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Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2839-2843, April 1989 Medical Sciences Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque (/) JOSIAH N. WILCOX*, KATHLEEN M. SMITH*, STEPHEN M. SCHWARTZt, AND DAVID GORDONt *Department of Cardiovascular Research, Genentech, 460 Point San Bruno Boulevard, South San Francisco, CA 94080; and tDepartment of Pathology, University of Washington, Seattle, WA Communicated by Earl P. Benditt, December 28, 1988

ABSTRACT Tissue factor (TF)-producing cells were iden- localizations using in situ hybridization and immunohis- tified in normal human vessels and atherosclerotic plaques by tochemistry. We report here the localization ofTF-producing in situ hybridization and immunohistochemistry using a spe- cells in the normal vessel wall and atherosclerotic plaques. cific riboprobe for TF mRNA and a polyclonal antibody directed against human TF . TF mRNA and protein were absent from endothelial cells lining normal internal METHODS mammary artery and saphenous vein samples. In normal Tissue Preparation. Normal human saphenous veins and vessels TF was found to be synthesized in scattered cells present internal mammary arteries were obtained during coronary in the tunica media as well as fibroblast-like adventitial cells bypass surgery. Human atherosclerotic plaques were ob- surrounding vessels. Atherosclerotic plaques contained many tained from patients undergoing carotid endarterectomy cells synthesizing TF mRNA and protein. Macrophages present surgery. as foam cells and adjacent to the cholesterol clefts The tissue samples were removed and immersed in freshly contained TF mRNA and protein, as did mesenchymal- prepared 4% (wt/vol) paraformaldehyde in 0.1 M sodium appearing intimal cells. Significant TF protein staining was phosphate (pH 7.4). The tissues were fixed at 40C for 3 hr to found deposited in the extracellular matrix surrounding overnight and then immersed in 15% (wt/vol) sucrose/iso- mRNA-positive cells adjacent to the cholesterol clefts and tonic phosphate-buffered saline for 2-4 hr at 40C to act as a within the necrotic cores. These results suggest that deposition cryoprotectant. The tissues were then embedded in optimal of TF protein in the matrix of the necrotic core of the cutting temperature compound (O.C.T., Miles Scientific) atherosclerotic plaque may contribute to the hyperthrombotic blocks and stored at -70°C. Finally, the tissues were sec- state of human atherosclerotic vessels. tioned at 10 Am thickness by using a cryostat, thaw-mounted onto polylysine-coated microscope slides, immediately re- Tissue factor (TF) is a membrane-bound that frozen, and stored at -70°C with desiccant. functions in the extrinsic pathway of blood by In Situ Hybridization. In situ hybridizations were carried acting as a cofactor for factor VII (1, 2). TF binds to co- out as described (16, 17). Prior to hybridization the sections agulation factor VII, and the resulting factor VIIa-TF complex were pretreated sequentially with paraformaldehyde (10 min) acts as a catalyst for the conversion offactors X to Xa and IX and with proteinase K at 1 ,g/ml (10 min) and prehybridized to IXa, leading to the formation of (3, 4). Thus, TF for 1-2 hr in 100 ,ul of prehybridization buffer [50% (vol/vol) facilitates both intrinsic and extrinsic pathways ofcoagulation formamide/0.3 M NaCI/20 mM Tris-HCl, pH 8.0/5 mM and is a key protein in the activation of the coagulation EDTA/0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bo- cascade. vine serum albumin/10% (wt/vol) dextran sulfate/10 mM TF activity has been isolated from a number of different dithiothreitol]. The hybridizations were started by adding tissues and has been cloned from adipose (5), fibroblast (6), 600,000 cpm ofthe 35S-labeled riboprobe in a small amount of and placental (7, 8) cDNA libraries. A 2.3-kilobase band prehybridization buffer. After hybridization the sections corresponding to human TF mRNA has been identified by were washed with 2 x SSC (two 