Many current animal models of heart failure are hampered by intrinsic methodological complexities, while other models yield only a subtle cardiac phenotype even after prolonged in vivo treatments. A new “chemogenetic” animal model of heart failure recapitulates a critical characteristic shared by many disease states that lead to heart failure in humans: an increase in redox stress in the heart. This “chemogenetic” approach exploits a recombinant yeast enzyme that can be dynamically and specifically activated in vivo to generate the reactive oxygen species (ROS) hydrogen peroxide (H2O2) in cardiac myocytes. Redox stress can be rapidly, selectively, and reversibly manipulated by chemogenetic generation of ROS in cardiac myocytes, yielding a new model of dilated cardiomyopathy. Treatment of animals with the angiotensin receptor blocker valsartan promotes recovery of ventricular function and resolution of adverse cardiac remodeling. This Mini-Review discusses in vivo chemogenetic approaches to manipulate and analyze oxidative stress in the heart.

Background and Purpose: Hypertensive vascular remodeling (VR) is responsible for end-organ damage and is the result of increased extracellular matrix accumulation and excessive vascular smooth muscle cell (VSMC) proliferation. MicroRNA-26a (miR-26a), a non-coding small RNA, is involved in multiple cardiovascular diseases. We aimed to validate the effect and mechanisms of miR-26a in hypertensive VR. Experimental Approach: Spontaneously hypertensive rats (SHRs) were injected intravenously with recombinant adeno-associated virus-miR-26a. In vitro experiments, angiotensin II (AngII)-induced VSMCs were transfected with miR-26a mimic or inhibitor. Key Results: We found miR-26a downregulated in the thoracic aorta and plasma of SHRs. Overexpression of miR-26a inhibited extracellular matrix deposition by targeting connective tissue growth factor (CTGF) and mitigated VSMC proliferation by regulating the enhancer of zeste homolog 2 (EZH2)/p21 pathway both in vitro and in vivo. AngII-mediated Smad3 activation suppressed miR-26a expression, which in turn promoted Smad3 activation via targeted regulation of Smad4, leading to further downregulation of miR-26a. Conclusion and Implications: Our study reveals that AngII stimulates a Smads/miR-26a positive feedback loop, which further reduces miR-26a expression, leading to collagen production and VSMC proliferation and consequently, VR. MiR-26a has an antagonistic effect on hypertensive VR and can be a strategy for treating hypertensive VR.

Background and Purpose Inactivation of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) by causing the accumulation of intracellular Ca2+ to activate calcineurin-mediated nuclear factor of activated T-lymphocytes (NFAT)/NF-κB pathways, resulted in the phenotypic modulation of smooth muscle cells (SMCs) to accelerate angiotensin II-induced aortic aneurysm. Our goal was to investigate the mechanism involved. Experimental Approach We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to represent partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis. KEY RESULTS Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. Overexpression of PPARγ2 inhibited the phenotypic modulation of SKI SMCs. Pioglitazone, the activator of PPARγ, blocked the activation of NFAT/NF-κB, inhibited SMC phenotypic modulation, and ameliorated angiotensin II-induced aortic aneurysms in SKI mice. CONCLUSIONS AND IMPLICATIONS The inactivation of SERCA2 C674 promotes the development of aortic aneurysm by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.

Targeting cancer metabolism has emerged as an attractive approach to improve therapeutic regimens in acute myeloid leukemia (AML). Mitochondrial proteases are closely related to cancer metabolism, but their biological functions have not been well characterized in AML. According to different catogory, we comprehensively reviewed the role of mitochondrial proteases in AML. This review highlights some ‘powerful’ mitochondrial protease targets, including their biological function, chemical modulators, and applicative prospect in AML.

