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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office Hungary Introduction Unexpected ischaemia-induced cardiac adverse events are major contributors to clinical trial discontinuation and drug attrition, and these effects remain undetected by routine preclinical cardiotoxicity assessment. On the other hand, the proarrhythmic effect of drug candidates is studied in healthy cells, tissues, or exclusively in healthy experimental animals. However, a number of experimental and clinical reports demonstrate that the proarrhythmic effects of most drugs are manifested only in pathological conditions. Thus, the aim is to develop a sensitized animal model that can reliably screen for the arrhythmogenic effects of a compound. Here we show that the selective COX- 2 inhibitor rofecoxib, which has been withdrawn over cardiovascular safety concerns, possesses cardiac electrophysiological adverse effects only revealed during ischaemia/reperfusion, a phenomenon we termed "hidden cardiotoxicity". Purpose Our group has previously reported the hidden cardiotoxic effect of rofecoxib on rat ventricular preparations. Given the significant differences in cardiac electrophysiological properties between rats and humans, the human extrapolation of arrhythmological results obtained from rats is limited, so our aim was to investigate the proarrhythmic effect of rofecoxib in a sensitized rabbit model. Methods Action potentials were registered from rabbit right ventricular papillary muscles using the conventional microelectrode technique. The following parameters were measured under normoxic and ischaemic conditions in the presence or absence of 10 μM rofecoxib: conduction time, action potential amplitude, action potential duration at 25%, 50%, 75% and 90% repolarization (APD25-90). The effects of rofecoxib upon test ischaemia and reperfusion were investigated. Results Rofecoxib (10 μM) did not alter electrophysiological parameters in normoxic conditions. However, following 30 minute ischaemia the APD90 was significantly decreased during reperfusion compared to APD90 in the vehicle-treated group: 161.96 ± 19.55 ms vs. 180.04 ± 23.21 ms (p<0.05, n=5), respectively. Following sI/R, a decrease in Vmax and AMPL parameters indicating impulse conduction velocity was also measured in the 10 μM rofecoxib- treated group, but the differences were not statistically significant. Conclusions Under pathological conditions, rofecoxib may increase the incidence of reperfusion arrhythmias due to the mechanism of delayed afterdepolarization (DAD) and reentrant arrhythmias. Consequently, the sensitized rabbit model detailed above may be suitable for investigating the "hidden cardiotoxic" effect of a drug candidate compound. Significant differences were observed in the effect of rofecoxib on repolarization between the rat and rabbit models. However, due to the well- known electrophysiological differences between the two species, the human relevance of the results obtained in rabbits is more reliable. [ABSTRACT FROM AUTHOR]

Purpose: We have shown previously that chloramphenicol (CAP) reduces infarct size, although its mechanism is not fully understood. It has also been evidenced that autophagy plays important role in cardioprotection. Therefore, our aim was to investigate whether autophagy is necessary for CAP-induced cardioprotection.Methods: Neonatal rat cardiomyocytes were isolated and treated for 1h as follows: (1) 300 μM CAP, (2) 10 μM chloroquine (CQ), (3) 300 μM CAP and 10 μM CQ, and (4) non-treated control. After the indicated treatments markers of autophagy were determined by Western blots. In ex vivo experiments isolated rat hearts were perfused with Krebs-Henseleit buffer containing 300 μM CAP initiated 15 min before the onset of 30 min global ischemia and 120 min of reperfusion. To inhibit early phases of autophagy, a group of CAP-treated hearts was perfused with 200 nM recombinant Tat-Atg5K130R protein (inhibiting autophagosome formation) before CAP administration. To investigate the role of lysosomal degradation in CAP-induced cardioprotection another group of isolated hearts was perfused with 5 μM CQ (inhibiting lysosomal degradation) before CAP administration. TTC staining was performed to determine infarct size in all groups.Results: CAP induces autophagy in neonatal cardiomyocytes by increasing the level of LC3-II and decreasing the phosphorylation of p70S6K. CAP significantly reduced infarct size in ex vivo perfused hearts, while inhibition of autophagosome formation (by Tat-Atg5K130R protein) was abolished CAP-induced cardioprotection. However, the inhibition of lysosomal degradation (by CQ) did not influence CAP -induced cardioprotection.Conclusions: Here we evidenced an induced autophagy and a downregulation in the mTOR pathway due to CAP treatment in cardiomyocytes. Our results demonstrate that formation of autophagosomes is essential for the cardioprotective effect of CAP, while it is unaffected by a block in the clearance of autophago-lysosomes. These results suggest that inducers of autophagy may form the basis of future cardioprotective therapies. [ABSTRACT FROM AUTHOR]

