Abstract 12715: Administration of Activated Protein C Stabilizes Membrane EPCR and Increases Resistance of Heart to Ischemic Insult in Aging

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Di Ren ◽  
Julia Fedorova ◽  
Jingwen Zhang ◽  
Zhibin He ◽  
Ji Li
Keyword(s):  
Protein Kinase ◽  
Protein C ◽  
Metabolic Response ◽  
Activated Protein C ◽  
Endothelial Protein C Receptor ◽  
Ischemic Insult ◽  
Cardiac Damage ◽  
Ampk Signaling ◽  
Cardioprotective Effects ◽  
Epcr Shedding

Introduction: Activated protein C (APC) is a circulating protease with anti-coagulant property. Endothelial protein C receptor (EPCR) mediates APC's downstream events. We have revealed that APC exerts cardioprotective effects during ischemia and reperfusion (I/R). Hypothesis: There is an impaired APC signaling in aging and administration of APC restores APC signaling of aged heart to improve cardiac tolerance to ischemic insult caused by I/R stress. Methods: Young C57BL/6J (3-4 months), aged C57BL/6J (24-26 months), and EPCR R84A/R84A (3-4 months, C57BL6/J background) transgenic mice without APC binding affinity were subjected to ischemia (45 min) and reperfusion (24 hrs) by ligation/release of left anterior descending coronary artery. APC, signaling selective APC-2Cys, or anticoagulant selective APC-E170A was injected through jugular vein five-minutes before reperfusion. Results: I/R stress triggers APC signaling in both young and aged hearts, but APC activity was significantly impaired in the aged versus young hearts. Serum EPCR measurement demonstrated that membrane EPCR's shedding into circulation was dramatically promoted during I/R stress. Intriguingly, administration of APC significantly reduced EPCR shedding caused by I/R stress in both young and aged hearts but not EPCR R84A/R84A heart. The echocardiography data showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction by I/R insult in both young and aged mice. Interestingly, APC and APC-2Cys restored aged heart's resistance to a comparable level as young heart to ischemic damage. The cardioprotective effects of APC and ACP-2Cys are diminished in EPCR R84A/R84A mice. Furthermore, metabolomics analysis and working perfusion heart results demonstrated that AMP-activated protein kinase (AMPK) signaling mediates acute adaptive metabolic response while protein kinase B (Akt) signaling pathway is involved in chronic metabolic programming in the heart with APCs treatments. Conclusions: I/R stress causes membrane EPCR shedding in the heart and APC's cardioprotection against I/R injury is not related with its anti-coagulant activity. Administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging

2021 ◽  
Author(s):  
Di Ren ◽  
Julia Fedorova ◽  
Kayla Davitt ◽  
Tran Ngoc Van Le ◽  
John H Griffin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein C ◽  
Ex Vivo ◽  
Activated Protein C ◽  
Wild Type ◽  
Endothelial Protein C Receptor ◽  
Cardiac Damage ◽  
Downstream Signaling ◽  
Cardioprotective Effects ◽  
Epcr Shedding

Background: Activated protein C (APC) is a plasma serine protease with anticoagulant and anti-inflammatory activities. Endothelial protein C receptor (EPCR) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. Methods: Young (3-4 months) and aged (24-26 months) wild type C57BL/6J mice, as well as EPCR point mutation (EPCR R84A/R84A ) knock-in C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. Results: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCR R84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMP-activated protein kinase (AMPK) mediates acute adaptive response while protein kinase B (AKT) is involved in chronic metabolic programming in the hearts with APC treatment. Conclusions: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

Activated protein C downregulates p38 mitogen-activated protein kinase and improves clinical parameters in an in-vivo model of septic shock

2007 ◽  
Vol 98 (11) ◽  
pp. 1118-1126 ◽  
Author(s):  
Claudia Nold-Petry ◽  
Doris Fischer ◽  
Bernd Richter ◽  
Roman Blaheta ◽  
Josef Pfeilschifter ◽  
...  
Keyword(s):  
Septic Shock ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Protein C ◽  
Apoptotic Cell ◽  
Endotoxic Shock ◽  
Mitogen Activated Protein Kinase ◽  
In Vivo Model ◽  
Endothelial Protein C Receptor ◽  
Mitogen Activated Protein

SummaryDespite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasmaTNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC’s clinical success may, in part, be due to p38MAPK inhibition.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice

Rheumatology ◽  
2019 ◽  
Vol 58 (10) ◽  
pp. 1850-1860 ◽  
Author(s):  
Meilang Xue ◽  
Suat Dervish ◽  
Kelly J McKelvey ◽  
Lyn March ◽  
Fang Wang ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Synovial Fibroblasts ◽  
Endothelial Protein C Receptor ◽  
Mouse Thymus ◽  
Tnf Α ◽  
Thymus Cells ◽  
Proliferation And Invasion

Abstract Objectives To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. Methods RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. Results In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. Conclusion APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

2005 ◽  
Vol 93 (04) ◽  
pp. 743-750 ◽  
Author(s):  
Sarah Horn ◽  
Siegfried Lang ◽  
Kenji Fukudome ◽  
Adriane Nahrup ◽  
Ursula Hoffmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Drotrecogin Alfa Activated ◽  
Cox 2 ◽  
Vitro Effect

SummaryProstacyclin (PGI2) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI2 in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI2 release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500ng/ml-20μg/ ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3–9h and caused a time- and dose-dependent release of 6-keto PGF1α, the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-α (TNF-α) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostagland-in I2 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6keto PGF1α-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA up-regulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

scholarly journals The Cardioprotective Signaling Activity of Activated Protein C in Heart Failure and Ischemic Heart Diseases

2019 ◽  
Vol 20 (7) ◽  
pp. 1762 ◽  
Author(s):  
Di Ren ◽  
Hemant Giri ◽  
Ji Li ◽  
Alireza R. Rezaie
Keyword(s):  
Heart Failure ◽  
Protein C ◽  
Heart Diseases ◽  
Ischemia Reperfusion ◽  
Activated Protein C ◽  
Ischemic Heart ◽  
Signaling Networks ◽  
Endothelial Protein C Receptor ◽  
Signaling Mechanism ◽  
Mtorc1 Signaling

Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.

Sign in / Sign up

Export Citation Format

Share Document