Background and Purpose: The study evaluated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a non-animal new approach methodology (NAM) for assessing cardiovascular repolarization risk in drug development. The research compared hiPSC-CM data with traditional methods (hERG assays and animal studies), to determine if hiPSC-CMs could potentially replace or reduce animal testing in regulatory decision-making, aligning with FDA Modernization Act 2.0 goals. Experimental Approach: The study analyzed 29 Investigational New Drug (IND) applications submitted to FDA/CDER between 2020 and 2023, comparing hiPSC-CM studies using multielectrode array (MEA) and voltage sensing optical (VSO) platforms with hERG assays, multi-channel assessments, and in vivo non-rodent QT studies. The analysis focused on concordance between these methods and their ability to predict clinical QT prolongation risk. Key Results: hiPSC-CM studies demonstrated higher overall accuracy in predicting clinical QT prolongation risk compared to standard hERG and in vivo non-rodent QT studies alone. Notably, to achieve comparable accuracy to hiPSC-CMs, additional animal studies beyond the standard in vivo QT assessment were needed. These additional studies included testing at higher exposures, using different animal species, or conducting repeated dose studies. The standard in vivo QT study had lower sensitivity, likely due to limitations in achieving high drug exposures in animals. hiPSC-CMs could evaluate effects at much higher exposure multiples relative to clinical concentrations. Conclusion and Implications: hiPSC-CMs offer a promising alternative to extensive animal testing for cardiovascular risk assessment. However, limitations include small sample size, early-phase INDs, variability in hiPSC-CM study rigor, and challenges in establishing exposure-response relationships. Despite these limitations, this research provides evidence supporting hiPSC-CMs as potential alternatives to animal studies in regulatory assessments. Further research is needed to establish robust protocols for integrating hiPSC-CM data into regulatory workflows, balancing the promise of reduced animal testing with the need for comprehensive safety assessment.

Background and Purpose: Mesenchymal stem cells (MSCs) are widely utilized in regenerative medicine due to their multipotency and immunomodulatory properties. Compared to conventional two-dimensional (2D) monolayer cultures, three-dimensional (3D) spheroid cultures better mimic the in vivo microenvironment, influencing the metabolic activity and therapeutic efficacy of MSCs. This study aims to evaluate how 2D and 3D culture conditions affect the behavior, proliferation, and functional properties of MSCs. Experimental Approach: Metabolomic and transcriptomic analyses were conducted on MSCs cultured under both 2D and 3D conditions. To assess metabolic differences between 2D and 3D cultured MSCs, polar metabolites were extracted and analyzed using ¹H-NMR spectroscopy. The data was processed with Chenomx and subjected to multivariate statistical analysis. For transcriptomic analysis, RNA sequencing was performed, followed by differential gene expression and gene set enrichment analysis. Key Results: The findings reveal that MSCs in 3D spheroids exhibit reduced proliferation, enhanced stemness, and distinct metabolic adaptations, including increased glycolysis and altered nutrient metabolism. Additionally, genes associated with ribosome biogenesis and cell cycle progression were downregulated in 3D MSCs. These changes promote a more quiescent state, favoring its applications on tissue repair and immune modulation. Conclusion and Implications: Understanding these metabolic adaptations offers valuable insights for optimizing culture conditions, improving MSC-based therapies, and identifying novel therapeutic targets and biomarkers.

Background: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients which in emergency situations requires platelet transfusion although there is ongoing debate on its effectiveness following ticagrelor therapy. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12R. Methods: Studies were performed in human platelets with both P2Y12R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were also undertaken to assess G protein subunit activation downstream of P2Y12R activation. Results: Initial studies revealed a range of P2Y12R ligands including ticagrelor displayed inverse agonist activity at the P2Y12R. Of these only ticagrelor was resistant to wash-out. In both human platelets and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12R function in contrast to other P2Y12R antagonists. The P2Y12R agonist 2MeSADP, which was also resistant to wash-out, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12R than other P2Y12R ligands. Conclusion: Ticagrelor binding to the P2Y12R is prolonged and more akin to that of an irreversible antagonist especially versus the endogenous P2Y12R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding and bleeding associated with urgent invasive procedures.

