© 2001 American Heart Association, Inc.
Integrative Physiology |
Mouse Carotid Artery Ligation Induces Platelet-Leukocyte–Dependent Luminal Fibrin, Required for Neointima Development
From the Center for Molecular and Vascular Biology (T.K., H.R.L., I.V., J.V., M.F.H.), University of Leuven and Center for Transgene Technology and Gene Therapy (M.D.), Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
Correspondence to Marc F. Hoylaerts, PhD, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail Marc.Hoylaerts{at}med.kuleuven.ac.be
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Abstract |
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Abstract—The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor–positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28±3.7% (n=11) and 42±5.1% (n=8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII-/- mice, stenosis was significantly lower 1 (11±3.6%, n=8) and 2 (21±4.7%, n=7) weeks after ligation (both P<0.01 versus background-matched controls). In u-PA-/- mice, luminal stenosis was significantly higher 1 (38±7.0%, n=7) and 2 (77±5.6%, n=6) weeks after ligation (P<0.05 and P<0.01, respectively, versus matched controls). In
2-AP-/- mice,
stenosis was lower at 1 week (14±2.6%, n=7,
P<0.01) but not at 2 weeks.
Responses in tissue-type plasminogen activator or plasminogen activator
inhibitor-1 gene–deficient mice equaled that in controls. Reducing
plasma fibrinogen levels in controls with ancrod or inducing partial
thrombocytopenia with busulfan resulted in significantly less
neointima, but inflammation was inhibited only in busulfan-treated
mice. We conclude that stasis induces platelet activation, leading to
microthrombosis and platelet-leukocyte conjugate formation, triggering
inflammation and tissue-factor accumulation on the carotid artery
endothelium. Delayed coagulation then results in formation of a fibrin
matrix, which is used by smooth muscle cells to migrate into the
lumen.
Key Words: platelets • fibrinogen • thrombosis • neointima • tissue factor
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Introduction |
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As early as the 18th century, Hewson demonstrated that blood within an isolated segment of a vein remains fluid for several hours, provided that the vessel is not injured.1 Blood containing veins, ligated for 30 to 60 minutes, attract leukocytes on the endothelium, but although stasis induces leukocyte migration through the endothelial layer, no platelet adherence, aggregation, or thrombus formation can be detected at this stage.2 Only when veins are ligated and left in situ for as long as 24 to 72 hours can fibrin and deposition of leukocytes, platelets, and erythrocytes be detected.3 The existence of hypoxemia in those sites where venous thrombosis commonly originates in humans4 has suggested that hypoxic endothelial damage is involved in the ultimate development of venous thrombosis.
In addition to causing delayed thrombosis, blood flow reduction also enhances intimal lesion formation in vascular grafts and balloon-injured vessels,5 6 7 implying that alterations in blood flow affect the proliferative response of smooth muscle cells. Kumar and Lindner8 have developed a model in which blood flow in the common carotid artery of the mouse is arrested by carefully ligating the vessel near the bifurcation. In this model, a neointima develops proximal to the ligation site as a consequence of partial blood stasis, reduced shear stress, enhanced arterial-wall tension proximal to the suture, and leukocyte activation in the absence of additional mechanical vessel injury. P-selectin gene deficiency in this model leads to dramatic reduction in lesion thickness and complete absence of inflammatory cells in carotid artery cross sections 3 and 7 days after ligation.9 This study concluded that P-selectin is involved in smooth muscle cell migration and proliferation, presumably by mediating leukocyte recruitment and platelet-leukocyte interactions.
Using this carotid artery ligation model, the present study investigates to what extent local coagulation is involved in neointima formation and how coagulation comes about. Using various gene-deficient mice and mice experimentally depleted in plasma fibrinogen or blood platelets, our findings reveal a central role for platelets in mediating tissue factor–dependent coagulation and show luminal fibrin formation to be essential for the development of a neointima.
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Materials and Methods |
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Wild-Type and Gene-Deficient Mice
All animal experiments were reviewed and approved by the Institutional Review Board of the University of Leuven and were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. FVIII gene–deficient mice were kindly provided by Dr Antonarakis (Division of Medical Genetics, University of Geneva, Switzerland).10 The urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and
2-antiplasmin
(2-AP) gene–deficient mice were generated
via homologous recombination in embryonic stem cells in our
department.11 12 13 14
Genotyping of mice was performed by polymerase chain reaction analysis
of tail-tip DNA.12
Experiments on gene-competent mice were all done in C57BL/6J mice. All
mice used in this study were between 8 and 12 weeks old, were of both
sexes, and weighed between 20 and 31 g.
