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Ferroptosis is a new form of cell death characterized by iron-dependent lipid peroxidation. Whether ferroptosis is involved in retinal microvascular dysfunction under diabetic condition is not known. Herein, the expression of ferroptosis-related genes in patients with proliferative diabetic retinopathy and in diabetic mice was determined with quantitative RT-PCR. Reactive oxygen species, iron content, lipid peroxidation products, and ferroptosis-associated proteins in the cultured human retinal microvascular endothelial cells (HRMECs) and in the retina of diabetic mice were examined. The association of ferroptosis with the functions of endothelial cells in vitro was evaluated. After administration of ferroptosis-specific inhibitor, Fer-1, the retinal microvasculature in diabetic mice was assessed. Characteristic changes of ferroptosis-associated markers, including glutathione peroxidase 4, ferritin heavy chain 1, long-chain acyl-CoA synthetase 4, transferrin receptor protein 1, and cyclooxygenase-2, were detected in the retinal fibrovascular membrane of patients with proliferative diabetic retinopathy, cultured HRMECs, and the retina of diabetic mice. Elevated levels of reactive oxygen species, lipid peroxidation, and iron content were found in the retina of diabetic mice and in cultured HRMECs. Ferroptosis was found to be associated with HRMEC dysfunction under high-glucose condition. Inhibition of ferroptosis with specific inhibitor Fer-1 in diabetic mice significantly reduced the severity of retinal microvasculopathy. Ferroptosis contributes to microvascular dysfunction in diabetic retinopathy, and inhibition of ferroptosis might be a promising strategy for the therapy of early-stage diabetic retinopathy., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: This is the first randomised controlled trial for assessment of the immunogenicity and safety of a candidate non-replicating adenovirus type-5 (Ad5)-vectored COVID-19 vaccine, aiming to determine an appropriate dose of the candidate vaccine for an efficacy study., Methods: This randomised, double-blind, placebo-controlled, phase 2 trial of the Ad5-vectored COVID-19 vaccine was done in a single centre in Wuhan, China. Healthy adults aged 18 years or older, who were HIV-negative and previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-free, were eligible to participate and were randomly assigned to receive the vaccine at a dose of 1 × 10 11 viral particles per mL or 5 × 10 10 viral particles per mL, or placebo. Investigators allocated participants at a ratio of 2:1:1 to receive a single injection intramuscularly in the arm. The randomisation list (block size 4) was generated by an independent statistician. Participants, investigators, and staff undertaking laboratory analyses were masked to group allocation. The primary endpoints for immunogenicity were the geometric mean titres (GMTs) of specific ELISA antibody responses to the receptor binding domain (RBD) and neutralising antibody responses at day 28. The primary endpoint for safety evaluation was the incidence of adverse reactions within 14 days. All recruited participants who received at least one dose were included in the primary and safety analyses. This study is registered with ClinicalTrials.gov, NCT04341389., Findings: 603 volunteers were recruited and screened for eligibility between April 11 and 16, 2020. 508 eligible participants (50% male; mean age 39·7 years, SD 12·5) consented to participate in the trial and were randomly assigned to receive the vaccine (1 × 10 11 viral particles n=253; 5 × 10 10 viral particles n=129) or placebo (n=126). In the 1 × 10 11 and 5 × 10 10 viral particles dose groups, the RBD-specific ELISA antibodies peaked at 656·5 (95% CI 575·2-749·2) and 571·0 (467·6-697·3), with seroconversion rates at 96% (95% CI 93-98) and 97% (92-99), respectively, at day 28. Both doses of the vaccine induced significant neutralising antibody responses to live SARS-CoV-2, with GMTs of 19·5 (95% CI 16·8-22·7) and 18·3 (14·4-23·3) in participants receiving 1 × 10 11 and 5 × 10 10 viral particles, respectively. Specific interferon γ enzyme-linked immunospot assay responses post vaccination were observed in 227 (90%, 95% CI 85-93) of 253 and 113 (88%, 81-92) of 129 participants in the 1 × 10 11 and 5 × 10 10 viral particles dose groups, respectively. Solicited adverse reactions were reported by 183 (72%) of 253 and 96 (74%) of 129 participants in the 1 × 10 11 and 5 × 10 10 viral particles dose groups, respectively. Severe adverse reactions were reported by 24 (9%) participants in the 1 × 10 11 viral particles dose group and one (1%) participant in the 5 × 10 10 viral particles dose group. No serious adverse reactions were documented., Interpretation: The Ad5-vectored COVID-19 vaccine at 5 × 10 10 viral particles is safe, and induced significant immune responses in the majority of recipients after a single immunisation., Funding: National Key R&D Programme of China, National Science and Technology Major Project, and CanSino Biologics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The incidence of atrial fibrillation (AF) is correlated with decreased levels of testosterone in elderly men. Late sodium current may exert a role in AF pathogenesis., Objective: The purpose of this study was to explore the effect of testosterone deficiency on AF susceptibility and the therapeutic effect of late sodium current inhibitors in mice., Methods: Male ICR mice (5 weeks old) were castrated to establish a testosterone deficiency model. One month after castration, dihydrotestosterone 5 mg/kg was administered subcutaneously for 2 months. Serum total testosterone level was assessed by enzyme-linked immunosorbent assay. High-frequency electrical stimulation was used to induce atrial arrhythmias. Whole-cell patch-clamp technique was used to for single-cell electrophysiologic study., Results: Serum dihydrotestosterone levels of castration mice declined significantly but recovered with administration of exogenous dihydrotestosterone. In comparison with sham mice, the number of AF episodes significantly increased by 13.5-fold, AF rate increased by 3.75-fold, and AF duration prolonged in castrated mice. Dihydrotestosterone administration alleviated the occurrence of AF. Action potential duration at both 50% and 90% repolarization were markedly increased in castrated mice compared to sham controls. The late sodium current was enhanced in castrated male mice. These alterations were alleviated by treatment with dihydrotestosterone. Systemic application of the I Na-L inhibitors ranolazine, eleclazine, and GS967 inhibited the occurrence of AF in castrated mice., Conclusion: Testosterone deficiency contributed to the increased late sodium current, prolonged action potential repolarization, and increased susceptibility to AF. Blocking of late sodium current is beneficial against the occurrence of AF in castrated mice., (Copyright © 2017. Published by Elsevier Inc.)

