Ischemia-reperfusion Injury Model Research Articles

Multicellular 3D models to study myocardial ischemia–reperfusion injury

Coronary heart disease is a major global health threat, with acute myocardial ischemia–reperfusion injury (IRI) being a major contributor to myocardial damage following an ischemic event. IRI occurs when blood flow to ischemic tissues is restored and exacerbates the cellular damage caused by ischemia/hypoxia. Although animal studies investigating IRI have provided valuable insights, their translation into clinical outcomes has been limited, and translation into medical practice remains cumbersome. Recent advancements in engineered three-dimensional human in vitro models could offer a promising avenue to bridge the “therapeutic valley of death” from bench to bedside, enhancing the understanding of IRI pathology. This review summarizes the current state-of-the-art cardiovascular 3D models, including spheroids, organoids, engineered cardiac microtissues, and organ-on-a-chip systems. We provide an overview of their advantages and limitations in the context of IRI, with a particular emphasis on the crucial roles of cell–cell communication and the multi-omics approaches to enhance our understanding of the pathophysiological processes involved in IRI and its treatment. Finally, we discuss currently available multicellular human 3D models of IRI.

Abstract 4140123: Real-time imaging of microvasculature obstruction and the vasculoprotection of nitric-oxide-donor nanoparticles during acute myocardial ischemia/reperfusion injury

Background/Introduction: Microvascular obstruction (MVO), due to damage to the coronary microvasculature, is a key determinant of infarct size, heart failure and poor outcomes following acute myocardial infarction, and there is currently no treatment for preventing MVO. Real-time in vivo imaging of MVO in the beating rodent heart is challenging due to the limited spatial and temporal resolution from movement artifacts. Here, we apply, for the first time, fiber-optic confocal laser endomicroscopy (CLM) for real-time imaging of the microvasculature in a beating murine heart with acute ischemia/reperfusion injury (IRI), and then monitoring the development of MVO. Methods: An in vivo murine acute myocardial IRI model (45 min ligation of left coronary artery (LCA) and 30 min reperfusion) was applied. At 10 min prior to ischaemia, 150 µl Dextran-FITC (150 kDa, 10 mg/ml) was injected retro-orbitally, and then CLM imaging with a flexible miniprobe (ProFlex S-1500 with CellVizio system) was applied to the epicardial surface at multiple sites at 5 min post-injection (baseline), 30 min post-ischemia and 30 min post-reperfusion. A nitric oxide donor(NO) nanoparticle (NONP) was synthesized and IV bolus injected into IRI mice 5min prior to reperfusion to prevent MVO. Results: We confirmed visualization of the macro- and microvasculature at various sites on the epicardial surface of the beating heart. Next, we observed reduced microvasculature blood flow below LCA ligature as evidenced by reduced or even totally absence of FITC within the vessels at 30min post-ischemia. The microvasculature at the non-ischemic myocardium was unaffected. Furthermore, at 30 min post-reperfusion, we visualised patchy areas of reduced FITC signal suggesting MVO, and damaged microvasculature as evidenced by leakage of FITC outside the vessel. Interestingly, NONP treatment preserved the microvascular network and prevented MVO at 30 min post-reperfusion with even greater FITC, suggesting increased microvascular blood flow and penetration into cardiac tissue because of the vasodilatory effect of NO in the ischemic area. Conclusion: With CellVizio CLM system, we have demonstrated the MVO development during IRI, and damage to the microvasculature with leakage of dye from vessels into cardiac interstitium, thereby providing a pre-clinical platform to test novel therapeutic agents for preventing MVO. Importantly, we have shown an effective MVO prevention with NO-donor nanoparticle following IRI in mice.

Circulating mtNFPs Are Associated with ARDS after CPB and Regulate Endothelial Barrier through FPR2.