Background: Remote ischemic preconditioning (RIPC) of the heart (i.e. brief ischemia/reperfusion cycles, subjected to a remote organ or a remote area, reduce the consecutive damage of a sustained cardiac ischemia. Although, its mechanism has been investigated thoroughly, exact interorgan transfer mechanisms of cardioprotection by RIPC are required to be further investigated. Exosomes and microvesicles/microparticles are vesicles of 30-100 nm and 100-1000 nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). As EVs are able to shuttle proteins, mRNAs and microRNAs, they are ideal candidates for inter-organ communication. However, whether EVs are involved in RIPC, is unknown.Purpose: Here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC.Methods: Hearts of male Wistar rats were isolated and perfused as described by Langendorff. Hearts were exposed to 3 × 5 min global ischemia/reperfusion cycles (IPC) or 30 min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these donor hearts were given to another set of recipient isolated hearts either unharmed or depleted of EVs by differential ultracentrifugation. Infarct size was determined by triphenyl tetrazolium chloride at the end of 30 min global ischemia and 120 min reperfusion period. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker hsp60 Western blot, and electron microscopy.Results: IPC markedly increased EV release from the heart as assessed by HSP60 immunoblot. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9 ± 1.6% vs. 25.0 ± 2.7%, respectively), similarly to cardioprotection afforded by IPC (7.3 ± 2.7% vs. 22.1 ± 2.9%, respectively) on the donor hearts. Furthermore, perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0 ± 2.3%).Conclusions: This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection. [ABSTRACT FROM PUBLISHER]

Introduction: We have previously shown that autophagy is necessary for the ischemic preconditioning (IPC) of the heart and that IPC is compromised in the setting of hypercholesterolemia (HC). The mTOR pathway, an upstream modulator of autophagy, has been shown to be activated in HC. The purpose of this study was to test the hypothesis that HC results in down regulation of autophagy due in part to activation of mTOR signalling and to investigate whether HC modulates programmed cell death mechanism such as apoptosis and necroptosis in the heart.Methods: Male Wistar rats were fed either normal chow or with 2% cholesterol and 1% cholic acid-enriched diet for 10 weeks. HC rats exhibited a 41% increase in plasma total cholesterol level over that of rats fed standard chow (2.89mmol/L vs. 4.09mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out, then left ventricles were snap-frozen for determination of autophagy, mTOR pathway, apoptosis, and necroptosis-related proteins by Western blot.Results: HC was associated with a significant reduction in LC3-II and in Beclin-1 expression compared to control values. Consistent with mTOR activation, S6 phosphorylation, an mTOR target, was increased in HC animals (p=0.021). Bax/Bcl2 ratio and cleaved caspase-3 signal increases in HC animlas, while no difference in the expression of RIP3 or MLKL was detected between treatments.Conclusion: These data indicate that hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an upregulated mTOR pathway; however the exact signaling mechanism is yet to be elucidated. Reduced autophagy was accompanied by signs of increased apoptosis, while cardiac necroptosis was not modulated by HC. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic states. [ABSTRACT FROM AUTHOR]

Background: Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability. Direct comparisons between the two methods are lacking from the literature., Methods and Results: In this study, we compared antibody- and lectin-based methods for the histological assessment of cardiomyocyte CSA (with the use of anti-laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti-CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy-to-use combined lectin histochemistry protocol (WGA and ILB4, 'CardiLect' protocol) for the histological assessment of cardiac remodelling. The lectin-based approach has been evaluated on a cross-species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script ('CardiLect Analyser') for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P = 0.0098, r S  = 0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P = 0.0402, r S  = -0.6364). Microvascular density showed significant negative correlation with LV mass (P = 0.0055, r S  = -0.7622) and significant positive correlation with EF (P = 0.0106, r S  = 0.7203)., Conclusions: The described combined lectin histochemistry protocol with the provided ImageJ script is an easy-to-use, cost-effective, cross-species approach for the histological assessment of cardiac remodelling., (© 2024 The Author(s). ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Numerous cardioprotective interventions have been reported to reduce myocardial infarct size (IS) in pre-clinical studies. However, their translation for the benefit of patients with acute myocardial infarction (AMI) has been largely disappointing. One reason for the lack of translation is the lack of rigor and reproducibility in pre-clinical studies. To address this, we have established the European IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) pig AMI network with centralized randomization and blinded core laboratory IS analysis and validated the network with ischemic preconditioning (IPC) as a positive control. Ten sites in the COST Innovators Grant (IG16225) network participated in the IMPACT network. Three sites were excluded from the final analysis through quality control of infarct images and use of pre-defined exclusion criteria. Using a centrally generated randomization list, pigs were allocated to myocardial ischemia/reperfusion (I/R, N = 5/site) or IPC + I/R (N = 5/site). The primary endpoint was IS [% area-at-risk (AAR)], as quantified by triphenyl-tetrazolium-chloride (TTC) staining in a centralized, blinded core laboratory (5 sites), or IS [% left-ventricular mass (LV)], as quantified by a centralized, blinded cardiac magnetic resonance (CMR) core laboratory (2 sites). In pooled analyses, IPC significantly reduced IS when compared to I/R (57 ± 14 versus 32 ± 19 [%AAR] N = 25 pigs/group; p < 0.001; 25 ± 13 versus 14 ± 8 [%LV]; N = 10 pigs/group; p = 0.021). In site-specific analyses, in 4 of the 5 sites, IS was significantly reduced by IPC when compared to I/R when quantified by TTC and in 1 of 2 sites when quantified by CMR. A pig AMI multicenter European network with centralized randomization and core blinded IS analysis was established and validated with the aim to improve the reproducibility of cardioprotective interventions in pre-clinical studies and the translation of cardioprotection for patient benefit., (© 2024. The Author(s).)

