Modulation of Tissue Factor Protein Expression in Experimental Venous Bypass Grafts
Abstract Vein graft failure is a major limitation of coronary artery and peripheral vascular surgery. Tissue factor (TF), a transmembrane glycoprotein, generates thrombin by initiating the extrinsic coagulation cascade and plays a major role in the response to arterial injury. This study was designed to examine changes in TF protein expression in response to venous bypass grafting. New Zealand White rabbits underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. The contralateral control jugular veins (n=6), early vein grafts (1 or 3 days after grafting, n=18), and late vein grafts (14 or 28 days after grafting, n=8) were examined by immunohistochemistry. The presence or absence of TF immunostaining in the intima was assessed in each vessel quadrant. In control veins, intimal TF staining was present in 5 of 24 vessel quadrants. In early vein grafts, TF staining was markedly increased in the intima (72 of 72 quadrants, P<.001 vs control veins), and TF immunostaining colocalized with CD18-positive leukocytes but not with endothelial cells, vascular smooth muscle cells, or RAM11-positive macrophages. In late vein grafts with intimal hyperplasia, TF expression was low or absent in the intima (6 of 32 quadrants, P<.001 vs early vein grafts; P=NS vs control veins), although medial smooth muscle cells expressed TF. Marked changes in TF expression occur in vein grafts. In early vein grafts, TF protein was greatly increased in the intima for at least 3 days and was associated with CD18-positive leukocytes. In late vein grafts with intimal hyperplasia, however, TF protein was not seen in the intima. These findings may have important implications for the development of therapeutic strategies to limit vein graft failure.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form in Circulation 1995;92(suppl I):I-354.
- Received April 23, 1996.
- Accepted September 26, 1996.
Coronary artery bypass grafting is a cornerstone of treatment for patients with coronary artery disease, with more than 400 000 CABG procedures performed annually in the United States alone. Although CABG is highly successful in relieving symptoms and prolonging survival in patients with severe coronary artery disease, the long-term success of CABG is limited by venous bypass graft failure, which results from two processes.1 2 First, acute vein graft thrombosis occurs early in the postoperative period in at least 5% of all vein grafts.3 Second, late vein graft failure occurs at a rate of 5% to 10% per graft per year due to a progressive atherosclerosis-like process.2 The substrate for the rapid development of atherosclerosis in venous bypass grafts is intimal hyperplasia resulting from SMC migration/proliferation, a characteristic feature of the accelerated atherosclerosis syndrome.4 5
TF is a transmembrane glycoprotein that binds to and activates factor VII.6 7 By catalyzing the rate-limiting step in the extrinsic coagulation cascade, TF is the major determinant of thrombin production in vivo.8 Immunohistochemical and in situ hybridization studies have shown that TF protein and mRNA are abundant in the adventitia of normal blood vessels.9 10 However, TF is consistently undetectable in the intima and endothelium of normal blood vessels, so that TF remains sequestered from circulating blood unless vessel injury occurs.9 10 In diseased vessels, however, TF expression has been demonstrated in carotid endarterectomy specimens10 and coronary artery lesions sampled by directional atherectomy.11
TF appears to play an important role in the response to vessel injury. Arterial balloon injury results in rapid upregulation of TF mRNA expression in medial smooth muscle, and TF mediates the increased procoagulant activity that has been observed in balloon-injured arterial segments.12 13 Coronary ECs express TF as a consequence of postischemic reperfusion injury.14 TF expression can be induced or upregulated in many cell types by agents or physiological stimuli that may play a role in atherosclerosis and in response to arterial injury.12 13 14 15 16 In addition, thrombin generation by TF also has pleiotropic actions,17 including stimulation of SMC proliferation,18 19 recruitment of inflammatory cells,20 and activation of ECs.21
The purpose of this study was to examine the changes in TF protein expression in a well established interposition rabbit jugular vein/carotid artery bypass graft model.22 23 In this model, venous bypass grafts undergo well characterized changes that can be divided into two temporal phases. In early vein grafts (postoperative days 1 to 3), nondenuding endothelial injury, inflammation, limited fibrin deposition, and platelet aggregation occur, but occlusive thrombosis is not a feature of this model.23 Prior studies using electron microscopy have demonstrated that polymorphonuclear leukocytes are the predominant cell type in the inflammatory infiltrate.23 In late vein grafts (postoperative days 14 to 28), the inflammatory infiltrate resolves, the functional integrity of the endothelium is restored, and SMC proliferation results in intimal hyperplasia.5 22 We hypothesized that modulation of TF protein might be an important feature of the injury response of veins to bypass grafting and might provide insights into the pathogenesis of vein graft failure.
