Pharmacological Research

BackgroundAtherosclerosis is driven by macrophage foam cell formation resulting from excessive oxidized low-density lipoprotein (oxLDL) accumulation and chronic vascular inflammation. This study evaluated the therapeutic potential of Aegeline, Atorvastatin, and their combined in mitigating oxLDL-induced inflammatory responses, cholesterol accumulation, and oxLDL uptake in human THP-1 macrophages. MethodsTHP-1 monocytes were differentiated into macrophages using a 72-hour differentiation protocol followed by a 48-hour resting period, confirmed via CD14 surface marker characterization. Macrophages were exposed to DiI-oxLDL and treated with Aegeline, Atorvastatin, or their combination. Key inflammatory cytokines and chemokines (CRP, TNF-, IL-6, and IL-8) were measured using ELISA. Cholesterol efflux capacity and cellular oxLDL uptake were quantitatively assessed using fluorescence retention assays and immunofluorescence imaging. ResultsDifferentiation of THP-1 monocytes to macrophages resulted in marked down-regulation of CD14 expression. DiI-oxLDL exposure triggered significant pro-inflammatory mediator secretion (p<0.001) and excessive intracellular cholesterol accumulation. Single-agent treatment with Aegeline or Atorvastatin significantly attenuated oxLDL-induced elevations of CRP, TNF-, IL-6, and IL-8. Atorvastatin alone strongly suppressed CRP expression back to physiological baseline levels (p=ns vs. control). Notably, the combination of Aegeline and Atorvastatin demonstrated enhanced, broad-spectrum anti-inflammatory efficacy, achieving superior suppression of TNF- (p=ns vs. control), IL-6, and IL-8 compared to monotherapies. Furthermore, both agents promoted cholesterol efflux and suppressed oxLDL uptake, with the combination treatment producing the lowest residual intracellular cholesterol levels (p<0.001). ConclusionAegeline and Atorvastatin effectively suppress oxLDL-induced macrophage inflammatory cascades and intracellular lipid overload. While Atorvastatin monotherapy exerts robust control over CRP and oxLDL loading, combining Aegeline with Atorvastatin provides synergistic efficacy, enhancing cholesterol efflux and restoring pro-inflammatory cytokine expression toward physiological levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/744794v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d90d88org.highwire.dtl.DTLVardef@1079202org.highwire.dtl.DTLVardef@2d659org.highwire.dtl.DTLVardef@4685af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Obesity-driven metabolic syndrome poses a critical global threat, yet standard therapies like GLP-1 receptor agonists trigger substantial lean mass wasting, with muscle loss accounting for up to 40% of reduced weight. Here we identify a non-canonical metabolic application for dronedarone hydrochloride, an anti-arrhythmic benzofuran derivative. In diet-induced and ob/ob obese mice, short-term dronedarone hydrochloride administration dose-dependently reduces food intake, clears visceral and subcutaneous adiposity, and reverses steatohepatitis. Head-to-head trials show that dronedarone hydrochloride achieves glycemic control and fat clearance non-inferior to semaglutide, tirzepatide, and empagliflozin, but uniquely and completely preserves skeletal muscle mass. Mechanistically, dronedarone hydrochloride operates independently of central hypothalamic appetite-regulating neuropeptides and the peripheral leptin pathway. By decoupling fat reduction from sarcopenia, our findings establish dronedarone hydrochloride as a muscle-sparing therapeutic candidate for metabolic syndrome.