Cardiopulmonary bypass (CPB) increases the risk of acute respiratory distress syndrome (ARDS) due to endothelial cell (EC) barrier dysfunction. However, the specific role of mitochondrial N-formyl peptides (mtNFPs) in ARDS following CPB remains unexplored. Here, we investigated the differential expression of circulating mtNFPs in patients after CPB, focusing on the novel role of FPR2 in ECs. Levels of circulating mtNFPs were assessed using enzyme-linked immunosorbent assay (ELISA). Several mtNFPs (ND4, ND5, ND6, and Cox1) were significantly upregulated in patients with ARDS at day 1 post-CPB compared to patients without ARDS. Higher levels of ND6 were correlated with worst PaO2/FiO2 (r=-0.2219 and P<0.0001) and cardiac Troponin T (r=2.107 and P<0.0001). Utilizing patient-derived serum and a rat lung ischemia reperfusion injury (LIRI) model, we observed a positive correlation between serum ND6 concentration and ARDS, which is also associated with EC barrier dysfunction. In vitro experiments, using trans-endothelial electric resistance (TEER) measurements and fluorescence microscopy with FITC-labeled VE-cadherin, demonstrated that ND6 disrupts the EC barrier through FPR2. Furthermore, FPR2 controls the release of ND6 out of mitochondria and cytoplasm under hypoxia reoxygenation (HR). Activated FPR2 leads to upregulation of nuclear transcription factor-kappa B (NF-κB) by inducing IκBα phosphorylation, promoting ICAM1 and VCAM1 expression, thereby compromising EC barrier integrity. Circulating pro-inflammatory and barrier-disruptive mtNFPs, particularly ND6, are associated with ARDS in patients undergoing CPB. The novel ND6-FPR2 axis regulates inflammation and EC permeability through the NF-κB pathway.

Molecular mechanisms of ferroptosis in renal ischemia-reperfusion injury Investigated via bioinformatics analysis and animal experiments.

Kidney transplantation is a pivotal treatment for end-stage renal disease. However, renal ischemia-reperfusion injury (IRI) during surgery significantly impacts graft function. Despite unclear molecular mechanisms, no specific therapies or preventative measures are available. Gene expression profiles from renal biopsies before and after IRI were downloaded from public databases. Differentially expressed genes were identified using the Wilcoxon rank-sum test and weighted gene co-expression network analysis. Ferroptosis-associated genes were screened using the FerrDb database. The genes with the highest connectivity were identified via the protein-protein interaction (PPI) network and upstream regulatory miRNAs were found through the gene-miRNA network. A mouse renal IRI model was constructed for transcriptome sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) validation to elucidate the relationship between key ferroptosis genes and regulatory miRNAs in renal IRI. Differential analysis identified 15 ferroptosis-associated genes (TNFAIP3, IL6, KLF2, EGR1, JUN, ZFP36, GDF15, CDKN1A, HSPB1, BRD2, PDK4, DUSP1, SLC2A3, DDIT3, and CXCL2) involved in renal IRI regulation. In animal experiments, ferroptosis-related genes were also upregulated in the model group. Enrichment analysis and hematoxylin-eosin pathological staining suggested these genes are primarily involved in renal inflammatory responses. PPI network analysis revealed IL6 as the gene with the highest connectivity, and the gene-miRNA network indicated IL6 might be regulated by miR-let-7a. Animal experiments revealed decreased miR-let-7a and increased IL6 levels in the model group, identifying potential therapeutic targets. MiR-let-7a regulates ferroptosis in renal IRI by targeting IL6, highlighting IL6 as a crucial gene in the ferroptosis process of renal IRI.

Severe ischemia and reperfusion injury induces epigenetic inactivation of LHX1 in kidney progenitor cells after kidney transplantation

Severe ischemia-reperfusion injury (IRI) causes acute and chronic kidney allograft damage. As therapeutic interventions to reduce damage are limited yet, research on how to promote kidney repair has gained significant interest. To address this question, we performed genome-wide transcriptome and epigenome profiling in progenitor cells isolated from urine of deceased (severe IRI) and living (mild IRI) donor human kidney transplants and identified LIM homebox-1 (LHX1) as an epigenetically regulated gene whose expression depends on the IRI severity. Using a mice model of IRI, we observed a relationship between IRI severity, LHX1 promoter hypermethylation and LHX1 gene expression. By functional studies we confirmed that LHX1 expression is involved in proliferation of epithelial tubular cells and podocyte differentiation from kidney progenitor cells. Our results provide evidence that severe IRI may reduce intrinsic mechanisms of kidney repair through epigenetic signaling.

Metformin Attenuates Myocardial Ischemia-Reperfusion Injury through the AMPK-HMGCR-ROS Signaling Axis.