As an ancient therapeutic method, acupuncture has been used to treat many diseases as an adjunctive therapy. However, its clinical efficacy remains controversial and the neural mechanisms have not been well understood. Accumulating studies have revealed that fMRI has made it possible to study brain responses to acupuncture. This review aims to provide scientific evidence to support the notion and discuss how these findings contribute to the neural mechanisms of acupuncture., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Previous studies suggested that platelet-activating factor (PAF) plays an important role in ischemic diseases. Apoptosis has been implicated in myocardial infarction-related cell death. The present study was designed to determine whether PAF could induce apoptosis in cardiac myocytes and the underlying mechanisms by which PAF causes apoptosis., Methods: H9c2 cardiac myocytes were used to investigate the effect of PAF on intracellular calcium concentration, cell viability and cell apoptosis. Signaling pathway of caspase-3, cytochrome c and MAPK (ERK, JNK, p38) was determined during the PAF induced apoptosis., Results: First, our results showed that treatment of H9c2 cardiomyocytes with PAF (0.2 to 20 microM) caused apoptosis in these cells and the apoptotic process was suppressed by either BN52021 (an antagonist of PAF receptor) or BAPTA/AM (an intracellular Ca2+ chelator), suggesting an involvement of PAF and its receptor mediated calcium-dependent signaling. Second, we found that activity of p38-MAPK (mitogen-activated protein kinase) and caspase-3 was elevated in the cells treated with PAF, without altering activity of ERK and JNK, and that PAF-induced enhancement of caspase-3 activity was attenuated by application of either BAPTA/AM or SB203580 (p38 inhibitor). Furthermore, PAF-induced apoptosis and release of cytochrome c from mitochondria was blunted by SB203580, and PAF-induced enhancement of p38 activity was also attenuated by BAPTA/AM., Conclusion: Our data implicate that a PAF and its receptor in triggering apoptosis occurs in cultured H9c2 cardiac myocytes via a calcium-dependent p38 MAPK activated cytochrome c/caspase-3 apoptosis signaling pathway., (Crown Copyright (c) 2009. Published by Elsevier Ireland Ltd. All rights reserved.)

