Thrombin and Phenotypic Modulation of the Endothelium

From the Research Center for Advanced Science and Technology (T.M., A.S., T.K.), University of Tokyo, Tokyo, Japan, and the Division of Molecular and Vascular Medicine (S.W., R.A., W.C.A.), Beth Israel–Deaconess Medical Center/Harvard Medical School, Boston, Mass.

Correspondence to William C. Aird, Beth Israel Deaconess Medical Center, RW-663, Boston, MA 02215. E-mail waird@bidmc.harvard.edu

    Abstract

Thrombin signaling in the endothelium is linked to multiple phenotypic changes, including alterations in permeability, vasomotor tone, and leukocyte trafficking. The thrombin signal is transduced, at least in part, at the level of gene transcription. In this review, we focus on the role of thrombin signaling and transcriptional networks in mediating downstream gene expression and endothelial phenotype. In addition, we report the results of DNA microarrays in control and thrombin-treated endothelial cells. We conclude that (1) thrombin induces the upregulation and downregulation of multiple genes in the endothelium, (2) thrombin-mediated gene expression involves a multitude of transcription factors, and (3) future breakthroughs in the field will depend on a better understanding of the spatial and temporal dynamics of these transcriptional networks.

Key Words: thrombin ? transcription ? DNA microarrays ? endothelial cells

    Introduction

Vascular diseases are among the most common causes of morbidity and mortality in the Western world. A remarkable feature of these disorders is the focal nature of their distribution. Indeed, there does not exist a single disease state that affects virtually every blood vessel type in the body. An important goal in vascular biology is to delineate mechanisms of vascular bed–specific pathology. One clue to these patterns is found within the endothelium. The endothelium serves as a "barometer" of the microenvironment, constantly sensing changes within the extracellular compartment and responding in ways that are usually beneficial, but sometimes harmful, to the host. As an extension of this model, the endothelium has been likened to a circuit board, 1 that is hard-wired to meet the demands of the underlying tissue and 1 that is highly vulnerable to "short-circuiting" as a mechanism of focal, vasculopathic disease states.1 The overall goal of this review is to explore thrombin signaling as a paradigm of endothelial cell circuitry. To that end, we will summarize the published literature, provide new insights into thrombin-associated transcriptional networks, and propose ways in which to integrate this information into the spatial and temporal context of the endothelium.

    Thrombin and Endothelial Cell Phenotypes

Thrombin is a multifunctional serine protease that is involved not only in mediating the cleavage of fibrinogen to fibrin in the coagulation cascade but also in activating a variety of cell types, including platelets and endothelial cells. Thrombin signaling in the endothelium might result in a multitude of phenotypic changes, including alterations in cell shape, permeability, vasomotor tone, leukocyte trafficking, migration, DNA synthesis, angiogenesis, and hemostasis (Table 1).

   TABLE 1. Thrombin and Endothelial Cell Phenotypes

Thrombin Signaling in Endothelial Cells

Thrombin signaling in the endothelium is mediated by a family of 7-transmembrane G protein–coupled receptors, termed protease-activated receptors (PARs).2 Currently, 4 members of the PAR family have been identified (PAR-1 through PAR-4; Table 2). PAR-1 and PAR-3 are thrombin receptors.3,4 Thrombin activation of PAR-4 requires PAR-3 as a thrombin-binding cofactor.5 Human umbilical vein endothelial cells (HUVECs) have been reported to express PAR-1, PAR-2, and, to a lesser extent, PAR-3, but not PAR-4.6,7 One study provided evidence for the existence of functional PAR-4 receptors (as well as those for PAR-1 and PAR-2 but not PAR-3) in the endothelium of human coronary artery ring segments.8 Of the various PAR family members, PAR-1 is the predominant thrombin receptor in endothelial cells.6 Thrombin activates PAR-1 by binding to a unique site in the extracellular domain of the receptor, resulting in cleavage between Arg41 and Ser42 and consequent exposure of a new N-terminus. The unmasked tethered ligand (SFLLRN) interacts with the extracellular loop 2 of the receptor (amino acids 248 to 268), resulting in receptor activation.9

   TABLE 2. Thrombin Receptors in Endothelial Cells (ECs)