To explore the role and mechanism of metformin (MET) in regulating myocardial injury caused by cardiac ischemia-reperfusion. A rat model of myocardial ischemia-reperfusion injury was established by ligation of the anterior descending branch of the left coronary artery. The myocardial area at risk and the infarction size were measured by Evans blue and 2,3,5‑triphenyltetrazole chloride (TTC) staining, respectively. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining was used to detect apoptosis of cardiomyocytes. The expression of 4‑hydroxynonenal (4‑HNE) was detected by immunohistochemical staining. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and expression of the Adenosine 5'-monophosphate-activated protein kinase (AMPK) - 3‑hydroxy-3‑methylglutaryl-CoA reductase (HMGCR) signaling pathway, respectively. MET treatment decreased the infarct size and the activity of the myocardial enzyme profile, thus demonstrating protection of ischemic myocardium. The number of TUNEL positive cells significantly decreased. Immunohistochemical results showed that MET decreased the expression of 4‑HNE in myocardial tissue and the content of malondialdehyde (MDA) in myocardial cells. Further experimental results showed that MET decreased HMGCR transcription and protein expression, and increased AMPK phosphorylation. In the model of hypoxia and reoxygenation injury of cardiomyocytes, MET increased the viability of cardiomyocytes, decreased the activity of lactic dehydrogenase (LDH), decreased malondialdehyde content and intracellular reactive oxygen species (ROS) concentrations, and regulate the AMPK-HMGCR signaling pathway through coenzyme C (ComC). MET inhibits the expression of HMGCR by activating AMPK, reduces oxidative damage and apoptosis of cardiomyocytes, and alleviates myocardial ischemia-reperfusion injury.

Ischemic Rescue Potential of Conditioned Medium Derived from Skeletal Muscle Cells-Seeded Electrospun Fiber-Coated Human Amniotic Membrane Scaffolds.

Revascularization procedures such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are crucial to restore blood flow to the heart and are used in the treatment of myocardial infarction (MI). However, these techniques are known to cause myocardial reperfusion injury in the ischemic heart. The present study aims to mimic ischemia-reperfusion injury in vitro on primary human cardiomyocytes (HCMs) and use the established injury model to study the rescue mechanism of skeletal muscle cell (SkM)-seeded electrospun fiber-coated human amniotic membrane scaffold (EF-HAM) on injured cardiomyocytes through paracrine secretion. An in vitro ischemia-reperfusion injury model was established by exposing the HCM to 5 h of hypoxia, followed by a 6 h reoxygenation period. Six different conditioned media (CM) including three derived from SkM-seeded EF-HAMs were introduced to the injured cells to investigate the cardioprotective effect of the CM. Cell survival analysis, caspase-3 and XIAP expression profiling, mitochondrial membrane potential analysis, and measurement of reactive oxygen species (ROS) were conducted to evaluate the outcomes of the study. The results revealed a significant increase in the viability of HCM exposed to H/R injury by 77.2% (p < 0.01), 111.8% (p < 0.001), 68.7% (p < 0.05), and 69.5% (p < 0.05) when supplemented with HAM CM, EF-HAM 3 min CM, EF-HAM 5 min CM, and EF-HAM 7 min CM, respectively. Furthermore, CM derived from SkM-seeded EF-HAM scaffolds positively impacted hypoxia-/reoxygenation-induced changes in caspase-3 expression, mitochondrial membrane potential, and reactive oxygen species generation, but not in XIAP expression. These findings suggest that EF-HAM composite scaffolds can exert antiapoptotic and cardioregenerative effects on primary human cardiomyocytes through the paracrine mechanism.

ND-13 preserves mitochondrial integrity via the RhoA/ROCK1/Drp1-S616 pathway providing acute cardioprotection in myocardial infarction

Abstract Background Ischemia-reperfusion injury (IRI) following acute myocardial infarction is a major contributor to heart failure and mortality worldwide. Drp1-driven mitochondrial fragmentation disrupts mitochondrial function, increases cardiomyocyte death, and aggravates cardiac dysfunction. We investigated whether ND-13, a novel DJ-1-derived peptide, can acutely protect the heart by maintaining mitochondrial homeostasis and modulating Drp1 activity during IRI. Methods The effects of ND-13 on Drp1 activity were assessed by studying mitochondrial dynamics, function and survival signalling pathways. Cardioprotective effects of ND-13 were assessed using in vitro, ex vivo and in vivo models of IRI. Results ND-13 demonstrated acute cardioprotective effects in murine adult cardiomyocytes subjected to simulated IRI by a 42% reduction in cell death. The excessive mitochondrial fission following IRI was attenuated by inhibiting Drp1 phosphorylation at the Ser616 residue by reduced activity of the RhoA/ROCK1 pathway. This in turn enhanced mitochondrial function as seen through increased mitochondrial respiration rates (basal, maximal and spare respiratory capacity), increased ATP production, maintained mitochondrial membrane potential and reduced reactive oxygen species levels. The acute cardioprotective effect of ND-13 was reproduced in both an ex vivo Langendorff based model of IRI with a 43% reduction infarct size and an in vivo murine model of IRI with a 35% reduction of infarct size. These results indicate the robust effect of the peptide and its potential for clinical translation through intravenous delivery. Conclusion In this study, we demonstrate ND-13 as a small peptide that modulates mitochondrial dynamics through the RhoA/ROCK1/DRP1-S616 pathway, which highlights a potential new therapeutic target for myocardial IRI.

