enhancing autophagy of UCMSC can improve pro-angiogenic efficacy of the conditioned medium, which may optimize therapeutic applications of UCMSCs-conditioned medium in wound healing and tissue repair.

Elevated Fibroblast Growth Factor 23 Impairs Endothelial Function through the NF-κB Signaling Pathway.

AIM: Roles of fibroblast growth factor 23 (FGF23) in endothelial dysfunction remain controversial, and evidence from population-based studies is lacking. The present study aimed to explore the effects of FGF23 on endothelial dysfunction on the basis of both clinical data of patients with coronary artery disease (CAD) and the in vitro research in human umbilical vein endothelial cells (HUVECs). METHODS: A total of 321 CAD patients were enrolled after coronary angiography, brachial artery flow-mediated dilation (FMD) was assessed using ultrasound equipment. Serum FGF23, nitric oxide (NO), and endothelin-1 (ET-1) were detected via enzyme-linked immunosorbent assay. Apoptosis was determined using the annexin V-fluorescein isothiocyanate/propidium lodide apoptosis detection kit. Cell migration was evaluated by wound healing and transwell migration assays. Reactive oxide species levels were determined using fluorescent probes, and NF-κB p65 nuclear translocation was assessed via immunofluorescence. RESULTS: Serum FGF23 was significantly increased in CAD patients combined with severe endothelial dysfunction (FMD ＜2%) compared to those with FMD ≥ 2% (P＜0.001). Furthermore, the levels of FGF23 were negatively correlated with NO, whereas positively correlated with ET-1 both in unadjusted analysis and multivariate-adjusted analysis. In HUVECs, FGF23 interfered with the bioavailability of NO via increased oxidative stress. Moreover, FGF23 directly impaired the endothelium by promoting HUVECs apoptosis and attenuating the migration of HUVECs. Additional experiments showed that FGF23 induced endothelial injury through activation of the NF-κB signaling pathway. CONCLUSIONS: Elevated FGF23 is clinically associated with endothelial dysfunction in CAD patients, and FGF23 impairs endothelial function through activation of the NF-κB signaling pathway.

Tafolecimab in Chinese Patients With Hypercholesterolemia (CREDIT-4): A Randomized, Double-Blind, Placebo-Controlled Phase 3 Trial.

Background: Tafolecimab is a novel fully human proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibody, developed for the treatment of hypercholesterolemia. Objectives: The purpose of this study was to assess the efficacy and safety of tafolecimab in Chinese patients at high or very high cardiovascular risk with hypercholesterolemia. Methods: Patients with diagnoses of heterozygous familial hypercholesterolemia (HeFH) by the Simon Broome criteria or at high or very high cardiovascular risk with nonfamilial hypercholesterolemia, with screening low-density lipoprotein cholesterol (LDL-C) level ≥1.8 mmol/L, were randomized 2:1 to receive tafolecimab or placebo 450 mg every 4 weeks (Q4W) in the 12-week double-blind treatment period. The primary endpoint was the percent change from baseline to week 12 in LDL-C levels. Results: A total of 303 patients were enrolled and received at least 1 dose of tafolecimab (n = 205) or placebo (n = 98). The least squares mean percent change in LDL-C level from baseline to week 12 was -68.9% (SE 1.4%) in the tafolecimab group and -5.8% (1.8%) in the placebo group (difference: -63.0%; [95% CI: -66.5% to -59.6%]; P < 0.0001). More patients treated with tafolecimab achieved ≥50% LDL-C reductions, LDL-C <1.8 mmol/L, and LDL-C <1.4 mmol/L at week 12 than did those in the placebo group (all P < 0.0001). Furthermore, tafolecimab markedly reduced non-HDL-C, apolipoprotein B, and lipoprotein(a) levels. During the double-blind treatment period, the most commonly reported adverse events included urinary tract infection (5.9% with tafolecimab vs 4.1% with placebo) and hyperuricemia (3.4% vs 4.1%). Conclusions: Tafolecimab was safe and showed robust lipid-lowering efficacy in Chinese patients at high or very high cardiovascular risk with hypercholesterolemia. (A Study of IBI306 in Participants With Hypercholesterolemia; NCT04709536).

