Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells

SummaryPleural mesothelial and vascular endothelial cells express protein C (PC) pathway components including thrombomodulin (TM) and endothelial protein C receptor (EPCR) and activate PC by the thrombin-TM dependent mechanism.We used these cells as model systems to identify molecules involved in endocytosis and degradation of PC. We find that mesothelial and endothelial cells can bind, internalize and degrade PC.Addition of thrombin markedly induced degradation of PC by these cells in a TM-dependent fashion, implicating the involvement of the thrombin-TM complex in internalization and degradation of PC. This observation defines a novel function for the thrombin-TM complex as a degradation receptor for PC and suggests that PC is degraded concurrent with its activation.A PC Gla-domain mutant, which is unable to bind to the EPCR, was degraded by the cells to a lesser extent than wild-type PC, implicating the PC degradation concurrent with its activation. Consistent with the role of thrombin-TM complex as a degradation receptor, the catalytically inactive thrombin-S195A also induced PC degradation though to a lesser extent than wild-type thrombin.This suggests that generation of activated PC (APC) can contribute to accumulation of degradation products, but is not essential for the thrombin-induced degradation of PC. The thrombin-TMmediated degradation of PC by both cell types suggest a previously unrecognized mechanism, which can contribute to PC consumption.This mechanism may be pathophysiologically relevant and can contribute to an acquired PC deficiency in conditions characterized by sustained thrombin generation.

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Thrombin inhibits nuclear factor κB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C

Thrombosis and Haemostasis ◽

10.1160/th08-09-0568 ◽

2009 ◽

Vol 101 (03) ◽

pp. 513-520 ◽

Cited By ~ 37

Author(s):

Jong-Sup Bae ◽

Alireza R. Rezaie

Keyword(s):

Endothelial Cells ◽

Adhesion Molecules ◽

Protein C ◽

Vascular Endothelial Cells ◽

Nuclear Factor Κb ◽

Small Gtpases ◽

Vascular Endothelial ◽

Nuclear Factor ◽

Endothelial Protein C Receptor ◽

Tnf Α

SummaryThe occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-α-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-κB (NF-κB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist in-fluenced the proinflammatory effects of TNF-α in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-α-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-κB pathways in response to TNF-α in cells pre-treated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-κB signalling cascades in vascular endothelial cells.

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Thrombin Inhibits NF-κb and RhoA Pathways in Cytokine-Stimulated Vascular Endothelial Cells When EPCR Is Occupied by Protein C.

Blood ◽

10.1182/blood.v112.11.1888.1888 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1888-1888

Author(s):

Alireza R. Rezaie ◽

Jong-Sup Bae

Keyword(s):

Endothelial Cells ◽

Adhesion Molecules ◽

Protein C ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Small Gtpases ◽

Vascular Endothelial ◽

Endothelial Protein C Receptor ◽

Signaling Specificity ◽

Natural Ligand

Abstract We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.

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Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases

AJP Cell Physiology ◽

10.1152/ajpcell.00003.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. C833-C842 ◽

Cited By ~ 22

Author(s):

Christophe Guitton ◽

Alice Cottereau ◽

Nathalie Gérard ◽

Thibaut Quillard ◽

Annabelle Chauveau ◽

...

Keyword(s):

Endothelial Cells ◽

Cross Talk ◽

Protein C ◽

Map Kinases ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Vascular Endothelial ◽

Endothelial Protein C Receptor ◽

Protein Levels ◽

Antiinflammatory Effects

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.

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Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant

Thrombosis and Haemostasis ◽

10.1160/th15-01-0018 ◽

2015 ◽

Vol 114 (11) ◽

pp. 1038-1048 ◽

Cited By ~ 30

Author(s):

Eveline A. M. Bouwens ◽

Ibai Tamayo ◽

Louise Turner ◽

Christian W. Wang ◽

Monique Stins ◽

...

Keyword(s):

Endothelial Cells ◽

Severe Malaria ◽

Protein C ◽

Cellular Response ◽

Malaria Parasite ◽

Vascular Endothelial Cells ◽

Protective Effects ◽

Endothelial Protein C Receptor ◽

Parasite Protein ◽

Soluble Epcr

SummaryThe Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.

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ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer

International Journal of Cancer ◽

10.1002/ijc.22882 ◽

2007 ◽

Vol 121 (8) ◽

pp. 1710-1716 ◽

Cited By ~ 16

Author(s):

Bon-Hun Koo ◽

Tiina Hurskainen ◽

Katrina Mielke ◽

Phyu Phyu Aung ◽

Graham Casey ◽

...

