Regulation of Brain Capillary Endothelial Thrombomodulin mRNA Expression

Tran, Nam; Wong, Vicky L.Y.; Schreiber, Sidney S.; Bready, James; Fisher, Mark

doi:10.1161/01.str.27.12.2304

Cited by 33 publications

(36 citation statements)

References 41 publications

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“…[31][32][33] Although myocardial infarction is most frequently the consequence of local thrombosis, ischemic strokes more frequently result from embolic disorders. 34 TM expression has been demonstrated in brain vessels, because TM mRNA expression is present in the blood-brain barrier 35 and protein C is activated in jugular vein blood during carotid artery occlusion in humans. 36 The endothelial TM expression in brain is constitutively downregulated by astrocytes and acutely downregulated by pathogenic mediators of ischemic stroke, such as interleukin-1␤ and tumor necrosis factor-␣.…”

Section: Erk2mentioning

confidence: 99%

“…36 The endothelial TM expression in brain is constitutively downregulated by astrocytes and acutely downregulated by pathogenic mediators of ischemic stroke, such as interleukin-1␤ and tumor necrosis factor-␣. 35 Interestingly, astrocyteinduced downregulation of TM endothelial expression occurs through transforming growth factor-␤ secretion, 37 known to enhance endothelial cell proliferation. 38 Overall, that TM …”

Section: Erk2mentioning

confidence: 99%

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Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Olivot

Estebanell

Lafay

et al. 2001

Circulation Research

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Abstract-On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin. T hrombin is a multifunctional serine protease generated at sites of vascular injury. Thrombin plays a key role in blood coagulation and thrombotic disorders. It acts as the central enzyme of the coagulation cascade by cleaving fibrinogen into fibrin and favoring its own production by activating several coagulation factors by limited proteolysis. Thrombin also regulates its own formation after binding to thrombomodulin (TM), an integral membrane-bound glycoprotein expressed on endothelial cells. TM acts as a cofactor of thrombin to activate protein C, a serine protease ensuring proteolytic inactivation of 2 coagulation factors, factor Va and factor VIIIa. Thrombin also interacts with a variety of cells mediating inflammatory and proliferative responses to vascular injury. 1 For all protein and cellular interactions, thrombin has a recognition site and a catalytic active site. By the former, called the anion-binding exosite (ABE1), thrombin binds to specific negatively charged sequences. By the catalytic site, thrombin exerts its proteolytic activity. 2 On vascular endothelial cells, thrombin ABE1 binds to TM. This binding occurs through the epithelial growth factor (EGF)-like domains 4 and 5 of TM. 3 Thrombin ABE1 also binds to the typical heptahelical thrombin receptor, the first member of pr...

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Section: Erk2mentioning

confidence: 99%

Section: Erk2mentioning

confidence: 99%

Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Olivot

Estebanell

Lafay

et al. 2001

Circulation Research

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“…Although TM is abundant in most systemic vasculature, immunohistochemical studies 4 -7 have revealed only limited, if any, TM expression in the human cerebrovasculature. A restricted functional role for PC activation in the central nervous system is further suggested by findings 8 that astrocyte coculture produces a 20-fold downregulation of endothelial cell TM mRNA production in vitro. Hence, the importance of the PC antithrombotic mechanism in brain has not yet been established, and it is unknown whether functional TM is present in sufficient quantity to generate APC during cerebrovascular insufficiency.…”

mentioning

confidence: 98%

Brain-Specific Protein C Activation During Carotid Artery Occlusion in Humans

Macko

Killewich

Fernández

et al. 1999

Stroke

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Background and Purpose-Activation of plasma protein C (PC) zymogen by thrombin-thrombomodulin at the endothelial surface is an important endogenous antithrombotic mechanism. It is unknown whether activated protein C (APC) is generated in vivo in the cerebrovasculature, because there is only limited thrombomodulin expression in human brain vascular endothelium. Therefore, we tested the hypothesis that carotid occlusion produces brain-specific PC activation. Methods-Blood samples were simultaneously collected from the ipsilateral internal jugular vein and radial artery before and during carotid cross-clamping and on "de-occlusion" in 8 awake patients undergoing routine carotid endarterectomy. Plasma PC zymogen and circulating APC levels were measured using enzyme immunocapture assay and expressed as percent of pooled plasma controls. Results-Internal jugular vein APC levels increased 28% exclusively during carotid occlusion and then decreased 32% with de-occlusion (Fϭ8.

