Published online before print February 3, 2003, doi: 10.1161/01.CIR.0000050149.22928.39
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2003;107:869.)
© 2003 American Heart Association, Inc.
Basic Science Reports |
Failure of Thrombus to Resolve in Urokinase-Type Plasminogen Activator Gene–Knockout Mice
Rescue by Normal Bone Marrow–Derived Cells
From St Thomas’ Hospital Campus, Kings College, London, UK (I.S., K.G.B., M.C., B.B., A.S.), and the Center for Transgene Technology and Gene Therapy, University of Leuven, Belgium (I.S., A.L., D.C.).
Correspondence to Dr Alberto Smith, Academic Department of Surgery, GKT Medical School, King’s College, St Thomas’ Hospital Campus, London SE1 7EH, UK. E-mail alberto.smith{at}kcl.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution.
Methods and Results— Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA-/-), and tPA gene-knockout (tPA-/-) mice in a flow model. Thrombi were harvested from wild-type and uPA-/- mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA-/- mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA-/- mice compared with wild-type controls (P<0.0001) but was unaffected in tPA-/- mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was 
4 times greater than in uPA-/- mice (P=0.0043). Thrombus size in uPA-/- mice transplanted with wild-type marrow (0.29±0.06 mm2) was significantly smaller than in uPA-/- mice given uPA-/- bone marrow (3.9±1.1 mm2) (P=0.0022). Donor bone marrow–derived cells expressing LacZ were present in the thrombus after transplantation.
Conclusions— The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA-/- animals, suggesting an important role for bone marrow–derived cells in this process.
Key Words: plasminogen activators • leukocytes • thrombus • veins
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Deep venous thrombosis can cause venous occlusion and valvular damage, which often results in pain, swelling, and ulceration in the affected limb. Leg ulcers affect 1% to 2% of Europeans at some time in their lives1 and are costly to treat. The current treatment of venous thrombosis with anticoagulants prevents thrombus propagation and embolization but has little if any effect on thrombolysis and has a small but significant risk of severe hemorrhage.2 Thrombolytic therapy does not consistently reduce the severity of postthrombotic syndrome and is also associated with an increased risk of hemorrhage.3 Venous thrombus resolves naturally by a slow process of organization and recanalization.4 Accelerating this process may prevent postthrombotic complications by reducing valvular damage and residual obstruction5 and would also be valuable when thrombolysis is contraindicated.
The formation of plasmin from plasminogen by the action of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators is thought to be the main pathway by which fibrin deposition is regulated in the vascular tree and by which pericellular fibrinolysis, required for cell migration in tissues, is activated.6–8 Natural thrombus resolution involves an organizational process similar to that found in wound healing.4,9 The migration and activity of monocytes are important components of the process of repair, because these cells are known to produce a variety of growth factors, chemotaxins, and matrix-degrading enzymes.10,11 We have previously demonstrated that venous thrombus resolution is linked to monocyte recruitment and activity. Monocytes enter the thrombus as resolution proceeds, and injection of monocyte chemotactic protein-1 into the thrombus results in enhanced resolution.13 We have also shown that the thrombus contains increasing quantities of both tPA and uPA activity as it resolves14 and that this activity is expressed by invading monocytes.15 These findings led us to speculate that monocytes orchestrate thrombus resolution and that this process is facilitated by the expression of both uPA and tPA.
Previous work in a model in which preclotted blood was injected into the jugular vein of uPA- and/or tPA-knockout mice suggested that tPA and not uPA is important for removal of the clot.14 Preclotted blood is formed in the tube under static conditions and is composed of red cells and platelets within a fine fibrin mesh, with leukocytes scattered randomly throughout the clot.16 A venous thrombus, however, forms in flowing blood and is composed of a laminar structure with layers of fibrin and associated platelets and leukocytes forming a strong meshwork that encapsulates the main red cell mass.9 Clots and thrombus may therefore resolve in a different manner.