Once activated, PAR-1 is coupled to a family of heterotrimeric G proteins, consisting of an -subunit and a ?-dimer. The G proteins are in turn linked to a number of signal intermediates that include, but are not limited to, mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidyl inositol 3-kinase (PI3K), and Akt (Table 3). Thrombin signaling might result in posttranscriptional changes, including calcium influx, cytoskeletal reorganization, and release of soluble mediators, growth factors, and matrix metalloproteinases (Table 4). In addition, thrombin signaling results in changes in downstream gene transcription (Table 5). For example, under in vitro conditions, thrombin has been shown to increase the expression of genes that are involved in cell proliferation, inflammation, leukocyte adhesion, vasomotor tone, and hemostasis (see Table 5 for references).

   TABLE 3. Thrombin Signaling in Endothelial Cells

   TABLE 4. Thrombin and Posttranscriptional Changes in Endothelial Cells

   TABLE 5. Thrombin and Transcriptional Changes in Endothelial Cells (Previously Published)

Uncovering Thrombin-Responsive Gene Programs

Most published studies have reported the effect of thrombin on specific target genes or clusters of genes. To more systematically catalog thrombin-responsive gene programs, we carried out DNA microarray experiments in control and thrombin-treated endothelial cells (please see online Figure I at http://atvb.ahajournals.org). In these experiments, HUVECs at passage 3 were serum-starved for 12 hours in medium containing 0.5% fetal bovine serum and then treated with 1.5 U/mL thrombin for varying periods of time. All microarray studies were carried out in duplicate with HUVECs from different donors. Only those genes that changed 2-fold in both experiments were considered significant. Several important themes emerged from these studies. First, although thrombin increased the expression of certain genes (74 of 8794 genes), thrombin signaling resulted in the downregulation of other transcripts (20 of 8794). Second, our studies revealed a time-dependent effect of thrombin on gene transcription. For example, of the genes that were induced by thrombin, 34 peaked at 1 hour, 21 at 4 hours, and 19 at 18 hours. It is noteworthy that 12 of the 34 thrombin-responsive genes that peaked at 1 hour were transcription factors (online Figure I; names denoted in red), compared with only 4 of 21 and 0 of 19 genes peaking at 4 and 18 hours, respectively. These results suggest that thrombin induces an early, temporally regulated transcriptional cascade. Third, the microarray results confirmed most, but not all, previous reports of thrombin-mediated gene expression. For example, the data support the results of a previous study that showed thrombin-mediated downregulation of endothelial nitric oxide synthase (eNOS) mRNA.10 Moreover, the microarray experiments demonstrated increased levels of angiopoeitin-2,11 platelet-derived growth factor (PDGF)-A,12 interleukin (IL)-8,13 intracellular adhesion molecule-1 (ICAM-1),14 vascular cell adhesion molecule-1 (VCAM-1),15 E-selectin,16 decay accelerating factor (DAF),17 early growth-response factor (Egr)-1,18 plasminogen activator inhibitor-1 (PAI-1),19 IL-6,13 monocyte chemoattractant protein-1 (MCP-1),13 and cyclooxygenase-2 (COX-2).20 Consistent with the published literature, the induction of Egr-1 was early18 and that of DAF, late.17 However, in contrast to previous reports,10,21–26 thrombin failed to induce Flk-1/KDR, Flt-1, endothelin-1, tissue factor (TF), endothelial protein C receptor (EPCR), and endothelin-converting enzyme-1, despite the use of duplicate microarray samples across multiple time points. Not all studies have shown a link between thrombin signaling and Flt-1 expression.21 Moreover, thrombin-mediated induction of Flk-1/KDR has been shown to require the presence of vascular endothelial growth factor (VEGF).21 Thrombin was reported to increase EPCR mRNA levels and/or EPCR promoter activity in rat and bovine aortic endothelial cells.25,26 The absence of EPCR induction in our studies might reflect differences in species and or vascular bed of origin. The discrepancy in TF response is more difficult to explain. Using ribonuclease protection assays, we have found that thrombin induces a high level of TF mRNA in multiple subtypes of human endothelial cells (including those derived from umbilical vein, pulmonary artery, and coronary artery), with maximal levels (>6-fold) occurring at 2 hours (Figure 1). Along with the published data, these latter findings suggest that the TF results in the microarray experiments represent a false-negative.