10-min washes; lx SSC = Northern blots using RNA from adipose tissue, small intes- 150 mM NaCl/15 mM sodium citrate, pH 7.0), treated with tine, placenta, kidney (5), and brain (8). Cultured endothelial RNase (20 ,g/ml for 30 min at room temperature), washed in cells produce low levels of TF mRNA (5). TF procoagulant 2x SSC (two 10-min washes), and washed at high stringency activity in endothelial cells is enhanced by the addition of in 0.1 x SSC at 520C for 2 hr. All SSC solutions up to this point endotoxin (9), thrombin (10), phorbol esters (9, 11), interleu- of the procedure contained 10 mM 2-mercaptoethanol and 1 kin 1, or (12, 13) to the culture medium. mM EDTA to help prevent nonspecific binding of the probe. TF mRNA has also been identified in the cell line The tissue was then washed in 0.5x SSC without 2-mer- U937 (8) and TF activity has been identified in normal captoethanol (two 10-min washes) and dehydrated by immer- monocytes after activation with endotoxin or phorbol esters sion in a graded alcohol series containing 0.3 M NH4Ac. The (14, 15). sections were dried, coated with NTB2 nuclear emulsion There is no information concerning the cellular distribution (Kodak), and exposed in the dark at 40C for 4-8 weeks. After of TF-producing cells within the tissues from which it has development, the sections were counterstained with hema- been isolated. It has been postulated that TF is not exposed toxylin and eosin. to blood elements but must be associated with the vascula- A cDNA probe specific to human TF subcloned in the SP64 ture where it could act quickly in response to vascular plasmid (Promega Biotec), provided by Karen Fisher and damage. With cDNA probes and antibodies specific for the Richard Lawn (Genentech), was labeled by transcription (18) TF protein, it is now possible to do more precise cellular using 35S-labeled UTP (specific activity, 1200 Ci/mmol; 1 Ci = 37 GBq; Amersham). This was a 1.3-kilobase probe and the The publication costs of this article were defrayed in part by page charge included entire coding sequence for human TF extending payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: TF, tissue factor. 2839 Downloaded by guest on September 25, 2021 2840 Medical Sciences: Wilcox et al. Proc. Nad. Acad. Sci. USA 86 (1989)

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FIG. 1. (Legend appears at the bottom of the opposite page). Downloaded by guest on September 25, 2021 ...... 1 .. Medical Sciences: Wilcox et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2841 from nucleotide 1 in the 5' flanking region to a HindIII site at anterior descending coronary artery, n = 1; right coronary nucleotide 1347 in the 3' untranslated region (5). The final artery, n = 2; left circumflex artery, n = 1). These arterial specific activity of this probe was 300 Ci/mmol. samples came from idiopathic dilated cardiomyopathy hearts Immunohistochemistry. TF antibody RD010 was prepared removed at the time of cardiac transplantation. These hearts by immunizing rabbits with recombinant human TF protein typically have no significant atherosclerotic disease, and this (19). The IgG fraction of the serum was purified by affinity was confirmed in our samples. All of these vessels showed chromatography on a recombinant human TF-Sepharose positive adventitial cells but at most a single medial cell column. This antibody was shown to react with a single band containing TF mRNA. at 42 kDa on a Western blot, neutralize TF activity, and In general, more cells were found to be positive in the immunoprecipitate TF protein (20). media of the saphenous vein by in situ hybridization than Immunohistochemistry was performed according to the could be detected by immunohistochemical staining. Visual manufacturer's direction using the Vectastain ABC alkaline comparison of the hybridization signal over the adventitial phosphatase system (Vector). The final reaction product was fibroblasts and that of positive cells in the media suggested stained with the alkaline phosphatase substrate I to give that these