The aim of this study was therefore to use a novel flow model of thrombosis17 in uPA and tPA gene–knockout mice to investigate the hypothesis that thrombus resolution is regulated by the fibrinolytic activity of monocytes.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Generation of Transgenic Mice
The tPA gene was deleted by use of a constructed targeted DNA vector, pNT.t-PA, containing a cassette expressing the neomycin-resistance gene (neo), which replaced the genomic sequences encoding most of the kringle-2 and part of the proteinase domains, including the active-site histidine residue. The targeting vector used to disrupt the uPA gene was pNT.u-PA, in which the neomycin cassette replaced the genomic sequences encompassing all but 23 amino acids of the coding sequence. Homologous recombinations (verified by Southern blot analysis of the genomic DNA) resulted in total inactivation of both plasminogen activator genes. Incorporation of the neomycin cassettes allowed the isolation of embryonic stem cells that had stably taken up the DNA vector. These pluripotent embryonic stem cells were subsequently introduced into host embryos by blastocyst injection.18
Mouse Model of Venous Thrombosis
Home Office approval was obtained for all the procedures performed. Thrombus was produced in the infrarenal vena cava of wild-type (uPA+/+/tPA+/+), uPA-/-, and tPA-/- adult mice (75% C57BL/6 25% 129 strain; weight, 18–32 g; age, 8 to 12 weeks; bred in Dr Collins’ lab) as previously described.17 Mice were anesthetized with isoflurane (Baxter Health Care Ltd), and the inferior vena cava was exposed below the renal veins through a midline laparotomy incision. The intestines were retracted, and retroperitoneal blunt dissection of the infrarenal vena cava was performed to mobilize a 5-mm segment distal to the left renal vein. A neurosurgical vascular clip (Braun Medical) was applied to the dissected vena cava for 15 seconds on 2 occasions, 30 seconds apart. A 5-0 Prolene suture was placed alongside the vena cava. A stenosis was produced in the vein by tying a 4-0 silk suture around the vena cava to include the Prolene suture. The Prolene was then pulled out to allow blood to continue to pass up the vein. The intestines were replaced, and the abdominal wall was sutured. The animals were then allowed to recover from the anesthesia. After surgery, the mice had access to water and chow (SDS; Lillico). The infrarenal vena cava containing thrombus was subsequently excised for analysis.
Tissue Harvesting
At fixed time intervals, cohorts of mice were reanesthetized, and a laparotomy was performed to excise the infrarenal vena cava, which was prepared for histology and image analysis.
Histology
The infrarenal vena cava containing the thrombus was fixed in 10% formal saline overnight and embedded in paraffin wax. Transverse sections 5 µm thick were cut at 300-µm intervals below the ligature and stained with Martius scarlet blue. This stains red cells yellow, fibrin red, mature fibrin gray/blue, extracellular matrix/collagen blue, and cell nuclei blue-black.13 Sections of tissues were viewed at 50x magnification and digitized using a microscope-mounted camera (Nikon Coolpix 990). The sizes of the thrombi were marked by use of a photo editor software (Satori Image, Spaceward Graphics). The area of thrombus in each section was measured with image analysis software (Image Pro Plus, Media Cybernetics). For ease of analysis, the thrombus was considered to be a cylinder, and therefore the sum of the cross-sectional area of all sections taken through the entire thrombus gave an approximate measure of the thrombus size. These measurements were calibrated and expressed in square millimeters. All the images were analyzed blindly. Thrombus size was also measured in a series of images (n=97) of sections taken from 10 randomly selected animals by a second "blinded" observer to determine the interobserver variability.
Thrombus Size
Thrombi were harvested from cohorts of wild-type and uPA-/- mice at 1, 7, and 10 days (n=6 per group); at 14, 21, and 28 days (n=12 per group); and at 60 days (n=6 per group) after thrombus induction. Thrombi were also harvested from wild-type and tPA-/- mice at 1, 14, and 28 days (n=8 per group).
Macrophage Content
Paraffin sections of the thrombus were taken at 1, 14, and 28 days after surgery from groups of wild-type, uPA-/-, and tPA-/- mice (n=6 per group) and were processed for immunohistochemistry to localize macrophages using the monoclonal antibody raised against the MAC3 antigen (Serotec). The antigen was retrieved by microwaving at 650 W for 10 minutes in citrate buffer at a pH of 6.0. Primary antibody binding was located using a biotinylated second antibody followed by a streptavidin-peroxidase complex and Vector SG substrate (Vector Laboratories). The percentage area of the thrombus containing the stained MAC3 antigen (macrophage density) was measured with the image analysis software described above.