Aims: The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis., Methods and Results: Wild type (WT), PCSK9 -/- , and LDLR -/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR -/- and PCSK9 -/- mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9 -/- mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR -/- mice showing high levels while PCSK9 -/- were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR -/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63 + EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63 + EVs were significantly depleted., Conclusions: This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD., (© 2024. The Author(s).)

Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute the only medication class that consistently prevents or attenuates human heart failure (HF) independent of ejection fraction. We have suggested earlier that the protective mechanisms of the SGLT2i Empagliflozin (EMPA) are mediated through reductions in the sodium hydrogen exchanger 1 (NHE1)-nitric oxide (NO) pathway, independent of SGLT2. Here, we examined the role of SGLT2, NHE1 and NO in a murine TAC/DOCA model of HF. SGLT2 knockout mice only showed attenuated systolic dysfunction without having an effect on other signs of HF. EMPA protected against systolic and diastolic dysfunction, hypertrophy, fibrosis, increased Nppa/Nppb mRNA expression and lung/liver edema. In addition, EMPA prevented increases in oxidative stress, sodium calcium exchanger expression and calcium/calmodulin-dependent protein kinase II activation to an equal degree in WT and SGLT2 KO animals. In particular, while NHE1 activity was increased in isolated cardiomyocytes from untreated HF, EMPA treatment prevented this. Since SGLT2 is not required for the protective effects of EMPA, the pathway between NHE1 and NO was further explored in SGLT2 KO animals. In vivo treatment with the specific NHE1-inhibitor Cariporide mimicked the protection by EMPA, without additional protection by EMPA. On the other hand, in vivo inhibition of NOS with L-NAME deteriorated HF and prevented protection by EMPA. In conclusion, the data support that the beneficial effects of EMPA are mediated through the NHE1-NO pathway in TAC/DOCA-induced heart failure and not through SGLT2 inhibition., (© 2024. The Author(s).)

