Simulated Ischemia-reperfusion Research Articles

Simulated ischaemia/reperfusion impairs trophoblast function through divergent oxidative stress- and MMP-9-dependent mechanisms.

Early-onset pre-eclampsia is believed to arise from defective placentation in the 1st trimester, leading to placental ischaemia/reperfusion (I/R) and oxidative stress. However, our current understanding of the effects of I/R and oxidative stress on trophoblast function is ambiguous in part due to studies exposing trophoblasts to hypoxia instead of I/R, and which report conflicting results. Here we present a model of simulated ischaemia/reperfusion (SI/R) to recapitulate the pathophysiological events of early-onset PE, by exposing 1st trimester cytotrophoblast HTR-8/SVneo cells to a simulated ischaemia buffer followed by reperfusion. We examined different ischaemia and reperfusion times and observed that 1h ischaemia and 24h reperfusion induced an increase in reactive oxygen species (ROS) production (p < 0.0001) and oxygen consumption rate (p < 0.01). SI/R-exposed trophoblast cells exhibited deficits in migration, proliferation and invasion (p < 0.01). While the deficits in migration and proliferation were rescued by antioxidants, suggesting a ROS-dependent mechanism, the loss of invasion was not affected by antioxidants, which suggests a divergent ROS-independent pathway. In line with this, we observed a decrease in MMP-9, the key regulatory enzyme necessary for trophoblast invasion (p < 0.01), which was similarly unaffected by antioxidants, and pharmacological inhibition of MMP-9 replicated the phenotype of deficient invasion (p < 0.01). Collectively, these data demonstrate that I/R impairs trophoblast migration and proliferation via a ROS-dependent mechanism, and invasion via a ROS-independent loss of MMP-9, disambiguating the role of oxidative stress and providing insights into the response of trophoblasts to I/R in the context of early-onset PE.

Cardioprotective microRNAs (protectomiRs) in a pig model of acute myocardial infarction and cardioprotection by ischaemic conditioning: MiR-450a.

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. 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. 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. 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.

The study on the role of O-GlcNAcylation of SIRT3 in regulating mitochondrial oxidative stress during simulate myocardial ischemia-reperfusion.

Myocardial ischemia-reperfusion injury (MIRI) is a significant complication following reperfusion therapy after myocardial infarction. Mitochondrial oxidative stress is a critical factor in MIRI, and Sirtuin 3 (SIRT3), as a major mitochondrial deacetylase, plays a key protective role, with its activity potentially regulated by O-GlcNAcylation. This study used the H9C2 cell line to establish a simulated ischemia/reperfusion (SI/R) model, we utilized co-immunoprecipitated to validate the relationship between O-GlcNAc transferase (OGT) and SIRT3, demonstrated SIRT3 O-GlcNAcylation sites through LC-MS/MS, and performed site mutations using CRISPR/Cas9 technology. The results were validated using immunoblotting. SIRT3 and superoxide dismutase 2 (SOD2) activities were detected using a fluorometric assay, while mitochondrial reactive oxygen species (MROS) levels and cellular apoptosis were assessed using immunofluorescence. We have identified an interaction between SIRT3 and OGT, where SIRT3 undergoes dynamic O-GlcNAcylation at the S190 site, facilitating SIRT3 deacetylase activity. During SI/R, elevated levels of O-GlcNAcylation activate SOD2 by promoting SIRT3 enzyme activity, thereby inhibiting excessive MROS production. This significantly mitigates the occurrence of malignant autophagy in myocardial cells during reperfusion, promoting their survival. Conversely, blocking SIRT3 O-GlcNAcylation at the S190 site exacerbates SI/R injury. We demonstrate that O-GlcNAcylation is a crucial post-translational modification (PTM) of SIRT3 during SI/R, shedding light on a promising mechanism for future therapeutic approaches.

Influence of Anesthetic Regimes on Extracellular Vesicles following Remote Ischemic Preconditioning in Coronary Artery Disease.

