Published online before print May 9, 2005, doi: 10.1161/CIRCULATIONAHA.104.492678
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(Circulation. 2005;111:2645-2653.)
© 2005 American Heart Association, Inc.
Vascular Medicine |
Endothelial Progenitor Cells Are Recruited Into Resolving Venous Thrombi
From the Academic Department of Surgery, Cardiovascular Division, King’s College, London, United Kingdom.
Correspondence to Dr Alberto Smith, Academic Department of Surgery, St Thomas’ Hospital, 1st Floor North Wing, Lambeth Palace Rd, London SE1 7EH, United Kingdom. E-mail alberto.smith{at}kcl.ac.uk
Received July 20, 2004; revision received January 12, 2005; accepted January 20, 2005.
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Abstract |
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Background— The purpose of this study was to determine whether endothelial cells of bone marrow origin are involved in thrombus recanalization.
Methods and Results— Irradiated mice were reconstituted with bone marrow from transgenic donors expressing green fluorescent protein (GFP) linked to the Tie2 promoter. Thrombi were formed in 2 groups of 6 mice. GFP-expressing cells were located and quantified in sections of the thrombi taken after 7 and 14 days. The cell markers Mac-3, F4/80, CD68 (macrophage), and vascular endothelial growth factor receptor 2 (VEGFR2; endothelial cells) were used to determine colocalization with GFP expression in tissue sections and peritoneal macrophages. The markers CD34 and VEGFR2 were used to quantify changes in circulating endothelial cells by flow cytometry of blood from 3 cohorts of wild-type animals that had either a thrombus induced (n=18), a sham operation (n=18), or no operation (n=10). The number of GFP-expressing cells was found to increase by 
3-fold in thrombi formed in transplanted animals between 7 and 14 days after induction (P=0.0022). No GFP-expressing cells were found lining the new vascular channels that formed at either time interval, but many of the GFP-expressing cells also expressed Mac-3, CD68, and VEGFR2. Approximately twice as many circulating CD34+/VEGFR2+ cells were found by day 3 in animals with thrombus compared with sham controls (CD45–, P=0.046 and CD45+, P=0.016).
Conclusions— Bone marrow–derived, Tie2-expressing cells were recruited into the thrombus during resolution but did not line the new vessels. Many of these cells expressed a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalization.
Key Words: thrombus • cells • revascularization • angiogenesis
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Introduction |
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Venous thrombi resolve by a process that is similar to the formation of granulation tissue in wound healing.1 Neutrophils, monocytes, and endothelial cells enter the thrombus as it organizes.2,3 In the initial stages of organization, the thrombus retracts away from the vein wall, which leads to the appearance of peripheral pockets and clefts that enlarge with time and eventually become lined by cells.4,5 These channels coalesce and enlarge, and blood flow is established through and around them.1 New vessels also appear within the body of the thrombus and contribute to restoration of a patent vein lumen (see Data Supplement Figure). This is associated with an increase in local expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, which are thought to drive neovascularization within the thrombus.6
Bone marrow–derived circulating endothelial progenitor cells appear to play an important part in physiological and pathological neovascularization in the adult.7–9 They may also have a role in thrombus resolution, because thrombus failed to resolve in urokinase-type plasminogen activator knockout mice but was restored to normal by transplantation of normal bone marrow.10
Tie-2 is a tyrosine kinase receptor that is expressed on endothelial cells and is essential for normal vascular development in the embryo and for stabilization of blood vessels in the adult.11 In FVB/N-TgN (Tie2/GFP) 287 Sato transgenic mice, the reporter gene green fluorescent protein (GFP) is expressed under transcriptional regulation by the Tie2 receptor promoter, and therefore green fluorescence can be used to identify cells that express the Tie2 receptor.12 In wild-type mice transplanted with bone marrow from the transgenic strain, the presence of GFP signifies a Tie2-positive cell that has been derived from the bone marrow rather than local tissues.
The purpose of the present study was to determine whether the new vessels that appear during thrombus resolution develop by angiogenesis from local endothelial cells in the vessel wall beneath the thrombus or originate from bone marrow–derived endothelial progenitors. Fluorescence-activated cell sorter (FACS) analysis was used to quantify changes in the numbers of circulating endothelial cells in animals with organizing venous thrombi.