Nephroprotective role of resveratrol in renal ischemia-reperfusion injury: a preclinical study in Sprague-Dawley rats.

Renal ischemia-reperfusion injury (IRI) is a significant contributor to renal dysfunction, acute kidney injury (AKI), and associated morbidity and mortality. Resveratrol, a polyphenol and phytoalexin, is known for its anti-inflammatory, antioxidant, and anti-cancer properties. This study investigates the nephroprotective potential of resveratrol in a rat model of renal IRI. Twenty-eight male Sprague-Dawley rats were divided into four groups: Sham, IRI, DMSO, and Resveratrol. The Sham group underwent identical procedures without renal pedicle clamping, while the IRI group experienced 30min of ischemia followed by 2h of reperfusion. The DMSO group received dimethyl sulfoxide (DMSO) intraperitoneally 30min before ischemia, and the Resveratrol group received 30mg/kg resveratrol intraperitoneally 30min before ischemia. Biochemical parameters (Urea, creatinine, IL-1β, NF-κβ, SOD, GSH, Bcl-2, and caspase-3) and histopathological changes were assessed. IRI caused a substantial increase in serum creatinine, Urea, IL-1β, NF-κβ, and caspase-3 levels, while simultaneously decreasing SOD, GSH, and Bcl-2 levels. Resveratrol treatment mitigated these effects by lowering inflammatory and apoptotic markers, enhancing antioxidant defenses, and improving histological outcomes. Resveratrol demonstrates significant nephroprotective effects in renal IRI, primarily through its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Early Cardiac Ischemia–Reperfusion Injury: Interactions of Autophagy with Galectin-3 and Oxidative Stress

Background: Cardiovascular diseases are the leading cause of death worldwide, including the United Arab Emirates. Ischemia–reperfusion (IR) injury results in the death of cardiac myocytes that were viable immediately before myocardial reperfusion. We aim to investigate the role of galectin-3 (Gal-3) in autophagy during ischemia–reperfusion injuries. Methods: Male C57B6/J and Gal-3 knockout (KO) mice were used for the murine model of IR injury. Heart samples and serum were collected 24 h post-IR and were processed for immunohistochemical and immunofluorescent labeling and an enzyme-linked immunosorbent assay. Results: There was a significant increase in left ventricle (LV) concentrations of Gal-3 in Gal-3 wild-type mice compared to sham mice. There were significantly higher concentrations of LV autophagy proteins and phospho-AMPK in IR Gal-3 KO mice than in IR Gal-3 wild-type mice, compared to lower concentrations of LV phospho-mTOR and p62 in IR Gal-3 KO than in IR wild-type mice. Antioxidant activities were higher in the LVs of IR Gal-3 wild-type mice, while oxidative stress was higher in the LVs of IR Gal-3 KO mice. Conclusions: Our study supports the interaction of Gal-3 with autophagy proteins, oxidative stress, and antioxidant proteins and demonstrates that the absence of Gal-3 can enhance autophagy in the heart after IR injury.

