Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium

Chen, Daxin; Γιαννόπουλος, Κωνσταντίνος; Shiels, Paul G.; Webster, Zoë; McVey, John H.; Kemball-Cook, Geoff; Tuddenham, Edward G. D.; Moore, Miranda S.; Lechler, Robert I.; Dorling, Anthony

doi:10.1182/blood-2003-12-4365

Cited by 52 publications

(67 citation statements)

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“…Results from complimentary but distinct experimental approaches, using either WT or PAR-1 KO hearts transplanted into defi binogenated rats or CD31-Hir-Tg hearts transplanted Thrombin generation during systemic infl ammation is highly dependent on the procoagulant changes induced on endothelium ( 26 ). Why mammals have evolved or maintained, as an integral part of EC activation, the switch from an anticoagulant to a procoagulant phenotype is interesting because if it results in intravascular thrombosis it can pose a direct threat to the survival of the individual, as often occurs during acute severe sepsis.…”

Section: Brief Definitive Reportmentioning

confidence: 99%

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Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1–dependent leukocyte recruitment in vivo

Chen¹,

Carpenter

Abrahams³

et al. 2008

The Journal of Experimental Medicine

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Thrombin and other coagulation proteases mediate a variety of eff ects independently of thrombosis through specifi c protease-activated receptors (PARs), stimulation of which can amplify infl ammation initiated by several diverse stimuli ( 1 ). Although the proinfl ammatory consequences of PAR stimulation have been implicated in several diseases ( 2 -4 ), it has yet to be established whether thrombin or PAR activation provides a unique stimulus responsible for a nonthrombotic manifestation of infl ammation.Working in a model of acute humoral xenograft rejection (mouse heart into rat), we previously reported that inhibiting thrombin generation or inhibiting thrombin itself inside the graft (using organs from transgenic [Tg] mice expressing endothelial cell [EC]-tethered anticoagulants) completely inhibited humoral rejection so that hearts were rejected by infi ltrating T lymphocytes ( 5 ). These fi ndings were surprising because the rejected hearts had signifi cant Ig and C deposition on graft ECs. Inhibiting thrombosis by depleting fi brinogen from the recipients (using a snake venom protein, ANCROD) failed to achieve the same degree of survival, and under these conditions, infi ltrating NK cells and macrophages (M ⌽ s), rather than T cells, were observed in rejected grafts ( 6 ). From these studies we hypothesized that thrombin was providing a stimulus in the humoral rejection process that was necessary for the infi ltration of NK cells and M ⌽ s. We have tested this hypothesis and confi rmed our conclusions in a second model of thioglycollateinduced M ⌽ recruitment into the peritoneum. RESULTS AND DISCUSSION Donor monocyte chemoattractant protein (MCP)-1 is required for NK cell and M ⌽ recruitmentWe hypothesized that infi ltration of NK cells and M ⌽ s into rejecting mouse hearts was due to the establishment of a chemokine gradient, most likely MCP-1, a CC chemokine known to be essential for NK cell and M ⌽ recruitment. Thrombin, acting through a family of protease-activated receptors (PARs), is known to amplify infl ammatory responses, but the in vivo importance of PARs in infl ammation is not fully appreciated. In a mouse heart-to-rat transplant model, where it is possible to distinguish graft (mouse) from systemic (rat) chemokines, we show that donor PAR-1 is required to generate the local monocyte chemoattractant protein (MCP)-1 needed to recruit rat natural killer cells and macrophages into the hearts. We have confi rmed the importance of this mechanism in a second model of thioglycollate-induced peritonitis and also show that PAR-1 is important for the production of MCP-3 and MCP-5. Despite the presence of multiple other mediators capable of stimulating chemokine production in these models, these data provide the fi rst evidence that thrombin and PAR activation are required in vivo to initiate infl ammatory cell recruitment. CORRESPONDENCE

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Section: Brief Definitive Reportmentioning

confidence: 99%

“…Cell culture. Mouse microvascular ECs were purifi ed and passaged as described previously ( 26 ) and used at passages 1 -3.…”