Carotid Artery Ligation
The carotid artery ligation model used was described
elsewhere.8 Dissected
arteries were embedded in OCT compound (Tissue-Tek, Miles Inc),
snap-frozen in precooled 2-methyl butane, and stored at -70°C until
additional use. Seven-micron-thick cross sections were made through the
whole frozen artery and stained with H&E. Approximately 100 sections,
28 µm apart, were analyzed per artery segment.
Induction of Thrombocytopenia in C57BL/6J
Mice
Before a 1-week study, thrombocytopenia was induced
in C57BL/6J mice via double-intraperitoneal injection of busulfan at 20
mg/kg, dissolved in polyethylene glycol 400 diluted with saline to 25%
just before the injection on days 0 and
3.15 Surgery and ligation
were performed on day 14. On days 14 and 21, circulating platelets,
leukocytes, and red blood cells were counted via tail bleeding on a
Cell Dyn 1300 (Abbott). Circulating platelet numbers in normal
C57BL/6J mice were 8.7±0.57x105/µL
(n=8). At day 14 after thrombocytopenia induction, platelet
numbers were 2.1±0.18x105/µL
(n=6), and at day 21, platelet numbers were
3.7±0.41x105/µL (n=6). Before a 2-week
study, thrombocytopenia was induced in C57BL/6J mice via
double-intraperitoneal injection of busulfan at 20 mg/kg on days 0 and
3 followed by intravenous injection on day 4 of 12.5 µL of a rabbit
antimurine platelet antiserum (Accurate Chemical and Scientific
Corporation) via the tail vein, diluted with saline to 100 µL just
before the injection. Surgery and ligation were then performed on day
5. On days 5, 12, and 19, the circulating platelet numbers equaled
3.9±0.48x104 (n=7),
2.3±0.24x105 (n=6), and
3.9±0.18x105 (n=6) platelets/µL,
respectively.
Fibrinogen Depletion by Ancrod in
C57BL/6J Mice
Fibrinogen depletion was induced with the specific
fibrinogen-degrading enzyme
ancrod,16 derived from snake
venom. The plasma fibrinogen concentration was measured via a fibrin
polymerization clotting
assay.17 In an acute or
early depletion experiment, a bolus of ancrod at 40 U/kg was injected
twice via the tail vein, 8 hours before and 2 days after the surgical
ligation procedure. In the chronic or late depletion experiment, the
same dose of ancrod was injected, starting 6 days after the ligation
procedure and repeated on days 8, 10, and 12. In both experiments,
animals were killed 14 days after the ligation procedure. Control
groups in each experimental protocol received vehicle (saline)
injection using the same schedule as the ancrod injections.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Microscopic Changes in Ligated Carotid Arteries
Shortly after ligation, carotid artery cross sections were stained with an antiplatelet antiserum. Figures 1a
and 1b show that the antiserum does not react with
red blood cells in buffy coat or murine spleen cells but strongly
stains a platelet-rich thrombus forming after vessel-wall injury
(Figure 1c). As early as 1 day after ligation, platelet
microaggregates and leukocytes decorated with platelets could be
observed adjacent to the endothelium
(Figure 1d). At 3 days, platelets and platelet-decorated
leukocytes (rosettes) were found lining the endothelium
(Figure 1e) in addition to clear vascular infiltration by
leukocytes
(Figure 2b). Platelet-rich thrombi were present both on the
ligated vessel wall and in the lumen after 1 week
(Figure 1f).
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At day 3, fibrin was mainly deposited on the vessel wall
around the accumulated leukocytes
(Figure 3a). By day 7, fibrin was also present within the
lumen of the vessel of wild-type (wt) mice, surrounding luminal
leukocytes
(Figure 3b), and was abundant in the lumen of
u-PA-/- mice
(Figures 3d through 3f).
Figures 3e and 3f illustrate massive smooth muscle cell
invasion of fibrin clots in these mice. After 2 weeks, the majority of
the proximal luminal space was occupied by migrated smooth muscle cells
(Figure 2d
), with residual narrow luminal channels. At this
time point, leukocyte-rich areas adjacent to the neointima were
positive for fibrin as well as the luminal leukocytes
(Figure 3c).