cell types do not differ greatly in their content of a final stain that appeared red. TF mRNA. However, a similar comparison of the immuno- The affinity-purified TF antibody RD010 was used at a histochemical staining intensity over these cells suggests that concentration of 4.4 ,ug/ml. An IgG fraction of the preim- the adventitial fibroblasts contain much more TF protein than mune serum was used as a control for the TF immunohis- do the medial cells. This could suggest that there is some tochemistry at the same IgG concentration as RD010. This alteration in TF translation or secretion in the medial cells was prepared by passing the preimmune serum over a protein resulting in the reduced protein content. Alternatively, anti- A-Sepharose column. Additional antibodies specific for hu- body RD010 may react differently with the TF in the medial man macrophages (ref. 21; HAM56, gift of Allen Gown, or adventitial cells due to some change in the TF protein University of Washington), smooth muscle cells (ref. 22; itself. HHF35, gift of Allen Gown), or human endothelial cells (ref. The morphology of TF-positive cells in the media of the 23; anti-Ulex lectin, Vector) were used on some serial saphenous vein differed from that of typical smooth muscle sections to aid in cell identification. cells. The cytoplasm of the TF-positive cells stained poorly with eosin and did not display a typical fusiform-shaped cytoplasm but rather appeared more cuboidal in shape, with RESULTS small dense nuclei. Cells with this morphology do not stain TF Localization in Normal Vessels. Normal human saphe- with smooth muscle a-actin antibodies (HHF35; data not nous vein and internal mamamary artery samples (nine shown) and must be considered undefined, since we lack a sections for each tissue, three sections from each of three positive immunochemical marker for these cells. individuals) were examined for the presence of TF. Endo- TF Localization in Atherosclerotic Plaques. Human athero- thelial cells were negative for TF mRNA and protein in all sclerotic plaques obtained from carotid endarterectomy sur- vascular tissues examined. Many scattered cells in the tunica gery were examined for TF mRNA and protein using the media of the saphenous vein contained TF mRNA as deter- above techniques. Extensive mRNA hybridization was seen mined by in situ cRNA hybridization (Fig. lA). Immunohis- in several regions of atherosclerotic plaque. Positive cells tochemical staining of these cells, however, was very weak were found scattered throughout the fibrous cap (Fig. 1C), but appeared to be cell-associated and correlated well with the base and shoulder region of the plaque, as well as in the the in situ results (Fig. 1B). The strongest labeling was seen necrotic core adjacent to the cholesterol clefts (Fig. 1F). Six over the adventitia where adventitial fibroblasts showed plaques were screened and cells showing TF mRNA hybrid- intense TF protein staining (Fig. 1B) and mRNA hybridiza- ization were seen in all ofthem. The normal media underlying tion. The internal mammary artery samples also showed the endarterectomy specimens did not contain any TF protein positive mRNA hybridization and intense protein labeling or mRNA-positive cells. over the adventitial fibroblasts. However, fewer medial cells The necrotic cores of the plaques were characterized by contained TF mRNA, as indicated by the in situ hybridiza- extensive TF protein localization in the extracellular matrix tion, and no protein could be detected in the media by particularly surrounding some cholesterol clefts (Fig. 1 E and immunohistochemistry. Additional normal arteries obtained G). Additional protein staining was seen in the macrophage- from hearts discarded from recipients of transplants were rich foam cell regions of many of the atherosclerotic plaques screened for TF mRNA by in situ hybridization (three examined (Fig. 1D). Such foam cell-rich regions often lay sections each from the following tissues: aorta, n = 2; left underneath the fibrous cap and adjacent to the necrotic cores.

