Infarct Area Research Articles

Macrophage transcriptome changes in response to cardiac injuries are determined by the type of lesion rather than their ontogenetic nature

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CRC1425 - DFG Introduction Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages complementing the original resident macrophage population, and together orchestrate cardiac remodeling. We aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time. Results and methods We use a reporter mouse line CX3CR1Yfp CreER/+:R26tdT/+ to visualize and quantify fluxes of resident and recruited macrophages and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a near 1:1 equilibrium at 4 weeks post I/R injury. In the remote myocardium the 1:1 equilibrium established already during the first week. In TAC-injured hearts, the 1:1 ratio of recruited and resident hearts also established within the first week post-surgery and persisted up to 8 weeks of follow-up even as cardiac function deteriorated. In the infarct, recruited macrophages showed increased cytokine synthesis, defensive response, and cell adhesion pathways during the first week after injury. Many inflammatory pathways remained activated up to day 28 post MI despite simultaneous activation of the anti-inflammatory response. Formerly resident macrophages and their progeny also mounted an immune response throughout the healing process. Unexpectedly, by day 28 post MI when a stable scar had formed, resident and recruited macrophage population transcriptomes converted onto a common, pro-inflammatory steady state phenotype, that differed from their originally divergent gene expression profiles in the healthy heart by over one thousand genes. In the remote zone, however, resident macrophages maintained cellular homeostasis function, unlike recruited macrophages that retained an inflammatory program. Following TAC surgery, transcriptional profiles remained differentially regulated at all time points tested, where recruited subset induced immune response and resident macrophages upregulated tissue remodeling genes. Conclusion Ischemic and non-ischemic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the changing tissue microenvironment determine partial or complete override of ontogenic cell programs.

Synergistic effect of chrysin and kaempferol in ameliorating Cerebral Ischemic Reperfusion injury in rat by controlling expression of proinflammatory mediators NF-κB and STAT3

ABSTRACT Objectives The two flavonoids kaempferol and chrysin are known to possess anti-inflammatory and antioxidant activities. In addition, these two flavonoids were reported to display synergistic effects against inflammation. The present study aims to provide an analysis of the combined effects of kaempferol and chrysin on ischemic rat brain induced by endothelin-1. Methods The neurological deficit score and infarct area of the brain were determined post drug treatment. Histopathological sections displayed the morphological changes in the brain tissue. The brain tissues were processed for assessing the antioxidant and anti-inflammatory activity by measuring superoxide dismutase activity, catalase activity, level of reduced glutathione, brain malondialdehyde, and amount of calcium. The expression level of inflammatory molecules was analyzed by western blotting and immunohistochemistry. Results The infarct area, neurological score and NF-κB and STAT3 expression levels were significantly reduced. Discussion The analysis of neuroprotective synergistic activity of kaempferol and chrysin indicated the therapeutic potential of the combination in alleviating cerebral ischemia by controlling expression of proinflammatory mediators.