Background and Purpose: Mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs) and corticotropin-releasing factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN) are implicated in the stress response. The present study investigated the role of GRs and MRs in the PVN in regulating depressive and anxiety-like behaviors. Experimental Approach: To model chronic stress, rats were exposed to chronic corticosterone treatment via drinking water for 21 days, and the GR antagonist RU486 and MR antagonist spironolactone, alone and combined, were directly injected in the PVN daily for 7 days before the behavioral tests. Depressive- and anxiety-like behaviors were evaluated in forced swim test, sucrose preference test, novelty-suppressed feeding test and social interaction test. The expression of GRs, MRs and CRF were detected by Western-Blot. Key Results: The rats exposed to corticosterone exhibited depressive- and anxiety-like behaviors. The expression of GRs and MRs decreased, and CRF levels increased in the PVN. The intra-PVN administration of RU486 increased the levels of GRs and CRF without influencing depressive- or anxiety-like behaviors. The spironolactone-treated group exhibited an increase in MRs without influencing GRs and CRF in the PVN, and improved anxiety-like behaviors. Interestingly, the intra-PVN administration of RU486 and spironolactone combined restored the expression of GRs, MRs, and CRF and improved depressive- and anxiety-like behaviors. Conclusion and Implications: These results suggest that the simultaneous restoration of GRs, MRs, and CRF in the PVN in this rat model of stress might play an important role in the treatment of depression and anxiety.

Background and purpose: The pathophysiology of coronary artery spasm ( CAS), with its associated ischaemic crises, is currently poorly understood, and treatment is frequently ineffective. In view of increasing evidence that platelet- platelet based defects may occur in CAS patients,. we investigated platelet reactivity in CAS patients and whether symptomatic crises reflect activation of platelet-endothelial interactions. Experimental approach: CAS patients were evaluated during acute and/or chronic symptomatic phases, and compared with healthy control subjects. Inhibition of platelet aggregation with ADP by the nitric oxide (NO) donor sodium nitroprusside (SNP), . and plasma levels of syndecan-1 (glycocalyx shedding marker), tryptase (mast cell activation marker), and platelet microparticles were measured. Key Results: Inhibition of aggregation by SNP was impaired in chronic CAS, and tended to deteriorate further during symptomatic crises, while plasma levels of syndecan-1, tryptase and platelet microparticles increased. Infusion of high dose N-acetylcysteine (NAC) plus glyceryl trinitrate rapidly restored platelet responsiveness to SNP and decreased plasma syndecan-1 levels. The effect of NAC on platelet responsiveness to SNP was mimicked in vitro by the H2S donor NaHS. Conversely, inhibition of enzymatic release of H2S attenuated NAC effect. Conclusion and Implications: CAS is associated with substantial impairment of platelet NO signaling. During acute symptomatic exacerbations, platelet resistance to NO is aggravated, together with mast cell activation and damage to both vasculature and platelets. NAC reverses platelet resistance to NO via release of H2S, and reverses glycocalyx shedding during symptomatic crises: this suggests that H2S donors may correct the pathophysiological anomalies underlying CAS.

Cholesterol (Chol) and oxysterol sulfates are important regulators of lipid metabolism, inflammation, cell apoptosis, and cell survival. Among the sulfate-based lipids, cholesterol sulfate (CS) is the most studied lipid both quantitatively and functionally. Despite the importance, very few studies have analysed and linked the actions of oxysterol sulfates to their physiological roles. Over expression of sulfotransferases confirmed the formation of a range of oxysterol sulfates and their antagonistic effects on liver X receptors (LXRs). It is therefore important to understand how further changes to oxysterol/oxysterol sulfate homeostasis can contribute to LXR activity in the physiological milieu. Here, we aim to bring together evidences for novel roles of oxysterol sulfates, the available techniques and the challenges for analysing them. Understanding the oxysterol/oxysterol sulfate levels and their physiological mechanisms could lead to new therapeutic targets for metabolic diseases.