   Figure 1. Ribonuclease protection assay of thrombin-treated endothelial cells. Endothelial cells were grown on 6-well plates and starved overnight in EBM-2 medium containing 0.5% fetal bovine serum, as described in the online supplement (please see http://atvb.ahajournals.org). The cells were treated in the absence or presence of 1.5 U/mL thrombin for the times indicated and were subsequently harvested for total RNA. RNase protection assays were carried out with [-32P]UTP-labeled TF, Egr-1, ICAM-1, urokinase-type plasminogen activator and ?-actin probes, as described in the online supplement. Results are representative of 3 independent experiments and were virtually identical in human pulmonary artery endothelial cells and HUVECs.

Finally, thrombin stimulated the expression of certain genes that have not previously been reported to be thrombin-responsive. Although the results will require validation by real-time polymerase chain reaction and/or RNase protection assays, the data reveal a number of interesting candidate genes, including CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a negative regulator of hypoxia-inducible factor-1,27 which, to our knowledge, has not been previously identified in endothelial cells; a disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1), a metalloprotease that has been shown to inhibit angiogenesis28,29; tumor necrosis factor (TNF) receptor superfamily member 12A, which has been reported to induce angiogenesis and migration30; and the CX3C chemokine fractalkine, a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes.31

Two other groups have used DNA microarrays to analyze thrombin or thrombin-receptor signaling in endothelial cells. In 1 study, treatment of serum-starved, confluent HUVECs with a PAR-1 agonist peptide (TFLLRNPNDK) for 90 minutes resulted in a reproducible upregulation of 1% of the 7000 genes represented on the HG-U95Av2 array (Affymetrix).32 There was no mention in this study of PAR-1–associated gene repression. In the other report, custom microarrays of 300 vascular cell–related gene fragments were used to probe the effect of various extracellular mediators, including 5 U/mL thrombin, on confluent HUVECs for 2, 6, and 24 hours.33 However, the authors did not comment on the nature and/or level of the thrombin-responsive transcripts.33

Thrombin-Mediated Transcriptional Networks

In theory, the effect of thrombin on mRNA expression in endothelial cells might be mediated at the level of mRNA stability or rate of transcription. Little is known about the effect of extracellular signals on mRNA stability in the endothelium. In contrast, there has been an increased understanding of the role for transcription factors in transducing the thrombin signal at the level of gene promoters (Figure 2 and Table 6).

   Figure 2. Schemata of thrombin-responsive transcriptional networks in endothelial cells. Shown are signaling pathways that couple thrombin with SRF–Egr-1, p65–NF-B with ICAM-1, p65–NF-B, GATA-2 with VCAM-1, and dbpB with PDGF-B and EPCR Abbreviations are as defined in text.

   TABLE 6. Thrombin and Transcriptional Networks in Endothelial Cells

DNA Binding Protein B (dbpB)

The first evidence for the existence of a thrombin-responsive transcriptional network in endothelial cells was derived from studies of the PDGF-B promoter. Thrombin-mediated expression of PDGF-B was shown to involve the inducible binding of a Y-box binding transcription factor (termed dbpB) to a 9-bp thrombin-response element (TRE) in the upstream promoter region.34,35 In response to thrombin, dbpB is cleaved and released from mRNA in the cytoplasm, resulting in nuclear translocation and transcriptional activation of downstream target gene(s).35 In addition to PDGF-B, dbpB has been implicated in the thrombin-mediated regulation of TF35 and EPCR genes.25 Recently, Esmon and colleagues used homologous recombination to delete the dbpB consensus motif from the endogenous EPCR gene. They showed that wild-type, but not mutant, EPCR was responsive to thrombin under in vivo conditions (C.T. Esmon, personal communication). These latter data support a role for the dbpB transcription factor in mediating thrombin response in the intact endothelium.