BACKGROUND: Subarachnoid hemorrhage (SAH) induces inflammatory responses and subsequent immune cell activation, which may contribute in cerebral vasospasm, microcirculatory impairment and poor neurological outcomes. Although cerebral vasospasm has traditionally been considered a major cause of delayed cerebral ischemia after SAH, therapies targeting angiographic vasospasm have not consistently improved functional outcomes. Early inflammatory responses may contribute to microcirculatory impairment, cerebral vasospasm, and subsequent neurological injury. Herein, we investigated whether interleukin-10 (IL-10), an anti-inflammatory cytokine, improves these outcomes in an experimental SAH model. METHODS: Mice received intramuscular injections of either an adeno-associated virus encoding IL-10 (AAV/IL-10) vector or an AAV expressing green fluorescent protein (AAV/GFP) vector (control). India ink angiography was performed to assess the diameter of the sphenoidal segment of the middle cerebral artery (MCA), the total length of the visible cortical arteries, and cortical staining intensity, as indices of cerebral vasospasm, microcirculatory impairment, and cerebral perfusion, respectively. Perivascular inflammatory cell infiltration and cytokine levels were assessed using immunohistochemistry and ELISA. We also evaluated the therapeutic efficacy of the AAV/IL-10 vector when administered immediately after SAH induction. RESULTS: IL-10 overexpression significantly improved neurological outcomes after SAH and was associated with attenuated cerebral vasospasm and microcirculatory impairment, as well as preservation of cerebral perfusion. It also significantly reduced neutrophil and macrophage infiltration around the internal carotid artery and attenuated SAH-induced elevations in IL-6 and matrix metalloproteinase-3 levels. Mice treated with the AAV/IL-10 vector immediately after SAH induction showed significant improvements in neurological scores and cerebral perfusion. CONCLUSIONS: AAV-mediated IL-10 overexpression improves neurological outcomes after SAH, likely by attenuating inflammatory responses, cerebral vasospasm, and microcirculatory impairment. These findings suggest that IL-10-based anti-inflammatory therapy is a promising therapeutic strategy for SAH.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Confounding the treatment options available to patients with triple-negative breast cancer (TNBC) are not only its purported lack of hormone receptor and growth factor receptor targets (ER-/ PR-/ HER2-), but its enrichment in plastic and chemoresistant breast cancer stem cells (BCSCs). Although descriptions of non-canonical PR expression in TNBC are rife in the literature, only canonical PR is considered in the definition of TNBC and is used to determine therapeutic strategy, not least because the utility of non-canonical PR modulation in TNBC chemoresistance is largely unexplored and poorly understood. Here we document the expression of three non-canonical PRs and the canonical PR (PGR) phosphorylated at Ser345 (p-PGR S345) in a panel of TNBC and luminal breast cancer cell lines, and employ combined PR agonists and antagonists to investigate the influence of PR activity on TNBC cell viability and PI3K inhibitor cytotoxicity. To examine the contributions of non-canonical membrane-associated PRs mPR{beta} and PGRMC1, we tested the agonist Org OD 02-0, a synthetic progestin targeted to mPRs; the PGRMC1 antagonist Ag-205; and the antagonist SPA70 against the cytosolic/nuclear PXR, in the background of pan-PI3K inhibition with Buparlisib (BUP). We also reveal that combinations of agonists and antagonists targeted to canonical and non-canonical PRs robustly potentiate the cytotoxic effects of PI3K inhibition, and also exhibit significant cytotoxicity on their own. Using functional assays, flow cytometry, immunocytochemistry and protein expression analyses, we found that simultaneously perturbing PRs and inhibiting PI3K function resulted in significantly greater cell death than vehicle control or BUP alone, and reduced the proportion of ALDH1+ BCSCs in two TNBC cell lines. We conclude that four types of PR are tractable targets in TNBC which participate in cell viability and enhance chemotherapy-induced cytotoxicity, and should be re-evaluated in an evolving definition of this challenging disease.