Background and Purpose: Diabetic nephropathy is one of the most common complications that is related to high morbidity and mortality in type 2 diabetic patients. We investigated ability of a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase inhibitor and as a cyclooxygenase-2 inhibitor against diabetic nephropathy. Experimental Approach: Sixteen-week-old type 2 diabetic and proteinuric obese ZSF1 rats were orally treated with vehicle, PTUPB, or enalapril for 8 weeks. Key Results: PTUPB alleviated diabetic nephropathy in obese ZSF1 rats by reducing albuminuria by 50%, renal tubular cast formation by 60-70%, renal fibrosis by 40-50%, glomerular injury by 55% and preserved glomerular nephrin expression. Enalapril demonstrated comparable effects and alleviated diabetic nephropathy in obese ZSF1 rats by reducing all kidney injury parameters by 30 to 50%. Diabetic renal injury in obese ZSF1 rats was accompanied by renal inflammation with 6-7-fold higher urinary MCP-1 level and renal infiltration of CD-68 positive cells. PTUPB and enalapril reduced renal inflammation but PTUPB demonstrated superior anti-inflammatory actions than enalapril. Obese ZSF1 rats were also hypertensive, hyperlipidemic, and exhibited liver injury. Interestingly, PTUPB but not enalapril decreased hyperlipidemia and liver injury in Obese ZSF1 rats. Conclusion and Implication: Overall, we demonstrate that a dual modulator PTUPB does not treat hyperglycemia, but can effectively alleviate hypertension, diabetic nephropathy, hyperlipidemia, and liver injury in type 2 diabetic rats. Therefore, we suggest that PTUPB has promising potential to be developed as a novel therapy for type 2 diabetic nephropathy and other complications.

Background and Purpose: Our previous study reported that erythroferrone (ERFE), a newly identified hormone produced by erythroblasts, responded to recombinant human erythropoietin (rHuEPO) sensitively but its dynamics was complicated by double peaks and circadian rhythm. This study intends to elucidate the underlying mechanisms for the double peaks of ERFE dynamics, and further determine whether early ERFE measurements can predict hemoglobin (HGB) responses to rHuEPO. Experimental Approach: By expressing recombinant rat ERFE protein and investigating its deposition in rats, the production of ERFE was deconvoluted. To explore the role of iron in ERFE production, we monitored short-term changes of iron status after injection of rHuEPO or deferiprone (DFP). Pharmacokinetic/pharmacodynamic (PK/PD) modelling was used to confirm the mechanisms and examine the predictive ability of ERFE for long-term HGB responses. Key Results: The rRatERFE protein was successfully expressed and purified. The production of ERFE was deconvoluted and showed two independent peaks (2 h and 8 h). Transient iron decrease was observed at 4h after rHuEPO injection and DFP induced significant increases of ERFE. Based on this mechanism, the PK/PD model could characterize the complex dynamics of ERFE. In addition, the model predictions further revealed a stronger correlation between ERFE and HGB peak values than that for observed values. Conclusions and Implications: The complex dynamics of ERFE should be composited by an immediate release and transient iron deficiency-mediated secondary production of ERFE. The early peak values of ERFE, which occur within a few hours, can predict HGB responses several weeks after ESA treatment.

Background and Purpose: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. Methods and results: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (eIF4G2) mRNA and eIF4G2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA upregulated the expression of eIF4G2 mRNA and eIF4G2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of eIF4G2 gene and triggered its transcriptional activity. Conclusion and Implications: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering eIF4G2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Background and Purpose: Hexokinase 2 (HK2) is a key enzyme linked to high tumor cell proliferation. Its inhibitors such as 3-bromopyruvic acid (3-BP) induce cancer cell death, highlighting HK2 modulation as potential anti-cancer treatment. However, standard chemotherapies often cause the emergence of cellular senescence, which goes along with cell metabolic reprogramming and treatment failure. This study explores whether targeting HK2 can induce cancer cell senescence and whether metabolic changes in senescent cancer cells are tied to the cellular HK2 status. Experimental Approach: The expression of hexokinase 1 (HK1) and HK2 was assessed using immunoblot and immunofluorescence analysis in cell lines and in primary murine breast cancer (BC) cells. The senescence inducing potential of HK2 inhibition, and the effect of chemotherapy-induced senescence on HK1 and HK2 expression was assessed. Cell-based approaches were complemented by analyzing single-cell RNA sequencing data from BC patients. Key Results: BC cell sensitivity to HK2 inhibition did not correlate with HK2 expression levels. Consistently, senescence was linked to a decrease in HK2, but an increase in HK1 expression. Moreover, genetic knockdown of HK2 induced senescence, indicating that a change in the HK2/HK1 ratio drives, rather than results from, cellular senescence. This shift in HK2/HK1 ratio was confirmed in single-cell RNA sequencing data of BC biopsies. Conclusion and Implications: Expressional shifts in the HK2/HK1 ratio may serve as a novel marker for BC cell senescence. While targeting HK2 shows promise in untreated cancers, senescence-inducing anti-cancer therapies may limit the effectiveness of HK2-targeted treatments in pre-treated cancer patients.

