Prevotella copri promotes vascular calcification via lipopolysaccharide through activation of NF-κB signaling pathway.

Emerging evidence indicates that alteration of gut microbiota plays an important role in chronic kidney disease (CKD)-related vascular calcification (VC). We aimed to investigate the specific gut microbiota and the underlying mechanism involved in CKD-VC. We identified an increased abundance of Prevotella copri (P. copri) in the feces of CKD rats (induced by using 5/6 nephrectomy followed by a high calcium and phosphate diet) with aortic calcification via amplicon sequencing of 16S rRNA genes. In patients with CKD, we further confirmed a positive correlation between abundance of P. copri and aortic calcification scores. Moreover, oral administration of live P. copri aggravated CKD-related VC and osteogenic differentiation of vascular smooth muscle cells in vivo, accompanied by intestinal destruction, enhanced expression of Toll-like receptor-4 (TLR4), and elevated lipopolysaccharide (LPS) levels. In vitro and ex vivo experiments consistently demonstrated that P. copri-derived LPS (Pc-LPS) accelerated high phosphate-induced VC and VSMC osteogenic differentiation. Mechanistically, Pc-LPS bound to TLR4, then activated the nuclear factor κB (NF-κB) and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome signals during VC. Inhibition of NF-κB reduced NLRP3 inflammasome and attenuated Pc-LPS-induced VSMC calcification. Our study clarifies a novel role of P. copri in CKD-related VC, by the mechanisms involving increased inflammation-regulating metabolites including Pc-LPS, and activation of the NF-κB/NLRP3 signaling pathway. These findings highlight P. copri and its-derived LPS as potential therapeutic targets for VC in CKD.

Fucoxanthin alleviated myocardial ischemia and reperfusion injury through inhibition of ferroptosis via the NRF2 signaling pathway.

Background: Myocardial ischemia and reperfusion injury (MIRI) is a severe complication of revascularization therapy in patients with myocardial infarction. Therefore, there is an urgent requirement to find more therapeutic solutions for MIRI. Recently, ferroptosis, which is characterized by lipid peroxidation, was considered a critical contributor to MIRI. Fucoxanthin (FX), a natural antioxidant carotenoid, which is abundant in brown seaweed, exerts protective effects under various pathological conditions. However, whether FX alleviates MIRI is unclear. This study aims to clarify the effects of FX on MIRI. Methods: Mice with left anterior descending artery ligation and reperfusion were used as in vivo models. Neonatal rat cardiomyocytes (NRCs) induced with hypoxia and reperfusion were used as in vitro models. TTC-Evans blue staining was performed to validate the infarction size. Transmission electron microscopy was employed to detect mitochondrial injury in cardiomyocytes. In addition, 4 weeks after MIRI, echocardiography was performed to measure cardiac function; fluorescent probes and western blots were used to detect ferroptosis. Results: TTC-Evans blue staining showed that FX reduced the infarction size induced by MIRI. Transmission electron microscopy showed that FX ameliorated the MIRI-induced myofibril loss and mitochondrion shrinkage. Furthermore, FX improved LVEF and LVFS and inhibited myocardial hypertrophy and fibrosis after 4 weeks in mice with MIRI. In the in vitro study, calcein AM/PI staining and TUNEL staining showed that FX reduced cell death caused by hypoxia and reperfusion treatment. DCFH-DA and MitoSOX probes indicated that FX inhibited cellular and mitochondrial reactive oxygen species (ROS). Moreover, C11-BODIPY 581/591 staining, ferro-orange staining, MDA assay, Fe2+ assay, 4-hydroxynonenal enzyme-linked immunosorbent assay, and western blot were performed and the results revealed that FX ameliorated ferroptosis in vitro and in vivo, as indicated by inhibiting lipid ROS and Fe2+ release, as well as by modulating ferroptosis hallmark FTH, TFRC, and GPX4 expression. Additionally, the protective effects of FX were eliminated by the NRF2 inhibitor brusatol, as observed from western blotting, C11-BODIPY 581/591 staining, and calcein AM/PI staining, indicating that FX exerted cardio-protective effects on MIRI through the NRF2 pathway. Conclusion: Our study showed that FX alleviated MIRI through the inhibition of ferroptosis via the NRF2 signaling pathway.

