Endothelial Protein C Receptor Shedding Research Articles

Circulating Soluble EPCR Levels Are Reduced in Patients with Ischemic Peripheral Artery Disease and Associated with Markers of Endothelial and Vascular Function.

(1) Background: Endothelial dysfunction initiates cardiovascular pathologies, including peripheral artery disease (PAD). The pathophysiology of impaired new vessel formation in the presence of angiogenic stimuli, such as ischemia and inflammation, is unknown. We have recently shown in mice that reduced endothelial protein C receptor (EPCR) expression results in defective angiogenesis following experimental hindlimb ischemia. (2) Purpose: To determine soluble (s)EPCR levels in the plasma of patients with PAD and to compare them with the protein C activity and biomarkers of endothelial function, inflammation, and angiogenesis. (3) Methods and Results: Clinical tests of vascular function and immunoassays of plasma from patients with PAD stage II were compared to age- and sex-matched individuals with and without cardiovascular risk factors or PAD stage III/IV patients. sEPCR levels were significantly lower in PAD stage II patients compared to subjects with risk factors, but no PAD, and further decreased in PAD stage III/IV patients. Plasma protein C activity or levels of ADAM17, a mediator of EPCR shedding, did not differ. Significant associations between sEPCR and the ankle-brachial index (p = 0.0359), age (p = 0.0488), body mass index (p = 0.0110), and plasma sE-selectin levels (p = 0.0327) were observed. High-sensitive CRP levels and white blood cell counts were significantly elevated in PAD patients and associated with serum glucose levels, but not sEPCR. In contrast, plasma TNFα or IL1β levels did not differ. Circulating levels of VEGF were significantly elevated in PAD stage II patients (p = 0.0198), but not associated with molecular (sE-selectin) or functional (ankle-brachial index) markers of vascular health. (4) Conclusions: Our findings suggest that circulating sEPCR levels may be useful as biomarkers of endothelial dysfunction, including angiogenesis, in persons older than 35 years and that progressive loss of endothelial protein C receptors might be involved in the development and progression of PAD.

Parmodulin, an Allosteric Inhibitor of Protease Activated Receptor 1 (PAR1) Protects Sickle Cell Mice from Thromboinflammation and Endothelial Dysfunction

Protease activated receptor 1 (PAR1) couples the coagulation cascade to endothelial inflammation in many diseases, including sickle cell disease (SCD). PAR1 is a GPCR activated by proteolytic cleavage of an extracellular N-terminus, and demonstrates biased signaling. Activation of PAR1 by thrombin stimulates prothrombotic and proinflammatory signaling, whereas activation by activated protein C (APC) stimulates cytoprotective and anti-inflammatory pathways. The ideal PAR-1 targeted therapy would inhibit detrimental signaling while preserving beneficial signaling. 3K3A-APC is a signaling selective variant of APC that activates cytoprotective PAR1 signaling with minimal anticoagulant effects, demonstrated to have a beneficial role in stroke models. Parmodulin 2 (PM2) is a small molecule allosteric inhibitor of PAR1 that simultaneously blocks detrimental signaling and activate beneficial cytoprotective signaling similar to that induced by APC. We previously demonstrated that endogenous APC-PAR1 signaling contributes to thrombin generation and inflammation in a mouse model of SCD, whereas thrombin-PAR signaling enhances these pathways. We hypothesize that inducing cytoprotective PAR1 signaling with either 3K3A-APC and PM2 will reduce thrombo-inflammation in sickle cell mice at steady state. Townes wild type (HbAA) and sickle (HbSS) mice (male and female, 4 months old) were used in the studies. In the first experiment, HbAA and HbSS (n=4-5 per group) mice were treated daily with either saline (SAL) or 3K3A-APC (0.2mg/kg, BW) for 4 days. Samples were collected 1 hour after final dose. As previously demonstrated, plasma levels of thrombin-anti thrombin (TAT) complexes, the proinflammatory cytokine IL-6, high mobility group box 1 (HMGB1), and, soluble vascular cell adhesion molecule (sVCAM), were increased in HbSS mice compared to HbAA controls. Surprisingly 3K3A-APC treatment did reduce any of these parameters in HbSS mice. APC and its variant 3K3A-APC require binding to the endothelial protein C receptor (EPCR) to activate PAR1; it is known that SCD causes increased EPCR shedding. Indeed, we found that both SAL- and 3K3A-APC treated HbSS mice had elevated sEPCR levels compared to HbAA controls. Together, these data indicate that 3K3A-APC does not affect thrombo-inflammation in HbSS mice at steady state most likely due to by reduced ECPR expression on the endothelium. In the second experiment, HbAA and HbSS (male and female, 4 months old) mice (n=8-11 per group) were treated with vehicle (20% DMSO, 20% PEG800, 60% PBS) or PM2 (10 mg/kg, BW) every other day for 1 week. The last dose was administered on the 8th day 1 hour prior to collecting plasma samples. As expected, HbSS mice had elevated plasma TAT levels compared to HbAA mice (11.04 ± 1.4 vs 45.19 ± 6.3, p<0.05) which was significantly attenuated by PM2 treatment in SS mice (16.17 ± 2, p<0.05). Similar results were observed for IL-6 (AA: 2.06 ± 0.7 vs SS: 29.12 ± 9.6 vs SS+PM2: 5.83 ± 2.9; p<0.05); sVCAM (AA: 330.6 ± 31.5 vs SS: 571.4 ± 50.4 vs SS+PM2: 403.3 ± 41.22; p<0.05) and HMGB1 (AA: 11.46 ± 1.1 vs SS: 25.37 ± 1.5 vs SS+PM2: 19.12 ± 1.7; p<0.05). PM2 had no effect on markers of anemia in HbSS mice including RBC, hematocrit, and reticulocyte counts. In the present study ,3K3A-APC did not attenuate thrombin generation and cytokine production in HbSS mice; this might be due to the shedding of EPCR which is important co-receptor for the protective APC-PAR1 signaling. In contrast, PM2-mediated allosteric inhibition of thrombin/PAR1 and simultaneous induction of APC-like cytoprotective signaling attenuated levels of TAT, IL-6, HMGB1, and sVCAM in HbSS mice. Future studies towards evaluating the effect of PM2 on downstream targets of PAR1 signaling in sickle mice will provide more insight into the mechanism of this protective effect.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging.