Cardioprotective potential of oleuropein, hydroxytyrosol, oleocanthal and their combination: unravelling complementary effects on acute myocardial infarction and metabolic syndrome

Abstract Background/Introduction Clinical studies have established the role of olive products in primary and secondary prevention of cardiovascular (CV) disease, but the single compounds or compound combinations responsible for the protective effects are still under investigation. Purpose We sought to explore and compare the cardioprotective potential of Oleuropein (OL), Hydroxytyrosol (HT), Oleocanthal (OC) and Oleanolic Acid (OA) and three combinational treatments after chronic oral administration in mice subjected to Ischemia/Reperfusion Injury (IRI) in the presence of metabolic syndrome (MS) and verify the effect of combination in a proof-of-concept clinical study in Chronic Coronary Artery Syndrome (CCAS) patients. Methods First, we explored the IRI-limiting potential of 6-weeks daily oral treatment with OL (20.6 mg/kg), HT (5.9 mg/kg), OC (11.6 mg/kg), OA (17.4 mg/kg) or vehicle (DMSO 5%) in healthy C57Bl6 mice. At week 6, myocardial ischemia/reperfusion (30 minutes/2 hours) was induced and infarct size was measured. Next, MS was established by 14 weeks of Western diet and the isolated compounds were administered for the last 6 weeks. At week 14, IRI was surgically induced, and infarct size was determined. Basic MS parameters (fasting blood glucose, plasma cholesterol, body weight and blood pressure) were recorded at baseline, weeks 8 and 14. According to the exerted benefits, three combinational treatments were designed and tested on the MS model of IRI. The underlying cardioprotective mechanism was investigated on the mixture with preponderant CV benefits (OL-HT-OC, Combo 2). ApoE-/- mice fed with Western diet for 12 weeks were treated with Combo 2 orally for the last 4 weeks and aortic atherosclerotic lesion extent was determined to detect possible antiatherosclerotic effects. After multi-organ toxicity evaluation, a Combo 2-based supplement was administered in CCAS patients for evaluation of cardiac function, inflammation and oxidative stress biomarkers. Results OL, OC and OA, but not HT, significantly reduced infarct size in vivo both in healthy and MS mice. OL was the only compound to reduce hyperglycemia, while OA significantly reduced plasma cholesterol. All mixtures (Combo 1: OL-HT-OA, Combo 2: OL-HT-OC, Combo 3: OL-HT-OC-OA) were cardioprotective, but only Combo 2 prevented MS progression by limiting hyperglycemia. Combo 2 protection against IRI was attributed to apoptosis suppression (reduced Bax/BcL-xL ratio) and increased expression of the antioxidant enzymes Superoxide Dismutase and Catalase in the ischemic myocardium. Combo 2 treatment also reduced atherosclerotic lesion extent. In our proof-of-concept clinical study, Combo 2 treatment ameliorated cardiac, vascular and endothelial function in CCAS patients. Conclusions Combinational treatment with OL-HT-OC exerts potent cardioprotective, antihyperglycemic and antiatherosclerotic properties in vivo, with remarkable and clinically translatable CV benefits in high-risk patients.

Complement factor D deficiency ameliorates adverse cardiac remodelling by inhibiting C5a-C5aR mediated ISGs+ neutrophil activation post myocardial infarction

Abstract Background Exaggerated inflammatory response after myocardial infarction (MI) exacerbates myocardial injury and adverse cardiac remodeling, leading to heart failure. Current management of MI involves myocardial protection and antifibrotic therapy but do not target the inflammatory response per se. Complement factor D (CFD) is an upstream rate-limiting enzyme of complement system activation, which is the early event of inflammatory response post MI. We hypothesize that CFD can be an important modulator of the inflammation-remodeling cascade post MI, but the specific mechanism remains elusive. Purpose To investigate the role and mechanism of CFD in regulating inflammatory expansion and adverse remodeling post MI. Methods We generated Cfd-AKO (Cfdfl/fl Adipoq-cre) mice using the Cre-LoxP recombination system, as CFD is mainly secreted by adipocytes. Immune status and cardiac remodeling progress were assessed by cardiovascular magnetic resonance (CMR), histology, flow cytometry, immunofluorescence, and ELISA. Single cell RNA sequencing (scRNA-seq) analyzed non­cardiomyocytes isolated from Cfd-AKO and WT mice post MI or sham operation. Genetic ablation and co-culture experiments dissected the functional molecular properties and intercellular communication. An CFD inhibitor ACH-4471 was used to explore the role of CFD in ischemia-reperfusion (IR) injury. Results The survival of Cfd-AKO mice was strongly improved, while ventricular arrhythmia induction rates and number of episodes were significantly reduced compared with those of WT mice. CMR imaging showed that Cfd-AKO mice had higher ejection fraction and reduced left ventricular dilation than WTs. Masson trichrome and picrosirius red staining revealed that besides smaller infarct size, the extent of fibrosis in the border and distal remote area decreased in Cfd-AKO mice. Cfd loss reduced myocardial injury, inhibited the release of inflammatory factors, and limits the amplification of complement components C3a and C5a post MI. ScRNA-seq revealed that CFD modulated neutrophil chemotactic ability and pro-inflammatory function by inducing interferon-stimulated genes expressing (ISG+) neutrophil CXCL10 expression. Mechanistically, CFD activates the ISG transcriptional profile via the C5a/C5aR dependent STAT1/2-IRF9 pathway. Activated neutrophils promote myofibroblast transformation through IL1β/IL1R dependent intercellular communication, thus facilitating pathological fibrosis. Early pharmacological blocking of CFD in mice IR injury models inhibited inflammation, reduced infarct size and ameliorated cardiac dysfunction. Conclusions We demonstrate the crucial role of CFD in cardiac remodeling post MI. This process is critically regulated by C5a-C5aR, an important component of complement-inflammation cascade. This CFD-C5a mediated neutrophil pro-inflammatory and pro-fibrotic dual activity represents a novel pathway and an attractive therapeutic window for heart failure post MI.CFD in cardiac remodelling post MI