Background and Purpose: The activation of the defense reaction inhibits the baroreflex response through the B3 and nucleus tractus solitarius (NTS) regions. Our aim was to determine whether and how baroreflex inhibition induced by the disinhibition of the rostral cuneiform nucleus, part of the defense pathway, involves serotonin cells in B3 and 5-HT3 receptors in the NTS. Experimental Approach: We performed immunohistochemistry and anatomical experiments to determine whether raphe serotonin cells expressing Fos were directly targeted by the rostral cuneiform nucleus. The effect of blocking raphe serotonin transmission and NTS 5-HT3 receptors, on cuneiform-induced inhibition of the baroreflex cardiac response, were also analyzed. Key Results: Bicuculline microinjected into the rostral cuneiform nucleus induced an increase of double labeled Fos-5-HT IR cells in both the LPGi and Raphe Magnus. The anterograde tracer Phaseolus vulgaris leucoaggutinin into the rostral cuneiform nucleus revealed a dense projection to the LPGi but not Raphe Magnus. Cuneiform-induced baroreflex inhibition was prevented by B3 injection of 8-OH-DPAT, a specific agonist for 5-HT1A receptors. Cuneiform disinhibition also failed to inhibit the baroreflex bradycardia after microinjection of a 5-HT3 receptor antagonist (granisetron) into the NTS or in 5-HT3 receptor knock-out mice. Conclusion and Implications: In conclusion, the rostral cuneiform nucleus participates in the defense inhibition of the baroreflex bradycardia via direct activation of the LPGi and a relay to the Raphe Magnus, to activate NTS 5-HT3 receptors and inhibit second-order baroreflex neurons. These data bring new insights in primary and secondary mechanisms involved in vital baroreflex prevention during stress.

Background and Purpose Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuro-inflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Experimental Approach Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcome were assessed. Abundance of target genes, proteins and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting and immunofluorescence. Key Results Stroke upregulates PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduces infarct size, apoptotic markers, macrophage accumulation and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Conclusions and Implications Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Editorial. Platelet purinergic receptors and non-thrombotic diseases.Simon C. Pitchford1* and Dingxin Pan.11Pulmonary Pharmacology Unit, Institute of Pharmaceutical Science, King’s College London, London, UK.*Author for correspondence and reprint requests:Dr Simon PitchfordPulmonary Pharmacology UnitInstitute of Pharmaceutical Science5.43 Franklin Wilkins Building150 Stamford StreetWaterloo CampusKing’s College LondonLondon UKSE1 9NHPhone: +44 2078484266Fax: +44 207 8484788Simon.pitchford@kcl.ac.uk

Background and Purpose: Chronic pain is a devastating problem affecting 1 in 5 individuals around the globe, with neuropathic pain the most debilitating and poorly treated type of chronic pain. Advances in transcriptomics and data mining have contributed to cataloging diverse cellular pathways and transcriptomic alterations in response to peripheral nerve injury but have focused on phenomenology and classifying transcriptomic responses. Experimental approach: Here, with the goal of identifying new types of pain-relieving agents, we compared transcriptional reprogramming changes in the dorsal spinal cord after peripheral nerve injury cross-sex and cross-species and imputed commonalities, as well as differences in cellular pathways and gene regulation. Key Results: We identified 93 transcripts in the dorsal horn that were increased by peripheral nerve injury in male and female mice and rats. Following gene ontology and transcription factor analyses, we constructed a pain interactome for the proteins encoded by the differentially expressed genes, discovering new, conserved signaling nodes. We interrogated the interactome with the Drug-Gene database to predict FDA-approved medications that may modulate key nodes within the network. The top hit from the analysis was fostamatinib, the molecular target of which is the non-receptor tyrosine kinase Syk, which our analysis had identified as a key node in the interactome. Conclusions & Implications : We found that intrathecally administrating the active metabolite of fostamatinib, R406, significantly reversed pain hypersensitivity in both sexes. Thus, we have identified and shown the efficacy of an agent that could not have been previously predicted to have analgesic properties.