Background and Purpose: Cardioprotective miRNAs (protectomiRs) are promising therapeutic tools. Here, we aimed to identify protectomiRs in a translational porcine model of acute myocardial infarction (AMI) and to validate their cardiocytoprotective effect., Experimental Approach: ProtectomiR candidates were selected after systematic analysis of miRNA expression changes in cardiac tissue samples from a closed-chest AMI model in pigs subjected to sham operation, AMI and ischaemic preconditioning, postconditioning or remote preconditioning, respectively. Cross-species orthologue protectomiR candidates were validated in simulated ischaemia-reperfusion injury (sI/R) model of isolated rat ocardiomyocytes and in human AC16 cells as well. For miR-450a, we performed target prediction and analysed the potential mechanisms of action by GO enrichment and KEGG pathway analysis., Key Results: Out of the 220 detected miRNAs, four were up-regulated and 10 were down-regulated due to all three conditionings versus AMI. MiR-450a and miR-451 mimics at 25 nM were protective in rat cardiomyocytes, and miR-450a showed protection in human cardiomyocytes as well. MiR-450a has 3987 predicted mRNA targets in pigs, 4279 in rats and 8328 in humans. Of these, 607 genes are expressed in all three species. A total of 421 common enriched GO terms were identified in all three species, whereas KEGG pathway analysis revealed 13 common pathways., Conclusion and Implications: This is the first demonstration that miR-450a is associated with cardioprotection by ischaemic conditioning in a clinically relevant porcine model and shows cardiocytoprotective effect in human cardiomyocytes, making it a promising drug candidate. The mechanism of action of miR-450a involves multiple cardioprotective pathways., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Aims: Heart failure with reduced ejection fraction (HFrEF) is a leading cause of death worldwide; thus, therapeutic improvements are needed. In vivo preclinical models are essential to identify molecular drug targets for future therapies. Transverse aortic constriction (TAC) is a well-established model of HFrEF; however, highly experienced personnel are needed for the surgery, and several weeks of follow-up are necessary to develop HFrEF. To this end, we aimed (i) to develop an easy-to-perform mouse model of HFrEF by treating Balb/c mice with angiotensin-II (Ang-II) for 2 weeks by minipump and (ii) to compare its cardiac phenotype and transcriptome to the well-established TAC model of HFrEF in C57BL/6J mice., Methods: Mortality and gross pathological data, cardiac structural and functional characteristics assessed by echocardiography and immunohistochemistry and differential gene expression obtained by RNA-sequencing and gene-ontology analyses were used to characterize and compare the two models. To achieve statistical comparability between the two models, changes in treatment groups related to the corresponding control were compared (ΔTAC vs. ΔAng-II)., Results: Compared with the well-established TAC model, chronic Ang-II treatment of Balb/c mice shares similarities in cardiac systolic functional decline (left ventricular ejection fraction: -57.25 ± 7.17% vs. -43.68 ± 5.31% in ΔTAC vs. ΔAng-II; P = 0.1794) but shows a lesser degree of left ventricular dilation (left ventricular end-systolic volume: 190.81 ± 44.13 vs. 57.37 ± 10.18 mL in ΔTAC vs. ΔAng-II; P = 0.0252) and hypertrophy (cell surface area: 58.44 ± 6.1 vs. 10.24 ± 2.87 μm 2 in ΔTAC vs. ΔAng-II; P < 0.001); nevertheless, transcriptomic changes in the two HFrEF models show strong correlation (Spearman's r = 0.727; P < 0.001). In return, Ang-II treatment in Balb/c mice needs significantly less procedural time [38 min, interquartile range (IQR): 31-46 min in TAC vs. 6 min, IQR: 6-7 min in Ang-II; P < 0.001] and surgical expertise, is less of an object for peri-procedural mortality (15.8% in TAC vs. 0% in Ang-II; P = 0.105) and needs significantly shorter follow-up for developing HFrEF., Conclusions: Here, we demonstrate for the first time that chronic Ang-II treatment of Balb/c mice is also a relevant, reliable but significantly easier-to-perform preclinical model to identify novel pathomechanisms and targets in future HFrEF research., (© 2024 The Author(s). ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

In recent years, various approaches have been undertaken to eliminate lipoproteins co-isolated with extracellular vesicles, as they were initially regarded as contaminating entities. However, novel discoveries are reshaping our perspective. In body fluids, these distinct particles not only co-exist, but also interactions between them are likely to occur. Extracellular vesicles and lipoproteins can associate with each other, share cargo, influence each other's functions, and jointly have a role in the pathomechanisms of diseases. Additionally, their association carries important implications for therapeutic and pharmacological aspects of lipid-lowering strategies. Extracellular vesicles and lipoprotein particles may have roles in the elimination of each other from the circulation. The objective of this minireview is to delve into these aspects. Here, we show that under certain physiological and pathological conditions, extracellular vesicles and lipoproteins are 'partners' rather than 'strangers' or 'rivals'., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Introduction: Ischemic conditionings (ICon) were intensively investigated and several protective signaling pathways were identified. Previously, we have shown the role of matrix metalloproteinases (MMP) in myocardial ischemia/reperfusion injury (MIRI) and the cardioprotective role of biglycan (BGN), a small leucine-rich proteoglycan in vitro. Here, we hypothesized that cardiac MMP and BGN signaling are involved in the protective effects of ICon., Methods: A reverse target-microRNA prediction was performed by using the miRNAtarget™ 2.0 software to identify human microRNAs with a possible regulatory effect on MMP and BGN, such as on related genes. To validate the identified 1289 miRNAs in the predicted network, we compared them to two cardioprotective miRNA omics datasets derived from pig and rat models of MIRI in the presence of ICons., Results: Among the experimentally measured miRNAs, we found 100% sequence identity to human predicted regulatory miRNAs in the case of 37 porcine and 24 rat miRNAs. Upon further analysis, 42 miRNAs were identified as MIRI-associated miRNAs, from which 24 miRNAs were counter-regulated due to ICons., Conclusions: Our findings highlight 24 miRNAs that potentially regulate cardioprotective therapeutic targets associated with MMPs and BGN in a highly translatable porcine model of acute myocardial infarction.

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements., (© 2024. The Author(s).)

Background: Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica , exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo ; and 2) to understand the molecular mechanism of ergolide action., Methods: Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data., Results: Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p <0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p <0.0001) in vivo , compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p <0.05; fold-change >1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs., Conclusions: Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Sundaramurthi H et al.)