Remote ischemic preconditioning (RIPC) reduces ischemia-reperfusion injury in aortocoronary bypass surgery, potentially via extracellular vesicles (EVs) and their micro-RNA content. Clinical data implicate that propofol might inhibit the cardioprotective RIPC effect. This prospective, randomized study investigated the influence of different anesthetic regimes on RIPC efficacy and EV micro-RNA signatures. We also assessed the impact of propofol on cell protection after hypoxic conditioning and EV-mediated RIPC in vitro. H9c2 rat cardiomyoblasts were subjected to hypoxia, with or without propofol, and subsequent simulated ischemia-reperfusion injury. Apoptosis was measured by flow cytometry. Blood samples of 64 patients receiving anesthetic maintenance with propofol or isoflurane, along with RIPC or sham procedures, were analyzed, and EVs were enriched using a polymer-based method. Propofol administration corresponded with increased Troponin T levels (4669 ± 435.6 pg/mL), suggesting an inhibition of the cardioprotective RIPC effect. RIPC leads to a notable rise in miR-21 concentrations in the group receiving propofol anesthesia (fold change 7.22 ± 6.6). In vitro experiments showed that apoptosis reduction was compromised with propofol and only occurred in an EV-enriched preconditioning medium, not in an EV-depleted medium. Our study could clinically and experimentally confirm propofol inhibition of RIPC protection. Increased miR-21 expression could provide evidence for a possible inhibitory mechanism.

Cardiac-targeted delivery of a novel Drp1 inhibitor for acute cardioprotection

Dynamin-related protein 1 (Drp1) is a mitochondrial fission protein and a viable target for cardioprotection against myocardial ischaemia-reperfusion injury. Here, we reported a novel Drp1 inhibitor (DRP1i1), delivered using a cardiac-targeted nanoparticle drug delivery system, as a more effective approach for achieving acute cardioprotection. DRP1i1 was encapsulated in cubosome nanoparticles with conjugated cardiac-homing peptides (NanoDRP1i1) and the encapsulation efficiency was 99.3 ± 0.1 %. In vivo, following acute myocardial ischaemia-reperfusion injury in mice, NanoDRP1i1 significantly reduced infarct size and serine-616 phosphorylation of Drp1, and restored cardiomyocyte mitochondrial size to that of sham group. Imaging by mass spectrometry revealed higher accumulation of DRP1i1 in the heart tissue when delivered as NanoDRP1i1. In human cardiac organoids subjected to simulated ischaemia-reperfusion injury, treatment with NanoDRP1i1 at reperfusion significantly reduced cardiac cell death, contractile dysfunction, and mitochondrial superoxide levels. Following NanoDRP1i1 treatment, cardiac organoid proteomics further confirmed reprogramming of contractile dysfunction markers and enrichment of the mitochondrial protein network, cytoskeletal and metabolic regulation networks when compared to the simulated injury group. These proteins included known cardioprotective regulators identified in human organoids and in vivo murine studies following ischaemia-reperfusion injury. DRP1i1 is a promising tool compound to study Drp1-mediated mitochondrial fission and exhibits promising therapeutic potential for acute cardioprotection, especially when delivered using the cardiac-targeted cubosome nanoparticles.

SIRT3 and RORα are two prospective targets against mitophagy during simulated ischemia/reperfusion injury in H9c2 cells