Keyword(s):

Colon Cancer ◽

Endothelial Cells ◽

Epithelial Cells ◽

Vascular Endothelial Cells ◽

Cell Types ◽

Vascular Endothelial ◽

Colorectal Tumors

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The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel

International Journal of Molecular Sciences ◽

10.3390/ijms21114024 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4024 ◽

Cited By ~ 2

Author(s):

Ilona Gróf ◽

Alexandra Bocsik ◽

András Harazin ◽

Ana Raquel Santa-Maria ◽

Gaszton Vizsnyiczai ◽

...

Keyword(s):

Cystic Fibrosis ◽

Endothelial Cells ◽

Epithelial Cells ◽

Sodium Bicarbonate ◽

Vascular Endothelial Cells ◽

Bronchial Epithelium ◽

Barrier Properties ◽

Vascular Endothelial ◽

Wild Type ◽

Bronchial Epithelial

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

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Factor X/Xa Elicits Protective Signaling Responses In Endothelial Cells Directly Via PAR-2 and Indirectly Via EPCR-Dependent Recruitment of PAR-1

Blood ◽

10.1182/blood.v116.21.2115.2115 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2115-2115

Author(s):

Alireza R. Rezaie ◽

Jong-Sup Bae ◽

Likui Yang

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Conflicts Of Interest ◽

Factor Viia ◽

Factor X ◽

Endothelial Protein C Receptor ◽

Caveolin 1 ◽

Gla Domain ◽

Protective Signaling ◽

Dependent Interaction

Abstract Abstract 2115 We recently demonstrated that the Gla-domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier protective response if endothelial cells were pre-incubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells, also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a crosstalk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla-domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells. Disclosures: No relevant conflicts of interest to declare.

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Axl Is Essential for in-vitro Angiogenesis Induced by Vitreous From Patients With Proliferative Diabetic Retinopathy

Frontiers in Medicine ◽

10.3389/fmed.2021.787150 ◽

2021 ◽

Vol 8 ◽

Author(s):

Wenyi Wu ◽

Huizuo Xu ◽

Zhishang Meng ◽

Jianxi Zhu ◽

Siqi Xiong ◽

...

Keyword(s):

Endothelial Cells ◽

Diabetic Retinopathy ◽

Proliferative Diabetic Retinopathy ◽

Tyrosine Kinases ◽

Vascular Endothelial Cells ◽

Specific Inhibitor ◽

Cell Types ◽

Specific Protein ◽

Vascular Endothelial

Proliferative diabetic retinopathy (PDR), characterized mainly with abnormal epiretinal angiogenesis forming fibrovascular membranes (FVMs), threatens vision of people with diabetes; FVMs consist of extracellular matrix and a variety of cell types including vascular endothelial cells. Axl, one of receptor tyrosine kinases, can be activated indirectly by vascular endothelial growth factor-A (VEGF-A) via an intracellular route for promoting angiogenesis. In this study, we revealed that growth arrest-specific protein 6 (Gas6), a specific ligand of Axl, was elevated in vitreous from patients with PDR and that Axl was activated in FVMs from patients with PDR. In addition, we demonstrated that in cultured human retinal microvascular endothelial cells (HRECs), Axl inhibition via suppression of Axl expression with Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9 or through inactivation with its specific inhibitor R428 blocked PDR vitreous-induced Akt activation and proliferation of HRECs. Furthermore, PDR vitreous-heightened migration and tube formation of HRECs were also blunted by restraining Axl. These results indicate that in the pathogenesis of PDR, Axl can be activated by Gas6 binding directly and by VEGF-A via an intracellular route indirectly, suggesting that Axl plays a pivotal role in the development of PDR and that Axl inhibition shows a bright promise for PDR therapy.

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Origin and diversity of embryonic endothelium/endocardium

10.1093/med/9780198757269.003.0005 ◽

2018 ◽

Author(s):

LeShana SaintJean ◽

H.S. Baldwin

Keyword(s):

Endothelial Cells ◽

Heart Development ◽

Vascular Endothelial Cells ◽

Coronary Vessels ◽

Cell Types ◽

Genetic Profile ◽

Vascular Endothelial ◽

Tremendous Progress

The endocardium represents a distinct population of endothelial cells that arises during the initiation of heart development. Endocardial cells can easily be distinguished from most of the other cardiac cell types. However, endocardial and vascular endothelial cells contain a similar genetic profile that limits the ability to study each group independently. Despite these limitations, tremendous progress has been made in identifying the different roles of endocardial cells throughout heart development. Initial studies focused on the origin of endocardial cells and their role in valvulogenesis, trabeculation, and formation of the ventricular and atrial septum. With the advancement of microscopy and the availability of endocardial specific reporter models (in vitro and in vivo) we have obtained more insight into the molecular, structural, and functional complexity of the endocardium. Additional studies have demonstrated how the endocardium is also involved in the development of coronary vessels within the compact myocardium and in heart regeneration.

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