Tissue Factor Overexpression in Rat Arterial Neointima Models Thrombosis and Progression of Advanced Atherosclerosis
Background—Tissue factor located in the atherosclerotic plaque might cause the clinically significant thrombotic events associated with end-stage disease. It might also affect intimal area by increasing matrix accumulation and stimulating smooth muscle cell (SMC) migration and proliferation. To test this hypothesis, we overexpressed tissue factor in a rat model of the human fibrous plaque.
Methods and Results—A neointima was generated by seeding tissue factor–overexpressing rat SMCs onto the luminal surface of a balloon-injured syngeneic rat carotid artery. The cells attached and expressed tissue factor over the long term. Mural thrombus accumulation was present at 4 and 7 days and increased neointimal SMC numbers and area by 2-fold at 2 and 4 weeks. Tissue factor overexpression accelerated reendothelialization compared with controls at 2 weeks and 1 month. Tissue factor–overexpressing SMCs exhibited increased migration both in vitro and in vivo. The increased migration by tissue factor–overexpressing SMCs in vitro was not dependent on activation of the coagulation cascade and could be blocked by an inhibitor of tissue factor.
Conclusions—These results suggest that tissue factor plays a direct role in neointimal development by coagulation-dependent and -independent pathways.
Tissue factor is a 45-kDa membrane-bound glycoprotein that is the primary initiator of coagulation.1 Tissue factor is found on the surface of a variety of cell types normally located outside the vasculature.2 Tissue factor binds factor VII/VIIa, and the TF:VIIa complex can proteolytically activate factor X, which in turn activates thrombin and generates fibrin. Recently, 3 groups3 4 5 have reported that tissue factor deficiency is lethal during murine embryogenesis. Because these embryos exhibit abnormal vasculature, tissue factor may play an essential role in vascular development and integrity. Other groups have demonstrated that inhibition of tissue factor can inhibit intimal hyperplasia, supporting a role for tissue factor or downstream factors in intimal biology.6 7
Tissue factor is present only in the adventitia of the normal vessel and can be transiently induced by injury.8 9 In advanced atherosclerosis, tissue factor is expressed in the plaque and might play a role in the thrombotic response associated with disruption of the luminal surface and the fibrous cap.10 Although advanced atherosclerotic lesions can be generated in animals by cholesterol feeding or genetic manipulation, they do not model the critical terminal events of wall disruption and thrombosis found in humans.
To model the thrombotic aspect of advanced atherosclerosis, we constructed a synthetic neointima containing smooth muscle cells (SMCs) that constitutively expressed rat tissue factor. Rat SMCs were transduced in vitro with a retroviral vector containing tissue factor, and then the transduced SMCs were seeded back onto the vessel wall, where they permanently attached on the luminal surface of the carotid artery.11 In this study, we report that tissue factor plays an important role in neointimal development by increasing mural thrombus, increasing intimal lesion size, and accelerating endothelial regrowth. This process may depend on coagulation-dependent and -independent mechanisms.
Construction of Rat Tissue Factor Retroviral Vector and SMC Transduction
The rat tissue factor coding sequence was introduced into the retroviral vector (LXSN) to construct the recombinant tissue factor vector (LTFSN) (Figure 1A⇓).12 The LXSN vector alone was used as a control. DNA sequencing was performed to verify the orientation and integrity of the retroviral construct.
Viral packaging and cell transduction were performed according to Miller and Rosman.12 Viral titers of ≈1×106 pfu/mL from PA317–tissue factor and vector alone were used to transduce rat SMCs enzymatically isolated from male Fischer 344 rat aortas.
Total cellular RNA was extracted13 and processed for Northern analysis.14 Blots were probed with a 700-bp EcoRI fragment of the rat tissue factor cDNA and then reprobed with 28s cDNA to document equal loading. Quantification was performed with a PhosphorImager and ImageQuant (Molecular Dynamics).