Background: Stroke remains a leading cause of death and disability worldwide. Matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-12, contribute to early blood-brain barrier (BBB) disruption, neuroinflammation, haemorrhagic transformation, and intracerebral haemorrhage (ICH). Intravenous thrombolysis is the only widely used pharmacological therapy for acute ischaemic stroke, but its utility is limited by narrow eligibility criteria and haemorrhagic risk. Inhibition of MMPs in the acute phase may offer a complementary neurovascular protective strategy. Methods: AZD1236, a selective dual MMP-9/-12 inhibitor, was evaluated in transient and permanent middle cerebral artery occlusion models and in a collagenase-induced ICH model in young, aged, obese, and female mice. Drug or vehicle was administered 2-6 hours after stroke onset. Outcomes included infarct or haematoma volume, BBB integrity, neurological function, and pain-related behaviours. Results: AZD1236 given within 2-4 hours after ischaemic or haemorrhagic insult significantly reduced infarct and haematoma volumes, improved short- and long-term neurological scores, and preserved BBB integrity, whereas treatment at 6 hours was largely ineffective. AZD1236 also attenuated the development of post-stroke mechanical allodynia and thermal hyperalgesia. Mechanistically, treatment reduced MMP-9 and MMP-12 activity, increased tight junction protein expression, and dampened inflammatory responses. Conclusions: Dual inhibition of MMP-9/-12 with AZD1236 confers robust neurovascular protection and mitigates post-stroke pain across clinically relevant models of ischaemic and haemorrhagic stroke. These findings provide a strong preclinical rationale for clinical evaluation of dual MMP-9/12 inhibition as an adjunctive neuroprotective strategy for acute stroke.

N,N-dimethyltryptamine (DMT) is an endogenous psychedelic tryptamine that has recently emerged as a promising therapeutic candidate for acute ischemic stroke. Although DMT consistently reduces infarct size, attenuates neuroinflammation, and improves functional outcome in experimental stroke, the cellular and receptor mechanisms underlying these effects remain poorly understood. Primary rat microglial cultures were used to examine the direct anti-inflammatory effects of DMT following lipopolysaccharide (LPS)-induced activation. Microglial morphology, phagocytosis, and proteomic alterations were analyzed. Radioligand binding assays determined the affinity of DMT for microglial sigma-1 receptors (Sig-1Rs). Pharmacological inhibition of Sig-1Rs and serotonin (5-HT) receptors was performed to define receptor-specific mechanisms. Translational relevance was evaluated in acute mouse brain slices subjected to mild oxygen-glucose deprivation (mOGD) and anoxic episodes, where microglial activation, spreading depolarizations (SDs), and neuronal injury were assessed. DMT directly suppressed LPS-induced microglial activation, promoted a homeostatic morphology, and reduced phagocytic activity. Proteomic profiling demonstrated that DMT selectively reprogrammed inflammatory pathways by suppressing proteins involved in cytokine and chemokine signaling and oxidative stress while largely preserving arachidonic acid-prostaglandin synthesis. DMT bound microglial Sig-1Rs with micromolar affinity comparable to that reported in whole-brain preparations. Pharmacological inhibition revealed that DMT-induced morphological reprogramming required both Sig-1R and serotonergic signaling, whereas suppression of phagocytosis was largely independent of either receptor pathway. In acute brain slices, DMT attenuated microglial activation, reduced SD propagation and ischemic neuronal injury, and tissue-level neuroprotection depended on serotonergic signaling. DMT directly targets microglia and selectively remodels inflammatory states rather than broadly suppressing microglial activation. The receptor mechanisms underlying its actions are context dependent, with Sig-1R and serotonergic signaling contributing differentially according to the cellular response and experimental model. These findings provide mechanistic insight into the neuroprotective actions of DMT and support its ongoing clinical translation as a potential therapy for ischemic stroke.