Tafolecimab in Chinese patients with non-familial hypercholesterolemia (CREDIT-1): a 48-week randomized, double-blind, placebo-controlled phase 3 trial.

Background: Tafolecimab, a fully human proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibody developed for the treatment of hypercholesterolemia, demonstrated robust lipid-lowering efficacy and favorable safety in previous short-term studies. We aimed to assess the long-term efficacy and safety of tafolecimab in Chinese non-familial hypercholesterolemia (non-FH) patients. Methods: Non-FH patients at high or very-high cardiovascular risk with screening low-density lipoprotein cholesterol (LDL-C) level ≥1.8 mmol/L or non-FH patients with screening LDL-C level ≥3.4 mmol/L and on stable lipid-lowering therapy for at least 4 weeks, were randomized in a 2:2:1:1 ratio to receive subcutaneous tafolecimab 450 mg Q4W, tafolecimab 600 mg Q6W, placebo 450 mg Q4W, or placebo 600 mg Q6W, respectively, in the 48-week double-blind treatment period. The primary endpoint was the percent change from baseline to week 48 in LDL-C levels. Findings: A total of 618 patients were randomized and 614 patients received at least one dose of tafolecimab (n = 411) or placebo (n = 203). At week 48, tafolecimab induced significant reductions in LDL-C levels (treatment differences versus placebo [on-treatment estimand]: -65.0% [97.5% CI: -70.2%, -59.9%] for 450 mg Q4W; -57.3% [97.5% CI: -64.0%, -50.7%] for 600 mg Q6W; both P < 0.0001). Significantly more patients treated with tafolecimab achieved ≥50% LDL-C reductions, LDL-C < 1.8 mmol/L, and LDL-C < 1.4 mmol/L than placebo group at both dose regimens (all P < 0.0001). Furthermore, tafolecimab significantly reduced non-HDL-C, apolipoprotein B, and lipoprotein(a) levels. The most commonly-reported treatment emergent adverse events in the tafolecimab groups included upper respiratory infection, urinary tract infection and hyperuricemia. Interpretation: Tafolecimab dosed at 450 mg Q4W and 600 mg Q6W was safe and showed superior lipid-lowering efficacy versus placebo, providing a novel treatment option for Chinese hypercholesterolemia patients. Funding: This study was sponsored by Innovent Biologics, Inc.

Liraglutide Attenuates Myocardial Fibrosis via Inhibition of AT1R-Mediated ROS Production in Hypertensive Mice.

BACKGROUND/AIMS: Glucagon-like peptide-1 receptor agonist liraglutide has been reported to exert cardioprotective effects, but its effect on cardiac fibrosis remains controversial. The aim of this study was to investigate the effects of liraglutide on cardiac fibrosis and potential mechanisms. METHODS: C57BL/6 mice (3-month old) were randomly divided into control, hypertension, and hypertension + liraglutide groups. The hypertensive state was created by infusion of Ang II (100 ng/kg·min) for 4 weeks through subcutaneously implanted osmotic pumps. The control mice were infused with saline. Mice were also given vehicle or liraglutide (400 µg/kg·day). Blood pressure (BP), blood sugar, myocardial fibrosis, AT1R expression, and reactive oxygen species (ROS) levels were measured. To further elucidate the mechanisms of fibrosis, mouse cardiac fibroblasts were isolated and treated with liraglutide (300 nM/L) or losartan (10 µM) for 3 hours, followed by Ang II (10-7 M) for additional 12 hours. Reactive oxygen species production and expressions of collagen-1 and -3 were measured. RESULTS: Liraglutide reduced BP and blood sugar but did not affect the body weight of the hypertensive mice. Liraglutide also inhibited collagen accumulation, AT1R expression, and ROS generation in the hearts of the hypertensive mice. In in vitro studies, pretreatment with liraglutide and losartan (as control) markedly inhibited Ang II-induced ROS production and collagen expression in the cultured cardiac fibroblasts. CONCLUSION: Liraglutide reduces myocardial fibrosis in the hypertensive mice, which appears to be dependent on at least in part inhibition of ROS production.