Bone Marrow Transplantation
This procedure was carried out as previously described.19 Femurs from 8-week-old uPA-/- mice and their wild-type littermates (in a 99.2% C57Bl/6 background) were isolated, and the marrow was flushed out with a syringe attached to a 26-gauge needle containing RPMI 1640 culture medium (Rosewell Park Memorial Institute) supplemented with 2% fetal bovine albumin (Life Technologies) and 10 U/mL heparin (Sigma). Monocellular suspensions were prepared by flushing the marrow up and down in a syringe attached to a 26-gauge needle. The bone marrow cells were centrifuged at 1200 rpm for 5 minutes, washed twice with RPMI 1640 without additives, resuspended in RPMI 1640 medium, and counted. Two groups of 8-week-old uPA-/- mice and a group of wild-type mice (n=14 per group) were lethally irradiated (9.5 Gy) with a linear accelerator 16 hours before bone marrow transplantation. A group of uPA-/- and wild-type mice were transplanted with uPA-/- bone marrow cells by tail-vein injection of 5x106 cells per mouse under aseptic conditions. The second group of uPA-/- mice were transplanted with wild-type bone marrow cells (5x106 cells per mouse). Thrombosis was then induced in the inferior vena cava of all mice 4 weeks after transplantation, by which time white blood cell counts had returned to levels comparable to the levels before transplantation (not shown). From the time of transplantation until thrombus induction, mice were housed under specific pathogen–free conditions in Thoren racks and had access to sterilized food and filtered water. The mice were euthanized after another 28 days, and the venae cavae containing thrombi were harvested.
Transplantation of ß-Galactosidase–Expressing Bone Marrow and X-Gal Staining
To demonstrate that monocytes within the thrombus might have originated from the bone marrow, we transplanted marrow from mice expressing the gene for the nonmammalian enzyme ß-galactosidase (LacZ+/+) into an irradiated uPA-/- mouse and a wild-type mouse. Thrombi were induced 4 weeks later, and the animals were euthanized at 11 days. The infrarenal vena cava containing thrombus was cryoembedded, and sections 10 µm thick were cut and stained for ß-galactosidase activity using the X-Gal staining kit (Invitrogen). Tissues from nontransplanted wild-type and LacZ+/+ mice were also stained for ß-galactosidase activity to act as negative and positive controls, respectively. The presence of blue stain (ß-galactosidase expression) was confirmed by light microscopy.
Statistics
The significance of intergroup differences was determined by Student’s t test, because all data were normally distributed. Differences between resolution curves were assessed with 2-way ANOVA. All results are expressed as means with SEM. Paired t test and the Pearson rank correlation coefficient were used to compare the measurement of thrombus size by the 2 observers.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The thrombus induced in the mice consisted of a laminated structure composed of red cells trapped between layers of fibrin (associated with platelets and neutrophils), similar to those we have previously observed in this model, in a flow model of venous thrombosis in the rat, and in humans.12,17
Thrombus Size
No difference was observed in thrombus size at day 1 between uPA-/- mice (11±2.3 mm2) and their wild-type controls (12±1.9 mm2) (P=0.86) (Figures 1a and 2). There was also no difference in thrombus size between the tPA-/- animals (17±2 mm2) and their wild-type controls (18±4 mm2) (P=0.81) (Figures 1b and 3). By 28 days, thrombus in the wild-type mice (3.1±0.86 mm2) and tPA-/- mice (2.7±0.35 mm2) had resolved to approximately one sixth of its original size (P=0.002 and P<0.0001, respectively) (Figures 1b and 3). Thrombus resolution was significantly impaired, however, in uPA-/- mice compared with their wild-type controls (P<0.0001) (Figures 1a and 2). By 60 days, thrombus had resolved in all 6 wild-type mice (1.0±0.25 mm2), but no resolution was observed in 3 of the uPA-/- mice (19±1.1 mm2) (P<0.0001). The remaining 3 uPA-/- mice died. There was a good correlation (R2=0.99, P<0.0001) and no difference in mean thrombus area (P>0.15) measured by the 2 observers.