BackgroundThe CXCR2 receptor pathway plays a major role in inflammatory and invasive angiogenesis in human disease. ObjectiveWe evaluated AZD5069, a selective CXCR2 antagonist, as an angiogenesis inhibitor in human cell culture. MethodsHuman Umbilical Venous Endothelial Cells (HUVECs), Human Aortic Endothelial Cells (HAECs), and Human Pulmonary Artery Endothelial Cells (HPAECs) were cultured with standard in vitro techniques. AZD5069 (0, 8, 16, 32, 64, 128, 256 M) was evaluated as an angiogenesis inhibitor with fluorescent-labeled 5-Ethynyl-2-deoxyuridine (EdU) uptake to quantify endothelial cell proliferation, scratch assay to quantify endothelial cell migration, and Geltrex assay to quantify endothelial cell tubule and hub formation. AZD5069 cytotoxicity was evaluated with in situ terminal deoxynucleotidyl transferase 2-Deoxyuridine triphosphate- 5 (dUTP) nick-end labeling (TUNEL) to quantify apoptosis and membrane-impermeable cyanine dye uptake to quantify necrotic cell death. ResultsAZD5069 significantly reduced HUVEC, HAEC, and HPAEC proliferation, migration, tubule count, total tubule length, and node count with a dose-response. AZD5069 did not cause apoptosis nor necrotic cell death. ConclusionsAZD5069 inhibited angiogenesis without cytotoxicity in human endothelial cell culture. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in cardiovascular, oncologic, and inflammatory disease. Condensed AbstractThe CXCR2 receptor pathway plays a major role regulating angiogenesis in inflammation and cancer. The CXCR2 receptor pathway has been evaluated in humans as a target for therapy in inflammatory disease and cancer but not as a therapeutic approach to block pathologic angiogenesis. AZD5069 is a clinical stage, direct CXCR2 antagonist. In human endothelial cell culture, AZD5069 inhibited angiogenesis without causing apoptosis or necrotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility as a novel angiogenesis blocker in human disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/731993v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ac8fb4org.highwire.dtl.DTLVardef@ea89forg.highwire.dtl.DTLVardef@607f94org.highwire.dtl.DTLVardef@157cec4_HPS_FORMAT_FIGEXP M_FIG Visual Abstract: AZD5069, a selective CXCR2 antagonist, significantly reduced endothelial cell proliferation, migration, and vascular tubule formation without causing necrotic or apoptotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in human cardiovascular, oncologic, and inflammatory disease with pathologic, dysregulated, or excessive angiogenesis. C_FIG

Mancozeb, a widely used fungicide composed of manganese ethylene-bis-dithiocarbamate with zinc salts, has raised concerns due to its potential neurotoxic effects. In this study, we investigated how chronic oral administration of mancozeb affects astrocyte function and neurobehavior in mice, focusing on store-operated Ca{superscript 2} entry (SOCE), mediated by Orai1 and STIM1. Mancozeb treatment at 0.5 {micro}g/kg/day for 4 weeks reduced glial fibrillary acidic protein (GFAP) expression in the hippocampus and corpus callosum of mice, indicating astrocyte atrophy. Further, administration at the human acceptable daily intake (30 {micro}g/kg/day) for 1 week induced hippocampal astrocyte atrophy and hyperlocomotor activity in open field tests. In vitro experiments revealed that mancozeb specifically inhibited SOCE in astrocytes by targeting the Orai1/STIM1 complex, as its inhibitory effect was abolished by short hairpin RNA (shRNA)-mediated knockdown of Orai1 or STIM1, but not by knockdown of TRPA1 or scramble shRNA. This demonstrates that mancozeb-mediated SOCE inhibition critically depends on the presence of Orai1 and STIM1, highlighting the molecular specificity of its action. Furthermore, mancozeb diminished endoplasmic reticulum (ER) Ca{superscript 2} stores and P2Y1 receptor agonist-induced Ca{superscript 2} transients. Electrophysiological analyses revealed that mancozeb selectively decreased the inhibitory postsynaptic current frequency without affecting excitatory currents, suggesting reduced astrocyte-mediated GABA release. Collectively, these findings demonstrate that mancozeb disrupts astrocytic Ca{superscript 2} homeostasis through Orai1/STIM1-dependent SOCE inhibition, leading to astrocyte atrophy and altered inhibitory neurotransmission, which may underlie the observed behavioral changes. These results highlight the potential neurotoxic risk posed by mancozeb via the impairment of astrocyte function and intracellular Ca{superscript 2} regulation. Importantly, these neurotoxic effects occurred at concentrations below current regulatory safety limits (ADI), indicating that mancozeb-induced disruption of astrocytic Ca{superscript 2} signaling provides a mechanistic basis for re-evaluating established human safety exposure standards. Environmental ImplicationsOur findings highlight that the widespread use of mancozeb has a significant impact on brain health. Mancozeb was shown to induce astrocyte atrophy even at low concentrations, amounting to six times the human acceptable daily intake. Mancozeb causes impairment of GABAergic synaptic transmission of neurons by disrupting the Ca{superscript 2} homeostasis via inhibition of Orai1 and STIM1 of astrocytes. These findings indicate that current regulatory standards significantly underestimate the risks of long-term mancozeb exposure to brain health. Therefore, this study underscores the risks of astrocyte-mediated neurotoxicity resulting from pesticide residue ingestion and emphasizes the need to rigorously re-evaluate current exposure limits from the perspective of brain health.