Rabbit Venous Bypass Grafting
New Zealand White rabbits (average weight, 2 to 2.5 kg) underwent right common carotid artery bypass grafting using a reversed, ipsilateral external jugular vein as previously described.23 Anesthesia was induced and maintained with subcutaneously injected ketamine hydrochloride (Ketastat, 60 mg/kg body wt, Bristol Laboratories) and xylazine (Anased, 6 mg/kg body wt, Lloyd Laboratories). Vein grafts were harvested 1, 3, 14, or 28 days after surgery. The contralateral external jugular veins (control vein) and carotid artery (control artery) were also harvested. Animals were killed under general anesthesia via an intravenous pentobarbital overdose (100 mg/kg body wt, Anthony Products). All excised vessels were handled in a similar manner and were gently washed in Ringer’s lactate solution, divided into 5-mm segments, and immediately fixed in ice-cold 4% paraformaldehyde for histology and immunohistochemistry or snap-frozen in liquid N2 for protein extraction. All animal care and procedures complied with the Principles of Laboratory Animal Care as formulated by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH publication No. 80-23, revised 1985).
After overnight fixation, tissues were prepared for both paraffin-embedded and frozen sections. Vessel segments were either dehydrated in a graded series of alcohol and embedded in paraffin (Paraplast, Oxford Labware) or placed in 30% sucrose in PBS for 2 hours, embedded in OCT compound (Miles Scientific), and frozen in liquid N2. Sections (6 μm) were prepared on silane-coated glass microscope slides. Samples of rabbit skin and lung served as control tissues for histologic and immunohistochemical stains and were handled in a similar manner.
Immunohistochemistry for TF was performed using the murine monoclonal anti-rabbit TF antibody AP-1 as previously described.24 In brief, sections were deparaffinized in xylene, rehydrated in a graded series of alcohol, and equilibrated in PBS. Blocking solution (10% horse serum in PBS) was applied for 1 hour at room temperature or overnight at 4°C. AP-1 was diluted in blocking solution to 4 μg/mL and applied to tissue sections for 1 hour at 37°C. Incubation with the primary antibody was followed by sequential incubation with biotinylated anti-mouse IgG and ABC reagent according to the manufacturer’s specifications (Vectastain ABC kit, Vector Laboratories). Levamisole was added to block endogenous alkaline phosphatase activity, and immune complexes were localized with the use of the chromogenic alkaline phosphatase substrate Vector Red (Vector Laboratories). The sections were counterstained with hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific). In all experiments, rabbit skin was included as a positive control. Two different negative controls were used. First, for each vessel examined, a serial section was incubated with a nonsense murine IgG monoclonal antibody or with a murine IgG monoclonal antibody against human TF (TF9-9C3,9 11 ). Second, we demonstrated the specificity of the AP-1 antibody by the elimination of TF immunostaining when AP-1 antibody was incubated overnight with rabbit brain thromboplastin (Organon Teknika) Immunostaining for TF did not differ between paraffin-embedded and frozen sections. Paraffin-embedded sections were therefore used for all analysis owing to improved preservation of tissue architecture and morphology.
To identify the cell types associated with TF expression in rabbit vessels, two approaches were used. First, serial sections were examined by using previously characterized mouse monoclonal antibodies. SMCs, ECs, leukocytes, or tissue macrophages were stained with antibodies directed against human smooth muscle actin (HHF35, Dako), human vWF (American Diagnostica), rabbit CD18 (Serotec), or rabbit RAM11 (Dako), respectively, at the supplier’s recommended concentration. The anti-CD18 antibody recognizes the β2 chain of the leukocyte–adherence glycoprotein complex.25 This integrin is present on activated monocytes and neutrophils and mediates endothelial adhesion and migration.26 We confirmed the specificity of the CD18 antibody by the consistent absence of staining in sections of control veins and carotid artery. RAM11 was originally raised against alveolar macrophages and has been widely used to detect macrophages in rabbit arteries and vein grafts.27 28 Accordingly, histologic sections of rabbit lung were used as a positive control. Immunostaining with these antibodies was performed as described for the AP-1 antibody; alternatively, sections were pretreated with 3% H2O2 and biotinylated secondary antibodies, followed by an avidin-biotin-peroxidase conjugate and NiCl2-enhanced 3,3′ diaminobenzidine (Vector Laboratories) to yield a black reaction product. In addition, early (day 1 or 3) vein grafts underwent cytochemical stains with PAS, which may be used to differentiate leukocyte types; the cytoplasm of PAS-positive cells stain red.29 Therefore, immunostaining for TF, CD18, or RAM11 with the peroxidase method was followed by histologic staining with PAS (Sigma Chemical Co).