|
) vs their wild-type (
). (*P<0.0001) and (b) tPA-/- (
) vs their wild-type
|
|
Thrombus Macrophage Content
The macrophage content of thrombus taken from the wild-type animals peaked at day 14 (5.3±1.0%) compared with day 1 (0.3±0.1%, P=0.0006) and day 28 (1.6±0.6%, P=0.01) (Figures 4a and 5a). There was a small but significant increase in the macrophage content of the thrombus between 1 (0.2±0.1%) and 28 (1.8±0.3%) days in the uPA-/- mice (P=0.0006) (Figure 4a). There was, however, a lower macrophage density in the thrombi of uPA-/- mice at day 14 (1.3±0.3%) compared with thrombi removed from their wild-type controls (P=0.0035) (Figures 4a and 5, a and b). There was no significant difference between the level and pattern of monocyte recruitment in tPA-/- mice and their wild-type controls (Figure 4b).
|
) vs their wild-type (
|
Bone Marrow Transplantation
The size of the thrombi in uPA-/- mice reconstituted with wild-type marrow (0.3±0.1 mm2, n=14) was significantly smaller than thrombus taken from uPA-/- mice transplanted with uPA-/- marrow (3.9±1.1 mm2, n=14, P=0.002) (Figure 6). The mean thrombus size in the wild-type mice reconstituted with uPA-/- marrow (2.0±0.6 mm2, n=14) was lower than that in the uPA-/- mice given uPA-/- marrow, but this did not reach statistical significance (P=0.12).
|
LacZ+/+ Bone Marrow Transplantation
ß-Galactosidase staining was observed in thrombi produced in both wild-type and uPA-/- mice transplanted with bone marrow from Lac-Z+/+ animals (Figure 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Venous thrombus resolves by a process of organization characterized by recruitment of leukocytes (mainly monocytes) and invasion of fibroblasts and endothelial cells, leading to recanalization.12 Urokinase is thought to be an important mediator of pericellular proteolysis, which is required for cell migration and tissue remodeling in wounds,20,21 whereas tPA is the main plasminogen activator responsible for the removal of fibrin from the vascular tree. Our previous work in the rat model showed that both plasminogen activators were present within the thrombus and that their levels increased as the thrombus resolved.14 These findings led us to postulate that absence of tPA and uPA might affect the resolution of venous thrombus.
In this study, thrombus resolution and recanalization were inhibited in the uPA gene–deficient mice. This process proceeded normally in wild-type mice and was almost complete by 28 days. By this time, wild-type thrombi were highly organized cellular structures containing many vascular channels and widespread collagen deposition. There was little evidence of organization and recanalization in the thrombi of uPA-/- mice at the same stage. In these animals, cell infiltration and collagen deposition were restricted primarily to the margins of the thrombus, and few vascular channels were present.
No temporal difference was observed in the degree of thrombus organization and resolution between wild-type and tPA-/- mice, suggesting that this plasminogen activator may not play a major part in the resolution of venous thrombi. Our results led us to the assumption that uPA is essential for normal thrombus resolution.
The results of the present study seem to contradict previously reported data showing that inactivation of the tPA gene impaired clot lysis, whereas the absence of uPA had no effect on this process.18 The previous model, however, was different from the one used in this study in that it involved injection of ex vivo clotted blood into the jugular vein to mimic thrombosis and is therefore be more analogous to studying the resolution of pulmonary emboli.