APC (activated protein C) is a plasma serine protease with anticoagulant and anti-inflammatory activities. EPCR (Endothelial protein C receptor) 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. Young (3-4 months) and aged (24-26 months) wild-type C57BL/6J mice, as well as EPCR point mutation (EPCRR84A/R84A) knockin 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. 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 EPCRR84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMPK (AMP-activated protein kinase) mediates acute adaptive response while AKT (protein kinase B) is involved in chronic metabolic programming in the hearts with APC treatment. 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.

Heme Induced Progressive Loss of Endothelial Protein C Receptor Promotes Development of Chronic Kidney Disease in Sickle Cell Disorders

Chronic kidney disease (CKD), widespread among the individuals with sickle cell disease (SCD), is a major contributor to early death in this patient population. The progressive deterioration of renal health in SCD is associated with chronic persistent intravascular hemolysis leading to anemia. We have previously reported that extracellular heme, released during acute intravascular hemolysis triggers clinically relevant acute kidney injury (AKI) in SCD mice (SS) (Blood (2020) 135 (13): 1044-1048). Although AKI is reversible, it is considered as a risk factor for CKD. The mechanistic approach elucidating the hemolysis driven pathogenesis of AKI-to-CKD transition in SCD is unknown. We found that CKD develops in the SS mice by progressive (1-10 months of age) increase in albuminuria (urinary albumin and creatinine ratio, uACR) and decrease in glomerular filtration rate (GFR) (n=5; p<0.001). Histopathology of the kidney showed age-dependent deterioration in renal peritubular vascular congestion in SS mice compared to that of the AA mice. Alongside, Evan's blue extravasation experiments showed that the SS mice are susceptible to vascular leakage that is correlated positively with age (Pearson r=0.98, p<0.001) and negatively with anemia (Pearson r= -0.46, p<0.05). We hypothesized that multiple acute hemolytic events may instigate persistent endothelial damage that ensues CKD development in SCD. To test this hypothesis, we intravenously infused vehicle or heme (14 μmoles/kg body weight; 5 times on alternate days) to 1-month old SS mice and monitored for 3 weeks following first heme injection. These mice developed severe albuminuria (n=5; p<0.01) with substantial loss of GFR (n=5; p<0.001), indicating heme induced CKD development. Next, we used ultrasound super-resolution (USR) imaging technology to determine renal microvascular changes in older SS mice (6-months) without heme challenge and in young SS mice (1-month) challenged with heme. Analysis of the USR data showed reduced renal blood volume (rBV) and substantial loss of vessel density in renal cortex as well as in corticomedullary areas of the older SS mice compared to the age-matched AA mice. Accordingly, multiple heme challenge reduced rBV and vessel density extensively in young SS mice comparable to the older SS mice without heme challenge. Since endothelial protein C receptor (EPCR) maintains vascular barrier integrity by activating protease activated receptor-1 (PAR1) signaling in the endothelium, we tested whether alterations in EPCR expression contribute to progressive endothelial damage in SS mice during CKD development. Using immunofluorescence microscopy, we determined that renal endothelium lacks expression of EPCR in older SS mice while younger SS mice retains EPCR cellular expression. In corroboration, infusion of heme in younger SS mice results in loss of renal endothelial EPCR. Shedding of EPCR from endothelium often results in a soluble form of EPCR (sEPCR). We found that SS mice had higher plasma levels of soluble EPCR (sEPCR) compared to their AA counterparts. While age dependent increase in plasma and urinary sEPCR were evident in SS mice (n=6; p<0.01), infusion of heme in younger SS mice results in significant increase