Promoters of other thrombin-responsive genes (eg, VCAM-1, thrombomodulin [TM], and Flk-1/KDR) contain consensus elements for dbpB. However, evidence for their functional role in thrombin signaling is lacking. For example, although the VCAM-1 promoter contains a putative TRE at -376 relative to the start site of transcription, a mutation of this site did not affect the ability of thrombin to induce promoter activity.15 Moreover, in mobility shift assays, thrombin did not induce the binding of nuclear protein to a radiolabeled probe that spans the motif.15 In DNA microarrays, thrombin failed to increase mRNA levels of Flk-1/KDR or TM, a finding that has been confirmed in Northern blot assays (C. Seguin and W.C. Aird, unpublished observations). These observations do not rule out a role for the TRE in mediating the thrombin response of VCAM-1, Flk-1/KDR, and/or TM under certain as-yet-undefined spatial and temporal conditions. However, on the basis of these findings, as well as those discussed later, we believe that the dbpb consensus motif should be considered as 1 member of an expansive family of TREs.

Nuclear Factor-B

There is increasing evidence for the importance of nuclear factor (NF)-B in mediating thrombin response in the endothelium. NF-B is a proinflammatory, rapidly inducible transcription factor that upregulates several target genes involved in endothelial cell activation. The NF-B/Rel family consists of 5 distinct DNA-binding proteins, including p50, p52, p65 (RelA), c-Rel, and RelB. In quiescent cells, NF-B is sequestered in the cytoplasm, complexed to the inhibitory protein IB. Stimulation of cells with inflammatory mediators results in the release of NF-B from IB. Once released, NF-B translocates into the nucleus, where it binds as heterodimers or homodimers to NF-B elements in the promoter regions of target genes.

Thrombin treatment of endothelial cells results in rapid translocation of NF-B to the nucleus (please see online Figure II at http://atvb.ahajournals.org). Thrombin-mediated induction of ICAM-1 has been shown to be mediated by the binding of p65 homodimers to a single NF-B site in the upstream promoter region.36 Similarly, thrombin stimulation of VCAM-1 involves the inducible binding of p65 NF-B to a tandem NF-B motif in the 5' flanking region. When the VCAM-1 NF-B motifs are linked in tandem to a thymidine kinase (TK) heterologous core promoter, they retain thrombin responsiveness.37 Taken together, these findings suggest that in the case of VCAM-1 and ICAM-1, p65 NF-B is both sufficient and necessary for transducing the thrombin response. These observations contrast to the more commonly reported involvement of p50-p65 heterodimers in mediating NF-B–inducible gene expression.38 In fact, the p50 subunit has been proposed to function as a negative transcriptional regulator of the VCAM-1 gene by virtue of its ability to inhibit the transcriptional potential of p65.39 RelB has also been shown to inhibit p65 DNA binding.40 Interestingly, the microarray studies of thrombin-treated endothelial cells demonstrated significant induction of p50 NF-B and RelB mRNA at 4 hours (online Figure IA), suggesting a possible negative-feedback mechanism in the thrombin–VCAM-1 signaling axis.

It is interesting to speculate that thrombin signaling is unique in its ability to induce binding of p65 homodimers to downstream target genes. An alternative explanation is that p65 homodimers are involved in transducing many signals at the level of the VCAM-1 and ICAM-1 promoters but not those of other endothelial cell genes. In support of this latter hypothesis, the addition of thrombin to porcine aortic endothelial cells was reported to induce the binding of both p65 and p50 to the NF-B binding site of the IB- promoter.19

A previous study showed that NF-B binding to the VCAM-1 promoter, but not to the E-selectin promoter, was dependent on the redox state of the endothelial cell.41 Although the latter study did not address the effect of reactive oxygen species on thrombin-mediated binding of NF-B, the results are important in that they demonstrate gene/promoter-specific differences in NF-B signaling. Given the potential influence of the NF-B consensus element or a surrounding promoter sequence in directing the binding of specific homodimers, heterodimers, and/or coactivators, it seems prudent to carry out studies of NF-B–mediated gene regulation with NF-B elements that are derived from the promoter of interest.

In studies of the VCAM-1 gene, thrombin has been shown to induce p65 homodimer binding through a PI3K-, PKC-–dependent, Akt-independent signaling pathway (Figure 2).15,37 PKC- was also implicated in thrombin-mediated induction of p65 NF-B binding to the ICAM-1 promoter. However, in contrast to VCAM-1, thrombin stimulation of ICAM-1 was shown to involve parallel PI3K and PKC- pathways, both converging at the level of Akt.42 It is not clear whether these differences reflect gene-specific signaling pathways or rather differences in experimental technique/design.