Autophagy during myocardial ischemia/reperfusion (MI/R) exacerbates cardiomyocyte injury. Melatonin (Mel) alleviates myocardial damage by regulating mitochondrial function and mitophagy, but the role of mitophagy in melatonin-induced cardioprotection remains unclear. This study aimed to explore the roles of sirtuin3 (SIRT3) and retinoid‐related orphan nuclear receptor‐α (RORα) in mitophagy during simulated ischemia reperfusion (SIR) in H9c2 cells. Our data showed that mitophagy was excessively activated after SIR injury, which was consistent with reduced cell survival, enhanced oxidative responses and mitochondrial dysfunction in H9c2 myocytes. Melatonin greatly enhanced cell viability, reduced oxidative stress and improved mitochondrial function. The effects of melatonin protection were involved in excessive mitophagy inhibition, as demonstrated by the reduced levels of mitophagy-linked proteins, including Parkin, Beclin1, NIX and BNIP3, and the LC3 II/LC3 I ratio and elevations in p62. Additionally, the decreases in SIRT3 and RORα in H9c2 myocytes after SIR were reversed by melatonin, and the above effects of melatonin were eliminated by small interfering RNA (siRNA)-mediated knockdown of SIRT3 and RORα. In brief, SIRT3 and RORα are two prospective targets in the cardioprotection of melatonin against mitophagy during SIR in H9c2 myocytes.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury

BackgroundCellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia–reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes.MethodsSCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs).ResultsAfter 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression.ConclusionAdding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

Low-intensity pulsed ultrasound of different intensities differently affects myocardial ischemia/reperfusion injury by modulating cardiac oxidative stress and inflammatory reaction.

The prevalence of ischemic heart disease has reached pandemic levels worldwide. Early revascularization is currently the most effective therapy for ischemic heart diseases but paradoxically induces myocardial ischemia/reperfusion (MI/R) injury. Cardiac inflammatory reaction and oxidative stress are primarily involved in the pathology of MI/R injury. Low-intensity pulsed ultrasound (LIPUS) has been demonstrated to reduce cell injury by protecting against inflammatory reaction and oxidative stress in many diseases, including cardiovascular diseases, but rarely on MI/R injury. This study was designed to clarify whether LIPUS alleviates MI/R injury by alleviating inflammatory reaction and oxidative stress. Simultaneously, we have also tried to confirm which intensity of the LIPUS might be more suitable to ameliorate the MI/R injury, as well as to clarify the signaling mechanisms. MI/R and simulated ischemia/reperfusion (SI/R) were respectively induced in Sprague Dawley rats and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). LIPUS treatment, biochemical measurements, cell death assay, estimation of cardiac oxidative stress and inflammatory reaction, and protein detections by western blotting were performed according to the protocol. In our study, both in vivo and in vitro, LIPUS of 0.1 W/cm2 (LIPUS0.1) and 0.5 W/cm2 (LIPUS0.5) make no significant difference in the cardiomyocytes under normoxic condition. Under the hypoxic condition, MI/R injury, inflammatory reaction, and oxidative stress were partially ameliorated by LIPUS0.5 but were significantly aggravated by LIPUS of 2.5 W/cm2 (LIPUS2.5) both in vivo and in vitro. The activation of the apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) pathway in cardiomyocytes with MI/R injury was partly rectified LIPUS0.5 both in vivo and in vitro. Our study firstly demonstrated that LIPUS of different intensities differently affects MI/R injury by regulating cardiac inflammatory reaction and oxidative stress. Modulations on the ASK1/JNK pathway are the signaling mechanism by which LIPUS0.5 exerts cardioprotective effects. LIPUS0.5 is promising for clinical translation in protecting against MI/R injury. This will be great welfare for patients suffering from MI/R injury.

Metformin protects against retinal ischemia/reperfusion injury through AMPK-mediated mitochondrial fusion