Increased perivascular fibrosis and pro-fibrotic cellular transition in intramyocardial blood vessels in patients with myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Myocardial infarction (MI) is pathologically characterized by irreversible myocardial necrosis caused by ischemia. Intramyocardial vascular dysfunction is believed to increase the risk of MI. In this study, we investigated intramyocardial blood vessels in deceased patients during the early and chronic phases of MI to explore the involvement of perivascular fibrosis and pro-fibrotic cellular transitions in the pathological process of MI. Methods Left ventricular tissue (LV) was obtained from the infarction area of autopsied patients with early-phase MI (3-6 hours old; n=23), chronic-phase MI (5-14 days old; n=12), and from non-infarcted controls (n=14). We quantified perivascular fibrosis using EVG staining and fibroblast activation protein (FAP) expression using immunohistochemistry. Co-localization of smooth muscle actin (SMA) or CD31 with fibroblast marker S100A4 indicative of transition of smooth muscle cells (SMC) and endothelial cells (EC) toward fibroblast-like cells in intramyocardial vessels was quantified using immunofluorescent microscopy. Results Compared to controls (61.03%), perivascular fibrosis was significantly elevated in MI patients (early-phase 77.20%; chronic-phase 82.58%)(p<0.0001), with the highest level observed in the infarct cores of chronic-phase patients (95.18%). Compared to controls, FAP expression was 3.5-fold higher in early-phase- and 15.3-fold higher in chronic-phase MI patients (p<0.05), wherein FAP expression again was highest in the infarct cores. Furthermore, in both early- and chronic-phase MI, SMC- and EC fibroblast-like cellular transition was increased. The fraction of SMA+S100A4+ vessels was significantly higher in early-phase MI (31.96%) and chronic-phase MI (21.9%: border zone; 37.25%: infarct core) compared to controls (8.95%)(p<0.05). Similarly, the fraction of CD31+S100A4+ area was significantly increased in early-phase MI (10.14%) and chronic-phase MI (8.31%: border zone 10.15%: infarct core) compared to controls (2.67%)(p<0.05). Conclusion We observed increased perivascular fibrosis, fibroblast activation and fibroblast-like cellular transition toward a pro-fibrotic phenotype of vascular cells in intramyocardial blood vessels in MI patients, that may contribute to the development of MI. The further increase of FAP expression and perivascular fibrosis, particularly in the infarct core, in the chronic-phase suggest that MI itself can exacerbate fibroblast activation and perivascular fibrosis, theoretically increasing the risk of reinfarction.FAP expression and perivascular fibrosispro-fibrotic vascular cellular transitio

Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Cardiovascular diseases are the leading cause of death and disability worldwide. Among them, ischemic heart disease, due to myocardial infarction (MI) remains the first cause of cardiovascular death. The anti-ageing protein Klotho is mainly expressed in membrane at renal level. However, Klotho also has a soluble form with different pleiotropic functions that are still undetermined, including a possible cardioprotective role that is currently the target of several studies. Purpose Firstly, we evaluated circulating Klotho levels related to cardiac damage in a cohort of 146 patients after ST-elevation myocardial infarction (STEMI) and in an experimental post-myocardial infarction (PMI) model. Secondly, we investigated soluble Klotho supplementation as a potential novel therapeutic strategy for the prevention of cardiac dysfunction, deleterious remodeling, cardiomyocyte calcium (Ca2+) mishandling and arrhythmias in PMI mice. Methods Study with human samples: we analysed Klotho and NT-proBNP plasma levels in STEMIpatients with preserved renal function. Experimental study: C57BL6 mice subjected to PMI by ligation of the left anterior descending coronary artery were treated with vehicle solution or recombinant Klotho (10 mg/Kg/day) for 15 days after MI. Cardiac function was studied through non-invasive methods including electrocardiogram (ECG) and cardiac magnetic resonance imaging (CMRI). Intracellular Ca2+ handling was analysed using confocal microscopy in isolated ventricular cardiomyocytes. Cardiac remodeling was studied through histological and biomolecular analysis of several markers of cardiac damage and target proteins involved in Ca2+ handling. Results Plasma Klotho levels decrease rapidly after MI in both STEMI patients and PMI mice. Additionally, MI patients in the lowest Klotho tertile showed significantly elevated NT-proBNP levels. Klotho treatment decreased the infarct area in PMI mice accompanied by a decrease in both cardiac hypertrophy and fibrosis. Moreover, reduction in ejection fraction and MI-related ECG alterations were reduced in PMI mice treated with Klotho, including increased QRS, JT, QTc intervals and occurrence of premature ventricular contractions. Klotho also prevented systolic Ca2+ release and cell shortening disturbances in cardiomyocytes from PMI mice without Klotho. Increased diastolic Ca2+ leak was prevented in mice treated with Klotho via CaMKII-dependent pathway by avoiding its phosphorylation and the RyR2 phosphorylation at CaMKII site. Conclusions Klotho supplementation protect against MI by avoiding deleterious cardiac remodeling and ameliorating one of the main complications of MI such as arrhythmias. Thus, recombinant Klotho prevents the intracardiomyocyte Ca2+ mishandling after MI. Our translational findings support the importance of Klotho as a novel biomarker of ventricular damage just after MI and as a novel therapeutic strategy for prevention cardiac events associated to ischemic heart disease.