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Myeloperoxidase and CD45 staining of cross sections taken on
days 3, 7, and 14 identified strongly stained cells primarily in the
adventitia, but positive cells were also found in the lumen as well as
lining the endothelium on day 3 and week 1 (not shown). Tissue factor
was found not only in cross sections of wt-mice carotid arteries in
between the elastic laminae but also within the early fibrin layers on
day 3, where it surrounded the adhering leukocytes. It was also found
associated with luminal monocytoid cells
(Figure 4a). The neointima stained for tissue factor after 2
weeks
(Figure 4d), whereas areas adjacent to the intima and luminal
cells
(Figures 4b and 4c) were found to be strongly positive 1 and 2
weeks after ligation. Tissue factor was strongly associated with
intraluminal monocytes but only weakly with intraluminal
granulocytes.
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Quantitation of Neointima Formation
Image analysis of cross sections of the ligated carotid
artery of control mice revealed no major remodeling of the outer vessel
wall over a period of 3 weeks. No significant change occurred of the
area within the external elastic lamina (EEL) in the immediate
neighborhood of the ligation, ie, in the cross-sectional area where the
degree of smooth muscle cell proliferation was maximal
(Figure 5). In contrast, a significant reduction of the area
within the internal elastic lamina (IEL) was already observed 1 week
after the ligation. Calculation of the media index confirmed that after
1 week, medial thickening had occurred, in agreement with a rapid onset
of smooth muscle cell activation. More importantly, a rapid reduction
of the luminal area was found, corresponding to a progressive and
intense luminal stenosis up to 75%
(Figure 5). These data suggested observation times of 1 and 2
weeks to be optimal for the study of smooth muscle cell proliferation
in response to the modulation of hemostasis in the various
gene-deficient mice. After 1 week of ligation, thrombotic occlusions
were present along with a developing neointima
(Figure 2c), which occupied most of the lumen after 2 weeks
(Figure 2d).
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Gene Deficiencies and Luminal Stenosis
Comparison of the degree of luminal stenosis as a
function of the genetic background identified small differences in the
degree of stenosis 1 and 2 weeks after ligation
(Figure 6). Because the variable degree of C57BL/6J
background limited direct comparisons between different strains, the
degree of stenosis measured in gene-deficient mice was always matched
to that in wt controls with identical genetic background. Thus,
hemophilia A mice showed strongly reduced stenosis at 1 week, when
stenosis was expressed as a percentage of the cross-sectional area
within the internal elastic membrane occupied by neointima (11±3.6%
versus 26±2.8% for matched controls,
P<0.01) and at 2 weeks
(21±4.7% versus 60±3.5% for matched controls,
P<0.01 by Student’s unpaired
t test) despite substantial
medial smooth muscle cell activation (mean media index in
FVIII-/- mice was 0.31±0.02 after 1week
and 0.34±0.02 after 2 weeks). Thus, overall, neointima formation was
3-fold lower as a consequence of the FVIII gene deficiency. In
contrast, neointima formation in u-PA-/-
mice was enhanced at 1 week (38±7.0% versus 19±2.7% for matched
controls, P<0.05) and at 2
weeks (77±5.6% versus 43±8.0% for matched controls,
P<0.01); ie, neointima
formation was increased almost 2-fold (media index in
u-PA-/- mice was 0.4±0.04 after 1 week
and 0.28±0.026 after 2 weeks). No differences were observed between
t-PA-/- mice and matched controls,
although a trend to higher luminal stenosis was found after 2 weeks.
Likewise, no differences were observed in mice deficient in PAI-1. Mice
deficient in the plasmin inhibitor 2-AP
revealed reduced neointima formation at 1 week (14±2.6% versus
26±2.8% for matched controls,
P<0.01) but showed identical
neointimas after 2 weeks
(Figure 6). In all of these cases, the media index was
increased and comparable with that of the matched controls (not shown).
Leukocyte adhesion to the luminal vessel wall in ligated
u-PA-/- and
FVIII-/-
(Figure 2f) mice on day 3 was comparable with that in control
mice, indicating that the early cellular inflammation did not depend on
the amount of fibrin formed per se.
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Neointimal Proliferation and Platelet
Activation
Because deficient luminal fibrinolysis in
u-PA-/- mice was associated with
enhanced smooth muscle cell migration and deficient coagulation in
FVIII-/- mice with a drop in smooth
muscle cell migration, the relation between coagulation and neointima
formation was additionally investigated via platelet depletion.
Figure 7 shows that as a consequence of partial
thrombocytopenia, neointima formation dropped from 30±1.5% in
untreated to 12±2.3% in busulfan-treated animals after 1 week and
from 45±3.9% to 25±4.8% after 2 weeks
(P<0.01, n=6 to 8). The media
index was increased in all cases and was comparable with that of the
control (not shown), confirming that the busulfan treatment had no
impact on smooth muscle cell activation, in agreement with earlier
findings.15 Analysis of
cellular infiltration in thrombocytopenic mice on day 3 revealed almost
complete absence of leukocytes adhering or infiltrating the endothelial
layer
(Figure 2e), in agreement with a critical role of platelets
in inflammation.