Retinal ischemia/reperfusion (I/R) injury is a common pathological process responsible for cellular damage in glaucoma, diabetic retinopathy and hypertensive retinopathy. Metformin is a biguanide drug that exerts strong effects on multiple diseases. This study aims to evaluate the protective effect of metformin against retinal I/R injury and its underlying mechanism. I/R induced reduction in retina thickness and cell number in ganglion cell layer, and metformin alleviated I/R-induced retinal injury. Both retinal I/R and simulated ischemia/reperfusion (SIR) in R28 cells down-regulated expression of mitochondrial fusion protein Mfn2 and OPA1, which led to mitochondrial fission. Metformin also alleviated damage in R28 cells, and reversed the alteration in Mfn2 and OPA1, mitochondrial fission and mitochondrial membrane potential (MMP) disruption-induced by I/R or SIR as well. Intriguingly, inhibition of AMPK by compound C or siRNA prevented metformin-mediated up-regulation of Mfn2 and OPA1. Compound C and knockdown of Mfn2 or OPA1 dramatically alleviated the protective effect of metformin against intracellular ROS generation, MMP disruption, mitochondrial fission and loss of RGCs in ganglion cell layer induced by SIR or I/R. Moreover, scavenging mitochondrial ROS (mito-ROS) by mito-TEMPO exerted the similar protection against I/R-induced retinal injury or SIR-induced damage in R28 cells as metformin. Our data show for the first time that metformin protects against retinal I/R injury through AMPK-mediated mitochondrial fusion and the decreased mito-ROS generation. These findings might also repurpose metformin as a therapeutic agent for retinal I/R injury.

Penehyclidine Hydrochloride Protects Rat Cardiomyocytes from Ischemia- Reperfusion Injury by Platelet-derived Growth Factor-B.

The lack of effective treatments for myocardial ischemiareperfusion (MI-R) injury severely restricts the effectiveness of the treatment of ischemic heart disease. In the present research, we aimed to investigate the protective effect and molecular mechanism of penehyclidine hydrochloride (PHC) on MI-R cells. Cell viability was quantified using CCK8. Cell apoptosis was analyzed using flow cytometry. Western blot and Elisa assays were used for the detection of target proteins. PHC pretreatment attenuated the inhibition of cell viability and decreased the percentage of apoptosis induced by simulated ischemia reperfusion (SIR). Platelet-derived growth factor B (PDGF-B) and its downstream AKT pathway were activated in PHC pretreated cells. After siRNAPDGF- B transfection, cell viability was inhibited and apoptosis was activated in PHC pretreated SIR cells, suggesting that PHC protected cells from SIR. PDGF-B knockdown also increased the levels of CK, LDH, IL-6 and TNF-α in PHC pretreated SIR cells. The effect of AKT inhibitor on H9C2 cells was consistent with that of PDGF-B knockdown. PHC pretreatment can protect cardiomyocytes from the decrease of cell activity and the increase of apoptosis caused by reperfusion through up-regulating PDGF-B to activate PI3K pathway. Our study indicates that PHC is a potential drug to protect cells from reperfusion injury and PDGF-B is a potential target for preventing MI-R injury.

Role of autophagy in simulated ischemic-reperfused left atrial myocardium

Background and aimAutophagy has recently emerged as a potential and promising therapeutic approach to maintain cardiac cellular homeostasis. The aim of the present study was to investigate the role of autophagy in the ischemic-reperfused atrial myocardium. MethodsIsolated rat left atria subjected to simulated ischemia-reperfusion were used. The bathing medium contained either 10 mM d-glucose or 10 mM d-glucose and 1.2 mM palmitate. 3-methyladenine (3-MA) was used as pharmacological autophagy inhibitor. ResultsLC3-II/LC3-I ratio, an indicator of autophagosome formation, was significantly enhanced during reperfusion, this increase being slowed by the exposure to high palmitate concentration and prevented by 3-MA. Beclin-1 was significantly increased during reperfusion period in both metabolic conditions, and pharmacological inhibition of AMPK partially prevented LC3-II/LC3-I ratio increase. Autophagy inhibition significantly increased mitochondrial damage and impaired mitochondrial ATP synthesis rate at reperfusion. Tissue ATP content recovery and contractile reserve were also reduced during this period, these effects being more pronounced either in 3-MA treated atria and ischemic-reperfused atria incubated with palmitate. Moreover, severe tachyarrhythmias were observed in the presence of 3-MA, in both metabolic conditions. This phenomenon was partially prevented by mitochondrial inner membrane ion channels blocker, PK11195. ConclusionPresent study provides new insights into the role of autophagy in ischemic-reperfused atrial myocardium. The observation of greater deterioration in mitochondrial structure and function when this process was inhibited, suggests an association between autophagy and the structural and functional preservation of mitochondria. Exogenous metabolic substrates, to which the myocardium is exposed during ischemia-reperfusion, might not affect this process.