Urokinase has been shown to be an important facilitator of cell migration and invasion18,22 and may be responsible for regulating the activation of other proteases, such as the metalloproteases, which sustain extracellular matrix turnover and tissue remodeling.23 We previously linked the uPA activity found in the thrombus to a monocyte infiltrate,14 and we therefore speculated that absence of uPA might affect the recruitment of monocytes that occurs during the natural resolution of venous thrombi. Analysis of the thrombus monocyte content demonstrated that monocyte entry into thrombi was delayed in uPA-/- mice. Wild-type thrombus monocyte numbers peaked at day 14, at which time the monocyte density was >5-fold higher than that found in the uPA-/- group. A study on the relationship between plasminogen activators and cardiac rupture in a model of acute myocardial infarction also showed that the absence of uPA reduces the migration of leukocytes, in particular monocytes, into tissue, whereas tPA deficiency had no effect on this process.24
The fact that significantly more monocytes had accumulated by 28 days in the thrombus of uPA-/- mice (compared with 1 day) in the same group suggests that these cells may also use plasmin-independent pathways to migrate. Monocytes can express a variety of proteases, including metalloproteases, that can degrade extracellular matrix to facilitate migration.11 Monocytes can also remove fibrin through receptor-mediated lysosomal degradation.25
Natural thrombus resolution involves the processes of organization and vascularization that also occur during wound healing. Monocytes are important mediators in wound healing, and inhibition of their migration is known to delay this process.21 They can regulate tissue organization by producing a variety of proteases and growth and chemotactic factors that regulate collagen turnover and tissue remodeling and may directly contribute to revascularization by "drilling tunnels" into wound tissue in a process similar to angiogenesis.26 In this context, it is interesting to note that we have previously observed thrombus revascularization in areas of high macrophage density in both human and experimental venous thrombi.12 Monocytes may therefore contribute to thrombus resolution by encouraging extracellular matrix remodeling and stimulating angiogenesis.
Our previous tracking studies have suggested that most monocytes that enter the thrombus are derived from the circulation and not from resident cells in the surrounding tissues.27 We therefore attempted to introduce normal circulating monocytes into uPA-/- mice by transplanting bone marrow from wild-type animals. This resulted in complete restoration of thrombus resolution in the uPA-/- mice. Impaired resolution occurred in animals given uPA-/- marrow. The small difference in thrombus resolution observed between uPA-/- mice transplanted with uPA-/- marrow and wild-type mice given uPA-/- marrow may have been the result of recruitment of residual macrophages present in tissues (eg, vessel wall) that were not destroyed by the irradiation. It has been previously reported that macrophages may survive in tissues for years.28
We have also shown, by transplanting LacZ-expressing bone marrow into wild-type mice, that bone marrow–derived cells are recruited into the thrombus. The lineage of these cells remains unclear, because our attempts with double-labeling techniques using specific markers for mouse macrophages have to date been unsuccessful.
Although bone marrow is a major source of monocytes, it is also a source of other cells, including endothelial progenitor cells. There is now considerable evidence to show that bone marrow–derived endothelial progenitor cells are present in the circulation.29,30 Endothelial progenitors have been implicated in postnatal vasculogenesis in both physiological and pathological conditions, including wound healing, in which revascularization is a vital process.31–33 It is therefore possible that endothelial progenitor cells were involved in the retrieval of thrombus resolution in uPA-/- mice transplanted with normal bone marrow.
This is the first study that directly indicates a central role for uPA in thrombus resolution. Enhancing the uPA activity in monocytes or enhancing their mobilization from the bone marrow may provide novel treatments for venous thrombosis. Studies of thrombus resolution using plasminogen activator inhibitor gene–deficient mice are under way. We are also using viral transduction methods to upregulate urokinase expression in monocytes34 and hope to reinfuse these transduced cells into our thrombosis models. The role of endothelial progenitor cells will also be investigated using specific endothelial cell markers and transgenic bone marrow donor mice expressing green fluorescent protein that is transcriptionally regulated by the endothelial cell–specific Tie-2 promoter.
|
Acknowledgments |
|---|
Dr Singh was supported by grants from the Royal College of Surgeons of England and the Guy’s and St Thomas’ Charitable Foundation.
Received July 16, 2002; revision received November 5, 2002; accepted November 6, 2002.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Browse N. Diseases of the Veins. Oxford, UK: Arnold; 1998.