Tissue Factor and Factor VIIa Assays In Vitro
Tissue factor activity was determined by the ability of tissue factor and exogenously added factor VIIa to convert factor X to Xa in a chromogenic assay.15
The assay for tissue factor activity described above was modified as follows to measure factor VIIa. Purified factor X (American Diagnostica) was added to cells cultured for 5 days in serum-free growth medium (SmBM 3 supplemented with human epidermal growth factor 10 ng/mL, human fibroblast growth factor 2 ng/mL, transforming growth factor-β1 0.5 ng/mL, and insulin 5 μg/mL, Clonetics Co). Samples were compared with identical wells given purified factor VIIa (0.1 to 1000 ng/mL, Zymogenetics).
Migration Assay for SMCs In Vitro
SMC migration in vitro was assayed with a 48-well modified Boyden microchemotaxis chamber (Neuro Probe) and polycarbonate filters (Nucleopore Corp) with 10-μm pores. The filters were precoated with 2.7 μg/well of basement membrane matrix (Matrigel, Collaborative Research) in 0.5× PBS and dried overnight. Thirty minutes before use, the matrix was reconstituted in 0.5× PBS and placed on top of the lower chamber containing 20 ng/well of platelet-derived growth factor (PDGF)-BB (Zymogenetics). SMCs were cultured in serum-free growth medium (SmBM 3 with growth factors described above) for 5 days, trypsinized, washed 3 times in serum-free medium, suspended at a concentration of 5×105/mL in serum-free medium, and added to the upper chamber. Recombinant hirudin (50 nmol/L, CIBA-Geigy) and FFR-VIIai (a catalytically inactive form of factor VIIa that retains full binding capacity to tissue factor, 4.8 μg/mL, Zymogenetics) were added to the cells 10 minutes before the chamber was loaded. The chemotaxis chamber was incubated for 5 hours at 37°C with 5% CO2. At the end of the assay, the cells that had migrated were stained with Diff-Quick (Baxter) and reported as mean number of cells per ×400 field.
Long-Term Overexpression of Tissue Factor in the Carotid
Male Fischer 344 rats (250 to 300 g) were seeded with the retrovirally transduced cells as previously described.11 To maintain vessel patency, both tissue factor–overexpressing and control cells were pretreated with a tissue factor inhibitor (10 μg FFR-VIIai/2.5×106 SMCs) for 10 minutes before seeding.
At various times, rats were killed, and the carotids were either removed or surgically exposed for further analysis. The rats were cared for according to the “Principles of Laboratory Animal Care” (formulated by the National Society of Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985).
Tissue Factor Activity Assay Ex Vivo
Tissue factor activity was assayed in isolated carotid segments. Rats were killed with an overdose of pentobarbital and exsanguinated through the abdominal aorta. The carotid artery was cannulated with Silastic tubing (0.012-in ID, Technical Products) both proximal and distal to the seeded area. Immediately after placement of the cannulas, the artery was flushed with 3 mL M199 (Sigma), and then 2 U/mL Proplex-T (Baxter) in M199 was infused and allowed to incubate in the vessel for 10 minutes. The luminal volume was collected by flushing the carotid artery with 200 μL M199, and the entire collected luminal volume was placed in a 96-well plate. S-2765 (10 μL) was added, and the OD was measured at 405 nm after a 20-minute incubation at 37°C. Contralateral carotids and LXSN control seeded carotid arteries were used as controls.
Migration of SMCs In Vivo
SMCs were genomically marked with bromodeoxyuridine (BrdU) in vitro before seeding to allow tracking. This strategy allows us to distinguish the seeded cells from endogenous SMCs by immunohistochemistry. Subconfluent SMCs were cultured with 0.06 μg/mL BrdU (Boehringer Mannheim) in DMEM with 10% FBS (Gibco-BRL) for 48 hours and seeded as previously described. This concentration of BrdU does not affect cell proliferation and labels >99% of SMCs. At various time points, carotids were perfusion-fixed with formalin and embedded in paraffin. Anti-BrdU antibody (Boehringer Mannheim) and alkaline phosphatase with DAB (Vectastain) or True Blue (Kirkegaard and Perry Laboratories), as described by the manufacturers, were used for visualization. The number of BrdU-positive cells were expressed as a percentage of total cells in each intimal quartile, starting with the region closest to the internal elastic lamina and progressing toward the lumen.
