Basic Sciences Clots Are Potent Triggers of Inflammatory Cell Gene Expression Indications for Timely Fibrinolysis Robert A. Campbell, Adriana Vieira-de-Abreu, Jesse W. Rowley, Zechariah G. Franks, Bhanu Kanth Manne, Matthew T. Rondina, Larry W. Kraiss, Jennifer J. Majersik, Guy A. Zimmerman, Andrew S. Weyrich 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 Downloaded from 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 http://atvb.ahajournals.org/ 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. Visual Overview—An online visual overview is available for this article. (Arterioscler Thromb Vasc Biol. 2017;37: 1819-1827. DOI: 10.1161/ATVBAHA.117.309794.) by guest on July 10, 2018 Key Words: fibrin◼ fibrinogen◼ inflammation◼ monocytes ◼ thrombin oagulation contributes to the pathogenesis of many dis- suggests that clots are ineffective triggers of gene expression Cease processes, including sepsis, myocardial infarction, by monocytes, but this assumption has not been rigorously and ischemic stroke.1,2 On vessel injury, blood is exposed tested. Accordingly, we determined whether intact clots alter to tissue factor, resulting in the formation of thrombin that gene expression patterns in monocytes using human models stems blood loss by activating coagulation proteases and of whole blood and plasma clot formation. Our data dem- platelets.3–5 Thrombin also induces the conversion of fibrin- onstrate that clots induce marked changes in the monocyte ogen to fibrin, resulting in the formation of clots that seal transcriptome, including increased expression of IL-8 (inter- wounds.6,7 leukin-8) and MCP-1 (monocyte chemoattractant protein-1). When clots form, they trap numerous blood cells, includ- Remarkably, these shifts were more intense than changes in ing monocytes. Monocytes play an important role in bridg- gene expression induced by LPS. ing hemostasis and inflammation because they generate After fibrin formation occurs under physiological condi- tissue factor and inflammatory cytokines in response to tions, tPA (tissue-type plasminogen activator) is generated external stimuli.8–10 Perhaps the most well-known stimulus of and immediately begins breaking down the fibrin matrix.18 monocyte gene expression is lipopolysaccharide (LPS).11,12 Iatrogenic fibrinolysis is used as a therapeutic intervention However, LPS is not present in most clinical situations where in pathological thrombosis.19,20 In the setting of stroke, tPA is clots form, and individual clotting factors, such as tissue fac- administered at high levels to quickly dissolve the clot and tor, thrombin,13,14 and fibrin,15–17 have variable and generally improve blood flow to ischemic brain. In either setting, the mild stimulatory effects on monocyte gene expression. This effect of fibrinolysis on cytokine expression has not been Received on: November 9, 2016; final version accepted on: July 21, 2017. From the Program in Molecular Medicine (R.A.C., J.W.R., Z.G.F., B.K.M., M.T.R., L.W.K., A.S.W.) and Departments of Internal Medicine (R.A.C., A.V.-d.-A., J.W.R., M.T.R., G.A.Z., A.S.W.), Surgery (L.W.K.), and Neurology (J.J.M.), University of Utah, Salt Lake City. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.309794/-/DC1. Correspondence to Robert Campbell, PhD, Eccles Institute of Human Genetics, Bldg 533, Rm 4280, University of Utah, Salt Lake City, Utah 84112. E-mail [email protected] © 2017 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.117.309794 1819 1820 Arterioscler Thromb Vasc Biol October 2017 I in the online-only Data Supplement). RNA-seq revealed that Nonstandard Abbreviations and Acronyms monocytes expressed over 10 000 transcripts and nearly 1000 FXIII factor XIII were differentially expressed (ie, >4-fold) in clot-retrieved IL-8 interleukin-8 monocytes compared with baseline monocytes (Figure 1A). LPS lipopolysaccharide Among the differentially expressed transcripts were IL-8 and MCP-1 monocyte chemoattractant protein-1 MCP-1, which were increased by >6- and 48-fold, respec- NF-κB nuclear factor kappa B tively, in clot-retrieved monocytes (Figure 1A and 1B). TLR4 toll-like receptor-4 Increased mRNA expression was confirmed by real-time tPA tissue-type plasminogen activator polymerase chain reaction (Figure II in the online-only Data Supplement) and was associated with elevated levels of IL-8 and MCP-1 protein, which accumulated in a time-dependent examined. Our results demonstrate that fibrinolysis signifi- fashion (Figure 1C). In addition to IL-8 and MCP-1, 13 other cantly downregulates IL-8 and MCP-1 synthesis by mono- proteins involved in inflammation were increased after whole cytes, depending on when lysis was initiated. These findings blood clot formation, and mRNA for these genes correlated have significant implications in ischemic stroke because with their protein expression levels (Figure III in the online- timely administration of tPA after stroke dictates outcomes. only Data Supplement). These cytokines were also expressed in thrombi extracted from patients (Figure IV in the online- Materials and Methods only Data Supplement), indicating that in vivo clot formation Downloaded from Materials and Methods is available in the online-only Data is also associated with the generation of inflammatory proteins. Supplement. Plasma Clot Formation Induces Inflammatory Results Gene Expression in Monocytes Whole Blood Clots Trigger Inflammatory Next we determined whether clotted plasma triggers gene http://atvb.ahajournals.org/ Gene Expression in Monocytes expression in a manner similar to whole blood. For these To test whether clots trigger inflammatory gene expression, studies, recombinant tissue factor was added to recalci- whole blood was clotted with recombinant tissue factor in the fied, cell-free plasma and allowed to clot before addition of presence of calcium for 2 hours, and gene expression patterns purified autologous monocytes (Figure V in the online-only were examined in purified monocytes. Isolated monocytes Data Supplement). Changes in monocyte gene expression were mostly CD14+C16− (>90%), and this population did in response to clot formation were then assessed. RNA-seq change significantly after whole blood clot formation (Figure analyses revealed that mRNAs were differentially expressed by guest on July 10, 2018 Figure 1. Whole blood clot formation markedly alters gene expression patterns in monocytes. A, RNA sequencing was performed in baseline or clot-retrieved monocytes, and relative expression of individual genes were plotted against each other. Red and green indicate increased and decreased gene expression, respectively, between clotted compared with baseline (4-fold difference or greater). IL-8 (inter- leukin 8) and MCP-1 (monocyte chemoattractant protein-1) mRNA expression compared with other genes is highlighted by arrows. B, Dis- tribution of RNA-seq reads across MCP-1 and IL-8 transcripts (with intron/exon structures depicted below each plot) in monocytes from unclotted (baseline) blood and clotted whole blood. C, Protein for IL-8 and MCP-1 protein was measured in the plasma of clotted whole blood at specific times postclot formation. The bars in this graph indicate mean±SEM of 3 independent experiments (n=3). The asterisk indicates P<0.05 compared with freshly isolated monocytes that were processed immediately. BL indicates baseline; and NT, not treated. Campbell et al Clots Trigger Inflammatory Cell Gene Expression 1821 in monocytes embedded within clots compared with freshly Monocytes embedded in plasma clots displayed increased isolated monocytes (baseline), unstimulated monocytes expression for IL-8 and MCP-1 mRNA compared with (untreated) that were left in suspension culture for equivalent all control groups as measured by RNA-seq (Figure 2D). time