Extracellular vesicles from dental pulp mesenchymal stem cells modulate macrophage phenotype during acute and chronic cardiac inflammation in athymic nude rats with myocardial infarction

Background/aimsExtracellular vesicles (EVs) derived from dental pulp mesenchymal stem cells (DP-MSCs) are a promising therapeutic option for the treatment of myocardial ischemia. The aim of this study is to determine whether MSC-EVs could promote a pro-resolving environment in the heart by modulating macrophage populations.MethodsEVs derived from three independent biopsies of DP-MSCs (MSC-EVs) were isolated by tangential flow-filtration and size exclusion chromatography and were characterized by omics analyses. Biological processes associated with these molecules were analyzed using String and GeneCodis platforms. The immunomodulatory capacity of MSC-EVs to polarize macrophages towards a pro-resolving or M2-like phenotype was assessed by evaluating surface markers, cytokine production, and efferocytosis. The therapeutic potential of MSC-EVs was evaluated in an acute myocardial infarction (AMI) model in nude rats. Infarct size and the distribution of macrophage populations in the infarct area were evaluated 7 and 21 days after intramyocardial injection of MSC-EVs.ResultsLipidomic, proteomic, and miRNA-seq analysis of MSC-EVs revealed their association with biological processes involved in tissue regeneration and regulation of the immune system, among others. MSC-EVs promoted the differentiation of pro-inflammatory macrophages towards a pro-resolving phenotype, as evidenced by increased expression of M2 markers and decreased secretion of pro-inflammatory cytokines. Administration of MSC-EVs in rats with AMI limited the extent of the infarcted area at 7 and 21 days post-infarction. MSC-EV treatment also reduced the number of pro-inflammatory macrophages within the infarct area, promoting the resolution of inflammation.ConclusionEVs derived from DP-MSCs exhibited similar characteristics at the omics level irrespective of the biopsy from which they were derived. All MSC-EVs exerted effective pro-resolving responses in a rat model of AMI, indicating their potential as therapeutic agents for the treatment of inflammation associated with AMI.

MiR-106a targets ATG7 to inhibit autophagy and angiogenesis after myocardial infarction.

Myocardial infarction (MI) is an acute condition in which the heart muscle dies due to the lack of blood supply. Previous research has suggested that autophagy and angiogenesis play vital roles in the prevention of heart failure after MI, and miR-106a is considered to be an important regulatory factor in MI. But the specific mechanism remains unknown. In this study, using cultured venous endothelial cells and a rat model of MI, we aimed to identify the potential target genes of miR-106a and discover the mechanisms of inhibiting autophagy and angiogenesis. We first explored the biological functions of miR-106a on autophagy and angiogenesis on endothelial cells. Then we identified ATG7, which was the downstream target gene of miR-106a. The expression of miR-106a and ATG7 was investigated in the rat model of MI. We found that miR-106a inhibits the proliferation, cell cycle, autophagy and angiogenesis, but promoted the apoptosis of vein endothelial cells. Moreover, ATG7 was identified as the target of miR-106a, and ATG7 rescued the inhibition of autophagy and angiogenesis by miR-106a. The expression of miR-106a in the rat model of MI was decreased but the expression of ATG7 was increased in the infarction areas. Our results indicate that miR-106a may inhibit autophagy and angiogenesis by targeting ATG7. This mechanism may be a potential therapeutic treatment for MI.