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Methods |
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Bone Marrow Harvest and Transplantation
The study was performed under the Animals (Scientific Procedures) Act 1986. Bone marrow cells were obtained from donor Tie2/GFP mice (Jackson Laboratory, Bar Harbor, Me) by flushing the femurs and tibias of age- and sex-matched (8-week-old male) mice. The cells were resuspended in cold PBS and counted on a hemocytometer. Background FVB/N wild-type mice (Harlan, Oxfordshire, UK) were lethally irradiated with 10.0 Gy and received 5x106 bone marrow cells by tail vein injection. Transplanted mice (Tie2/GFP/BMT) were left for 4 weeks to allow complete reconstitution of the bone marrow.
Mouse Model of Venous Thrombosis
Laminated thrombus was produced in the inferior vena cava (IVC) of mice with a flow model of venous thrombosis described previously.10 A midline laparotomy was performed to expose the infrarenal portion of the IVC. A 5-mm segment of the IVC just below the left renal vein was dissected. A neurosurgical vascular clip (Braun Medical) was applied to this portion of the vein for 15 seconds on 2 occasions, 30 seconds apart, to induce endothelial damage. A length of 5-0 polypropylene suture was then placed alongside the IVC. A 4-0 silk ligature (Ethicon Ltd), passed around the IVC just below the left renal vein, was tightened and tied, incorporating the polypropylene suture. The polypropylene was then withdrawn, which left a stenosis in the IVC while allowing some flow within it.
Tissue Harvesting and Histology
Animals were reanesthetized at 1- and 2-week intervals (n=6 per group) and perfusion fixed transcardially with 4% paraformaldehyde after a saline flush. The IVC was harvested from just above the ligature to the confluence of the common iliac veins. This tissue was placed in 4% paraformaldehyde overnight followed by 24-hour fixation in 18% sucrose. Samples were embedded in OCT compound, snap-frozen in liquid nitrogen, and stored at –80°C. Transverse frozen sections (5 µm) of the IVC containing thrombus were obtained at 200-µm intervals throughout the length of the sample. This produced between 10 and 13 sections of each thrombus for analysis (some 60 sections per group). Tissue sections were mounted with Vectashield mounting medium that contained the nuclear stain DAPI (Vector Laboratories).
Image Acquisition and Analysis
Sections were viewed at x400 magnification with a DAPI and a GFP filter set (Leica). Images obtained with each filter set were captured with a microscope-mounted CoolSnap-Pro CF color digital camera and ProScan motorized stage (Datacell). High-magnification images were obtained of the whole section area and tiled to make a single composite with image-analysis software (Image-Pro Plus, Media Cybernetics). The area of the section that contained DAPI or GFP fluorescence within each thrombus section was quantified with the same software. These values were expressed as a fraction of the total thrombus area at each level, and an average value was calculated for the entire thrombus.
Colocalization of GFP With Endothelial and Macrophage Markers
Sections of thrombus and peritoneal macrophages from transplanted mice were used to colocalize GFP expression with endothelial and macrophage specific antigens. The thrombi were formed in Tie2/GFP/BMT mice as already described and harvested after 2 weeks. Thrombi were fixed in 4% paraformaldehyde for 1 hour and processed as described above. Macrophages were obtained from 8-week-old mice by intraperitoneal injection of 3 mL of thioglycolate medium.13,14 After 4 days, macrophages were harvested from the peritoneal cavity by lavage with 10 mL of PBS. The cells were counted, cytospun onto glass slides, and fixed in 4% paraformaldehyde. GFP-positive cells were identified with either rabbit anti-GFP (ab290; Abcam Ltd) or mouse anti-GFP (Molecular Probes). Tissue sections were immunostained with rat anti-Mac-315 (BD Pharmingen) and mouse anti-CD68 (Dako Cytomation) antibodies to identify macrophage markers. Endothelial cells were colocalized with rabbit anti-VEGFR2 (Santa Cruz Biotechnology). Peritoneal macrophages were stained with rat anti-Mac-3 and rat anti-F4/8016 (Serotec) antibodies. Alexa Fluor 568-conjugated (goat anti-rat and goat anti-rabbit) and Alexa Fluor 488 (goat anti-mouse and goat anti-rabbit) were used as secondary antibodies (Molecular Probes). Rabbit IgG (Dako Cytomation), mouse IgG (relevant isotype), and rat IgG (Sigma) were used as negative controls.