Guiding ablation strategies for ventricular tachycardia in patients with structural heart disease by analyzing links and conversion patterns of traceable abnormal late potential zone.

BACKGROUND: Substrate-based ablation can treat uninducible or hemodynamically instability scar-related ventricular tachycardia (VT). However, whether a correlation exists between the critical VT isthmus and late activation zone (LAZ) during sinus rhythm (SR) is unknown. OBJECTIVE: To demonstrate the structural and functional properties of abnormal substrates and analyze the link between the VT circuit and abnormal activity during SR. METHODS: Thirty-six patients with scar-related VT (age, 50.0 ± 13.7 years and 86.1% men) who underwent VT ablation were reviewed. The automatic rhythmia ultrahigh resolution mapping system was used for electroanatomic substrate mapping. The clinical characteristics and mapping findings, particularly the LAZ characteristics during SR and VT, were analyzed. To determine the association between the LAZ during the SR and VT circuits, the LAZ was defined as five activation patterns: entrance, exit, core, blind alley, and conduction barrier. RESULTS: Forty-five VTs were induced in 36 patients, 91.1% of which were monomorphic. The LAZ of all patients was mapped during the SR and VT circuits, and the consistency of the anatomical locations of the LAZ and VT circuits was analyzed. Using the ultrahigh resolution mapping system, interconversion patterns, including the bridge, T, puzzle, maze, and multilayer types, were identified. VT ablation enabled precise ablation of abnormal late potential conduction channels. CONCLUSION: Five interconversion patterns of the LAZ during the SR and VT circuits were summarized. These findings may help formulate more precise substrate-based ablation strategies for scar-related VT and shorter procedure times.

Amine oxidase copper-containing 3 aggravates cardiac remodelling by generating hydrogen peroxide after myocardial infarction.

Amine oxidase copper-containing 3 (AOC3) is a member of the semicarbazide-sensitive amine oxidase enzyme family. It acts as an ectoenzyme catalysing the oxidative deamination of primary amines and generating hydrogen peroxide (H2 O2 ). While AOC3 is implicated in cardiovascular diseases such as atherosclerosis, its role in cardiac remodelling after myocardial infarction (MI) is unclear. In this study, we first confirmed a long-term upregulation of AOC3 in both cardiac myofibroblasts after MI in vivo and angiotensin II (ANGII)-treated cardiac fibroblasts in vitro. AOC3 knockdown not only inhibited the activation of cardiac fibroblasts induced by ANGII but also alleviated cardiac fibrosis in mice after MI. Using sh-AOC3 lentiviruses, exogenous recombinant AOC3 (r-AOC3), semicarbazide (an AOC3 inhibitor), and catalase (a hydrogen peroxide scavenger) treatments, we also demonstrated that AOC3 promoted H2 O2 generation, increased oxidative stress, and enhanced ERK1/2 activation, which were responsible for the activation of cardiac fibroblasts. In particular, AOC3 knockdown also improved cardiac function and hypertrophy after MI. Through a coculture system, we confirmed that AOC3 expressed on cardiac myofibroblasts was able to enhance oxidative stress and induce hypertrophy of cardiomyocytes by promoting H2 O2 generation. Similarly, r-AOC3 promoted H2 O2 generation and resulted in oxidative stress and hypertrophy of cardiomyocytes, which were almost inhibited by both semicarbazide and catalase. In conclusion, AOC3 plays a critical role in cardiac fibrosis and hypertrophy after MI by promoting the generation of H2 O2 . AOC3 is a promising therapeutic target against cardiac remodelling. © 2023 The Pathological Society of Great Britain and Ireland.