Comparative efficacy of sweated and non-sweated Salvia miltiorrhiza Bge. extracts on acute myocardial ischemia via regulating the PPARα/RXRα/NF-κB signaling pathway

Salvia miltiorrhiza Bge. (S. miltiorrhiza) is a well-known traditional Chinese medicine for the treatment of cardiovascular diseases. The processing of S. miltiorrhiza requires the raw herbs to sweat first and then dry. The aim of this study was to investigate the anti-acute myocardial ischemia (AMI) of S. miltiorrhiza extracts (including tanshinones and phenolic acids) before and after sweating, and to further explore whether the “sweating” primary processing affected the efficacy of S. miltiorrhiza. The AMI animal model was established by subcutaneous injection of isoprenaline hydrochloride (ISO). After treatment, the cardiac function of rats was evaluated by electrocardiogram (ECG), biochemical, and histochemical analysis. Moreover, the regulation of S. miltiorrhiza extracts on the peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor α (RXRα)/nuclear transcription factor-kappa B (NF-κB) signaling pathway of rats was assessed by the Western blotting. The results showed that sweated and non-sweated S. miltiorrhiza extracts including tanshinones and phenolic acids significantly reduced ST-segment elevation in ECG and the myocardial infarction area in varying degrees. Meanwhile, sweated and non-sweated S. miltiorrhiza reversed the activities of aspartate transaminase (AST), lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), and superoxide dismutase (SOD), as well as the levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in AMI rats. Concurrently, the results of Western blotting revealed that S. miltiorrhiza extracts regulated the PPARα/RXRα/NF-κB signaling pathway to exert an anti-inflammatory effect. Most importantly, sweated S. miltiorrhiza tanshinones extracts are more effective than the non-sweated S. miltiorrhiza, and the anti-inflammatory efficacy of tanshinones extract was also better than that of phenolic acid extract. Although phenolic acid extracts before and after sweating were effective in anti-AMI, there was no significant difference between them. In conclusion, both tanshinones and phenolic acids extracts of sweated and non-sweated S. miltiorrhiza promote anti-oxidative stress and anti-inflammatory against AMI via regulating the PPARα/RXRα/NF-κB signaling pathway. Further, the comparations between sweated and non-sweated S. miltiorrhiza extracts indicate that sweated S. miltiorrhiza tanshinones extracts have better therapeutic effects on AMI.

Predictors of decremental conduction after extrastimulus in post-infarction ventricular tachycardia substrate maps

Abstract Introduction Ventricular tachycardia (VT) substrate ablation techniques attempt to identify areas of decremental conduction (DC) within the scar, given their close relationship with VT isthmuses. DC identification requires the systematic use of extrastimuli during substrate mapping. This can be time consuming and induce ventricular arrhythmias during mapping. Objective This study aims to identify predictors of decremental response after extastimulus that allow recognizing areas with higher probability of DC conduction in order to focus functional mapping of the substrate on them. Methods Sixteen consecutive ischemic patients (93.8% male, 66.3±12.4 years) undergoing VT substrate ablation were included. A detailed substrate map during right ventricular (RV) pacing was obtained in all of them. Systematic analysis of electrograms (EGMs) recorded from the infarct area was performed during RV pacing at 600-500 ms, and during a short-coupled RV extrastimulus. Electrograms during RV pacing exhibiting decremental evoked conduction delay after an extrastimulus (DECD-AE) were compared to those not showing DECD-AE. Results Mean low voltage (<1.5 mV) area was 45±18.6 cm2. A total of 1744 EGMs were analyzed. Of them, 453 (26%) showed DECD-AE. Bipolar EGMs showing DECD-AE exhibited a higher number of deflections [4(interquartile range 3-5) vs 2(1-3); p<0.0001], a higher amplitude [0.52(0.27-1) vs 0.3(0.14-0.7) mV; p=0.003] and similar duration [114(102-135) vs 117(100-133 ms); p=0.492] compared to EGMs without DECD-AE. The area under curve was 0.64 for bipolar voltage and 0.8 for number of bipolar deflections. The presence of <3 bipolar deflections excluded DECD-AE with a negative predictive value of 98.5%. Conclusions In areas of myocardial scar, bipolar EGMs with <3 deflections are unlikely to present DECD-AE. Focusing functional mapping on signals with ≥3 deflections could simplify procedures and save time.