in plasma sEPCR (n=6; p<0.01). In consistent with the mouse data, we discovered that the plasma sEPCR was significantly elevated in SCD patients compared to normal individuals (n=8-16; p<0.05). Moreover, the plasma sEPCR was significantly associated with the baseline albuminuria in a cohort of SCD patients (n=16; Pearson r=0.64; p<0.01). This study supports the conclusion that multiple hemolytic events may trigger CKD development in SCD by gradual loss of renal microvascular EPCR expression. Clinically, the sEPCR can be developed as risk factor for sickle CKD. Finally, our data suggest that restoration of EPCR function may protect SCD patients from CKD. DisclosuresNo relevant conflicts of interest to declare.

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

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.

An ADAM-10 dependent EPCR shedding links meningococcal interaction with endothelial cells to purpura fulminans.

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N. meningitidis pathogenesis is its ability to colonize microvessels endothelial cells via type IV pili. Importantly, endothelial cells are key in controlling the coagulation cascade through the activation of the potent anticoagulant Protein C (PC) thanks to two endothelial cell receptors among which the Endothelial Protein C Receptor (EPCR). Considering that congenital or acquired deficiencies of PC are associated with purpura fulminans, we hypothesized that a defect in the activation of PC following meningococcal adhesion to microvessels is responsible for the thrombotic events observed during meningococcemia. Here we showed that the adhesion of N. meningitidis on endothelial cells results in a rapid and intense decrease of EPCR expression by inducing its cleavage in a process know as shedding. Using siRNA experiments and CRISPR/Cas9 genome edition we identified ADAM10 (A Disintegrin And Metalloproteinase-10) as the protease responsible for this shedding. Surprisingly, ADAM17, the only EPCR sheddase described so far, was not involved in this process. Finally, we showed that this ADAM10-mediated shedding of EPCR induced by the meningococcal interaction with endothelial cells was responsible for an impaired activation of Protein C. This work unveils for the first time a direct link between meningococcal adhesion to endothelial cells and a severe dysregulation of coagulation, and potentially identifies new therapeutic targets for meningococcal purpura fulminans.

Unfractionated heparin protects the protein C system against lipopolysaccharide-induced damage in vivo and in vitro.

Activation of protein C is greatly enhanced by the presence of thrombomodulin (TM) and endothelial protein C receptor (EPCR) on the endothelial surface. Impairment of the anticoagulant protein C system occurs during endotoxemia and contributes to sepsis-associated hypercoagulability. Previous studies have demonstrated that unfractionated heparin (UFH) can attenuate coagulation in endotoxemic mice. However, whether UFH has an effect on the protein C system remains to be elucidated. The current study evaluated the therapeutic effect of UFH on the protein C system in a mouse model of lipopolysaccharide (LPS)-induced sepsis, and further investigated the effect of UFH on the expression of TM and EPCR in vitro using human umbilical vein endothelial cells (HUVECs). The in vivo data indicated that UFH preconditioning attenuated the decline in circulating activated protein C following LPS administration, and also reduced LPS-induced shedding of TM and EPCR. In HUVECs, LPS stimulation led to the downregulation of TM and EPCR expression, and UFH dose-dependently restored the mRNA and protein levels of TM and EPCR. In addition, UFH inhibited the LPS-induced activation of mitogen-activated protein kinase 14, proto-oncogene tyrosine-protein kinase Src and nuclear factor κB signaling in HUVECs. In summary, these results suggest that UFH has a protective effect on the protein C system during sepsis. Thus, UFH may be a candidate therapeutic agent for the treatment of patients with sepsis.