Trimethylamine-N-oxide (TMAO) promotes balloon injury-induced neointimal hyperplasia via upregulating Beclin1 and impairing autophagic flux.

BACKGROUND AND AIMS: TMAO is a microbiota-dependent metabolite associated with increased risk of various cardiovascular diseases. However, the relationship between TMAO and vascular injury-related neointimal hyperplasia is unclear. This study aimed to explore whether TMAO promotes neointimal hyperplasia after balloon injury and elucidate the underlying mechanism. METHODS AND RESULTS: Through hematoxylin and eosin staining and immunohistochemistry staining, we found that supplementary TMAO promoted balloon injury-induced neointimal hyperplasia, while reducing TMAO by antibiotic administration produced the opposite result. TMAO showed limited effect on rat aortic vascular smooth muscle cells (RAOSMCs) proliferation and migration. However, TMAO notably induced dysfunction of rat aortic vascular endothelial cells (RAOECs) in vitro and attenuated reendothelialization of carotid arteries after balloon injury in vivo. Autophagic flux was measured by fluorescent mRFP-GFP-LC3, transmission electron microscopy, and western blot. TMAO impaired autophagic flux, as evidenced by the accumulation of p62 and LC3II and high autophagosome to autolysosome ratios. Furthermore, we confirmed that Beclin1 level increased in TMAO-treated RAOECs and carotid arteries. Knocking down Beclin1 alleviated TMAO-induced autophagic flux impairment and neointimal hyperplasia. CONCLUSIONS: TMAO promoted neointimal hyperplasia through Beclin1-induced autophagic flux blockage, suggesting that TMAO is a potential target for improvement of vascular remodeling after injury.

Elabela ameliorates doxorubicin-induced cardiotoxicity by promoting autophagic flux through TFEB pathway.

Doxorubicin (DOX) is a widely used and effective antineoplastic drug; however, its clinical application is limited by cardiotoxicity. A safe and effective strategy to prevent from doxorubicin-induced cardiotoxicity (DIC) is still beyond reach. Elabela (ELA), a new APJ ligand, has exerted cardioprotective effect against multiple cardiovascular diseases. Here, we asked whether ELA alleviates DIC. Mice were injected with DOX to established acute DIC. In vivo studies were assessed with echocardiography, serum cTnT and CK-MB, HW/BW ratio and WGA staining. Cell death and atrophy were measured by AM/PI staining and phalloidin staining respectively in vitro. Autophagic flux was monitored with Transmission electron microscopy in vivo, as well as LysoSensor and mRFP-GFP-LC3 puncta in vitro. Our results showed that ELA improved cardiac dysfunction in DIC mice. ELA administration also attenuated cell death and atrophy in DOX-challenged neonatal rat cardiomyocytes (NRCs). Additionally, we found that ELA restored DOX-induced autophagic flux blockage, which was evidenced by the reverse of p62 and LC3II, improvement of lysosome function and accelerated degradation of accumulated autolysosomes. Chloroquine, a classical autophagic flux inhibitor, blunted the improvement of ELA on cardiac dysfunction. At last, we revealed that ELA reversed DOX-induced downregulation of transcription factor EB (TFEB), and silencing TFEB by siRNA abrogated the effects of ELA on autophagic flux as well as cell death and atrophy in NRCs. In conclusion, this study indicated that ELA ameliorated DIC through enhancing autophagic flux via activating TFEB. ELA may become a potential target against DIC.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

Association Between Postoperative Long-Term Heart Rate Variability and Postoperative Delirium in Elderly Patients Undergoing Orthopedic Surgery: A Prospective Cohort Study.