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Neointimal Proliferation and Fibrin
Formation
To finally confirm a role for fibrin in smooth muscle
cell migration, fibrinogen-depletion studies were undertaken. Both an
early depletion of fibrinogen (injections of ancrod just before
ligation and on day 2) as well as a later and more chronic fibrinogen
depletion (injection of ancrod on days 6, 8, 10, and 12) reduced
neointima formation, analyzed 2 weeks after ligation
(Figure 8). After early fibrinogen depletion, luminal
stenosis equaled 29±4.4%, compared with 53±8.4% for
vehicle-injected animals
(P<0.01). Thus, early
fibrinogen depletion reduced neointima formation by 
50%. Although
some fluctuation was observed between the early and late control
groups, stenosis in these control groups was not significantly
different (P=0.163), and both
groups did not differ from the ligated noninjected C57BL/6J mice,
analyzed at week 2 (42±5.1%). Delayed ancrod-induced fibrinogen
depletion also was associated with a significant drop in neointima
formation, from 38±5.3% in the vehicle-injected controls to 17±3.4%
(P<0.01) or 45% of the
control value. Fibrinogen depletion had no effect on medial
proliferation (not shown). Likewise, the early fibrinogen depletion did
not interfere with leukocyte adhesion and infiltration on day 3 (not
shown).
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Discussion |
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Our present findings are in line with older observations3 ; ie, blood stasis does not provoke immediate blood coagulation but rather induces delayed thrombosis over 1 to 7 days, coinciding with inflammatory responses in the blood vessel. Therefore, in the present study, we have investigated how platelets and leukocytes participate in the inflammatory response and how cellular inflammation is linked to thrombosis and smooth muscle cell proliferation.
Hypoxia causes initial endothelial cell activation, with a rise in endothelial intracellular Ca2+ concentrations18 and cellular production of, for example, platelet-activating factor.19 Thus, hypoxia can trigger inflammation, because changes in adhesive and inflammatory characteristics of activated endothelial cells result in the recruitment of leukocytes, a process mediated via adhesion molecules such as E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-120 and also P-selectin.21 That P-selectin–mediated cellular interactions are important in the genesis of ligation-induced inflammation is beautifully illustrated by the findings of Kumar et al,9 which show that in P-selectin gene–deficient mice, inflammatory cells do not accumulate in the vessel wall at 3 days after ligation.
Our present findings show that during stasis, activated platelets adhere to leukocytes and that platelet-leukocyte rosettes interact with endothelial cells, interactions which are mediated via platelet P-selectin both for platelet-granulocyte rosettes22 and platelet-monocyte rosettes.23 The critical role of platelets in mediating vessel-wall inflammation by leukocytes was clear from the almost complete absence of adhering leukocytes 3 days after ligation in thrombocytopenic wt mice, indicating that the crucial role of P-selectin in inflammation9 largely pertains to platelet P-selectin–assisted leukocyte deposition on activated endothelium.
Although endothelial cells can be induced to express tissue factor, these observations largely pertain to cell cultures.24 Recent convincing evidence indicates that leukocytes are a source of active blood-borne tissue factor.25 Østerud et al24 have shown that addition of platelets to activated monocytes markedly enhances the tissue factor activity of the monocytes. They suggest that in platelet-monocyte rosettes, the monocyte provides the tissue factor apoprotein and the platelets provide the phosphatidyl serine environment required for tissue-factor activity. This interpretation then explains why inflammation via deposition of platelet-monocyte rosettes delivers tissue factor and causes fibrin formation at day 3 in a fine layer overlying the endothelium, enveloping adherent leukocytes. Luminal monocytes also were positive for tissue factor, explaining why fibrin was found surrounding these cells but not granulocytes. Tissue factor in those cells was upregulated over the 2-week observation period, a finding in agreement with the knowledge that hypoxia upregulates tissue-factor expression in monocytes,26 additionally substantiating that stasis-induced hypoxia is involved in cellular activation processes.