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca(2+) stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca(2+) imaging, we found that the depletion of ER/SR Ca(2+) stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca(2+) ([Ca(2+)](i)), followed by sustained increase depending on extracellular Ca(2+). But, TRP channels inhibitor (SKF96365), not L-type channels or the Na(+)/Ca(2+) exchanger inhibitors, inhibited [Ca(2+)](i) relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl(3)) or by an increased extracellular Ca(2+)([Ca(2+)](o)) increased the concentration of intracelluar Ca(2+), whereas, the sustained elevation of [Ca(2+)](i) was reduced in the presence of SKF96365. Similarly, the duration of [Ca(2+)](i) increase was also shortened in the absence of extracellular Ca(2+). Western blot analysis showed that GdCl(3) increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl(3). Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca(2+)-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis., (2010 Elsevier Inc. All rights reserved.)

Objectives:   The analgesic effect of electroacupuncture (EA) stimulation has been proved. However, its mechanism of action is not clear. It has been well-known that cholecystokinin-8 (CCK-8) is a neuropeptide which is mainly related to the mediation of pain. The caudate nucleus was selected to determine if the release of CCK and the neural activity in this nucleus were involved in producing EA analgesia., Materials and Methods:   Radiant heat focused on the rat-tail was used as the noxious stimulus. The pain threshold of rats was measured by tail-flick latency (TFL). EA stimulation at the bilateral Zusanli (ST 36) acupoints of rats was used to investigate the effects of EA analgesia. The electrical activities of pain-excited neurons (PEN) and pain-inhibited neurons (PIN) in the caudate nucleus were recorded with a glass microelectrode. The present study examined the antagonistic effects of the intracerebral ventricular injection of CCK-8 on EA analgesia and reversing effects of CCK-B receptor antagonist (L-365,260) injection into the caudate nucleus on CCK-8., Results:   The radiant heat focused on the tail of rats caused an increase in the evoked discharge of PEN and a reduction in the evoked discharge of PIN. EA stimulation at the bilateral ST 36 acupoints of rats resulted in the inhibition of PEN, the potentiation of PIN, and prolongation of TFL. The analgesic effect of EA was antagonized when CCK-8 was injected into the intracerebral ventricle of rats. The antagonistic effect of CCK-8 on EA analgesia was reversed by injection of CCK-B receptor antagonist (L-365,260) into the caudate nucleus of rats., Conclusions:   Our results suggest that CCK-8 antagonize EA analgesia through its B receptor., (© 2009 International Neuromodulation Society.)