BACKGROUND: Postoperative delirium (POD) is a common complication after orthopedic surgery in elderly patients. The elderly may experience drastic changes in autonomic nervous system (ANS) activity and circadian rhythm disorders after surgery. Therefore, we intend to explore the relationship between postoperative long-term heart rate (HR) variability (HRV), as a measure of ANS activity and circadian rhythm, and occurrence of POD in elderly patients. METHODS: The study population of this cohort was elderly patients over 60 years of age who scheduled for orthopedic surgery under spinal anesthesia. Patients were screened for inclusion and exclusion criteria before surgery. Then, participants were invited to wear a Holter monitor on the first postoperative day to collect 24-h electrocardiographic (ECG) data. Parameters in the time domain [the standard deviation of the normal-to-normal (NN) intervals (SDNN), mean of the standard deviations of all the NN intervals for each 5-min segment of a 24-h HRV recording (SDNNI), and the root mean square of successive differences of the NN intervals (RMSSD)] and frequency domain [heart rate (HR), high frequency (HF), low frequency (LF), very low frequency (VLF), ultra low frequency (ULF), and total power (TP)] were calculated. Assessment of delirium was performed daily up to the seventh postoperative day using the Chinese version of the 3-Min Diagnostic Interview for CAM-defined Delirium (3D-CAM). The relationship between HRV and POD, as well as the association between HRV and duration of POD, was assessed. RESULTS: Of the 294 cases that finally completed the follow-up, 60 cases developed POD. Among the HRV parameters, SDNNI, VLF, and ULF were related to the occurrence of POD. After adjustment for potential confounders, the correlation between HRV indices and POD disappeared. Through stratified analysis, two significant negative correlations emerged: ULF in young-old participants and SDNNI, VLF, and ULF in male patients. CONCLUSION: The lower HRV parameters may be related to the occurrence of POD, and this correlation is more significant in young-old and male patients. ANS disorders and rhythm abnormalities reflected by HRV changes may represent a possible mechanism that promotes POD.

Nkx2-3 induces autophagy inhibiting proliferation and migration of vascular smooth muscle cells via AMPK/mTOR signaling pathway.

Vascular remodeling and restenosis are common complications after percutaneous coronary intervention. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in intimal hyperplasia-induced vascular restenosis. NK2 Homeobox 3 (Nkx2-3), a critical member of Nkx family, is involved in tissue differentiation and organ development. However, the role of Nkx2-3 in VSMCs proliferation and migration remains unknown. In this study, we used carotid balloon injury model and platelet-derived growth factor-BB (PDGF)-treated VSMCs as in vivo and in vitro experimental models. EdU assay and CCK-8 assay were used to detect cell proliferation. Migration was measured by scratch test. Hematoxylin and eosin staining and immunohistochemistry staining were used to evaluate the intimal hyperplasia. The autophagy level was detected by fluorescent mRFP-GFP-LC3 in vitro and by transmission electron microscopy in vivo. It was shown that Nkx2-3 was upregulated both in balloon injured carotid arteries and PDGF-stimulated VSMCs. Adenovirus-mediated Nkx2-3 overexpression inhibited intimal hyperplasia after balloon injury, and suppressed VSMCs proliferation and migration induced by PDGF. Conversely, silencing of Nkx2-3 by small interfering RNA exaggerated proliferation and migration of VSMCs. Furthermore, we found that Nkx2-3 enhanced autophagy level, while the autophagy inhibitor 3-MA eliminated the inhibitory effect of Nkx2-3 on VSMCs proliferation and migration both in vivo and in vitro. Moreover, Nkx2-3 promoted autophagy in VSMCs by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. These results demonstrated for the first time that Nkx2-3 inhibited VSMCs proliferation and migration through AMPK/mTOR-mediated autophagy.

Persistence and outcomes of non-vitamin K antagonist oral anticoagulants versus warfarin in patients with non-valvular atrial fibrillation.