FIG. 1. Localization of TF in the normal human saphenous vein (A and B) and in human carotid endarterectomy specimens (C-G). (A) In situ hybridization using a specific 35S-labeled TF cRNA probe indicated that there were scattered TF-producing cells in the tunica media and adventitia. (B) Cells containing TF protein were detected by immunocytochemistry using antibody RD010 and the Vectastain alkaline phosphatase method (positive cells stain red). Scattered cells in the tunica media were lightly stained by antibody RD010, whereas strong immunohistochemical staining was always seen in the adherent adventitial fibroblasts. Endothelial cells lining the lumen of the normal vessel were always negative for TF protein and mRNA. L, lumen; M, media; A, adventitia. (C) Localization ofTF in the human atherosclerotic plaque by in situ hybridization. Carotid endarterectomy specimens were hybridized to an 35S-labeled TF riboprobe and revealed many cells producing TF in the fibrous cap ofthe atherosclerotic plaque. (D) Localization oftissue factor protein in macrophage foam cells ofthe atherosclerotic plaque by immunohistochemistry with antibody RD010. (E and F) Colocalization of TF protein and mRNA in the same cells of the plaque. (E) Immunohistochemistry with TF antibody RD010 indicated strong staining ofthe necrotic core region ofthe plaque particularly in areas adjacent to the cholesterol clefts. (F) In situ hybridization of a section immediately adjacent to the one shown in E indicated that cells containing TF mRNA were found adjacent to the cholesterol clefts, suggesting local synthesis of the TF protein detected in this region. Not all of the protein detected here was cell associated. Arrows point to two cholesterol clefts followed on the serial sections. (G and H) Identification of cells producing TF in the necrotic core of the atherosclerotic plaque. Serial sections stained with the TF antibody RD010 (G) (same section as shown in E) or with a monoclonal antibody (HAM-56) to human macrophages (H) were compared and indicated that the TF-protein-producing cells in the necrotic core may be macrophages. Additional adjacent sections in the series were screened with markers specific for smooth muscle cells (HHF-35), endothelial cells (Ulex europaeus lectin binding), or T cells (anti-Leu4), all of which were negative in this region but labeled the appropriate cells elsewhere in the section. Arrows point to two cholesterol clefts followed on the serial sections (E-H). (Magnifications are as follows: for A, x760; for B, x615; for C, G, and H, x310; for D, x1230; for E, x75; for F, x760.) Downloaded by guest on September 25, 2021 2842 Medical Sciences: Wilcox et al. Proc. Natl. Acad Sci. USA 86 (1989) Finally, as with the normal vessels, no TF mRNA or protein The production of TF by macrophages has been demon- was detected in either the lining the vascular strated by other investigators (27,28). The immunostaining of surface or the small vessels within the plaques. macrophage foam cells suggests that in these cells TF is It was possible to confirm that the protein staining and the intracellular as well as possibly cell surface associated. It is in situ hybridization labeled the same regions on serial not clear to what extent such stores of TF are macrophage- sections (see Fig. 1 E-G). In afew instances we could identify derived or whether this protein originates from phagocytosis the same cell on two adjacent serial sections and show that of surrounding necrotic core debris. It is interesting to note this cell was positive with both the TF antibody and the in situ that Levy et al. (28), have found that certain lipoprotein mRNA hybridization. This observation served as a control fractions can induce procoagulant activity originating from for both the immunohistochemistry and hybridization anal- monocytes/macrophages. ysis. Additional controls were done on serial sections in Evidence indicates (29) that TF-initiated coagulation is the every experiment. The in situ hybridizations were controlled major in vivo coagulant pathway. It is a highly thrombogenic by hybridization of serial sections with -derived protein and requires only phospholipid and factor VII/VIa growth factor A chain or platelet-derived growth factor for its activity. The TF-factor VIIa complex directly acti- receptor-specific riboprobes (17). Different patterns of hy- vates factor IX and X leading to the generation of thrombin. bridization were seen with these probes compared to TF. TF Factor VII is normally present in blood but requires binding immunohistochemistry was always controlled by incubation to TF for activation offactors IX and X. Since normally there of serial sections with preimmune serum that failed to label is no in vivo coagulation in the absence of vascular damage, any cells at all. it is reasonable to assume that TF is not exposed to blood (7, All of the carotid endarterectomy specimens examined (n 29). This is consistent with our findings, since normal vessel = 16) showed positive TF protein staining in some region of endothelial cells in direct contact with the blood do not the plaque. Prominent staining of regions surrounding the TF. TF is found in scattered smooth cholesterol clefts in the necrotic core was a feature observed synthesize