The expression of GFP and endothelial (VEGFR2) and macrophage (CD68 and Mac-3) markers by cells in the thrombus was quantified by counting cells in 5 high-power fields (x400 magnification) in serial sections taken from the thrombus. The mean percentage of cells coexpressing the various markers was calculated.
Detection of Circulating Endothelial Cells
Thrombus was formed in 8-week-old FVB/N wild-type mice. A sham-operated group of animals underwent the same procedure, in which a silk ligature was passed around the IVC but left untied. One set of animals from each cohort (n=18) was killed 3 days after operation and another (n=18) at 7 days. Samples (1 mL) of whole blood were collected from each animal by cardiac puncture. Blood was also obtained in an identical fashion from a group (n=10) that had not undergone any procedures.
Flow Cytometry
The effect of operation and the presence of thrombus within the IVC on circulating endothelial cells were studied by FACS analysis with antibodies raised against CD45,17 CD34, and VEGFR2 (BD Biosciences). Previous evidence from murine experiments has shown that a proportion of CD34-positive cells are endothelial cells.7,18,19 Location of the vascular endothelial growth factor receptor-2 (VEGFR2, FLK-1) was used to confirm an endothelial cell phenotype.9,20
PerCP-CY5.5–conjugated rat anti-mouse antibody was used to detect CD45+ hematopoietic cells. Endothelial cells within both the CD45-positive and -negative cell populations identified with anti-VEGFR2 and anti-CD34 were detected with a R-phycoerythrin–conjugated and fluorescein isothiocyanate-conjugated rat anti-mouse antibody, respectively. A mouse Fc receptor blocker was added to each whole blood sample and incubated for 5 minutes before addition of each of the antibodies and incubation for 20 minutes at room temperature. After they were washed in PBS, red blood cells were lysed by addition of ammonium chloride lysing reagent and incubation for 10 minutes. Cells were then fixed by resuspension in 0.1% BSA in DPBS containing 0.04% paraformaldehyde. The stained cells were enumerated with 3-color flow cytometry on a FACScan flow cytometer (Becton Dickinson). Gates were used to exclude debris, platelets, and dead cells before acquisition of 1 million events per sample and recording of the percentage of the total number of cells counted that were CD45–/CD34+/VEGFR2+ and CD45+/CD34+/VEGFR2+.
Statistical Analysis
Differences in GFP or DAPI expression at the 2 time intervals chosen were quantified by a blinded observer and compared with the Mann-Whitney U test. All results quoted are expressed as means with SEM. The flow cytometry data were normally distributed, and therefore, an unpaired t test was used to compare the percentage of cells in each of the groups.
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Results |
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Tie2/GFP Cells in Resolving Thrombus
A laminated thrombus had developed in all 12 animals. This structure was composed of layers of fibrin interspersed with platelets and red cells and was consistent with previous observations using this model in the mouse, rat, and human.3,10 The development of channels in and around the thrombus began as early as 7 days after thrombus formation (Figure 1).
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Sections of the thrombus taken from all of the transplanted animals contained cells that expressed GFP. The majority of these cells were situated around the perimeter of the thrombus at day 7. By day 14, a larger number of Tie2-positive cells were seen, and some had penetrated the entire thrombus (Figure 2). The quantity of GFP fluorescence was 1.00±0.13% at day 7 compared with 3.04±0.66% by day 14 (n=6, P=0.0022). This was associated with an increase in total cellularity of the thrombus, confirmed by an increase in DAPI fluorescence at day 14 (15.90±2.5%) compared with day 7 (6.30±0.75%, n=6, P=0.0087; Figure 3).
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The peripheral channels seen around the thrombus at 1 week were not lined by GFP-positive cells. Additional areas of recanalization were apparent within and around the thrombus at 2 weeks, and although GFP positive cells were situated near some of these, they again did not line the new channels.