Analysis of Tissue Sections
For analysis, vessels were categorized as control artery, control vein, early vein graft (day 1 or 3 after grafting), or late vein graft (day 14 or 28 after grafting). The intima, media, and adventitia of representative vessel sections were defined by using sections stained with hematoxylin-eosin and Masson’s trichrome as previously described.22 23 The adventitia of all vessels showed abundant TF staining. To compare TF protein expression between vessel categories, TF immunostaining in the intima of the vessel was graded as present or absent in each vessel quadrant. The numbers of quadrants with TF immunostaining were compared by a χ2 test. A value of P<.05 was considered significant.
TF Activity and Western Blot Analysis
Protein was extracted from frozen tissue samples by grinding the tissue to a fine powder in a mortar and pestle in liquid N2, followed immediately by sonication in 0.5 mL ice-cold Tris-saline buffer (0.05 mol/L Tris, pH 7.4, 150 mmol/L NaCl) as previously described.9 Insoluble debris was pelleted in a microcentrifuge at 14 000g for 5 minutes. Total protein concentration in the supernatant was determined with the Bradford assay (Bio-Rad Laboratories).
TF activity in vessel protein extracts was determined by a one-step recalcification assay as previously described.30 In brief, duplicate portions of vessel protein extract were added to citrate-anticoagulated, pooled, normal human platelet-poor plasma. The time taken to produce a fibrin clot after addition of CaCl2 at 37°C was determined with a fibrometer (BBL Fibrosystems). TF activity in vessel extracts was calculated by comparison with standard dilutions of a partially purified rabbit brain thromboplastin preparation (100 000 mU thromboplastin activity per milliliter).
For Western blot analysis, equal samples of vessel protein extract (50 μg total protein) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat milk and incubated with AP-1 (2 μg/mL) for 1 hour at room temperature. Protein bands were visualized with an anti-mouse IgG horseradish peroxidase–conjugated antibody followed by chemiluminescence (Dupont-NEN).
A total of 32 control veins or vein grafts from 26 rabbits were studied. Vessels harvested 1 (n=3) or 3 (n=15) days after surgery were categorized collectively as early vein grafts, and all showed the common features of endothelial injury and inflammation. Vessels harvested after 14 (n=2) or 28 (n=6) days were categorized as late vein grafts, because they all demonstrated intimal hyperplasia with an intact endothelium and no inflammation. All animals survived until their designated time of harvest, and all vein grafts were patent at the time of harvest.
TF Expression in Control Artery, Control Veins, and Early Vein Grafts
Localization of TF Protein
Representative histologic and immunohistochemical sections of rabbit control vein, early vein grafts, and control carotid artery are shown in Fig 1⇓. In control veins (the contralateral external jugular vein not used in the bypass operation), intense TF staining was present throughout the adventitia and SMCs of the thin media but not in ECs of the intima (Fig 1A⇓). In control veins (n=6), TF immunostaining in the intima was detected in only 5 of 24 vessel quadrants. In contrast, all early vein grafts (n=18) displayed TF staining in the intima (72 of 72 vessel quadrants, P<.001 versus control veins; Fig 1B⇓ and 1C⇓).
Surgical manipulation alone was not responsible for the increased TF expression in early vein grafts. The wall of the carotid artery segment that formed the surgical anastomosis in early vein grafts demonstrated TF staining in the adventitia but not in the smooth muscle media or intima (Fig 1F⇑) and was similar to control carotid artery (the contralateral artery not used in the bypass operation; Fig 1D⇑).