Carbon monoxide-releasing molecule-2 suppresses thrombomodulin and endothelial protein C receptor expression of human umbilical vein endothelial cells induced by lipopolysaccharide in vitro.

Objective:The aim of this study was to observe the counter-effect of carbon monoxide-releasing molecule-2 (CORM-2) on lipopolysaccharide (LPS)-suppressed thrombomodulin (TM) and endothelial protein C receptor (EPCR) expressions from human umbilical vein endothelial cell (HUVEC), and to reveal its mechanisms.Methods:HUVECs were divided into 5 treatment groups, wherein reagents were added simultaneously. TM and EPCR proteins of the cells and the culture medium levels of soluble TM, soluble EPCR, and matrix metalloproteinase-2 (MMP-2) were detected after administration, whereas mRNA levels of TM and EPCR, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity among groups, were also evaluated.Results:No significant difference was observed in any indicator between CORM-2 and sham groups. Addition of LPS produced drastic increase in MMP-2 expression, NF-κB activity, shedding of TM and EPCR (into the culture medium), as well as remarkable decrease in both mRNA and protein expressions of TM and EPCR, and cell viability. LPS + CORM-2 treatment significantly reduced the increase in MMP-2, NF-κB activity, and TM/EPCR shedding, whereas maintained both mRNA and protein levels of TM and EPCR, and preserved cell viability.Conclusions:CORM-2 protects HUVEC from LPS-induced injury, by way of suppressing NF-κB activity, which downregulates TM and EPCR mRNAs. It also decreases MMP-2 expression and prevents the shedding of TM and EPCR from the surface of endothelial cells, so as to preserve their protective effect.

Targeted Gene Sequencing Identifies Variants in the Protein C and Endothelial Protein C Receptor Genes in Patients With Unprovoked Venous Thromboembolism

The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine whether mutations that impair PC-EPCR interactions are associated with an increased risk of VTE. We sequenced exon 3 of PROC and exons 2 and 3 of PROCR (the exons that encode the protein-protein binding domains of PC and EPCR) in 653 patients with unprovoked VTE and in 627 healthy controls. Five single nucleotide variants, each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, and Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). We did not detect any nonsynonymous coding variants in the controls. When the PC variants were expressed in human embryonic kidney 293 cells, all exhibited decreased synthesis, and 2 of the variants had reduced capacity for activated PC generation. When expressed on the surface of human embryonic kidney 293 cells, the EPCR variants showed reduced affinity for fluorescently labeled PC. In addition, the previously reported EPCR A3 haplotype, which promotes cellular shedding of EPCR, is over-represented in the patient group (P=0.001). This is the first targeted DNA sequencing analysis of PROC and PROCR in a large group of patients with unprovoked VTE. Our data suggest that mutations that impair PC-EPCR interactions may be associated with an increased risk of VTE.

Inhibitory effects of epi-sesamin on endothelial protein C receptor shedding in vitro and in vivo

Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation by the thrombin-thrombomodulin complex. The activity of EPCR is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). The EPCR shedding is mediated by the tumor necrosis factor-α converting enzyme (TACE). Epi-sesamin (ESM), from the roots of Asarum siebodlii, is known to exhibit anti-allergic and anti-fungal activities. However, little is known about the effects of ESM on EPCR shedding. We investigated this issue by monitoring the effects of ESM on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-mediated EPCR shedding. Data showed that ESM induced potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding, likely through suppression of TACE expression. In addition, treatment with ESM resulted in a reduction of PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, ESM should be viewed as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of EPCR shedding.

Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo

The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.