GATA-2

GATA-binding proteins were initially characterized as constitutively active transcription factors involved in mediating cell type–specific gene expression and lineage determination.43,44 However, recent studies have uncovered a potentially important role for GATA proteins in temporal gene regulation. GATA DNA-binding activity and/or GATA mRNA expression have been shown to increase in response to a number of mediators, including insulinlike growth factor 1,45 follicle-stimulating hormone,46 endothelin-1,47 IL-3,48 and IL-4.49 GATA activity has been reported to decrease in response to other mediators, such as estrogen50 and transforming growth factor (TGF)-?.51

Thrombin-mediated induction of VCAM-1 was shown to involve the inducible binding of GATA-2 to a tandem GATA motif in the upstream promoter.15 The GATA elements were both sufficient and necessary for thrombin-mediated induction of the VCAM-1 gene. Interestingly, the effect of thrombin on GATA-2 DNA binding and transcriptional activity was found to be mediated by a PI3K-, PKC-–dependent signaling pathway37 (Figure 2). The mechanism by which thrombin induces the binding of GATA-2 to the VCAM-1 promoter remains to be established. In preliminary studies, the addition of thrombin to endothelial cells did not induce GATA-2 mRNA and protein level, nor did it alter the total level of serine phosphorylation (T. Minami and W.C. Aird, unpublished results). Of note, thrombin treatment of HUVECs reduced the expression of GATA-3 mRNA (online Figure I). Whether the relative levels of GATA-2 and GATA-3 play a role in mediating the thrombin response of VCAM-1 and the extent to which GATA transcription factors are involved in mediating the thrombin response of other genes remains to be determined.

Early Growth-Response Factor-1

Egr-1 (also known as zif268, TIS 8, NFGI-A, and Krox 24) is a member of the immediate-early gene family that includes c-fos, c-jun, and early growth-response genes.52–57 Egr-1, which encodes a serum-inducible zinc finger nuclear phosphoprotein, is rapidly induced in cultured cells by a wide variety of mitogenic and nonmitogenic stimuli. In endothelial cells, Egr-1 has been shown to be activated by acidic fibroblast growth factor,58 basic fibroblast growth factor,59,60 VEGF,61 epidermal growth factor (EGF),62,63 shear stress,64–66 cyclical strain,67 and hypoxia.68,69 Under in vivo conditions, the administration of VEGF and EGF results in vascular bed–specific changes in Egr-1 expression.63 Moreover, elevated Egr-1 levels have been reported in human atherosclerotic lesions.70

The murine and human Egr-1 promoters contain 5 functional serum-response elements (SREs) organized into 2 clusters: a 5' cluster of 3 SREs and a 3' cluster of 2 SREs. Collectively, the 5 SREs are responsible for transducing most, if not all, signals that activate Egr-1 expression. The 5' SRE cluster has been shown to mediate response to the majority of extracellular signals, including EGF,62 shear stress,64 growth hormone,71 urea,72 hypotonicity, lipopolysaccharide (LPS),73 granulocyte-colony stimulating factor,74 and hypoxia.69 In nonendothelial cells, the 3' cluster has been implicated in the response to glucose-induced depolarization75 and granulocyte-macrophage stimulating factor.76,77

Recently, thrombin has been shown to induce the expression Egr-1 via the most proximal SRE (SRE-1).18 Although previous studies have shown that the response of the 5' cluster of SREs to extracellular signals is mediated by the coordinate action of serum-response factor (SRF) and ternary complex factor,64,69,71,73 SRE-1 was shown to transduce the thrombin signal by an SRF-dependent, TCF-independent mechanism.18

There is a growing appreciation that thrombin signaling contributes to a proinflammatory state. Egr-1 has been reported to induce a number of downstream genes, including PDGF-A, PDGF-B, TF, Flt-1, TGF-?, TNF-, urokinase-type plasminogen activator, and metalloproteinases.61,78–80 Endothelial expression of Egr-1 is increased in response to injury.81 The effect of Egr-1 on an individual target gene is likely to vary according to the cell type and extracellular signal.82 An important goal for future studies will be to characterize the nature of the Egr-1–responsive gene program in thrombin-treated endothelial cells and to determine the extent to which these gene products contribute to the cellular phenotype.