Identification of Cell Types Associated With TF Protein in Early Vein Grafts
Serial sections were immunostained for TF and with a series of antibodies against different cell types (Fig 2⇓). RAM11-positive macrophages were present in the adventitia but not the intima and did not colocalize with TF staining (Fig 2D⇓). HHF35-positive SMCs were present in the media, which was thinned and partially disrupted (Fig 2E⇓). TF staining mainly colocalized with areas characterized by CD18-positive leukocyte infiltration (see insets to Fig 2A⇓ and 2B⇓). Indeed, the entire vessel wall of early vein grafts was heavily infiltrated by CD18-positive leukocytes, which were adherent to the endothelial surface and formed aggregates in the subendothelial space (Figs 1B⇑ and 2B⇓). In many places the integrity of the endothelium was disrupted by infiltrating CD18-positive leukocytes (Fig 2B⇓ and 2C⇓). The nuclei of the majority of the cells in the inflammatory infiltrate were multilobed (Fig 1B⇑ and 1C⇑). In addition, we found that these cells were PAS-positive and that CD18 and TF colocalized in these PAS-positive cells (not shown). These results demonstrate that increased TF protein expression in early vein grafts is spatially associated with PAS-positive, CD18-positive leukocytes.
TF Expression in Late Vein Grafts
There was a marked contrast in TF expression between late vein grafts and early vein grafts. In late vein grafts (day 14 or 28), intimal hyperplasia had developed, which was clearly defined on histologic sections and had stained heavily for SMCs (Fig 3C⇓).22 23 The luminal surface was lined with an intact endothelium (Fig 3D⇓). RAM11-positive macrophages and CD18-positive leukocytes were rarely detected in late vein grafts. Staining for TF was present in the adventitia and to a lesser extent in SMCs of the media, but the intima was practically devoid of TF staining (Fig 3B⇓). In late vein grafts (n=8), TF staining in the intima was present in only 6 of 32 vessel quadrants (P<.001 versus early vein grafts, P=NS versus control vein).
TF Activity and Protein Expression in Control Veins and Vein Grafts
TF activity in whole-vessel protein extracts was similar in control veins and early and late vein grafts (mean±SD: control vein, 0.27±0.07; early vein grafts, 0.25±0.10; and late vein grafts, 0.42±0.08; all in units per milligram protein). Western blot analysis of vessel total protein revealed no differences in the quantity of TF protein (as a proportion of total protein) between control veins, early vein grafts, and late vein grafts (data not shown).
This report provides the first description of the changes in TF expression induced by experimental venous bypass grafting. We found that (1) TF expression was markedly increased in the intima in early vein grafts for at least 3 days; (2) in late vein grafts, TF staining was present in medial SMCs but was not associated with intimal hyperplasia; and (3) in early vein grafts, TF protein in the intima was associated with PAS-positive, CD18-positive leukocytes.
Increased TF Protein Expression in the Intima of Early Vein Grafts
The first major finding of this study was that TF protein was increased in the intima of early vein grafts compared with control veins. Importantly, the wall of the carotid artery that formed the arteriovenous anastomosis with early vein grafts did not show TF expression in the media or intima. Thus, increased TF expression in the intima of early vein grafts appears to be a specific characteristic of the injury sustained by a vein after arterial bypass grafting and is not a general result of surgical manipulation.
Our results showed that TF protein was present in the intima of vein grafts 24 hours after surgery and lasted for at least 3 days thereafter. This time course is prolonged compared with arterial injury models, wherein TF mRNA, protein, and procoagulant activity are detectable within 2 hours of balloon injury but return to baseline after 24 hours.12 13 The prolonged increase in TF expression in the intima of vein grafts may be the result of sustained vessel injury in veins subjected to the arterial circulation in contrast with the single episode of balloon injury in arteries. Also, in arterial injury, TF expression is upregulated in medial smooth muscle,12 whereas SMCs in early vein grafts have not been found to be consistently associated with TF expression. These findings may result from the fundamental differences between arterial balloon injury and venous bypass grafting, or they may reflect biological differences between arteries and veins.