Effect of human heart valve-derived ECM and NP/PCL electrospun nanofibrous sheet on mice bone marrow mononuclear cells and cardiac repair

BackgroundBiomaterials can improve cardiac repair combined with transplantation of bone marrow mononuclear cells (BMMNCs). In this study, we compared the phenotype and cardiac repair between human heart valve-derived scaffold (hHVS) and natural protein/polycaprolactone (NP/PCL) anchored BMNNCs. Methods and resultsBMMNCs were obtained from mice five days following myocardial infarction. Subsequently, BMMNCs were separately cultured on hHVS and PCL. Proliferation and cardiomyogenic differentiation were detected in vitro. Cardiac function was measured after transplantation of cell-seeded cardiac patch on MI mice. After that, the BMMNCs were collected for mRNA sequencing after culturing on the scaffolds. Upon anchoring onto hHVS or PCL, BMMNCs exhibited an increased capacity for proliferation in vitro, however, the cells on hHVS exhibited superior cardiomyogenic differentiation ability. Moreover, both BMMNCs-seeded biomaterials effectively improved cardiac function after 4 weeks of transplantation, with reduced infarction area and restricted LV remodeling. Cell-seeded hHVS was superior to cell-seeded PCL. ConclusionBMMNCs on hHVS showed better capacity in both cell cardiac repairing and improvement for cardiac function than on PCL. Compared with seeded onto PCL, BMMNCs on hHVS had 253 genes up regulated and 189 genes down regulated. The reason for hHVS’ better performance than PCL as a scaffold for BMMNCs might be due to the fact that optimized method of decellularization let more cytokines in ECM retained.

#1331 PCSK9 inhibitor addition during hypothermic perfusion protects kidney from ischemic damage in a rat model of donation after circulatory death

Abstract Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8 (PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p < 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p < 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p < 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p < 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p < 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p < 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p < 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p < 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

#1446 PCSK9 inhibitor addition during hypothermic perfusion protects kidney from ischemic damage in a rat model of donation after circulatory death

Abstract Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8(PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p < 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p < 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p < 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p < 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p < 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p < 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p < 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p < 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

PRDX1 Interfering Peptide Disrupts Amino Acids 70-90 of PRDX1 to Inhibit the TLR4/NF-κB Signaling Pathway and Attenuate Neuroinflammation and Ischemic Brain Injury.

Ischemic stroke ranks among the leading causes of death and disability in humans and is accompanied by motor and cognitive impairment. However, the precise mechanisms underlying injury after stroke and effective treatment strategies require further investigation. Peroxiredoxin-1 (PRDX1) triggers an extensive inflammatory cascade that plays a pivotal role in the pathology of ischemic stroke, resulting in severe brain damage from activated microglia. In the present study, we used molecular dynamics simulation and nuclear magnetic resonance to detect the interaction between PRDX1 and a specific interfering peptide. We used behavioral, morphological, and molecular experimental methods to demonstrate the effect of PRDX1-peptide on cerebral ischemia-reperfusion (I/R) in mice and to investigate the related mechanism. We found that PRDX1-peptide bound specifically to PRDX1 and improved motor and cognitive functions in I/R mice. In addition, pretreatment with PRDX1-peptide reduced the infarct area and decreased the number of apoptotic cells in the penumbra. Furthermore, PRDX1-peptide inhibited microglial activation and downregulated proinflammatory cytokines including IL-1β, IL-6, and TNF-α through inhibition of the TLR4/NF-κB signaling pathway, thereby attenuating ischemic brain injury. Our findings clarify the precise mechanism underlying PRDX1-induced inflammation after ischemic stroke and suggest that the PRDX1-peptide can significantly alleviate the postischemic inflammatory response by interfering with PRDX1 amino acids 70-90 and thereby inhibiting the TLR4/NF-κB signaling pathway. Our study provides a theoretical basis for a new therapeutic strategy to treat ischemic stroke.

