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Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis.

ABSTRACT: OBJECTIVE:Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine whether clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases. APPROACH AND RESULTS:We performed next-generation RNA sequencing on monocytes extracted from whole blood clots and using a purified plasma clot system. Numerous mRNAs were differentially expressed by monocytes embedded in clots compared with unclotted controls, and IL-8 (interleukin 8) and MCP-1 (monocyte chemoattractant protein-1) were among the upregulated transcripts in both models. Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in lipopolysaccharide-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (ie, 1-2 hours) reduced the synthesis of IL-8 and MCP-1, whereas delayed fibrinolysis was far less effective. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. CONCLUSIONS:These findings demonstrate that clots are potent inducers of monocyte gene expression and that timely fibrinolysis attenuates inflammatory responses, specifically IL-8 and MCP-1. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

SUBMITTER: Campbell RA

PROVIDER: S-EPMC5620127 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis.

Campbell Robert A RA Vieira-de-Abreu Adriana A Rowley Jesse W JW Franks Zechariah G ZG Manne Bhanu Kanth BK Rondina Matthew T MT Kraiss Larry W LW Majersik Jennifer J JJ Zimmerman Guy A GA Weyrich Andrew S AS

Arteriosclerosis, thrombosis, and vascular biology 20170803 10

<h4>Objective</h4>Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine whether clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases.<h4>Approach and r ...[more]

PMID: 28775073

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Project description:BackgroundThrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro.Design and methodsTissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator.ResultsLPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/microL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (approximately 50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity.ConclusionsOur data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins.

Project description:BackgroundThe NF-?B signaling pathway orchestrates many of the intricate aspects of neuroinflammation. Astrocytic ??-adrenergic receptors have emerged as potential regulators in central nervous system inflammation and are potential targets for pharmacological modulation. The aim of this study was to elucidate the crosstalk between astrocytic ??-adrenergic receptors and the TNF-? induced inflammatory gene program.MethodsProinflammatory conditions were generated by the administration of TNF-?. Genes that are susceptible to astrocytic crosstalk between ??-adrenergic receptors (stimulated by clenbuterol) and TNF-? were identified by qPCR-macroarray-based gene expression analysis in a human 1321 N1 astrocytoma cell line. Transcriptional patterns of the identified genes in vitro were validated by RT-PCR on the 1321 N1 cell line as well as on primary rat astrocytes. In vivo expression patterns were examined by intracerebroventricular administration of clenbuterol and/or TNF-? in rats. To examine the impact on the inflammatory cell content of the brain we performed extensive FACS analysis of rat brain immune cells after intracerebroventricular clenbuterol and/or TNF-? administration.ResultsParallel transcriptional patterns in vivo and in vitro confirmed the relevance of astrocytic ??-adrenergic receptors as modulators of brain inflammatory responses. Importantly, we observed pronounced effects of ?2-adrenergic receptor agonists and TNF-? on IL-6, CXCL2, CXCL3, VCAM1, and ICAM1 expression, suggesting a role in inflammatory brain cell homeostasis. Extensive FACS-analysis of inflammatory cell content in the brain demonstrated that clenbuterol/TNF-? co-administration skewed the T cell population towards a double negative phenotype and induced a shift in the myeloid brain cell population towards a neutrophilic predominance.ConclusionsOur results show that astrocytic ??-adrenergic receptors are potent regulators of astrocytic TNF-?-activated genes in vitro and in vivo, and ultimately modulate the molecular network involved in the homeostasis of inflammatory cells in the central nervous system. Astrocytic ??-adrenergic receptors and their downstream signaling pathway may serve as potential targets to modulate neuroinflammatory responses.

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Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis.

Publications

Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis.

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