Colocalization of GFP With Endothelial and Macrophage Markers
Tissue Sections
The distribution of Mac-3–positive and CD68-positive cells was similar to that observed previously during natural thrombus resolution in both rat and mouse models.3,10 Macrophages were present mostly at the periphery of the thrombus at 1 week and throughout the thrombus at 2weeks. Almost half of the Mac-3–positive (45.2±1.9%) and CD68-positive (49.6±1.4%) cells also expressed GFP (Figures 4 and 5
). The majority of GFP-expressing cells (92.0±0.7%) in the thrombus also stained positively for VEGFR2 (Figure 6). The proportion of GFP-positive cells expressing Mac-3 and CD68 was 78.8±2.6% and 74.6±3.2%, respectively. The thrombus also contained Mac-3–positive cells (43.4±2.0%) that coexpressed VEGFR2 (Figure 7).
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Peritoneal Lavage
Some of the macrophages (F4/80 and Mac-3 positive) obtained from peritoneal irritation in Tie2/GFP mice coexpressed GFP (Figure 8).
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Circulating Endothelial Cells
FACS analysis demonstrated an almost 2-fold increase in the number of circulating VEGFR2+/CD34+ cells in the CD45– fraction at day 3 in animals with thrombus in situ (0.581±0.097%) compared with those after sham operation (0.33±0.072%, P=0.046; Figure 9, A and C). By day 7 after operation, there was no difference in either of the 2 groups. The mean percentage of VEGFR2+/CD34+ circulating cells in animals with thrombus in situ (at day 3 and 7) was 9-fold higher than in nonoperated mice (0.064±0.021%, P=0.0006 at day 3 and P=0.022 at day 7).
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The number of VEGFR2+/CD34+ cells in the CD45+ fraction at day 3 was also higher in animals with thrombi in situ (11.89±2.2%) than in sham-operated animals (5.89±0.99, P=0.016; Figure 9, B and D). Again, by day 7, there was no detectable difference between the 2 groups.
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Discussion |
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Cells expressing the fluorescent protein GFP under regulation by the endothelial cell–specific Tie2 promoter were seen entering the periphery of the thrombus 7 days after thrombus induction and had migrated through the entire body of the thrombus by day 14. This suggests that Tie2 (which denotes endothelial lineage)–expressing bone marrow progenitors are recruited into organizing thrombus, but these cells express an unexpected macrophage phenotype. Bone marrow–derived progenitors, however, did not line the new recanalizing channels that form either between the thrombus and the vein wall or within the thrombus itself in any of the sections taken throughout the length of the thrombus. This leads to the speculation that the revascularizing processes that take place during thrombus resolution involve mature endothelial cells derived from the vena venora (microvessels that supply the vein wall) and that the stimulus for this may be provided by the bone marrow–derived progenitors found within the thrombus. This concept appears to be in accordance with the work of Ziegelhoeffer et al,21 who showed that bone marrow–derived progenitors that are recruited into areas of ischemia do not integrate into newly formed vessels but express a variety of cytokines (including VEGF and fibroblast growth factor-2) that may stimulate angiogenesis.
The presence of bone marrow–derived endothelial progenitor cells in resolving thrombus has been reported previously.22,23 These cells were found in the thrombus at 7 days, and in contrast to the findings in the present study, some were able to differentiate into mature endothelium and eventually line the vascular channels that appeared; however, an occlusive model of thrombosis was used in these experiments. This model produces a clot that may resolve in a manner different from the laminar thrombus induced in the flow model of thrombosis used in the present study. The latter is more appropriate, because it produces thrombi with a structure consistent with that seen in humans.
The majority of GFP-positive cells that were recruited into the thrombus were large mononuclear cells and did not have the flattened, spindle-shaped morphology typical of endothelial cells. The shape and distribution pattern of GFP-positive cells at 1 and 2 weeks resembled that of macrophages. The vast majority of GFP-expressing cells in the thrombus expressed the endothelial cell marker VEGFR2.24,25 GFP also colocalized with the mouse macrophage–specific antigen, Mac-3,15,26 and CD68 (a classic macrophage marker) in approximately three fourths of the fluorescent cells. Analysis of the numbers of macrophages (CD68- and Mac-3–positive cells) expressing GFP and VEGFR2 revealed that GFP and VEGFR2 were coexpressed in just less than half of these. This suggests that the bone marrow–derived cells that are recruited into thrombus express a mixed macrophage (Mac-3+,CD68+) and endothelial (Tie2+, VEGFR2+) phenotype. Expression of Tie2 by macrophages outside the thrombus environment was also found in these animals by demonstration of colocalization of GFP and the macrophage markers F4/80 and Mac-3 in cells harvested from the peritoneum after stimulation with thioglycolate.