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia.

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15min followed by 30min stop-flow ischemia at 37 degrees C. Amobarbital (2.5mM) or azide (5mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.

Kinetic alterations of collagen and elastic fibres and their association with cardiac function in acute myocardial infarction.

The present study aimed to investigate kinetic alterations of collagen and elastic fibres and their association with cardiac function in the acute myocardial infarction (AMI) heart. AMI was generated in Sprague-Dawley rats by ligation of the left anterior descending coronary artery. Cardiac function was determined using B­ultrasonography, AMI was verified using histopathology. The kinetics of collagen type I/III and elastic fibre were evaluated using immunohistochemistry and western blotting at 1 week (1 w), 2 weeks (2 w), 3 weeks (3 w) and 4 weeks (4 w) post­AMI. Cardiac function was decreased by 78, 70, 50 and 38% at weeks 1, 2, 3 or 4 post­AMI, respectively, compared with the normal heart. Elastic fibre was decreased gradually, demonstrating alterations of 2, 77, 86 or 97% reduction, respectively, at weeks 1, 2, 3 or 4 in the AMI heart. Collagen I fibre was increased 1.4­, 1.5­, 2.9­ or 3.9­fold at weeks 1, 2, 3 or 4 respectively, compared with the normal heart. Similarly, collagen III was increased 1.2­, 1.7­, 2.8­ or 3.9­fold, following AMI. Kinetics of increased collagen I/III, in combination with decreased elastic fibre in infarcted area following AMI, provided evidence that compromised cardiac function following AMI was due to graduate wound healing/scar formation in the infarcted zone, increased stiffness and reduced flexibility of the heart.

SYK protects cardiocytes against anoxia and hypoglycemia-induced injury in ischemic heart failure.

Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is reported to be related to cell survival after A/H (anoxia/hypoglycemia) insult. However, the role of SYK in cardiocyte survival under A/H injury remains unclear. In this study, we aimed to gain insight into the role and molecular mechanism of SYK in cardiocytes exposed to A/H stress. The mRNA and protein expressions of SYK in H9c2 cardiocytes exposed to A/H injury, separately detected by real-time quantitative PCR and Western blot, were both robustly up-regulated. Then we overexpressed SYK in H9c2 with A/H injury, and found that cell viability was significantly increased and LDH leakage was decreased. Moreover, apoptosis measured by annexin V-fluorescein isothiocyanate/propidium iodide and reactive oxygen species (ROS) identified by 2', 7'-dichlorofluorescin diacetate were markedly inhibited in H9c2 with A/H injury following SYK overexpression. Furthermore, we observed that SYK could induce HO-1 expression by regulating the Akt phosphorylation level in H9c2 with A/H injury, protecting H9c2 from the injury induced by A/H treatment.

Metabolic regulation of magnolol on the nuclear receptor, liver X receptor.

The aim of the present study was to investigate whether magnolol, the essential component of the traditional Chinese medicine, Magnolia officinalis, can pass through liver X receptor α (LXRα), to subsequently play an important role in the lipid metabolic balance. Using a HepG2 human hepatoma cell line, mammalian cellular one-hybridization and mammalian cell transcriptional activation experiments were performed to detect the combination degree of magnolol at different concentrations with LXRα, and assess the transcriptional activity. In addition, using a THP-1 human monocytic cell line, quantitative polymerase chain reaction was performed to assess the effect on the expression levels of downstream genes. Magnolol was shown to dose-dependently combine with LXRα, and subsequently regulate the transcriptional activity of LXRα. In addition, magnolol was found to adjust the expression of associated LXRα downstream genes in the macrophages. In conclusion, magnolol was demonstrated to affect LXRα, which may outline a new molecular mechanism through which magnolol exerts a lipid-lowering function.