Bergenin alleviates myocardial ischemia‐reperfusion injury via SIRT1 signaling

Myocardial ischemia-reperfusion (MI/R) is a major risk factor for cardiovascular disease. At present, reducing oxidative stress and apoptosis is a crucial therapeutic strategy for ameliorating MI/R injury. However, there is a lack of drugs targeting oxidative stress and apoptosis for the clinical therapy of MI/R. Bergenin is a reportedly effective agent with antioxidative and antiapoptotic activity against acute injury. Nevertheless, the roles and potential mechanisms of bergenin against MI/R injury remain unknown. Here, we hypothesized that bergenin attenuated MI/R-induced apoptosis and reactive oxygen species (ROS) production via SIRT1. Mice were subjected to MI/R and treated with bergenin, after which the cardiac function, cardiomyocyte apoptosis, LDH release, and MDA content were evaluated. In vitro, myocardial injury model of H9c2 cells was induced by simulated ischemia/reperfusion (SI/R), apoptosis and oxidative stress was decreased after treated with bergenin. Bergenin significantly reduced myocardial apoptosis and ROS generation in vitro and improved cardiac function in vivo. Intriguingly, bergenin remarkably decreased apoptosis in cardiac tissue accompanied by SIRT1 upregulation following MI/R injury. Further studies showed that inhibiting SIRT1 blocked bergenin’s beneficial impact against apoptosis following SI/R injury through excessive oxidative stress and depression of the Bcl2 to Bax ratio. Collectively, these findings indicate that bergenin alleviates MI/R injury by ameliorating myocardial apoptosis and oxidative damage via the SIRT1 signaling pathway.

Role of cardiac mitofusins in cardiac conduction following simulated ischemia–reperfusion

Mitochondrial dysfunction induced by acute cardiac ischemia–reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.

Salidroside attenuates myocardial ischemia/reperfusion injury via AMPK-induced suppression of endoplasmic reticulum stress and mitochondrial fission

Ischemic heart disease (IHD) is the primary cause of death worldwide. Salidroside (Sal), the major active compound derived from Rhodiola rosea, is believed to have cardioprotective effects. AMP-activated protein kinase (AMPK), is a pivotal AMP-activated protein kinase in energy metabolism. Whether Sal plays an anti-endoplasmic reticulum stress/mitochondrial fission role through AMPK remains elusive. In this study, we established a myocardial ischemia/reperfusion (I/R) rat model. Rat hearts exposed to Sal with or without compound C were then subjected to I/R. Further, H9c2 cardiomyocytes were subjected to simulated ischemia/reperfusion (SIR) by hypoxia-reoxygenation. The rats and cardiomyocytes were pretreated with Sal, followed by Compound C and AMPK-siRNA to block AMPK activity. We found that Sal significantly ameliorated cardiac function, mitigated infarct size and serum content of lactate dehydrogenase and creatine kinase, improved mitochondrial function, and reduced mitochondrial fission and apoptosis. Furthermore, in cultured H9c2 cardiomyocytes, Sal increased the cell viability and inhibited SIR-induced myocardial apoptosis and mitochondrial fission. Furthermore, the translocation of Drp1 from the cytoplasm to mitochondria induced by salidroside was confirmed both in vivo and in vitro. However, the use of Compound C or AMPK siRNA to block AMPK activity leads to blockade of the protective effects of Sal. In summary, protects against myocardial I/R by activating the AMPK signaling pathway, inhibiting ER stress, and reducing mitochondrial fission and apoptosis.