In vitro와 in vivo에서 라이코펜이 EPCR 탈락에 미치는 영향

내피세포 단백질 C 수용체(EPCR)가 트롬빈-트롬보모듈린 복합체에 의한 단백질 C (PC) 활성 증가에 중요한 역할을 한다. EPCR의 활성은 ecodomain의 분열과 수용성 단백질(sEPCR)로 분비함으로써 현저하게 변화한다. EPCR의 탈락은 tumor necrosis factor-<TEX>${\alpha}$</TEX> converting enzyme (TACE)에 의해 매개된다. 토마토에서 발견된 라이코펜은 항산화 효과, 항암 효과, 항염증 효과를 가지고 있다. 그러나 EPCR 탈락에서의 라이코펜의 효과는 알려지지 않았다. 우리는 라이코펜이 PMA, TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>와 CLP에 의해 유도된 EPCR 탈락에 미치는 영향을 연구했다. 그 결과, 라이코펜은 TACE의 발현을 억제시켜 PMA, TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>와 CLP에 의해 매개된 EPCR 탈락을 저해함을 보여준다. 또한 라이코펜은 PMA가 유발한 p38, ERK1/2, JNK의 인산화를 감소시켰다. 이러한 결과를 토대로, 라이코펜은 EPCR 탈락의 저해를 통해 다양한 중증 혈관 염증 질병 치료를 위한 후보 물질이 될 수 있을 것이다. Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation through the thrombin-thrombomodulin complex. EPCR activity is markedly changed by ectodomain cleavage and released as the soluble protein (sEPCR). EPCR shedding is mediated by tumor necrosis factor-<TEX>${\alpha}$</TEX> converting enzyme (TACE). Lycopene found in tomatoes and tomato products has anti-oxidant, anti- cancer and anti-inflammatory effects. However, little is known about the effects of lycopene on EPCR shedding. We investigated this issue by monitoring the effects of lycopene on the phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-<TEX>${\alpha}$</TEX>, interleukin (IL)-<TEX>$1{\beta}$</TEX> and on the cecal ligation and puncture (CLP)-mediated EPCR shedding. Data showed that lycopene potently inhibited the PMA, TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX> and CLP-induced EPCR shedding by suppressing TACE expression. Furthermore, lycopene reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2 and c-Jun N-terminal kinase (JNK). Given these results, lycopene should be viewed as a candidate therapeutic agent for the treatment of various severe vascular inflammatory diseases via inhibition of the EPCR shedding.

Down-regulation of endothelial protein C receptor shedding by persicarin and isorhamnetin-3-O-galactoside

Increasing evidence has shown that beyond its role in coagulation, endothelial protein C receptor (EPCR) plays an important role in the cytoprotective pathway. Previous reports have shown that EPCR can be shed from the cell surface, and that this is mediated by tumor necrosis factor-α converting enzyme (TACE) and that sEPCR levels are increased in patients with systemic inflammatory diseases. Persicarin and isorhamnetin-3-O-galactoside (I3G) are active compounds from Oenanthe javanica, which has been widely studied for its neuroprotective, antioxidant, and barrier protective activities. However, little is known of the effects of persicarin on EPCR shedding. Here, we investigated this issue by monitoring the effects of persicarin and I3G on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. According to the results, persicarin and I3G induced potent inhibition of PMA and CLP-induced EPCR shedding by suppressing expression of TACE. In addition, persicarin and I3G reduced PMA-stimulated phosphorylation of p38MAPK, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, persicarin and I3G could be used as a candidate therapeutic for treatment of severe vascular inflammatory diseases.

Emodin-6-O-β-d-glucoside down-regulates endothelial protein C receptor shedding

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway and in the cytoprotective pathway. Previously, EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). Soluble EPCR levels are increased in patients with systemic inflammatory diseases. Recently, we reported that a new active compound, emodin-6-O-β-D-glucoside (EG) from Reynoutria japonica, has anti-inflammatory activities. However, little is known of the effects of EG on EPCR shedding. Here, we investigated this issue by monitoring the effects of EG on the phorbol-12-myristate 13-acetate (PMA) or the cecal ligation and puncture (CLP)-mediated EPCR shedding and its underlying mechanisms. Data showed that EG potently inhibited the PMA and CLP-induced EPCR shedding by suppressing TACE expression. Given these results, EG could be used as a candidate therapeutic for the treatment of vascular inflammatory diseases.