RIP1 inhibition protects retinal ganglion cells in glaucoma models of ocular injury

AbstractReceptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear. This study demonstrates that RIP1 inhibition protects retinal ganglion cells (RGCs) in preclinical glaucoma models. Genetic inactivation of RIP1 improves RGC survival and preserves retinal function in the preclinical glaucoma models of optic nerve crush (ONC) and ischemia–reperfusion injury (IRI). In addition, the involvement of necroptosis in ONC and IRI glaucoma models was examined by utilizing RIP1 kinase-dead (RIP1-KD), RIP3 knockout (RIP3-KO), and MLKL knockout (MLKL-KO) mice. The number of RGCs, retinal thickness, and visual acuity were rescued in RIP1-kinase-dead (RIP1-KD) mice in both models, while wild-type (WT) mice experienced significant retinal thinning, RGC loss, and vision impairment. RIP3-KO and MLKL-KO mice showed moderate protective effects in the IRI model and limited in the ONC model. Furthermore, we confirmed that a glaucoma causative mutation in optineurin, OPTN-E50K, sensitizes cells to RIP1-mediated inflammatory cell death. RIP1 inhibition reduces RGC death and axonal degeneration following IRI in mice expressing OPTN-WT and OPTN-E50K variant mice. We demonstrate that RIP1 inactivation suppressed microglial infiltration in the RGC layer following glaucomatous damage. Finally, this study highlights that human glaucomatous retinas exhibit elevated levels of TNF and RIP3 mRNA and microglia infiltration, thus demonstrating the role of neuroinflammation in glaucoma pathogenesis. Altogether, these data indicate that RIP1 plays an important role in modulating neuroinflammation and that inhibiting RIP1 activity may provide a neuroprotective therapy for glaucoma.

The renoprotective efficacy and safety of genetically-engineered human bone marrow-derived mesenchymal stromal cells expressing anti-fibrotic cargo

BackgroundKidney fibrosis is a hallmark of chronic kidney disease (CKD) and compromises the viability of transplanted human bone marrow-derived mesenchymal stromal cells (BM-MSCs). Hence, BM-MSCs were genetically-engineered to express the anti-fibrotic and renoprotective hormone, human relaxin-2 (RLX) and green fluorescent protein (BM-MSCs-eRLX + GFP), which enabled BM-MSCs-eRLX + GFP delivery via a single intravenous injection.MethodsBM-MSCs were lentiviral-transduced with human relaxin-2 cDNA and GFP, under a eukaryotic translation elongation factor-1α promoter (BM-MSCs-eRLX + GFP) or GFP alone (BM-MSCs-eGFP). The ability of BM-MSCs-eRLX + GFP to differentiate, proliferate, migrate, produce RLX and cytokines was evaluated in vitro, whilst BM-MSC-eRLX + GFP vs BM-MSCs-eGFP homing to the injured kidney and renoprotective effects were evaluated in preclinical models of ischemia reperfusion injury (IRI) and high salt (HS)-induced hypertensive CKD in vivo. The long-term safety of BM-MSCs-RLX + GFP was also determined 9-months after treatment cessation in vivo.ResultsWhen cultured for 3- or 7-days in vitro, 1 × 106 BM-MSCs-eRLX + GFP produced therapeutic RLX levels, and secreted an enhanced but finely-tuned cytokine profile without compromising their proliferation or differentiation capacity compared to naïve BM-MSCs. BM-MSCs-eRLX + GFP were identified in the kidney 2-weeks post-administration and retained the therapeutic effects of RLX in vivo. 1–2 × 106 BM-MSCs-eRLX + GFP attenuated the IRI- or therapeutically abrogated the HS-induced tubular epithelial damage and interstitial fibrosis, and significantly reduced the HS-induced hypertension, glomerulosclerosis and proteinuria. This was to an equivalent extent as RLX and BM-MSCs administered separately but to a broader extent than BM-MSCs-eGFP or the angiotensin-converting enzyme inhibitor, perindopril. Additionally, these renoprotective effects of BM-MSCs-eRLX + GFP were maintained in the presence of perindopril co-treatment, highlighting their suitability as adjunct therapies to ACE inhibition. Importantly, no major long-term adverse effects of BM-MSCs-eRLX + GFP were observed.ConclusionsBM-MSCs-eRLX + GFP produced greater renoprotective and therapeutic efficacy over that of BM-MSCs-eGFP or ACE inhibition, and may represent a novel and safe treatment option for acute kidney injury and hypertensive CKD.