or store was muscle-like cells in the tunica media and adventitial cells in five out of seven plaques in which this feature present. adherent to the vessel. Vessel wall rupture into these areas The others showed variable staining in monocytes, macro- stores of phage foam cells, or mesenchymal-appearing intimal cells would be required to expose the blood to significant (17). A femoral plaque obtained by endarterectomy was procoagulant TF activity. Zaugg (30) has shown that damaged examined and showed TF protein staining consistent with human aorta exhibits factor VII-dependent procoagulant that seen in the carotid plaques. The mRNA hybridizations activity, in support of this hypothesis. were in agreement with the immunohistochemistry and all six Advanced human atherosclerosis is characterized by inti- carotid plaques examined by in situ hybridization had posi- mal smooth muscle cell proliferation accompanied by accu- tive intimal cells. mulation of fats, inflammatory cells including macrophages, and T cells within the atherosclerotic plaque (24, 31, 32). Commonly, the critical event that converts an asymptomatic DISCUSSION atherosclerotic plaque into a symptomatic one is thrombosis The purpose of these experiments was to localize sites of TF (33-35), whereas nondiseased arteries rarely become throm- biosynthesis in both the normal and atherosclerotic vessel. In botic. It has been suggested that plaque rupture is the integral the normal vessel, we have shown by immunohistochemistry event that precipitates clot formation (36-39). An occlusive and in situ hybridization that TF is synthesized by scattered mural accompanies most cases ofacute myocardial cells in the tunica media and fibroblasts in the adventitia infarctions (40-43). Plaque rupture or cracking that exposes surrounding the vessels. We did not find any evidence of TF the necrotic core region to the lumen is usually found to mRNA or protein localization in endothelial cells of any underlie such thrombi in both the coronary (33, 44, 45) and vessel studied. Previous cell culture work has suggested that cerebral arteries (46). The source of the thrombogenicity of induction of TF synthesis by endothelial cells represents a the plaque has not previously been determined, but it has major procoagulant mechanism by which endothelial cells been suggested that coagulation occurs when blood compo- participate in homeostasis (24). However, cultured fibro- nents come into contact with fats or the collagen matrix blasts (24-26) and vascular smooth muscle cells (25) have within the plaque. Our studies clearly demonstrate that there been shown to produce TF at much higher levels. It is not is significant synthesis of TF in atherosclerotic plaques clear if induction of endothelial TF biosynthesis is a normal accompanied by strong staining of TF protein in the necrotic in vivo mechanism by which the endothelium modifies core and in foam cell-rich regions ofthe plaque. These results homeostasis or represents the response of the endothelium to suggest that overproduction and/or trapping of TF protein in infection and endotoxin stimulation. the atherosclerotic plaque may play a significant role in the Atherosclerotic plaques were examined and found to have thrombosis associated with plaque rupture. many more TF mRNA-containing cells and stronger TF protein staining than the media of normal saphenous veins, We thank Gary Kollman for excellent technical assistance, the internal mammary artery, or regions of normal media under- Department ofVascular Surgery at the University ofWashington for lying the plaque. TF mRNA was found in mesenchymal-like their help in obtaining the human tissues used in this study, Karen intimal cells in the atherosclerotic intima as well as in Fisher for providing the TF probe, and Gordon Vehar, Stuart macrophages and cells adjacent to the cholesterol clefts. Bunting, and Richard Lawn for their helpful discussions. S.M.S and Immunohistochemistry indicated that there was a consider- D.G. were supported by Grant P01-HL-03174 from the National able amount of TF protein trapped in the extracellular matrix Institutes of Health. D.G. was also supported by a Robert Wood of the necrotic core of the atherosclerotic plaque. This was Johnson Minority Faculty Development Award. but was not cell-associated probably synthesized locally 1. Nemerson, Y. & Bach, R. (1982) Prog. Thromb. 6, since cells adjacent to these regions contained TF mRNA. 237-261. There is no evidence that TF is a secreted protein (5, 8); 2. Nemerson, Y. (1983) Haemostasis 13, 150-155. however, it is possible that the TF protein found in the 3. Nemerson, Y. (1966) Biochemistry 5, 601-608. necrotic core may be shed from the surfaces of the synthe- 4. Osterud, B. & Rapaport, S. I. (1977) Proc. Nati. Acad. Sci. sizing cells and trapped in the surrounding matrix. Alterna- USA 74, 5260-5264. tively, the TF protein in this region may originate from cells 5. Fisher, K. L., Gorman, C. M., Vehar, G. A., O'Brian, D. P. & that have died and left TF-rich membranes behind. Lawn, R. L. (1987) Thromb. Res. 48, 89-99. Downloaded by guest on September 25, 2021 Medical Sciences: Wilcox et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2843

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