While chemotherapy is effective in killing cancer cells, its non-specific cytotoxicity harms peripheral neurons, leading to chemotherapy-induced peripheral neuropathy (CIPN). Lacking effective interventions, clinicians often reduce or discontinue treatment. However, these adjustments do not necessarily reverse neuropathy and may jeopardize cancer control. Growing evidence now indicates that chemotherapy not only exerts neurotoxic effects but also modulates immune responses. The degree to which chemotherapy dosing regimens shape CD4+ T cell responses in the dorsal root ganglia (DRG), and how these cells influence mechanical hypersensitivity, remains poorly understood. In this study, female mice were administered a single, high dose (sHD) or multiple low doses (mLD) of paclitaxel (PTX). The mLD group, which received the higher cumulative dose, exhibited an earlier T cell response in the DRG and attenuated mechanical hypersensitivity with fewer ATF3+ DRG neurons compared to the sHD group. This regimen promoted a focused CD4+ T cell response while driving a broad and diversified CD8+ T cell expansion. In contrast, the sHD PTX regimen elicited a delayed, polyfunctional CD4+ T cell response but generated limited CD8+ effector differentiation. To directly assess the contribution of CD4+ T cells to CIPN pathogenesis, we administered PTX to mice lacking CD4+ T cells. CD4 deficient mice were significantly less hypersensitive than CD4+ sufficient mice with a stronger reduction in the sHD group. Although higher cumulative doses are associated with increased CIPN risk, our results suggest that the concentration and frequency of PTX more directly influence DRG immune programming, and that this immune shaping modulates CIPN severity.

Cannabidiol (CBD) is a non-psychoactive component of cannabis that has been studied as a potential therapy for chronic pain. CBD attenuates behavioral hypersensitivities in models of neuropathic pain, and promotes production of bioactive lipids (e.g., anandamide), altering lipid signaling. However, a lack of understanding of the mechanisms underlying the therapeutic effects of CBD has hindered development and application of CBD to mechanism-based therapies for pain in people. We asked whether the analgesics effects of CBD were dependent upon the enzyme NAPE-PLD. We used a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) to evaluate the acute and chronic antinociceptive effects of CBD and investigate its mechanisms. Pharmacological specificity was tested with antagonists targeting CB1, CB2, PPAR{gamma}, and PPAR receptors. Mechanisms were further examined using NAPE-PLD and GPR55 knockout mice. We also assessed repeated CBD dosing during both the development and maintenance of paclitaxel-induced CIPN in wild-type, GPR55 KO, and NAPE-PLD KO mice. CBD suppressed paclitaxel-induced behavioral hypersensitivities; these effects were attenuated by a PPAR and PPAR{gamma} antagonists, but not CB1 or CB2 antagonists. CBD reduced both the development and maintenance of neuropathic nociception in a model CIPN in wild-type mice, but these effects were absent in NAPE-PLD KO mice. By contrast, anti-allodynic efficacy of CBD was fully preserved in GPR55 KO mice. Pharmacological blockade of the PPAR receptor and genetic deletion of NAPE-PLD abolished the antinociceptive effects of CBD in a model of CIPN, suggesting a pivotal role for NAPE-PLD and PPAR receptors in CBD-mediated analgesia in chemotherapy-induced neuropathic pain.