Bidirectional Impact of Varying Severity of Acute Kidney Injury on Calcium Oxalate Stone Formation.

Acute Kidney Injury (AKI) is a prevalent renal disorder. The occurrence of AKI may promote the formation of renal calcium oxalate stones by exerting continuous effects on renal tubular epithelial cells. We aimed to delineate the molecular interplay between AKI and nephrolithiasis. A mild (20 min) and severe (30 min) renal ischemia-reperfusion injury model was established in mice. Seven days after injury, calcium oxalate stones were induced using glyoxylate (Gly) to evaluate the impact of AKI on the formation of kidney stones. Transcriptome sequencing was performed on tubular epithelial cells (TECs) to elucidate the relationship between AKI severity and kidney stones. Key transcription factors (TF) regulating differential gene transcription levels were identified using motif analysis, and pioglitazone, ginkgetin, and fludarabine were used for targeted therapy to validate key transcription factors as potential targets for kidney stone treatment. Severe AKI led to increased deposition of calcium oxalate crystals in renal, impaired kidney function, and upregulation of kidney stone-related gene expression. In contrast, mild AKI was associated with decreased crystal deposition, preserved kidney function, and downregulation of similar gene expression. Transcriptomic analysis revealed that genes associated with inflammation and cell adhesion pathways were significantly upregulated after severe AKI, while genes related to energy metabolism pathways were significantly upregulated after mild AKI. An integrative bioinformatic analysis uncovered a TF regulatory network within TECs, pinpointing that PKNOX1 was involved in the upregulation of inflammation-related genes after severe AKI, and inhibiting PKNOX1 function with Pioglitazone could simultaneously reduce the increase of calcium oxalate crystals after severe AKI in kidney. On the other hand, motif analysis also revealed the protective role of STAT1 in the kidneys after mild AKI, enhancing the function of STAT1 with Ginkgetin could reduce kidney stone formation, while the specific inhibitor of STAT1, Fludarabine, could eliminate the therapeutic effects of mild AKI on kidney stones. Inadequate repair of tubular epithelial cells after severe AKI increases the risk of kidney stone formation, with the upregulation of inflammation-related genes regulated by PKNOX1 playing a role in this process. Inhibiting PKNOX1 function can reduce kidney stone formation. Conversely, after mild AKI, effective cell repair through upregulation of STAT1 expression can protect TEC function, reduce stone formation, and activating STAT1 function can also achieve the goal of treating kidney stones.

Phosphatidylserine-mediated uptake of extracellular vesicles by hepatocytes ameliorates liver ischemia-reperfusion injury.

Compelling evidence suggests that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) promote regeneration in animal models of liver injury by delivering signaling molecules. However, their target cells and uptake mechanism remain elusive. In this study, MSC-EVs were intravenously administered in a mouse model of liver ischemia-reperfusion injury (IRI). Our results revealed that MSC-EVs exhibit enhanced liver targeting in IRI mice, and injured hepatocytes display a greater capacity for MSC-EV uptake. We found that phosphatidylserine (PS) displayed on the exterior of injured hepatocytes promotes MSC-EV internalization, possibly by binding to MFGE8, a protein expressed on the MSC-EV membrane. Furthermore, the therapeutic effect of MSC-EVs on liver IRI is highly dependent on this PS-mediated uptake pathway. Our findings provide evidence that MSC-EVs preferentially target injured hepatocytes, relying on a PS-dependent uptake route to exert hepatoprotective effects, which are critical for the future design of EV-based therapeutic strategies for liver IRI.