AIMS AND OBJECTIVES: To compare persistence and outcomes of non-vitamin K antagonist oral anticoagulants (NOACs) versus warfarin in Chinese patients with non-valvular atrial fibrillation (AF). BACKGROUND: Given the unpredictable warfarin response and the costliness of NOACs, more research is needed to clarify which drug enjoys better persistence and outcomes, helping to provide personalised care for patients. DESIGN: A prospective cohort study. METHODS: Chinese patients taking NOACs or warfarin from March 2016-April 2018 were followed up by telephone or outpatient visit at 3, 6 months and half a year thereafter. Anticoagulant persistence and outcomes including stroke and bleeding were collected. We used Cox regression to analyse data. This study was reported according to the STROBE guideline. RESULTS: A total of 344 patients were enrolled; 146 patients received NOACs including dabigatran and rivaroxaban, and 198 patients received warfarin. Persistence with anticoagulants was low and dropped sharply at the third month. Patients on NOACs had worse persistence at 3, 6 and 12 months than those on warfarin. There was no difference in the incidence of ischaemic stroke and bleeding between groups, although ischaemic stroke and major bleeding occurred less frequently in the NOACs group. Paroxysmal AF, no heart failure and no stroke were predictors of NOACs non-persistence. Prior catheter ablation and no diabetes were associated with poor persistence of warfarin. The main reason for anticoagulant cessation was patient preference. CONCLUSIONS: Chinese patients taking NOACs had lower persistence, similar rate of ischaemic stroke and bleeding compared with those on warfarin. Further inventions are needed to improve persistence in Chinese patients on NOACs. RELEVANCE TO CLINICAL PRACTICE: Anticoagulation should highlight both persistence and outcomes emphasising personalised care of different drugs. Further interventions to improve persistence should be developed based on causes and risk factors and carried out in the third month of therapy.

Traditional vs. genetic pathogenesis of arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), a predominantly familial and autosomal dominant inherited heart muscle disorder, is pathologically characterized by progressive right ventricular myocardial atrophy and fibrofatty replacement and clinically by ventricular arrhythmias with left bundle branch block morphology. Symptoms poorly reflect disease severity, with disease commonly first manifesting as sudden death among the young. The inflammatory and apoptotic theories first put forth to explain ARVC pathogenesis do not explain all cases, and advances in genetic technology have allowed to elucidate genetic mechanisms, with desmosomal mutations attracting much attention. As reviewed here, various non-mutually exclusive pathogenetic mechanisms therefore appear to underlie ARVC.

[Changes in serum level of carboxy-terminal telopeptide of type I collagen in patients with coronary heart disease].

OBJECTIVE: To investigate the serum level of carboxy-terminal telopeptide of type I collagen (ICTP) and explore its correlation with MMP-2 and MMP-9 in patients with coronary artery disease (CHD). METHODS: A total of 103 CHD patients treated in our hospital between October, 2013 and May, 2014 were enrolled, including 39 with stable angina pectoris (SAP), 39 with unstable angina (UA), and 25 with acute myocardial infarction (AMI), with 38 non-CHD volunteers as the control group. The serum levels of ICTP, MMP-2, and MMP-9 were detected in all the subjects using enzyme-linked immunosorbent assay (ELISA). RESULTS: No significant difference in serum levels of MMP-2, MMP-9, or ICTP was found between the control and SAP groups or between UA and AMI groups (P>0.05), but the latter two groups had significantly higher serum levels of MMP-2, MMP-9, and ICTP than the former two groups (P<0.05). Serum ICTP level was found to negatively correlated with the fibrotic area and positively with the lipid component in the plaques (P<0.05). Regression analysis revealed significant positive correlations of serum ICTP with MMP-2 and MMP-9 (P<0.05). CONCLUSION: An elevated serum ICTP level is indicative of the presence of unstable plaques in CHD patients. Serum ICTP is more strongly correlated with MMP-2 than with MMP-9, and can be used as a non-invasive marker for assessing vulnerable plaques in patients with acute coronary syndrome.