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“…We have demonstrated that astrocytes regulate endothelial expression of both tPA and TM in vitro. 36,37 The mechanism of this action has been uncertain. Astrocytes can secrete a number of cytokines and growth factors known to modulate endothelial function, including transforming growth factor-␤ (TGF-␤).…”

mentioning

confidence: 99%

Transforming Growth Factor-β Mediates Astrocyte-Specific Regulation of Brain Endothelial Anticoagulant Factors

Tran

Correale

Schreiber

et al. 1999

Stroke

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Background and Purpose-Astrocytes are potent regulators of brain capillary endothelial cell function. Recently, astrocytes were shown to regulate brain capillary endothelial expression of the fibrinolytic enzyme tissue plasminogen activator (tPA) and the anticoagulant thrombomodulin (TM). To study the mechanism of this process, we examined the hypothesis that astrocyte regulation of endothelial tPA and TM is mediated by transforming growth factor-␤ (TGF-␤). Methods-Brain capillary endothelial cells were grown in blood-brain barrier models. We examined astrocyte-endothelial cocultures, endothelial monocultures, and astrocyte-conditioned media (ACM) for the expression of TGF-␤. We also incubated endothelial cells with ACM to determine the role of TGF-␤. Following 24 hours of incubation, we assayed for tPA and TM mRNA, as well as tPA and TM activity. Results-Astrocyte-endothelial cocultures and ACM exhibited significantly higher levels of active TGF-␤ than brain endothelial monocultures and endothelial cells grown in nonconditioned media, respectively. Brain endothelial cells incubated with ACM exhibited reduced tPA and TM mRNA and activity. Treatment with exogenous TGF-␤ produced dose-dependent reductions in tPA and TM. The effects of ACM on both tPA and TM were blocked by TGF-␤ neutralizing antibody. tPA is a critical circulating fibrinolytic enzyme that proteolytically activates plasminogen to plasmin. 1 Intravenous tPA improves neurological outcome in clinical stroke. 2 Reduced expression of brain capillary tPA is associated with increased infarct size following transient middle cerebral artery occlusion in diabetic and nicotine stroke models. 3,4 Brain capillary endothelial expression of tPA is limited, [5][6][7] and understanding mechanisms underlying this limited expression has potential therapeutic value. Conclusions-TheseTM, an important antithrombotic protein, functions as a cofactor for the activation of circulating protein C. 8,9 Both TM and activated protein C offer protection against a variety of thrombotic events. Treatment with purified or recombinant TM protects against thromboembolism in animal models, 10 -14 See Editorial Comment, page 1677 while TM neutralizing antibodies potentiate thrombininduced thromboembolism. 10 Treatment with activated protein C also provides protection against thromboembolic events. 15,16 Resistance to the effects of activated protein C is closely linked to venous thrombosis in humans, 17 including cerebral venous thrombosis. 18 Low levels of circulating activated protein C are present in certain forms of ischemic stroke. 19 Moreover, brain-specific protein C activation has been demonstrated in humans in vivo 20 ; brain capillaries ex vivo also exhibit protein C activation. 21 Brain TM expression is limited, [22][23][24][25][26] and, like tPA, identification of mechanisms underlying this limited expression would be expected to have therapeutic value.Astrocytes are regulators for a wide variety of brain capillary endothelial functions. Astrocytes are responsible for ...

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Regulation of Brain Capillary Endothelial Thrombomodulin mRNA Expression

Abstract: These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.

Cited by 33 publications

References 41 publications

Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Brain-Specific Protein C Activation During Carotid Artery Occlusion in Humans

Transforming Growth Factor-β Mediates Astrocyte-Specific Regulation of Brain Endothelial Anticoagulant Factors

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