Background Patients with advanced pancreatic ductal adenocarcinoma (aPDAC) often experience general health decline at diagnosis due to a high-symptom burden. The optimal management of symptoms and/or poor performance status (PS) in these patients remains an unmet medical need. Patients and Methods In this multicenter study, patients with PS[&ge;]2 and pathologically confirmed or imaging-suspected aPDAC were included at first oncology visit in a personalized 14-day emergency integrative supportive care program (14-EISCP) to manage pain, nutrition, diagnostics, and stenting procedures. The primary endpoint was the 14-EISCP success in feasibility of planned procedures and clinical benefit defined as post-EISCP PS[&le;]1, [&ge;]5 points improvement in fatigue, pain, global health-related quality of life (HRQoL) scores (EORTC QLQ-C15-PAL), or chemotherapy initiation within 30 days. Results A total of 106 patients were included; 93 evaluable patients considered for primary endpoint analysis (median age: 76 years [68-80], PS3: 20.9%, metastases: 61.3%). The median overall survival was 4.1 months (IC95% 2.6-5.6). The 14-EISCP was successful in 59.1% (n=55) of patients, meeting the primary objective (clinically relevant). The 14-EISCP feasibility was achieved in 70.9% of cases. Post-EISCP clinical benefit was observed in 79.6% of patients, with PS improvement to 0/1 in 13.2%, HRQoL improvement in 23.9%, and chemotherapy initiation [&le;]30 days in 73.1%. Among evaluable patients, 17.2% received mFOLFIRINOX or gemcitabine-nab-paclitaxel, 35.4% received FOLFOX, 25.3% had gemcitabine or 5-fluorouracil alone, and 22.2% received best supportive care. In patients with PS2 at baseline, the administration of doublet/triplet chemotherapy was associated with improved overall survival compared to single-agent. Discussion These results offer a promising framework for improving outcomes in aPDAC patients, bridging the gap between symptom management and systemic therapy administration. Conclusions In patients with PS[&ge;]2 and aPDAC, the personalized 14-EISCP was feasible and lead to meaningful clinical benefit, allowing doublet or triplet chemotherapy in half the patients.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Mcl1 is a major driver of therapeutic resistance across hematologic malignancies, but direct Mcl1 inhibition has been limited by on-target cardiotoxicity. Here, building on our development of an Mcl1-targeting autophagy-targeting chimera (AUTAC), we show that AUTAC-mediated degradation creates a tumor-selective therapeutic window that spares the heart. AUTAC induced robust cytotoxicity and Mcl1 degradation in multiple myeloma models, while showing minimal toxicity in cardiac cell lines, primary cardiomyocytes, and murine heart tissue. In vivo, AUTAC reduced tumor Mcl1 without measurably affecting cardiac Mcl1. Mechanistically, this selectivity was associated with lower expression of the p62/SQSTM1, TRAF6, and UBC13 machinery required for AUTAC activity in cardiac cells, together with lower intracellular AUTAC accumulation relative to tumor cells. AUTAC also enhanced the antitumor activity of carfilzomib and venetoclax, including in resistant models, without worsening cardiotoxicity or promoting cardiac Mcl1 loss. Compared with classical Mcl1 inhibitors, AUTAC caused markedly less cardiomyocyte death, mitochondrial depolarization, and apoptotic signaling. These findings identify AUTAC-mediated Mcl1 degradation as a cardiac-sparing strategy to target an otherwise clinically constrained vulnerability and support tumor-selective lysosomal degradation as a path to safer Mcl1-directed therapy.

BackgroundOwing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. MethodsMale C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x106 or 6x106 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. ResultsIn contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. ConclusionsImmune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

CD47 delivers a dominant "Dont Eat Me" signal that inhibits macrophage-mediated clearance of tumour cells. Using immune competent, chemoresistant neuroblastoma (NB) models, we tested a Fc-silent CD47 blocker (ALX301) with anti-GD2 antibody alone and in combination with a clinically aligned temozolomide/irinotecan chemoimmunotherapy backbone. Tumours expressed GD2 and CD47, and bound ALX301. In macrophage coculture assays, anti-GD2 antibody induced dose-dependent phagocytosis, whereas ALX301 or an anti-CD47 antibody alone did not. CD47 blockade in combination with a suboptimal concentration of anti-GD2 antibody showed an additive effect on phagocytosis in vitro. In vivo, however, ALX301 failed to improve tumour control or survival when added to anti-GD2 or to chemoimmunotherapy in two models. Toxicity was acceptable, showing only mild, expected red-cell changes without organ injury. This form of CD47 inhibition is therefore mechanistically active in vitro but insufficient to enhance anti-GD2 antibody-based therapy in immune competent mice bearing a chemoresistant NB, highlighting the potential need for myeloid-reprogramming partners.