Early rise in circulating endothelial protein C receptor correlates with poor outcome in severe sepsis

The endothelial protein C receptor (EPCR) negatively regulates the coagulopathy and inflammatory response in sepsis. Mechanisms controlling the expression of cell-bound and circulating soluble EPCR (sEPCR) are still unclear. Moreover, the clinical impact of EPCR shedding and its potential value to predict sepsis progression and outcome remain to be established. We investigated the time course of plasma sEPCR over the 5 first days (D) of severe sepsis in 40 patients. No significant difference was observed when comparing sEPCR at admission (D1) to healthy volunteers and to patients with vasculitis. We report that the kinetics profile of plasma sEPCR in patients was almost stable at the onset of sepsis with no change from D1 to D4 and then a significant decrease at D5. This pattern of release was consistently observed whatever the level of sEPCR at D1. Characteristics of patients or of infections (except Gram negative) had no or little critical influence on the sEPCR profile. However, we found that sEPCR kinetics was clearly associated with patient's outcome (D28 survival). We demonstrate that a significant but moderate (<15% of basal level) and transient increase in sEPCR level at D2 is associated with poor outcome at D28. Severe sepsis, at the onset, only triggers moderate quantitative changes in plasma sEPCR levels. Our findings suggest that in severe sepsis, an early (at D2), transient but significant increase in circulating sEPCR may be detrimental suggesting that sEPCR could provide an early biological marker of sepsis outcome.

Expression and shedding of endothelial protein C receptor in prostate cancer cells

BackgroundIncreasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.ResultsEPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.ConclusionsThe study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.

Activation of endothelial intrinsic NF-κB pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice

AbstractAlthough the role of systemic activation of the nuclear factor κB (NF-κB) pathway in septic coagulation has been well documented, little is known about the contribution of endothelial-specific NF-κB signaling in this pathologic process. Here, we used transgenic mice that conditionally overexpress a mutant I-κBα, an inhibitor of NF-κB, selectively on endothelium, and their wild-type littermates to define the role of endothelial-specific NF-κB in septic coagulation. In wild-type mice, lipopolysaccharide (LPS) challenge (5 mg/kg intraperitoneally) caused markedly increased plasma markers of coagulation, decreased plasma fibrinogen level, and widespread tissue fibrin deposition, which were abrogated by endothelial NF-κB blockade in transgenic mice. Endothelial NF-κB blockade inhibited tissue factor expression in endothelial cells, but not in leukocytes. Endothelial NF-κB blockade did not inhibit LPS-induced tissue factor expression in heart, kidney, and liver. Endothelial NF-κB blockade prevented LPS down-regulation of endothelial protein C receptor (EPCR) and thrombomodulin protein expressions, inhibited tissue tumor necrosis factor-α converting enzyme activity, reduced EPCR shedding, and restored plasma protein C level. Our data demonstrate that endothelial intrinsic NF-κB signaling plays a pivotal role in septic coagulation and suggests a link between endothelial-specific NF-κB activation and the impairment of the thrombomodulin-protein C-EPCR anticoagulation pathway.

Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interferon-γ and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1β and TNF-α correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl- l-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1β and TNF-α, and downstream by MAP kinase signaling pathways and metalloproteinases.

Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor-alpha converting enzyme/ADAM17.

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor-alpha converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol-12-myristate 13-acetate (PMA)-stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA-stimulated EPCR shedding is completely blocked in fibroblasts from TACE-deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAG sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

Novel Role of the Human Alveolar Epithelium in Regulating Intra-Alveolar Coagulation

Intra-alveolar fibrin deposition is a common response to localized and diffuse lung infection and acute lung injury (ALI). We hypothesized that the alveolar epithelium modulates intra-alveolar fibrin deposition through activation of protein C. Our objectives [corrected] were to determine whether components of the protein C activation pathway are present in the alveolar compartment in ALI and whether alveolar epithelium is a potential source. In patients with ALI, pulmonary edema fluid levels of endothelial protein C receptor (EPCR) were higher than plasma, suggesting a source in the lung. To determine whether alveolar epithelial cells are a potential source, protein C activation by A549, small airway epithelial, and primary human alveolar epithelial type II cells was measured. All three cell types express thrombomodulin (TM) and EPCR, and activate protein C on the cell surface. Activation of protein C was inhibited by cytomix (TNF-alpha, IL-1beta, and IFN-gamma). Release of EPCR and TM into the conditioned medium was inhibited by the metalloproteinase inhibitors tumor necrosis factor protease inhibitor (TAPI) and GM6001, indicating that the shedding of EPCR and TM from the alveolar epithelium is mediated by a metalloproteinase. These findings provide new evidence that the alveolar epithelium can modulate the protein C pathway and thus could be an important determinant of alveolar fibrin deposition. Local fibrin deposition may be a fundamental mechanism for the lung to localize and confine injury, thus limiting the risk of dissemination of injury or infection to the systemic circulation.