The regulation of Tie2 expression on bone marrow progenitor cells is poorly understood. It is thought that the hematopoietic and endothelial lineages develop in close proximity,27 and therefore, the Tie2/GFP population of cells may be heterogenous. Experiments with the FVB/N-TgN (Tie2/GFP) 287 Sato mouse have revealed a subset of Tie2/GFP cells that coexpress the hematopoietic marker CD45, as well as the endothelial markers CD34, VEGFR2, and CD31.28 A number of recent studies have suggested that circulating endothelial progenitor cells and hematopoietic stem cells, rather than exhibiting strict lineage commitment, maintain a high degree of plasticity and are able to transdifferentiate under appropriate stimulation in different microenvironments. Pluripotent stem cells derived from peripheral blood monocyte lineage express endothelial cell receptors and take on their morphology when cultured with VEGF in vitro.29 A population of "circulating angiogenic cells" derived from the monocyte-macrophage lineage but capable of expressing endothelial cell characteristics has also been described.30 Stimulation of bone marrow–derived monocyte lineage cells with VEGF causes them to transdifferentiate into endothelial-type cells, with a marked increase in expression of the Tie2 receptor on the surface of the cells.31 Treatment of peripheral blood monocytes with proangiogenic cytokines promotes transdifferentiation into cells that express endothelial markers such as von Willebrand factor, vascular endothelial cadherin, and endothelial cell nitric oxide synthase.32 We have previously shown that VEGF and basic fibroblast growth factor levels (produced mainly by a monocytic cell infiltrate) increase during thrombus resolution.6 We have also shown that monocyte infiltrates (CD68-positive cells) are generally found in areas of dense microvascular channel formation in human venous thrombi.3 One could speculate, therefore, that the thrombus microenvironment promotes the transdifferentiation of bone marrow–derived precursors into cells with a mixed phenotype (macrophage and endothelial) that promote neovascularization. Bone marrow–derived cells with macrophage characteristics (F4/80+ and CD45+) have been located around areas of collateral vessel growth in animal models of tissue ischemia.21,33 These cells were found to secrete proangiogenic cytokines (VEGF, fibroblast growth factor-2, and monocyte chemotactic protein-1), and it was suggested that the cells have a role in orchestrating neovascularization.
An increase in circulating endothelial cell (CD34+/VEGFR2+) numbers was also observed during thrombus resolution in the present study. Previous reports have analyzed endothelial cells in both the CD45+ and CD45– populations.34–36 However, the differences in circulating endothelial cell numbers in the present study were similar whether the CD45– or CD45+ populations were analyzed. Approximately double the number of circulating endothelial cells were found 3 days after thrombus formation compared with sham-operated animals. These circulating cells may represent a population of both progenitor and mature endothelial cells. Circulating mature endothelial cells have been identified,37 but little is known about how long they persist in the circulation. The greater number of circulating endothelial cells observed in animals with a thrombus compared with those having a sham operation may simply reflect the degree of injury to the vena cava caused by the formation of a substantial thrombus. The findings of the present study confirm those of our previous investigations10 and suggest that bone marrow–derived cells are mobilized in the presence of thrombus and are subsequently recruited from the circulation into the thrombus. The mechanisms by which these cells are mobilized remains to be elucidated.
The data from the present study support the concept that circulating progenitor cells may play an important part in the resolution of venous thrombi, but a direct association between endothelial progenitor cells and thrombus neovascularization was not found. Stem cell plasticity, however, is an emerging concept that may account for the unexpected location and receptor expression of the bone marrow–derived cells found in organizing venous thrombus.
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Acknowledgments |
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Mr Modarai was supported by grants from The British Heart Foundation and the Guy’s and St Thomas’ Charitable Foundation.
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Footnotes |
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The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.104.492678/DC1.
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References |
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