5-Methyltetrahydrofolate Attenuates Oxidative Stress and Improves Kidney Function in Acute Kidney Injury through Activation of Nrf2 and Antioxidant Defense.

Oxidative stress is a major mediator of adverse outcomes in acute kidney injury (AKI). Deficiency of micronutrients, such as folate, is common in AKI. Our previous study reported that AKI impaired kidney reabsorption of folate and decreased plasma folate level in rats. The present study investigated the effect of 5-methyltetrahydrofolate (5-MTHF), a biologically active form of folate/folic acid, on AKI-impaired kidney function and oxidative stress. Sprague-Dawley rats developed AKI after kidney ischemia (45 min) and reperfusion (24 h). Injection of 5-MTHF (3 µg/kg body weight) improved kidney function and attenuated oxidative stress with a restoration of glutathione and a reduction of lipid peroxidation in the kidney. Injection of 5-MTHF activated transcription factor Nrf2 and increased the expression of glutathione synthesizing enzymes, superoxide dismutase-1 and heme oxygenase-1 in the kidney. Simulated ischemia-reperfusion through hypoxia-reoxygenation increased oxidative stress in proximal tubular cells. Incubation of cells with 5-MTHF alleviated cell injury and increased antioxidant enzyme expression and intracellular glutathione levels. Inhibition of Nrf2 expression through siRNA transfection abolished the effect of 5-MTHF against oxidative stress. These results suggest that low-dose folic acid can improve kidney function through activation of Nrf2 and restoration of antioxidant defence. Micronutrient supplements may improve clinical outcomes in AKI.

Effect of Pre‐Conditioning with Copolymer‐Based Cell Membrane Stabilizer Poloxamer 188 on Adult Mouse Coronary Artery Endothelial Layer Integrity after Prolonged Hypoxia/Reoxygenation

Prolonged ischemia followed by reperfusion can cause significant tissue damage and cell death. This ischemia/reperfusion (IR) injury can be alleviated by administration of Copolymer‐based Cell Membrane Stabilizers (CCMS), like the tri‐block Poloxamer (P)188, thought to be able to seal gaps in cell membranes and/or tight junctions and, thus, repair leaks attributable to severe cellular stress. We have previously tested post‐conditioning, i.e. the administration of P188 upon reperfusion/reoxygenation, to attenuate IR injury in various models. Here, we hypothesized that pre‐conditioning, i.e. its administration before simulated ischemia (hypoxia), has a protective effect on endothelial cell layer integrity as assessed by Trans‐Endothelial Electrical Resistance (TEER).Confluent adult mouse coronary artery endothelial cell (ECs) layers were pre‐treated with 0, 30, 100, 300, 1,000, or 2,000 µM P188 during plating of cells before simulated IR with a 24‐hour hypoxic followed by a 2‐hour reoxygenation (HR) period. EC monolayer barrier integrity was assessed by TEER.In contrast to mere post‐conditioning, pre‐conditioning dose‐dependently attenuated HR injury compared to control/normoxia (Fig 1) even after prolonged HR for 24+2 hrs.These results suggest that CCMS may indeed elicit its cardioprotective effect, in part, through protection of the EC layer. However, more research is needed to determine optimal dosage and timing for CCMS administration.

Parathyroid Hormone-Related Peptide and Its Analog, Abaloparatide, Attenuate Lethal Myocardial Ischemia-Reperfusion Injury.