Elevated serum GDF15 level as an early indicator of proximal tubular cell injury in acute kidney injury

Acute kidney injury (AKI) is a high-burden medical condition, and current diagnostic criteria can only assess AKI after full manifestation. Stress marker growth differentiation factor 15 (GDF15) was reported to have a role in kidney injury of critical patients. Herein, we evaluated dynamic changes in GDF15 across diverse AKI scenarios and explored the underlying mechanisms of its induction. Serum parameters and renal lesions were analyzed in mouse models of unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO). The human proximal tubular (HK−2) cell line was stimulated with various conditions, and induction of GDF15 expression was determined. Serum GDF15 levels were rapidly induced within hours after injury in both animal models and declined thereafter. Renal GDF15 expression exhibited a temporary and early increased induction and was mainly located in aquaporin 1-positive proximal tubules in both unilateral AKI model tissues. In cell experiments, rapid GDF15 production was highly induced by t-BHP and CoCl2. Treatment with either an antioxidant or mitogen-activated protein kinase inhibitors abolished t-BHP- and CoCl2-mediated GDF15 expression. In addition, silencing nuclear factor erythroid 2-related factor 2 expression also reduced the basal and t-BHP- or CoCl2-mediated GDF15 expression level in HK-2 cells. Our data showed that elevated serum GDF15 levels could be detected early in unilateral AKI models without notable alterations in kidney function parameters. GDF15 expression was associated with oxidative stress- and hypoxia-mediated proximal tubular cell injury. These data document that elevated serum GDF15 can possibly serve as an early biomarker for proximal tubular cell injury in AKI.

Bioinformatics Identification and Validation of Angiogenesis-Related Genes in Myocardial Ischemic Reperfusion Injury.

Angiogenesis plays a critical protective role in myocardial ischemia-reperfusion injury (MIRI); however, therapeutic targeting of associated genes remains constrained. To bridge this gap, we conducted bioinformatics analysis to identify pivotal angiogenesis-related genes in MIRI, potentially applicable for preventive and therapeutic interventions. We collected two mouse heart I/R expression datasets (GSE61592 and GSE83472) from Gene Expression Omnibus, utilizing the Limma package to identify differentially expressed genes (DEGs). Angiogenesis-related genes (ARGs) were extracted from GeneCards, and their overlap with DEGs produced differentially expressed ARGs (ARDEGs). Further analyses included Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and disease ontology to explore biological functions. Weighted gene correlation network analysis (WGCNA) was used to investigate molecular modules linked to MIRI. Additionally, a protein-protein interaction (PPI) network was constructed to pinpoint hub genes relevant to MIRI. Receiver operating characteristic curves were used to assess the diagnostic efficacy of these hub genes for MIRI. An ischemia-reperfusion injury model was established using human cardiac microvascular endothelial cells (HCMECs), with the expression of hub genes validated within this experimental framework. We identified 47 ARDEGs, 41 upregulated and 6 downregulated. PPI network analysis revealed suppressor of cytokine signaling 3 (Socs3), C-X-C motif chemokine ligand 1 (Cxcl1), interleukin 1 beta (Il1b), and matrix metallopeptidase 9 (Mmp9) as hub genes. Receiver operating characteristic (ROC) curve analysis demonstrated strong diagnostic potential for Socs3, Cxcl1, Il1b, and Mmp9. In vitro validation corroborated the mRNA and protein expression predictions. Our study highlights the pivotal role of Socs3, Cxcl1, Il1b, and Mmp9 in MIRI development, their significance in immune cell infiltration, and their diagnostic accuracy. These findings offer valuable insights for MIRI diagnosis and treatment, presenting potential molecular targets for future research.

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis

BackgroundIntestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PurposeTo investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. ResultsOur results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. ConclusionThis study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

Differential Effects of HDAC6 Inhibition Versus Knockout During Hepatic Ischemia-Reperfusion Injury Highlight Importance of HDAC6 C-terminal Zinc-finger Ubiquitin-binding Domain.

Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage ( P < 0.01 for AST and P < 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI ( P < 0.01 for AST and P < 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia ( P < 0.01 for AST and P < 0.01 for ALT). Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.