RETROSPECTIVE ANALYSIS OF CLINICAL, EPIDEMIOLOGY AND PATHOMORPHOLOGICAL FEATURES OF MYOCARDIAL INFARCTION

The aim of this study is to conduct a retrospective analysis of the clinical, epidemiological, and pathomorphological features of myocardial infarction. Materials and methods: A retrospective analysis was conducted using data from medical records and autopsy results from the pathology department for the year 2023. Results. In 2023, the pathology department performed 182 autopsies, with 123 patients diagnosed with acute, repeated, or recurrent myocardial infarction (MI), accounting for 67.6% of all autopsies. The average age of the deceased was 74.4±0.9 years, with 81.3% being persons of incapacitated age. Women comprised the majority at 55.3% (T=2784.5; p=0.001). The highest proportion of MI-related deaths occurred in September, accounting for 12.2%. In the pathological diagnosis, myocardial infarction (MI) was the primary disease, with varying localization of the pathological process in the left ventricle. Transmural MI was detected in 95.9% of the deceased, while subendocardial MI was found in 4.1%. Acute MI developed against the background of a previously experienced MI in 43.9% of cases, and in 5.5% of cases, it was a repeated acute recurrent MI. Changes in the aorta, coronary vessels, and basal vessels of the brain were observed in 100.0% of the cases, with hypertensive disease detected in 95.9% of cases. Acute cardiopulmonary failure with the development of pulmonary edema was the immediate cause of death in 95.9% of cases. Macroscopic examination revealed hypertrophy of the left ventricle. The myocardium appeared reddish-brown and elastic, with white layers. Brown-gray infarct areas of different sizes were present in the left ventricular wall, while in cases of repeated acute myocardial infarction, the affected area was gray-white and hard to the touch. Microscopically, hypertrophy of cardiomyocytes of the left ventricle with perinuclear lipofuscinosis was observed in all deceased individuals. Sections of the infarct zone contained homogenized, anucleated, fragmented cardiomyocytes, dilated full blood vessels with small perivascular hemorrhages, and neutrophilic-macrophage infiltrates of varying intensity. Large centers of cardiosclerosis were detected in cases of repeated MI, while signs of angiogenesis with the formation of granulation tissue were observed in cases of recurrent MI. Conclusion. In 2023, 123 autopsies revealed a diagnosis of acute, repeated, or recurrent myocardial infarction (MI) in 67.6% of all cases. The average age of the deceased was 74.4±0.9 years, with 81.3% being individuals of incapacitated age. There was a predominance of women, accounting for 55.3% of cases. Acute MI developed against the background of a previously experienced one in 43.9% of the deceased, while repeated acute recurrent MI was detected in 5.5% of cases. Pathomorphological changes corresponded to the typical features of MI depending on its age.

Exosomes derived from TNF-α-treated bone marrow mesenchymal stem cells ameliorate myocardial infarction injury in mice

ABSTRACT Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) exhibit considerable therapeutic potential for myocardial regeneration. In our investigation, we delved into their impact on various aspects of myocardial infarction (MI), including cardiac function, tissue damage, inflammation, and macrophage polarization in a murine model. We meticulously isolated the exosomes from TNF-α-treated BMSCs and evaluated their therapeutic efficacy in a mouse MI model induced by coronary artery ligation surgery. Our comprehensive analysis, incorporating ultrasound, serum assessment, Western blot, and qRT-PCR, revealed that exosomes from TNF-α-treated BMSCs demonstrated significant therapeutic potential in reducing MI-induced injury. Treatment with these exosomes resulted in improved cardiac function, reduced infarct area, and increased left ventricular wall thickness in MI mice. On a mechanistic level, exosome treatment fostered M2 macrophage polarization while concurrently suppressing M1 polarization. Hence, exosomes derived from TNF-α-treated BMSCs emerge as a promising therapeutic strategy for alleviating MI injury in a mouse model.