In contrast to arterial bypass conduits, acute thrombosis is a significant cause of vein graft failure.2 3 As a key initiator of the extrinsic coagulation cascade, TF plays a pivotal role in intravascular thrombosis and mediates the increased procoagulant activity after arterial injury.8 11 12 13 In the model used in our study, endothelial fibrin deposition and platelet aggregation occur in early vein grafts.23 Therefore, TF may play a role in thrombosis in vein grafts, which supports the hypothesis that strategies aimed at inhibiting TF may be beneficial in reducing acute thrombosis in venous bypass grafts.31
TF Protein Expression in Late Vein Grafts With Intimal Hyperplasia
The second major finding of this study was that TF protein was not present in the intimal hyperplasia of late vein grafts. TF protein expression therefore precedes the development of intimal hyperplasia in vein grafts. Intimal hyperplasia is the universal response of veins that are placed in the arterial circulation and is the precursor of accelerated vein graft atherosclerosis.32 33 TF per se or TF-mediated thrombin generation may play a role in the development of intimal hyperplasia after arterial injury.6 12 13 Thrombin is a potent mediator of platelet aggregation, resulting in the release of smooth muscle mitogens such as platelet-derived growth factor.17 34 In addition, thrombin is a direct mitogen for SMCs.17 18 19
The complex intravascular coagulation cascade offers several potential sites for pharmacological or molecular strategies to modulate the effects of thrombin on intimal hyperplasia.31 However, although TF is the major source of thrombin, which likely plays an important role in the development of intimal hyperplasia, TF may also have effects independent of thrombin production that can affect the response to vessel injury. In tumors, overexpression of TF increases vascular endothelial growth factor activity and thereby appears to modulate EC growth and development, despite the inhibition of thrombin.35 36 This model of venous bypass grafting provides the opportunity to study in vivo the effects of inhibiting TF or thrombin and to observe these effects on the development of intimal hyperplasia.
Cell Types Associated With TF Expression in Early Vein Grafts
The third major finding of the present study was the close association of CD18-positive leukocytes with areas of increased TF expression in the intima of early vein grafts. Some of these leukocytes had multilobed nuclei and a majority were PAS-positive. A number of different cell types in the vessel wall have been shown to express TF when activated in vitro or in models of arterial injury, including ECs, SMCs, and monocytes.6 7 12 13 14 15 16 Our findings do not exclude the possibility of some TF expression in early vein grafts by ECs, RAM11-negative macrophages, or vascular SMCs. Nevertheless, CD18-positive leukocytes were consistently associated with areas of intense TF staining and appeared to be responsible for the increased TF expression in the intima of early vein grafts.
Mechanism of TF Expression by CD18-Positive Leukocytes in Vein Grafts
The results of this study further support the link between endothelial injury and the pathogenesis of intimal hyperplasia.4 22 23 The CD18 complex on leukocytes interacts specifically with integrins such as intercellular adhesion molecule 1, which is present on ECs only after activation.26 There is evidence that TF expression by mononuclear cells is signaled by endothelial adhesion through E-selectin37 and P-selectin,38 suggesting that TF expression by CD18-positive leukocytes in vein grafts results from leukocyte adherence to and migration across the injured endothelium.
Some limitations of this study need to be considered. First, spatial changes in TF protein expression were localized by immunohistochemistry, which is subject to inherent limitations. However, all studies were performed with negative and positive controls, and the antibody used in this study (AP-1) has been previously characterized and functions as an inhibitor of TF activity.14 24 We further showed the specificity of this antibody by demonstrating a single dominant protein band on Western blots of total vessel protein extracts. Second, owing to the overwhelming TF protein expression in the adventitia, we were not able to demonstrate differences in overall vessel TF protein or procoagulant activity in early vein grafts compared with control vein or late vein grafts. In arteries, a thick muscular media permits enzymatic separation of the intima/media from the adventitia and the quantification of proteins in different regions.14 In control veins and early vein grafts, the extremely thin media would make this approach difficult.
Selected Abbreviations and Acronyms
|CABG||=||coronary artery bypass graft|
|SMC||=||smooth muscle cell|
|VWF||=||von Willebrand factor|
K.M.C. is a British Heart Foundation Clinical Scientist Fellow. G.J.F. holds a Traveling Fellowship from Trinity College, Dublin, Ireland. This research was funded in part by a grant from the American Heart Association, North Carolina Affiliate, Inc, NC-96-GS-61 (to B.H.A.), and from the National Institutes of Health, Bethesda, Md (grant HL-15448 to P.O.H.). We are grateful to Dr Charles Greenberg (Duke University) for performing the TF activity assays.
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