External Seeding Method for Measuring SMC Migration In Vivo
A second method was developed to measure migration in vivo from the adventitia into the carotid wall. Carotid arteries were surgically exposed and stripped of loose connective tissue surrounding the adventitia, and a Teflon sheet (≈1.5×3 cm) was placed under the carotid to protect the surrounding tissue. The carotid artery was decellularized by gentle touching of liquid nitrogen–cooled forceps to the carotid artery until frozen. Freeze-thaw cycles were repeated 3 times. The Teflon sheet was removed, BrdU-labeled SMCs (1.25×106 SMCs in 400 μL) were seeded onto the adventitia of the carotid artery and incubated for 10 minutes, and then the neck wound was closed. An Alzet osmotic minipump (model 2 ML1, Alza Co) containing either the tissue factor inhibitor FFR-VIIai (2.2 mg/mL) or saline was placed subcutaneously in the rat. Silicone medical grade tubing (0.012-in ID, Technical Products) was used to deliver FFR-VIIai (10 μL/h for 1 week) to the periadventitial region. The total number of BrdU-positive cells migrating into the media and intima per cross section was quantified in histochemical cross sections.
Tissue Preparation, Morphometry, and Endothelial Staining
At various time points, tissue factor–overexpressing (LTFSN) and control (LXSN) seeded carotids were flushed with lactated Ringer’s solution (Baxter) and perfusion-fixed with 4% formalin, pH 7.4 (Fisher Scientific) at 120 mm Hg pressure. Vessels were excised and embedded in paraffin for histology and immunohistochemistry. Cross-sectional areas were analyzed at 2 sites with a digitizing pad (Opelco) and camera lucida. Endothelial regeneration was visualized by Evans blue staining. Evans blue dye (60 mg/kg) (E-2129, Sigma) was injected into the tail vein 60 minutes before euthanasia.16
Carotid arteries were flushed and perfusion-fixed at 120 mm Hg with 4% paraformaldehyde for 3 minutes. Tissue intended for scanning electron microscopy was pinned out to expose the luminal surface and fixed in 2% osmium tetroxide before sputter-coating.
All values are expressed as mean±SD. Comparisons between tissue factor and control groups and corrections for multiple comparisons were made with combined Wilcoxon tests for blocked data.17 Comparisons between tissue factor and control groups at individual time points were made with Mann-Whitney nonparametric tests. Statistical significance was set at P<0.05.
Construction and Overexpression of Rat Tissue Factor in SMCs In Vitro
Tissue factor–overexpressing cells had growth curves similar to those of controls (Figure 1B⇑). RNA from these cells was analyzed by Northern blotting to verify expression of tissue factor (Figure 1C⇑). A 4.0-kB tissue factor transcript composed of 3.1 kB of the retroviral vector sequence and 0.9 kB coding sequence of rat tissue factor was easily identified in tissue factor–overexpressing cells.
There was a 7-fold increase in tissue factor activity in vitro in tissue factor–overexpressing cells compared with controls (Figure 2⇓). Although uninjured SMCs in vivo do not normally express tissue factor, SMCs in vitro do have low-level expression.
Migration In Vitro
The migration of tissue factor–overexpressing SMCs in vitro through a basement membrane matrix toward the potent chemoattractant PDGF-BB was increased compared with control SMCs (Figure 3⇓). SMCs used in this assay were cultured in the absence of serum and coagulation factors for 5 days; no residual factor VIIa (≪1 pmol/L) was detected. Tissue factor–overexpressing SMCs had an ≈2-fold increase in directed chemotaxis, with no increase in chemokinesis. Addition of recombinant hirudin did not reduce migration of tissue factor–overexpressing or control SMCs. Addition of catalytically inactive factor VIIa reduced migration of tissue factor–overexpressing SMCs but not control SMCs (Figure 3⇓). Factor VIIa or Proplex-T, a preparation of factor VIIa and X, also reduced migration of tissue factor–overexpressing SMCs but not control SMCs (data not shown). These experiments suggest that tissue factor can increase migration independently of coagulation in response to chemotactic factors.