Section: Animalsmentioning

confidence: 99%

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1–dependent leukocyte recruitment in vivo

Chen¹,

Carpenter

Abrahams³

et al. 2008

The Journal of Experimental Medicine

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“…It has been recently reported that platelets accumulate in the livers and lungs of LPS-treated animals. 21 In Figure 5B-C, platelet accumulation is clearly shown in ␤-gal stained livers, lungs, and spleens, but only in mice under Tet-off conditions. No significant platelet accumulation in the above tissues was observed in mice that were not treated with LPS compared with LPS-treated mice (data not shown).…”

Section: Inducible Expression Of ␤-Galactosidase and Generation Of A mentioning

confidence: 90%

“…Double-transgenic mice were anaesthetized with isoflurane (40 g/g body weight) and intraperitoneally injected with lipopolysaccharide (LPS) (E coli serotype 0127;B8; Sigma), at a dosage of 60 g/g of body weight, or saline in a total volume of up to 100 L (control), essentially as described in Chen et al 21 Mice were killed 2 hours after LPS or saline administration and perfused for 5 minutes with 1 ϫ PBS, followed by 2% paraformaldeldyde for 15 minutes and, finally, 1 ϫ PBS for 10 minutes. The livers and lungs were surgically removed immediately after fixation and subjected to staining for ␤-galactosidase as described above.…”

Section: Lps-induced Acute Inflammation and Tissue Sectionsmentioning

confidence: 99%

Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

Nguyen

Makitalo

et al. 2005

Blood

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Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally overexpress genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic ␤-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by ␤-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4 - IntroductionMegakaryocytes (MKs) progress through distinct developmental stages, including lineage commitment, expansion, polyploidization, maturation, and fragmentation into platelets. 1,2 To investigate mechanisms that control megakaryocytic development, gene targeting is required to manipulate levels of a potential regulatory protein of interest. More than a decade ago, the rat platelet factor 4 (PF4) gene promoter 3 was used as a tool to specifically overexpress genes in early committed megakaryocytes in vivo. Since then, PF4 and other related promoters have been used to examine effects of deregulating the expression of cell-cycle genes, antiapoptotic proteins, receptors, thrombosis-modulating proteins, and others on megakaryocyte/platelet development and/or activity. 4-14 PF4-driven constitutive expression is limited, depending on the transgene used, due to occasional lethality during development or adverse effects during adulthood. For instance, Kufrin et al demonstrated that PF4-driven expression of urokinase-type plasminogen activator in mice exhibited a bleeding diathesis, which may have limited the potential number of founder lines that were available for analysis. 10 In addition, if a given transgene is not expressed due to regulatory elements within the site of genomic integration, increased numbers of founders need to be generated. 13 Tetracycline-inducible promoters and transactivating mouse lines have been used throughout the literature to circumvent such limitations. A successful application of this system was reported for targeted expression in myeloid cells and hematopoietic progenitors in vivo, [15]...

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“…Targeting coagulation cascade proteins could be an effective way to modulate inflammation. Indeed, A. Dorling from Imperial College London showed that targeted expression of the anticoagulants hirudin and tissue factor pathway inhibitor effectively inhibited intravascular thrombosis in a model of murine endotoxemia and markedly inhibited vascular remodeling after injury [30]. In addition to hirudin and tissue factor, the ubiquitous stress-responsive gene heme oxygenase-1 (HO-1) and carbon monoxide (CO) also control inflammatory responses.…”

Section: Alternative Targets and New Trends For Immunosuppressionmentioning

confidence: 99%

New trends in immunosuppression and immunotherapy

Jiang

Lombardi

2006

International Immunopharmacology

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Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium

Abstract: We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-

Cited by 52 publications

References 28 publications

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1–dependent leukocyte recruitment in vivo

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1–dependent leukocyte recruitment in vivo

Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

New trends in immunosuppression and immunotherapy

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