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

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
zhu li ◽  
Matthew J Hampton ◽  
Matthew B Barajas ◽  
Matthias L Riess
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Myocardial Ischemia ◽  
Ischemia Reperfusion Injury ◽  
Calcium Influx ◽  
Ischemia Reperfusion ◽  
Cell Types ◽  
Membrane Function ◽  
Ldh Release

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.

Abstract 423: Proteasome Priming by Protein Kinase G Protects Against Myocardial Ischemia-reperfusion Injury

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Xuejun Wang ◽  
Erin J Terpstra ◽  
Eduardo Callegari ◽  
Chengjun Hu ◽  
Hanming Zhang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Myocardial Ischemia ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Underlying Mechanism ◽  
26S Proteasome ◽  
Transgenic Mouse Line ◽  
Pde5 Inhibition ◽  
Myocardial Ischemia Reperfusion

Cardiac proteasome functional insufficiency is implicated in a large subset of heart disease and has been experimentally demonstrated to play an essential role in cardiac proteotoxicity, including desmin-related cardiomyopathy and myocardial ischemia-reperfusion (I-R) injury. Pharmacological inhibition of phosphodiesterase 5 (PDE5) via sildenafil for example, which can stabilize cGMP and thereby increase cGMP-dependent protein kinase (PKG) activity, is consistently reported to protect against I-R injury; however, the underlying mechanism is not fully understood. We have recently discovered that PKG activation enhances proteasomal degradation of misfolded proteins (Ranek, et al. Circulation 2013), prompting us to hypothesize that proteasome-priming may contribute to cardioprotection-induced by PDE5 inhibition. Here we used a cardiomyocyte-restricted proteasome inhibition transgenic mouse line (Tg) and non-Tg (Ntg) littermates to interrogate the action of sildenafil on I-R injury created by left anterior descending artery (LAD) ligation (30 min) and release (24 hr). Sildenafil was administered 30 min before LAD ligation. Results showed that (1) the 26S proteasome activity of the Ntg I-R hearts was significantly elevated by sildenafil but this elevation was blocked in the Tg line; (2) the infarct size reduction by sildenafil treatment in Ntg mice was completely abolished in the Tg mice with the same treatment; and (3) systolic and diastolic function impairment after I/R was markedly attenuated in sildenafil-treated Ntg mice, but not in the sildenafil-treated Tg mice. Additionally, immunoprecipitation assays show that PKG interacted with the proteasome in cultured cardiomyocytes, and this interaction appeared to be augmented by sildenafil treatment. Moreover, in vitro incubation of active PKG with purified human 26S proteasomes increased proteasome peptidase activities and the phosphorylation at specific serine residues of a 19S proteasome subunit as revealed by “gel-free” nano-LC-MS/MS. We conclude that active PKG directly interacts with, phosphorylates, and increases the activities of, the proteasome and that proteasome priming mediates to cardioprotection of PDE5 inhibition against I-R injury.

scholarly journals Inhibition of BRD4 Reduces Neutrophil Activation and Adhesion to the Vascular Endothelium Following Ischemia Reperfusion Injury

2020 ◽  
Vol 21 (24) ◽  
pp. 9620
Author(s):  
Shelby Reid ◽  
Noah Fine ◽  
Vikrant K. Bhosle ◽  
Joyce Zhou ◽  
Rohan John ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neutrophil Adhesion ◽  
Neutrophil Accumulation ◽  
Iri Model ◽  
Brd4 Inhibition

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.

scholarly journals Mangiferin Attenuates Myocardial Ischemia-Reperfusion Injury via MAPK/Nrf-2/HO-1/NF-κB In Vitro and In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Kun Liu ◽  
Fei Wang ◽  
Shuo Wang ◽  
Wei-Nan Li ◽  
Qing Ye
Keyword(s):  
Oxidative Stress ◽  
Coronary Artery ◽  
Myocardial Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cells ◽  
Myocardial Ischemia Reperfusion Injury ◽  
Myocardial Ischemia Reperfusion

The aim of this study was to investigate the cardioprotective effect of mangiferin (MAF) in vitro and in vivo. Oxidative stress and inflammatory injury were detected in coronary artery ligation in rats and also in hypoxia-reoxygenation- (H/R-) induced H9c2 cells. MAF inhibited myocardial oxidative stress and proinflammatory cytokines in rats with coronary artery occlusion. The ST segment of MAF treatment groups also resumed. Triphenyltetrazolium chloride (TTC) staining and pathological analysis showed that MAF could significantly reduce myocardial injury. In vitro data showed that MAF could improve hypoxia/reoxygenation- (H/R-) induced H9c2 cell activity. In addition, MAF could significantly reduce oxidative stress and inflammatory pathway protein expression in H/R-induced H9c2 cells. This study has clarified the protective effects of MAF on myocardial injury and also confirmed that oxidative stress and inflammation were involved in the myocardial ischemia-reperfusion injury (I/R) model.

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