Tissue Factor Overexpression in the Carotid Artery
Tissue factor overexpression in the carotid artery resulted in immediate thrombosis of >90% of the seeded vessels (data not shown). To study the long-term effects of tissue factor overexpression, it was necessary to temporarily attenuate tissue factor activity during the seeding process. This was done by treating the tissue factor–overexpressing cells with a catalytically inactive form of factor VIIa that binds tissue factor but does not allow conversion of factor X to Xa. Treatment with the inactive factor VIIa completely blocked tissue factor activity for ≥4 hours after seeding (Figure 2⇑). Tissue factor activity remained elevated in the vessels seeded with tissue factor–overexpressing SMCs at all time points tested after 4 hours and was ≈5-fold greater than in vessels seeded with control SMCs (Figure 2⇑). This increased activity was maintained even as the neointima became progressively filled with a mix of endogenous and seeded SMCs. The level of tissue factor expression after seeding of tissue factor–overexpressing SMCs is higher than the increase seen after injury alone.
Neointimal Area, Mural Thrombus, Endothelial Regeneration
Tissue factor overexpression in the carotid artery resulted in increased neointimal areas (Figures 4⇓ and 5⇓). At early time points, this was due to a large increase in mural thrombus (Figure 4⇓). At 4 days, there was a clear boundary between the seeded SMCs and an acellular area of fibrin accumulation, which accounted for ≈50% of the neointimal area. At 1 week, the fibrin-rich mural thrombus was invaded by SMCs (Figure 4⇓).
One week after seeding, the entire region of the tissue factor–overexpressing but not control carotid artery was covered in platelets (Figure 6⇓). By 2 and 4 weeks, the surface was free of platelets and partly covered by regenerating endothelium derived from the proximal and distal uninjured regions adjoining the denuded area. Tissue factor–overexpressing carotids had a 3-fold increase in endothelial coverage of the seeded area at 2 weeks and a 2-fold increase at 4 weeks compared with control seeded vessels (Figure 7⇓). The control LXSN cell–seeded vessels were reendothelialized at the same rate as the balloon-injured vessels not seeded with cells (data not shown).
At later time points, there was a significant increase in the number of neointimal SMCs in the tissue factor–overexpressing carotids and no significant differences in cell-to-matrix ratio (2-week cell-to-matrix ratio: tissue factor 0.59±0.33, control 0.63±0.30, P=NS, n≥5). SMC proliferation measured by BrdU labeling index did not show any significant differences between experimental control groups at 4 days, 1 week, 2 weeks, and 1 month (data not shown). The absence of differences at late times suggests that increased migration into the layer of thrombus might contribute in part to this increased neointimal area.
Migration In Vivo
To confirm that tissue factor–overexpressing cells have increased migration compared with control seeded cells in vivo, a labeling strategy was used. Tissue factor–overexpressing cells or LXSN controls were cultured in vitro with BrdU before cell seeding. The BrdU serves as a genomic tag for subsequent identification of the seeded cells in the carotid. The concentration of BrdU was not toxic, and the labeled and unlabeled cells grew at the same rate (data not shown). By 2 weeks, tissue factor–overexpressing SMCs tended to be located closer to the lumen than control seeded SMCs (Figure 8⇓). The LXSN control SMCs do not migrate as rapidly as the tissue factor–overexpressing cells and tend to be located closer to the internal elastic lamina.
Seeding tissue factor–overexpressing SMCs in the lumen creates a large mural thrombus that hinders comparisons of their migration with that of controls. We have developed a novel migration assay to overcome this problem. SMCs are seeded onto the adventitial side of a decellularized carotid artery. This method does not require pretreatment with tissue factor inhibitor and does not result in mural thrombus. Using this method, we found that tissue factor–overexpressing SMCs migrate more rapidly toward the lumen of the carotid (Figure 9⇓). Increased migration of tissue factor–overexpressing SMCs was reduced by continuous infusion of catalytically inactive factor VIIai. Carotid arteries externally seeded with tissue factor–overexpressing SMCs had an average of 21±15 tissue factor–overexpressing cells per field in the media and intima at 1 week, compared with 4.5±3.5 cells when tissue factor inhibitor was infused (n=5, P≤0.05).