Injectable Self-Oxygenating Cardio-Protective and Tissue Adhesive Silk-Based Hydrogel for Alleviating Ischemia After Mi Injury.

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy.

Preconditioning with selective autoretroperfusion: In vivo and in silico evidence of washout hypothesis.

Prompt reperfusion of coronary artery after acute myocardial infarction (AMI) is crucial for minimizing heart injury. The myocardium, however, may experience additional injury due to the flow restoration itself (reperfusion injury, RI). The purpose of this study was to demonstrate that short preconditioning (10min) with selective autoretroperfusion (SARP) ameliorates RI, based on a washout hypothesis. AMI was induced in 23 pigs (3 groups) by occluding the left anterior descending (LAD) artery. In SARP-b (SARP balloon inflated) and SARP-nb (SARP balloon deflated) groups, arterial blood was retroperfused for 10min via the great cardiac vein before releasing the arterial occlusion. A mathematical model of coronary circulation was used to simulate the SARP process and evaluate the potential washout effect. SARP restored left ventricular function during LAD occlusion. Ejection fraction in the SARP-b group returned to baseline levels, compared to SARP-nb and control groups. Infarct area was significantly larger in the control group than in the SARP-b and SARP-nb groups. End-systolic wall thickness was preserved in the SARP-b compared to the SARP-nb and control groups. Analyte values (pH, lactate, glucose, and others), measured every 2min during retroperfusion, suggest a "washout" effect as one important mechanism of action of SARP in reducing infarct size. With SARP, the values progressively approached baseline levels. The mathematical model also confirmed a possible washout effect of tracers. RI can be ameliorated by delaying restoration of arterial flow for a brief period of time while pretreating the infarction with SARP to restore homeostasis via a washout mechanism.

Acute Combined Cerebrolysin and Nicotinamide Administration Promote Cognitive Recovery Through Neuronal Changes in the Hippocampus of Rats with Permanent Middle Cerebral Artery Occlusion

Stroke is one of the leading causes of disability worldwide, where the Hippocampus (HPC) is affected. HPC organizes memory, which is a cognitive domain compromised after a stroke, where cerebrolysin (CBL) and Nicotinamide (NAM) have been recognized as potentially therapeutic. In this study, we aimed to evaluate the efficacy of a combined administration of CBL and NAM in a rat stroke model. Male Sprague-Dawley rats (n = 36) were divided into four groups: saline (pMCAO − Saline), CBL (pMCAO + CBL), NAM (pMCAO + NAM), and experimental (pMCAO + CBL-NAM) (n = 9 per group). A permanent middle cerebral artery occlusion (pMCAO) was induced through electrocauterization of the middle cerebral artery, followed by the administration of CBL (2.5 ml/kg), NAM (500 mg/kg) or combined immediately after skin suture, as well as at 24, 48, and 72 h post-surgery. The rats were evaluated in the novel object recognition test; hippocampal infarct area measurement; reconstruction of neurons from CA1 for Sholl analysis; and, measurement of brain-derived neurotrophic factor (BDNF) levels near the infarct zone. Our findings revealed that the administration of CBL or NAM induced infarct reduction, improved cognition, and increased BDNF levels. Moreover, a combination of CBL and NAM increased dendritic intersection in CA1 pyramidal neurons. Thus, the combined administration of CBL and NAM can promote cognitive recovery after a stroke, with infarct reduction, cytoarchitectural changes in HPC CA1 neurons, and BDNF increase. Our findings suggest that this combination therapy could be a promising intervention strategy for stroke.