1.Cancer-induced bone pain (CIBP) occurs in a majority of patients when primary or metastatic cancer develops within the bone. This pain has a significant impact on quality of life, yet there are limited effective treatment options available. Nerve sprouting is a complex mechanism that has been implicated in CIBP. Netrin-1 is a neuronal guidance molecule that is produced by numerous cell types, including cancer cells. In this study we aimed to determine whether netrin-1 inhibition (with NP137 - a humanized IGg1 monoclonal antibody) could ameliorate nerve sprouting, and nociception by extension, in three models of CIBP - osteosarcoma, metastatic breast cancer, and metastatic prostate cancer. Sustained administration of NP137 failed to produce an anti-nociceptive effect in these models, but a delayed onset was observed in the osteosarcoma model. NP137 did not produce a disease-modifying effect, as micro-computed tomography did not reveal reduced bone destruction in the NP137-treated groups. Additionally, there was no nerve fibre density reduction in any of the groups at the late-stage of the disease, suggesting that nerve sprouting occurs in early- to mid-stage CIBP development. Investigation of NP137 exposure indicated that serum levels of NP137 were comparable between the sham and cancer groups. Our study indicates that netrin-1 may play a role in early-stage CIBP development, but inhibition of this mechanism does not produce robust anti-nociception.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.

AimsCardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment for refractory ventricular tachycardia (VT). Intriguingly, the clinical benefit of SBRT often occurs within days of treatment, preceding the development of radiation-induced fibrosis, suggesting alternative underlying mechanisms. This study aimed to investigate the acute and persistent effects of ionizing radiation on cardiac bioenergetics and mitochondrial function, providing mechanistic insights into early cardiac responses to radiation exposure. Methods and resultsWe employed a translational multi-model approach, including HL-1 mouse cardiomyocytes and ex vivo mouse left ventricular living myocardial slices (LMS). Bioenergetic profiling, assessment of mitochondrial respiration and calcium handling were performed following exposure to clinically relevant radiation doses (10 Gy and 25 Gy). In HL-1 cardiomyocytes, 10 Gy induced acute bioenergetic stress, characterized by reduced adenylate energy charge, cytoskeletal disorganization, and impaired mitochondrial respiration, accompanied by increased calcium oscillation amplitude. 25 Gy exposure led to NAD+ depletion but paradoxically enhanced mitochondrial respiratory capacity, suggesting an adaptive metabolic response. Murine myocardial slices demonstrated reduced creatine content while preserving energy balance as indicated by phosphocreatine/ATP ratio, indicating tissue-level metabolic resilience. These findings reveal model-specific metabolic perturbations induced by cardiac irradiation, underscoring the importance of tissue complexity in modulating the cardiac response to radiation. ConclusionThis study demonstrates that ionizing radiation at 10 Gy and 25 Gy induced dose- and model-dependent bioenergetic alterations in cardiac cells and tissues, including changes in mitochondrial respiration, nucleotide levels, and redox balance. While 10 Gy exacerbated metabolic disruption, 25 Gy triggered partial recovery, highlighting differential responses across cellular and tissue levels. These metabolic changes may contribute to the immediate effects of cardiac SBRT and potentially to long-term cardiotoxicity. Translational PerspectiveOur study provides novel mechanistic insights into the metabolic effects of cardiac irradiation, revealing acute mitochondrial stress, redox imbalance and alterations in calcium homeostasis in cardiomyocytes. These early bioenergetic changes may contribute to both the immediate anti-arrhythmic effects and the potential long-term cardiotoxicity of stereotactic body radiation therapy. Understanding these molecular responses is essential to optimize the therapeutic window of cardiac radioablation and minimize adverse effects. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/730816v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@d963d3org.highwire.dtl.DTLVardef@28e03dorg.highwire.dtl.DTLVardef@19a0165org.highwire.dtl.DTLVardef@1d1ca34_HPS_FORMAT_FIGEXP M_FIG C_FIG