Increased expression of tissue factor in the intima might account for the thrombotic properties of advanced atherosclerosis. In this study, we demonstrate that tissue factor may influence lesion development through coagulation-dependent and -independent mechanisms. Tissue factor overexpression increased neointimal area by increasing mural thrombus and SMC migration as well as increasing endothelial regeneration. Tissue factor–overexpressing SMCs migrated more rapidly than control SMCs both in vitro and in vivo. Taken together, we conclude that tissue factor expression can play an important role in neointimal formation.
Tissue Factor Increases Neointimal Formation
The increase in neointimal area could be due to 3 mechanisms: increased matrix accumulation, increased proliferation or decreased cell death, or increased migration of SMCs. Because SMC density and proliferation were not altered in tissue factor–overexpressing neointimas, it is likely that enhanced SMC migration contributed to increased intimal thickening. Sato et al18 19 showed that the catalytically active tissue factor complex can induce chemotaxis in SMCs. Recent work by Ott et al20 defined coagulation-independent and ligand (factor VIIa)–dependent roles for tissue factor in cell adhesion and migration. We have shown that tissue factor–overexpressing cells themselves exhibit increased ability to migrate in vitro and in vivo. Although we cannot exclude the possibility that tissue factor–overexpressing SMCs also induce endogenous SMCs to migrate, we conclude that tissue factor expression is able to directly facilitate migration in an autocrine manner.
Tissue Factor Increases Migration In Vitro and In Vivo
Tissue factor–overexpressing SMCs have an increased rate of migration in response to the chemoattractant PDGF-BB in vitro. This assay was conducted in the absence of all coagulation cascade components and thus rules out the possibility that factors such as factor Xa or thrombin are responsible for tissue factor–induced migration in vitro. Determining the cause of migration in vivo is complicated by the massive platelet-rich mural thrombus that forms as a result of tissue factor overexpression. The increased migration in vivo could be due to a combination of factors derived from the thrombus. Therefore, we propose that tissue factor in vivo facilitates increased migration by coagulation-dependent and coagulation-independent mechanisms. The coagulation-independent mechanisms have not been defined but might include intracellular signaling pathways or association with cytoskeletal components.20 21
Tissue Factor Accelerates Endothelial Regeneration
Tissue factor overexpression is associated with accelerated endothelial regeneration. This finding is consistent with observations made by Lindner et al22 showing that increased mural thrombus is associated with accelerated endothelial regeneration. In other experiments in which we have limited fibrinolytic activity by overexpressing plasminogen activator inhibitor-1, we have found a similar increase in endothelial regeneration.23 From these experiments, we cannot determine whether the increased endothelial regeneration is a direct result of the fibrin or of components derived from or attached to fibrin.
Relevance of the Rat Model to Advanced Atherosclerosis
Overexpression of tissue factor in the intima models several aspects of the human lesion, including prolonged elevation of tissue factor localized to the intima and increased mural thrombus. Tissue factor might perform the same functions in the atherosclerotic plaque as in the intima generated by cell seeding. It might also contribute to intimal hyperplasia by increasing SMC migration into the intima. After the fibrous cap ruptures, tissue factor would be expected to increase thrombus formation at the site of plaque rupture. In addition, it might encourage endothelial regeneration over the disrupted lesion. In summary, tissue factor expression might encourage rapid repair at the risk of increasing thrombosis at sites of vascular injury.
This work was supported by NIH grant HL-52459. Dr Hasenstab was supported by NIH training grant T32-HL-07312.
- Received July 6, 1999.
- Revision received December 16, 1999.
- Accepted January 11, 2000.
- Copyright © 2000 by American Heart Association
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