2. Haas S. Deep vein thrombosis: beyond the operating table. Orthopedics. 2000; 23 (6 suppl): s629–s632.[Medline] [Order article via Infotrieve]
3. Wells PS, Forster AJ. Thrombolysis in deep vein thrombosis: is there still an indication? Thromb Haemost. 2001; 86: 499–508.[Medline] [Order article via Infotrieve]
4. Sevitt S. The mechanisms of canalisation in deep vein thrombosis. J Pathol. 1973; 110: 153–165.[CrossRef][Medline] [Order article via Infotrieve]
5. Meissner MH, Manzo RA, Bergelin RO, et al. Deep venous insufficiency: the relationship between lysis and subsequent reflux. J Vasc Surg. 1993; 18: 596–605.[CrossRef][Medline] [Order article via Infotrieve]
6. Tabrizi P, Wang L, Seeds N, et al. Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in a murine stroke model: studies in tPA-deficient mice and wild-type mice on a matched genetic background. Arterioscler Thromb Vasc Biol. 1999; 19: 2801–2806.
7. Bugge TH, Flick MJ, Danton MJ, et al. Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1996; 93: 5899–5904.
8. Ploplis VA, French EL, Carmeliet P, et al. Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood. 1998; 91: 2005–2009.
9. Cox JST. The maturation of canalisation of thrombi. Surg Gynaecol Obstet. 1963; 116: 153–165.
10. Leibovich SJ, Wiseman DM. Macrophages, wound repair and angiogenesis. Prog Clin Biol Res. 1988; 266: 131–145.[Medline] [Order article via Infotrieve]
11. Lijnen HR. Elements of the fibrinolytic system. Ann N Y Acad Sci. 2001; 936: 226–236.[Medline] [Order article via Infotrieve]
12. McGuinness CL, Humphries J, Waltham M, et al. Recruitment of labelled monocytes by experimental venous thrombi. Thromb Haemost. 2001; 85: 1018–1024.[Medline] [Order article via Infotrieve]
13. Humphries J, McGuinness CL, Smith A, et al. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg. 1999; 30: 894–899.[CrossRef][Medline] [Order article via Infotrieve]
14. Northeast AD, Soo KS, Bobrow LG, et al. The tissue plasminogen activator and urokinase response in vivo during natural resolution of venous thrombus. J Vasc Surg. 1995; 22: 573–579.[CrossRef][Medline] [Order article via Infotrieve]
15. Soo KS, Northeast AD, Happerfield LC, et al. Tissue plasminogen activator production by monocytes in venous thrombolysis. J Pathol. 1996; 178: 190–194.[CrossRef][Medline] [Order article via Infotrieve]
16. Quarmby J, Smith A, Collins M, et al. A model of in vivo human venous thrombosis that confirms changes in the release of specific soluble cell adhesion molecules in experimental venous thrombogenesis. J Vasc Surg. 1999; 30: 139–147.[CrossRef][Medline] [Order article via Infotrieve]
17. Singh I, Smith A, Vanzieleghem B, et al. Antithrombotic effects of controlled inhibition of factor VIII with a partially inhibitory human monoclonal antibody in a murine vena cava thrombosis model. Blood. 2002; 99: 3235–3240.
18. Carmeliet P, Schoonjans L, Kieckens L, et al. Physiological consequences of loss of plasminogen activator gene function in mice. Nature. 1994; 368: 419–424.[CrossRef][Medline] [Order article via Infotrieve]
19. Fazio S, Babaev VR, Murray AB, et al. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl Acad Sci U S A. 1997; 94: 4647–4652.
20. van Hinsbergh VW, Koolwijk P, Hanemaaijer R. Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells. EXS. 1997; 79: 391–411.[Medline] [Order article via Infotrieve]
21. Rohovsky S, D’Amore PA. Growth factors and angiogenesis in wound healing. In: Zeigler TR, Pierce GF, Herndon DN, ed. Growth Factors and Wound Healing. New York, NY: Springer; 1997.
22. Blasi F. Proteolysis, cell adhesion, chemotaxis, and invasiveness are regulated by the u-PA-u-PAR-PAI-1 system. Thromb Haemost. 1999; 82: 298–304.[Medline] [Order article via Infotrieve]
23. Cuzner ML, Opdenakker G. Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system. J Neuroimmunol. 1999; 94: 1–14.[CrossRef][Medline] [Order article via Infotrieve]
24. Heymans S, Luttun A, Nuyens D, et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999; 5: 1135–1142.[CrossRef][Medline] [Order article via Infotrieve]
25. Simon DI, Ezratty AM, Francis SA, et al. Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway. Blood. 1993; 82: 2414–2422.