Recently, fibrin has been demonstrated to directly support
smooth muscle cell migration in
vitro.27 Because the present
ligation model, in addition to producing vessel-wall
inflammation–dependent coagulation, also triggers fast and
reproducible smooth muscle cell migration and proliferation, we have
additionally studied the link in this model between coagulation and
smooth muscle cell activity. In contrast to previous
investigations,8 9
we did not analyze the ligated vessel longitudinally but analyzed the
entire vessel cross-sectionally, focusing on the area with the highest
degree of neointima formation, close to the ligation. This may explain
the lack of inward remodeling of the EEL in our model compared with
these studies. We studied neointimal proliferation in various mice
strains with well-characterized gene deficiencies of the coagulation
factor VIII, the fibrinolytic enzymes t-PA and u-PA, and the
fibrinolytic enzyme inhibitors PAI-1 and
2-AP, taking care to correct for differences
in murine genetic backgrounds.
In u-PA-/- mice, ligation induced enhanced fibrin formation, findings in agreement with the occurrence and persistence of mural thrombosis, also in electrically injured arteries of u-PA gene–deficient mice.28 In the injury model, u-PA significantly enhanced arterial neointima formation,28 but in the present study without medial damage, a larger neointima was found in u-PA-/- mice than in wt controls 2 weeks after ligation, at which time point thrombi were completely invaded by smooth muscle cells. Thus, in the present ligation model, u-PA is not required to mediate smooth muscle cell migration, and the larger neointima in u-PA-/- mice rather coincides with enhanced luminal fibrin formation, observed histochemically in the carotid arteries of these mice.
The opposite finding, that reduced fibrin formation in the
ligated carotid artery of hemophilia A mice coincides with strongly
reduced neointima formation, leads us to conclude that fibrin is
required for smooth muscle cell migration into the vessel wall lumen.
Histologically, smooth muscle cells were indeed seen migrating over
fibrin matrix in u-PA-/- mice. These
data support a model in which fibrinolytic modifications only affect
neointima formation by modulating the luminal fibrin content. Thus, the
absence of plasma 2-AP strongly reduced
neointima formation, probably as a consequence of enhanced luminal
fibrinolysis. However, this effect was no longer detectable 2 weeks
after ligation. The absence of t-PA had no impact on smooth muscle cell
activation and proliferation, suggesting that luminal t-PA is
efficiently inhibited in wt mice, in contrast to luminal u-PA. The
absence of PAI-1 had no impact on the degree of smooth muscle cell
activation and proliferation. This finding agrees with our recent
observation that murine PAI-1 is essentially confined to the vessel
wall and present in plasma and platelets only at extremely low
levels.29
Platelet-depletion experiments confirmed that in addition to a reduction in inflammation, there is less neointima formation. Sirois et al,15 studying rats pretreated with busulfan, have already shown that inhibition of neointima formation is mediated by the absence of platelets rather than by drug-related inhibition of smooth muscle cell proliferation. A possible decrease in leukocyte count after busulfan, although statistically not significant, may also have contributed to a reduction in neointima development.
Despite their very different outcome in terms of neointima formation, u-PA-/- and FVIII-/- mice and fibrinogen-depleted mice showed comparable vessel wall inflammation at day 3, confirming that the platelet-leukocyte–dependent inflammation precedes coagulation reactions and neointima development.
Tissue factor is expressed in the newly forming intima, suggestive of continued activation of coagulation throughout the observation period. The experiments with early or delayed fibrinogen depletion resulting in a partial reduction in neointima formation suggest that the persistent presence of fibrin remains crucial. These data confirm that smooth muscle cells use fibrin matrices to migrate. Together with the larger neointima in u-PA-/- mice, these fibrinogen-depletion experiments performed in wt mice additionally show that a deficient generation of thrombin per se is not responsible for the weak neointima development in FVIII-/- mice and that a diminished endothelial cell activation by thrombin30 would not be the basis for reduced neointima formation.
In conclusion, this in vivo study shows that blood stasis and hypoxia cause activation of platelets, endothelial cells, and monocytes. It additionally shows that stasis-induced inflammation largely results from the adhesion to endothelium of rosettes between activated platelets and leukocytes. The concentration on the endothelium of monocytic tissue factor and platelet phospholipids triggers blood coagulation, resulting in the formation of endothelial fibrin layers together with the inflammation. The fibrin matrix, formed over an interval of several days, is then actively used as a support by migrating smooth muscle cells during formation of a neointima. Interruption of inflammation and tissue factor deposition via platelet depletion and reduction of the coagulation efficiency via fibrinogen depletion or a FVIII deficiency all result in reduced fibrin deposition and ultimately in diminished neointima formation.
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Acknowledgments |
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This work was supported by Interuniversitaire Attractiepolen research grant P4/34 and by Flemish Fund for Scientific Research grant G.0306.98N.
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Footnotes |
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Original received July 10, 2000; resubmission received November 7, 2000; accepted December 1, 2000.
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