Isoliquiritigenin alleviates myocardial ischemia-reperfusion injury by regulating the Nrf2/HO-1/SLC7a11/GPX4 axis in mice

Ischemia-reperfusion (I/R) injury, a multifaceted pathological process, occurs when the prolongation of reperfusion duration triggers ferroptosis-mediated myocardial damage. Isoliquiritigenin (ISL), a single flavonoid from licorice, exhibits a wide range of pharmacological impacts, but its function in ferroptosis caused by myocardial I/R injury remains unclear. This study delved into the protective effect of ISL on myocardial I/R injury-induced ferroptosis and its mechanism. Neonatal mouse cardiomyocytes (NMCM) underwent hypoxia/reoxygenation (H/R) to simulate the pathological process of myocardial I/R. ISL significantly attenuated H/R-triggered production of reactive oxygen species in NMCM, reduced the expression of malondialdehyde and the activity of lactate dehydrogenase, enhanced superoxide dismutase and catalase activity, and increased the expression of nuclear factor E2-related factor 2 (Nrf2) and its downstream heme oxygenase 1 (HO-1), thereby mitigating oxidative stress damage. CCK8 experiment revealed that the ferroptosis inhibitor Ferrostatin-1 significantly improved myocardial cell viability after 24 h of reoxygenation, and ISL treatment showed a similar effect. ISL reduced intracellular free iron accumulation, up-regulated glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression, and inhibited lipid peroxidation accumulation, thereby alleviating ferroptosis. The Nrf2-specific inhibitor ML385 counteracted ISL's defensive role against H/R-triggered oxidative stress damage and ferroptosis. In vivo experiments further confirmed that by regulating the translocation of Nrf2 into the nucleus, ISL treatment increased the levels of HO-1, GPX4, and SLC7A11, inhibited the expression of ACSL4, Drp1 to exert the antioxidant role, alleviated mitochondrial damage, and ferroptosis, ultimately reducing myocardial infarction area and injury induced by I/R. ML385 nearly abolished ISL's protective impact on the I/R model by inhibiting Nrf2 function. In summary, ISL is capable of mitigating oxidative stress, mitochondrial damage, and cardiomyocyte ferroptosis caused by I/R, thereby reducing myocardial injury. A key mechanism includes triggering the Nrf2/HO-1/SLC7A11/GPX4 pathway to prevent oxidative stress damage and cardiomyocyte ferroptosis caused by I/R.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Biomarker Periodic Repolarization Dynamics Indicates Enhanced Risk for Arrhythmias and Sudden Cardiac Death in Myocardial Infarction in Pigs.

Periodic repolarization dynamics (PRD) is an electrocardiographic biomarker that captures repolarization instability in the low frequency spectrum and is believed to estimate the sympathetic effect on the ventricular myocardium. High PRD indicates an increased risk for postischemic sudden cardiac death (SCD). However, a direct link between PRD and proarrhythmogenic autonomic remodeling has not yet been shown. We investigated autonomic remodeling in pigs with myocardial infarction (MI)-related ischemic heart failure induced by balloon occlusion of the left anterior descending artery (n=17) compared with pigs without MI (n=11). Thirty days after MI, pigs demonstrated enhanced sympathetic innervation in the infarct area, border zone, and remote left ventricle paralleled by altered expression of autonomic marker genes/proteins. PRD was enhanced 30 days after MI compared with baseline (pre-MI versus post-MI: 1.75±0.30 deg2 versus 3.29±0.79 deg2, P<0.05) reflecting pronounced autonomic alterations on the level of the ventricular myocardium. Pigs with MI-related ventricular fibrillation and SCD had significantly higher pre-MI PRD than pigs without tachyarrhythmias, suggesting a potential role for PRD as a predictive biomarker for ischemia-related arrhythmias (no ventricular fibrillation versus ventricular fibrillation: 1.50±0.39 deg2 versus 3.18±0.53 deg2 [P<0.05]; no SCD versus SCD: 1.67±0.32 deg2 versus 3.91±0.63 deg2 [P<0.01]). We demonstrate that ischemic heart failure leads to significant proarrhythmogenic autonomic remodeling. The concomitant elevation of PRD levels in pigs with ischemic heart failure and pigs with MI-related ventricular fibrillation/SCD suggests PRD as a biomarker for autonomic remodeling and as a potential predictive biomarker for ventricular arrhythmias/survival in the context of MI.