26. Moldovan NI, Goldschmidt-Clermont PJ, Parker-Thornburg J, et al. Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium. Circ Res. 2000; 87: 378–384.
27. Burnand KG, Gaffney PJ, McGuinness CL, et al. The role of the monocyte in the generation and dissolution of arterial and venous thrombi. Cardiovasc Surg. 1998; 6: 119–125.[CrossRef][Medline] [Order article via Infotrieve]
28. Issekutz TB, Issekutz AC, Movat HZ. The in vivo quantitation and kinetics of monocyte migration into acute inflammatory tissue. Am J Pathol. 1981; 103: 47–55.[Abstract]
29. Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood. 1998; 92: 362–367.
30. Rafii S, Heissig B, Hattori K. Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther. 2002; 9: 631–641.[CrossRef][Medline] [Order article via Infotrieve]
31. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999; 85: 221–228.
32. Marchetti S, Gimond C, Iljin K, et al. Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo. J Cell Sci. 2002; 115: 2075–2085.
33. Hillebrands JL, Klatter FA, van Dijk WD, et al. Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis. Nat Med. 2002; 8: 194–195.[CrossRef][Medline] [Order article via Infotrieve]
34. Schleef RR, Olman MA, Miles LA, et al. Modulating the fibrinolytic system of peripheral blood mononuclear cells with adenovirus. Hum Gene Ther. 2001; 12: 439–445.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  R. Jaffe, G. Leung, N. R. Munce, A. S. Thind, H. Leong-Poi, K. J.T. Anderson, X. Qi, J. Trogadis, A. Nadler, D. Shiff, et al. Natural History of Experimental Arterial Chronic Total Occlusions. J. Am. Coll. Cardiol., March 31, 2009; 53(13): 1148 - 1158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-D. Li, B.-Q. Ye, S.-X. Zheng, J.-T. Wang, J.-G. Wang, M. Chen, J.-G. Liu, X.-H. Pei, L.-J. Wang, Z.-X. Lin, et al. NF-{kappa}B Transcription Factor p50 Critically Regulates Tissue Factor in Deep Vein Thrombosis J. Biol. Chem., February 13, 2009; 284(7): 4473 - 4483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Modarai, J. Humphries, J.A. Gossage, M. Waltham, K.G. Burnand, G.S. Kanaganayagam, A. Afuwape, E. Paleolog, A. Smith, and A. Wadoodi Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution Arterioscler Thromb Vasc Biol, October 1, 2008; 28(10): 1753 - 1759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bonderman, J. Jakowitsch, B. Redwan, H. Bergmeister, M.-K. Renner, H. Panzenbock, C. Adlbrecht, A. Georgopoulos, W. Klepetko, M. Kneussl, et al. Role for Staphylococci in Misguided Thrombus Resolution of Chronic Thromboembolic Pulmonary Hypertension Arterioscler Thromb Vasc Biol, April 1, 2008; 28(4): 678 - 684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. K. Henke, C. G. Pearce, D. M. Moaveni, A. J. Moore, E. M. Lynch, C. Longo, M. Varma, N. A. Dewyer, K. B. Deatrick, G. R. Upchurch Jr, et al. Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model. J. Immunol., September 1, 2006; 177(5): 3388 - 3397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Modarai, K.G. Burnand, B. Sawyer, and A. Smith Endothelial Progenitor Cells Are Recruited Into Resolving Venous Thrombi Circulation, May 24, 2005; 111(20): 2645 - 2653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Henke, A. Varga, S. De, C. B. Deatrick, J. Eliason, D. A. Arenberg, P. Sukheepod, P. Thanaporn, S. L. Kunkel, G. R. Upchurch Jr, et al. Deep Vein Thrombosis Resolution Is Modulated by Monocyte CXCR2-Mediated Activity in a Mouse Model Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1130 - 1137. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||