Other Transcriptional Networks

Thrombin has been shown to induce the activity of activator protein-1 in endothelial cells in some, but not all, studies.23,83–85 Moreover, thrombin signaling in endothelial cells results in phosphorylation of the forkhead transcription factors FKHR and AFX (please see online Figure III at http://atvb.ahajournals.org). The results of the microarray experiments reveal upregulation and downregulation of many transcription factors (online Figure I). These results await validation at the level of both mRNA and DNA binding activity. Thrombin signaling in vascular smooth muscle cells (VSMCs) has been linked to NF-B, Sp1, cAMP-responsive element binding protein,86,87 activator protein-1,88 hypoxia-inducible factor-,89,90 signal transducers and activators of transcription (STATs),91 and nuclear factor of activated T cells92; in fibroblasts to Elk-1 and cyclicAMP regulatory element-binding protein (CBP)/p30093,94; and in mesangial cells to STAT-1 and -3.95 A role for these latter transcription factors in transducing the thrombin signal in endothelial cells has yet to be established.

Transcriptional Network Clustering

Common bioinformatic approaches for analyzing the results of microarray experiments include unsupervised learning techniques (eg, hierarchical clustering) and supervised learning techniques, such as assigning genes to predefined biologically meaningful classes (functional clustering). An alternative approach is to link the microarray data with an existing genomic database to gain insight into thrombin-responsive transcriptional networks. This strategy entails 2 steps: (1) identifying promoter sequences of genes that are induced by thrombin and (2) scanning a predetermined length of the promoter for established or conserved DNA motifs. In preliminary studies, we have used such an approach to analyze our microarray data (Figure 3). A preponderance of promoters of genes that were maximally upregulated at 1 hour contained SREs, which is what one would expect for an immediate-early gene. In contrast, there was a greater representation of NF-B, Egr-1, and GATA-binding elements in the promoters of genes that were induced at 4 hours. Finally, consensus dbpB binding elements were found on the promoters of growth related oncogen ? (GRO-?) and Egr-3 (maximally induced at 1 hour); VCAM-1, ICAM-1, fractalkine, and NF-B1 (maximally induced at 4 hours); and DAF, chemokine (CXC motif) receptor 4, myosin IE, PDGF-A, PDGF-B, and TNF (ligand) superfamily member 15 (maximally induced at 18 hours).

   Figure 3. Scanning the promoters of highly induced genes for established or conserved transcription factor–binding motifs. Promoter sequences (-500 to +100) from 8 of the highest thrombin-induced genes at 1 and 4 hours were retrieved from GenBank and analyzed with Chip2 promoter version 1.0 of GenomatixSuite software (CTC Laboratory systems). Please see http://atvb.ahajournals.org for additional details. GLI indicates glioma; MGSA indicates melanoma growth stimulating activity; and TRAF indicates TNF- receptor-associated factor. Other abbreviations are as defined in text.

Cell Type–Specific Nature of Thrombin Signaling

Thrombin receptors are present on many different cell types, including endothelial cells, platelets, VSMCs,96–100 fibroblasts,93,101,102 mast cells,103,104 macrophages,104 microglia,105 tumor cells, keratinocytes,106 and leukocytes.107,108

Thrombin signaling varies between cell types. For example, whereas thrombin induces expression of VCAM-1 in endothelial cells, it has no such effect in VSMCs.37 In contrast, IL-4 increased VCAM-1 mRNA levels in VSMCs, suggesting that the VCAM-1 gene is inducible under other conditions. Moreover, thrombin (as well as the PAR-1 agonist peptide) induced the expression of TF in VSMCs, confirming that VSMCs possess functional thrombin receptors.37