Parathyroid hormone-related peptide (PTHrP) is well-known to play a role in bone formation, and abaloparatide, an analog of PTHrP(1-34), is approved for the treatment of osteoporosis in post-menopausal women. PTHrP has also been reported to have cardiovascular effects, with recent data demonstrating that exogenously administered PTHrP can limit the death of isolated cardiomyocytes subjected to oxidative stress via upregulation of classic ‘survival kinase’ signaling. Our aim in the current study was to extend this concept and, employing both in vitro and in vivo models, establish whether PTHrP(1-36) and abaloparatide are cardioprotective in the setting of lethal myocardial ischemia-reperfusion injury. We report that preischemic administration of PTHrP(1-36) and abaloparatide attenuated cell death in HL-1 cardiomyocytes subjected to simulated ischemia-reperfusion, an effect that was accompanied by the augmented expression of phospho-ERK and improved preservation of phospho-Akt, and blocked by co-administration of the MEK-ERK inhibitor PD98059. Moreover, using the translationally relevant swine model of acute coronary artery occlusion-reperfusion, we make the novel observation that myocardial infarct size was significantly reduced in pigs pretreated with PTHrP(1-36) when compared with placebo-controls (13.1 ± 3.3% versus 42.0 ± 6.6% of the area of at-risk myocardium, respectively; p < 0.01). Taken together, these data provide the first evidence in support of the concept that pretreatment with PTHrP(1-36) and abaloparatide renders cardiomyocytes resistant to lethal myocardial ischemia-reperfusion injury.

Antioxidant Activity Of Rat Liver With A Low Resistance To Hypoxia After Systemic Ischemia Reperfusion

Objective — To assess the antioxidant activity of rat liver after systemic ischemia reperfusion (IRP). Material and Methods — The study was conducted on 70 male rats. For all animals of the treatment group (n=35) under ether anesthesia, we were stopping stopping systemic circulation for five minutes. After that, the animals were given an external cardiac massage and artificial lung ventilation. We did not perform circulatory arrest after ether anesthesia in animals of the control group (n=35). In all animals, we were measuring the levels of serum hormones (corticosterone, aldosterone), the content of glucocorticoid and mineralocorticoid receptors in liver homogenates, and the activity of enzymes of the antioxidant system (superoxide dismutase and catalase). We were making control measurements on days 1, 3, 5, 7, 14, 21, and 35 after the simulated IRP. Results — On day 1 after simulation of IRP development, the levels of cortisol and aldosterone in the serum of treatment group rats were significantly higher, by 14.3% and 33.5%, respectively, compared with the control group. In response to stress (IRP), we observed the highest concentration of cortisol in the blood of treatment group rats on day 3 (p=0.0002), which decreased afterwards. On day 1 after IRP, there was a reduction in the activity of superoxide dismutase and catalase in treatment group rats, by 50.3% and by 29%, respectively (p&lt;0.0001). The lowest antioxidant activity in the rat liver after IRP was observed on days 3-7. Conclusion — Systemic IRP is associated with pronounced changes in the dynamics of corticosteroid receptors in the liver, which leads to a reduction in the activity of key antioxidant enzymes.

Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

LncRNA TUG1 aggravates cardiomyocyte apoptosis and myocardial ischemia/reperfusion injury.

Cardiomyocyte apoptosis is a fundamental pathogenic factor leading to myocardial ischemia/reperfusion (MI/R) injury. The long non-coding RNA (IncRNA) TUG1 regulates apoptosis in various cell types. We report here that TUG1 expression is induced in mouse heart following MI/R injury as well as in cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R) in vitro. Clinically, TUG1 expression is also elevated in plasma from patients with acute myocardial infarction (AMI), which implies its potential application as a disease biomarker. Functionally, TUG1 overexpression promotes, and its knockdown reduces SI/R-induced lactate dehydrogenase (LDH) release and caspase-3 activity in cardiomyocytes in vitro, illustrating that TUG1 exacerbates SI/R-induced apoptosis. Furthermore, in vivo, TUG1 aggravates MI/R injury in a mouse model, and subsequent observations show concurrent increased apoptosis of cardiomyocytes. Hence, this study unveils a clinical relevance and functional role of TUG1 in MI/R injury, and also implicates that targeting TUG1 may have therapeutic effects in treating MI/R injury.