It was previously reported that excessive arsenic trioxide would produce cardiovascular toxicity. Bone marrow mesenchymal stem cells (BMSCs) have been shown to play a supporting role in cardiovascular functions. The increasing apoptosis of BMSCs commonly would promote the development of cardiovascular diseases. Thus we hypothesize that arsenic trioxide caused apoptosis in BMSCs, which provided a better understanding of arsenic toxicity in hearts. The present study was designed to investigate the proapoptotic effects of arsenic trioxide on BMSCs and explore the mechanism underlying arsenic trioxide-induced BMSCs apoptosis. We demonstrate that arsenic trioxide significantly inhibited survival ratios of BMSCs in a concentration-dependent and time-dependent manner. The Annexin V/PI staining and terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling (TUNEL) assay also showed that arsenic trioxide markedly induced the apoptosis of BMSCs. The caspase-3 activity was obviously enhanced in the presence of arsenic trioxide in a concentration-dependent manner in BMSCs. Additionally, arsenic trioxide caused the increase of intracellular free calcium ([Ca(2+)](i)) in rat BMSCs. BAPTA pretreatment may attenuate the apoptosis of BMSCs induced by arsenic trioxide. Taken together, arsenic trioxide could inhibit the proliferation and induce the apoptosis of BMSCs by modulating intracellular [Ca(2+)](i), and activating the caspase-3 activity., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The present study was conducted to identify effective factors that could induce differentiation of pancreatic ductal epithelial cells (PDECs) into insulin-producing cells. We examined the effect of hepatocyte growth factor (HGF) and betacellulin-delta4 (BTC-delta4) on PDECs. We isolated PDECs from adult rats and stimulated them with HGF and BTC-delta4 for 28days. We observed several characteristics of PDECs after induction, including functional changes in PDECs, differentiation rate, and expression levels of critical transcription factors. We observed that insulin content and glucose-induced insulin secretion were significantly higher in induced PDECs compared to control PDECs. The expression of markers specific to mature beta-cells, i.e., insulin and glucose transporter-2 (GLUT2) was also observed. An analysis of the expression of different transcription factors (PDX-1, Ngn3, NeuroD, and Pax4) revealed that cell-specific gene expression patterns varied from 7 to 28days after treatment. Hence, we conclude that treatment with a combination of HGF and BTC-delta4 is effective in inducing the differentiation of PDECs into insulin-secreting cells.

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophy remains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release of intracellular calcium. Moreover, Ca(2+)-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy. Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects of CaSR activated by GdCl(3) on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophy and up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca(2+)](i)) and activated CaN hypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl(3). But the effects of GdCl(3) on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggest that CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.

Protein kinase C (PKC) plays a role in cardioprotection through reduction of intracellular Ca(2+) concentration [Ca(2+)](i) during ischemic preconditioning (IPC). Cardioprotection against ischemic post-conditioning (PC) could be associated with reduced [Ca(2+)](i) through PKC. The calcium-sensing receptor (CaR), G protein-coupled receptor, causes accumulation of inositol phosphate (IP) to increase the release of intracellular Ca(2+). However, this phenomenon can be negatively regulated by PKC through phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that the prevention of cardiomyocyte damage by PC is associated with [Ca(2+)](i) reduction through an interaction of PKC with the CaR. Isolated rat hearts were subjected to 40min of ischemia followed by 90min of reperfusion. The hearts were post-conditioned after the 40min of ischemia by three cycles of 30s of reperfusion and 30s of re-ischemia applied before the 90min of reperfusion. Immunolocalization of PKCepsilon in the cell membrane was observed with IPC and PC, and in hearts exposed to GdCl(3) during PC. CaR was expressed in cardiac cell membrane and interacted with PKC in IPC, PC, and exposure to GdCl(3) during PC groups. On laser confocal microscopy, intracellular Ca(2+) was significantly decreased with IPC, PC, and exposure to GdCl(3) during PC compared with the I/R and PKC inhibitor groups, and cell structure was better preserved and promoted the recovery of cardiac function after reperfusion in the same groups. These results suggested that PKC is involved in cardioprotection against PC through negative feedback of a CaR-mediated reduction in [Ca(2+)](i).

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.

Volume-sensitive outwardly rectifying (VSOR) Cl- channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl- channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl- channels in oxidative stress-induced mesangial cell apoptosis. H2O2-induced Cl- currents showed phenotypic properties of VSOR Cl- channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl- channel blockers. Moreover, blockage of VSOR Cl- channels by DIDS (100 microM), NPPB (10 microM) or niflumic acid (10 microM) rescued mesangial cell apoptosis induced by H2O2. Treatment with 150 microM H2O2 for 2h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl- channel blockers. We conclude that VSOR Cl- channels are involved in H2O2-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.