The discordance in thrombin response between endothelial cells and other cell types is not unique to VCAM-1. For example, although previous studies have shown that thrombin and/or PAR-1 singling induces VEGF expression in fibroblasts, endometrial cells, tumor cells, mesothelial cells, and VSMCs,100,101,109–111 there are no reports of thrombin stimulation of VEGF in endothelial cells, an observation that is supported by our microarray data. In contrast, other extracellular signals, such as hypoxia, have been shown to increase VEGF mRNA levels in endothelial cells (largely through stabilizing mRNA).112,113 We have recently demonstrated that thrombin induces expression of PDGF and ICAM-1 in cultured endothelial cells, but not in VSMCs (S.Q. Wu and W.C. Aird, unpublished observations). Other studies have suggested that PAR-1 levels (and therefore, sensitization to thrombin) are differentially regulated in endothelial cells and VSMCs.114,115 Differences also exist in PAR-1 signaling between endothelial cells and platelets. For example, in platelets, thrombin stimulates cdc42 and Rac1 activity, whereas in endothelial cells, thrombin has no effect on cdc42 activity and inhibits Rac1.116

Taken together, these results suggest that thrombin signaling varies between different cell types and that these differences are mediated by differential expression and/or activity of PARs or downstream signal intermediates. It follows that thrombin signaling should be studied in the context of the cell type of interest and that data obtained from 1 cell lineage should not be extrapolated to another.

Not All Inflammatory Mediators Are Created Equal

Endothelial cell activation is not an all-or-nothing phenomenon. Indeed, the notion of the endothelium existing in a binary state (quiescent or activated) has given way to an appreciation of the endothelium as a nonlinear system that displays phenotypic heterogeneity in space and time.117 Any notion that inflammatory mediators have identical effects on endothelial cells is an oversimplification.

Although there are similarities in the way inflammatory mediators, such as thrombin, TNF-, IL-1, and LPS, modulate endothelial cell phenotype, there are also important qualitative and quantitative differences. For example, in 1 study with DNA microarrays, treatment of HUVECs with TNF- and IL-1? resulted in overlapping but nonidentical patterns of gene expression.33 Thrombin, but not TNF- or LPS, induced PDGF-A in human endothelial cells derived from umbilical vein, pulmonary artery, or coronary artery (S.Q. Wu and W.C. Aird, unpublished observations). Compared with TNF-, thrombin signaling in porcine aortic endothelial cells results in lower and delayed induction of NF-B binding activity.19 In HUVECs, TNF-–and IL-?–mediated induction of ICAM-1 peaks at 2 hour, whereas thrombin stimulation is maximal at 4 hours.36 Moreover, in the same cell type, thrombin induces ICAM-1 via a PKC-–p65–NF-B signaling pathway,36 whereas TNF-–mediated induction of the ICAM-1 gene involves a PKC-–p65–NF-B signaling pathway.118 Inhibitors of mitogen-activated protein kinase p38 completely abrogate thrombin-, but not TNF-–, induced leukocyte recruitment in HUVECs.119 As a final example of mediator-specific responses, treatment of HUVECs with TNF-, but not LPS, resulted in a biphasic change in PAR-1 mRNA levels, with an initial decrease and a subsequent rebound above baseline.120 Together, these and other studies emphasize that although inflammatory mediators induce overlapping changes in endothelial phenotypes, each mediator engages the endothelium in its own unique way.

Temporal and Spatial Control of Thrombin Signaling

There is no question that in vitro studies of thrombin signaling have advanced our understanding of this important mediator. However, cell culture studies have their limitations. When endothelial cells are removed from their native tissue environment, they are uncoupled from critical extracellular cues and undergo phenotypic drift. As a result, signaling pathways and the transcriptional control machinery might change in ways that are difficult to control or account for. Under in vivo conditions, endothelial cells are differentially regulated in space and time.121 Stated another way, the intact endothelium represents a nonlinear dynamic, in that the whole is far greater than the sum of the parts.117 Studies of the individual parts (eg, cultured endothelial cells), however important and informative they might be, do not provide insight into higher-order behavior.

Consider, for example, the thrombin–VCAM-1 signaling pathway.15,37 The results of in vitro studies are interesting in that they (1) establish a role for GATA-2 as a signal transducer in endothelial cells, (2) imply a novel link between an atypical PKC isoform and GATA-2, and (3) provide a potentially valuable model for dissecting the mechanisms of cell type–specific thrombin signaling (endothelial cells vs VSMCs). However, there are many gaps in our knowledge. Our results are derived from cells (namely, HUVECs) that originate from a vascular bed, which, although readily accessible and historically favored, is poorly understood in health and disease. In the context of the blood vessel wall, the intact endothelium is not surrounded by tissue-culture medium and plastic but rather by free-flowing blood on 1 side and extracellular matrix and parenchymal cells on the other. How does the thrombin–VCAM-1 signaling pathway normally behave under in vivo conditions? Or, for that matter, under states of activation, such as sepsis, in which blood might be acidic, hypoxic, and delivered under low perfusion pressures; in which there might be local accumulation of inflammatory mediators and thrombin and secondary changes in leukocyte trafficking, fibrin deposition, and barrier function, all of which might impact in their own way on endothelial cell phenotype?122

In addition to these temporal considerations and in keeping with the theme of endothelial cell heterogeneity, there is every reason to believe that the expression and/or activity of the various components of the thrombin–VCAM-1 signaling axis vary between different sites of the vascular tree. For example, at any given point in time, regional differences might exist in the amount of thrombin being generated in the intravascular space, the number or ratio of PARs on the cell surface,123–125 the expression and/or activity of PKC- and - isoforms, and/or the level or transcriptional potential of p65 NF-B and GATA-2.126

In summary, current in vitro models of endothelial cell biology fall short in capturing the complex, dynamic, and emergent nature of the intact endothelium. At best, the results of the in vitro studies provide a first approximation of the true biology and might be used as a road map for exploring the system in vivo. An important goal for the future will be to learn how to better leverage the advantages that are inherent in the reductionist (in vitro) and holistic (in vivo) approaches for both mechanistic and therapeutic gain.

Thrombin Signaling—A 2-Edged Sword

Although our understanding of the molecular basis of thrombin–PAR-1 interactions and downstream signaling is rapidly evolving, we have relatively little insight about the role of PAR signaling in health and disease. As a general rule, thrombin is considered to activate endothelial cells, leading to cellular "dysfunction." Thrombin is present in increased concentrations at sites of vascular injury,127 in the vicinity of a thrombus,127 in patients with acute coronary syndromes,128 and in primate models of sepsis.129 Thrombin and/or PAR signaling has been implicated in rheumatoid (or collagen-induced) arthritis130,131 and preeclampsia.108 Despite these associations, it has been difficult to prove a cause-and-effect relation between thrombin generation and pathology or to quantify the relative contributions of thrombin-induced fibrin formation versus thrombin-mediated activation of platelets, endothelial cells, or other cell types.

There is increasing evidence that thrombin signaling also plays a protective role in endothelial cell biology. PAR-1–null mice might be rescued with endothelial cell–specific expression of PAR-1, suggesting that activation of PAR-1 and its signaling pathway in endothelial cells are essential for vascular integrity.132 Moreover, thrombin signaling might play an important role in angiogenesis.22,133,134 Thrombin also promotes the production and secretion of extracellular matrix proteins135 and positively influences remodeling processes.136,137 Other studies have shown that thrombin protects against cell death.138,139 In a recent report, PAR-1 stimulation in HUVECs resulted in increased expression of the antiapoptotic genes BCL2-related protein A1 and inhibitor of apoptosis 1, and a variety of negative regulators of proinflammatory pathways.32 Taken together, these studies demonstrate the multifaceted role of thrombin signaling at the level of the endothelium.

If one is to believe that thrombin (and/or PAR) signaling is a 2-edged sword (as we do), then it should theoretically be possible to tease out signaling pathways or transcriptional networks whose inhibition will lead to a preferential loss of proinflammatory response while retaining the protective function. To test this hypothesis, we are currently using microarray experiments of control or thrombin-treated HUVECs that have been pretreated in the absence or presence of various inhibitors of signal intermediates or transcription factors. By mining the resulting data, we will be able to assign downstream thrombin target genes to distinct signaling pathways. Our goal is to identify signal transduction components that are amenable to selective therapeutic targeting.

    Conclusions

Thrombin not only represents an interface between coagulation and inflammation but is also a powerful tool for exploring "gray" areas in vascular biology, namely, (1) the spatial and temporal dynamics of endothelial cell phenotypes, (2) the nonbinary nature of endothelial cell activation, (3) the double-edged sword of endothelial cell signaling, and (4) the emergent properties of the endothelium. Continued research in thrombin signaling promises to advance our understanding in each of these areas.

    Acknowledgments

 

This work was supported by National Institutes of Health grants HL60585, HL63609, HL65216, and HL36028.

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