(Circulation. 2000;102:332.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Thrombomodulin Overexpression to Limit Neointima Formation
From the Department of Cell Biology (J.M.W., J.L.-H., S.L.C.W.), Division of Plastic Surgery (E.Y., P.N.C., S.M.S.), and Department of Comparative Medicine (R.G.), Baylor College of Medicine, Houston, Tex; Stanford Institute of Bioengineering and Molecular Medicine and Department of Radiology, Stanford University School of Medicine, Stanford, Calif (J.M.W., M.D.K., P.R.H., M.D.D.); Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas (J.L.-H.); and Department of Radiology, MD Anderson Cancer Center, Houston, Tex (M.C., J.T.).
Correspondence to Savio L.C. Woo, PhD, Institute for Gene Therapy and Molecular Medicine, Mt Sinai School of Medicine, Box 1496, One Gustave L. Levy Place, New York, NY 10029. E-mail swoo{at}smtplink.mssm.edu
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Abstract |
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Background—These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury.
Methods and Results—An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76±0.06%, whereas viral controls reached 0.77±0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47±0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals.
Conclusions—This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.
Key Words: gene therapy • extracellular matrix • viruses • inflammation • thrombosis
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Introduction |
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Clinically significant restenosis occurs in 40% of balloon angioplasty patients within 6 months and exacts high tolls in survival and medical costs.1 Although processes leading to restenosis are complex, several factors prove critical in underlying neointima formation. Thrombosis represents both an initiator of neointima formation and a significant complication of angioplasty itself. Patients with significant thrombus formation at the time of balloon dilation consistently demonstrate more extensive late lesions,2 3 4 and therapy to decrease platelet aggregation by use of antibodies to the platelet GP IIb/IIIa receptor decreases rates of restenosis.5 Thrombin activates thrombin receptors on platelets, smooth muscle cells (SMCs), and endothelial cells and results in phenotypic cellular activation, growth factor release, and proliferation of each cell type.6 7 8 The thrombotic cascade affects phenotypes of endothelial cells and SMCs,2 9 potentially altering responses of each cell type to mechanical damage and subsequent exposure to growth factor.10 11 Preliminary studies suggest that therapies that use antithrombotic moieties, such as tissue factor pathway inhibitor,12 hirudin,12 NO,13 or prostacyclin,2 14 or inhibiting prothrombotic elements, such as factor VIIa or Xa12 or thrombin,12 limit neointima formation.
Thrombin mediates thrombosis and activates endothelial cells, SMCs, platelets, and macrophages directly to yield enhanced local inflammation and enhanced matrix proteolysis.6 7 8 The primary physiological buffer for thrombin activity in normal vessels is thrombomodulin (THM).
The surface glycoprotein THM, by binding thrombin on a 1:1 or a 2:1 basis15 and increasing inactivation via antithrombin III,16 removes a catalytically active procoagulant. Bound thrombin undergoes a conformational change altering its specificity and allowing conversion of protein C to its active form, a powerful anticoagulant.17 18 In addition to the antithrombotic and anti-inflammatory effects of THM in blocking thrombin, protein C itself adds potent antithrombotic and anti-inflammatory activity. Thrombotic disorders have been found to result from relative deficiency of THM19 or protein C/S,20 21 and homozygous deficiency of THM has been shown be lethal in mice.22 THM is normally present at very high levels on the surface of unperturbed endothelial cells. However, injured endothelial cells express dramatically decreased levels of THM expression, predisposing to thrombotic complications and enhancements of local inflammation and matrix proteolysis. We have previously demonstrated that adenovirus-mediated local overexpression of THM is sufficient to maintain functional THM expression despite certain types of local perturbation.23 In this work, we examine the impact of local overexpression of THM on the processes leading to neointima formation after mechanical dilation balloon injury in the rabbit common femoral artery.
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Methods |
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Recombinant adenovirus containing the Escherichia coli ß-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter (Adv/RSV-ßgal) was kindly provided by Dr Michel Perricaudet (Institut Gustave Roussy, Ville-Juif, France). Recombinant adenovirus expressing human THM under the control of the RSV promoter (Adv/RSV-THM) was constructed as previously described.23 All animal experiments were performed in accordance with NIH and institutional guidelines.
Mechanical Overdilation and In Vivo Adenovirus Delivery
The distal left common femoral artery of each New Zealand White
rabbit was cannulated. A 2-mmx2-cm Cordis SAVVY angioplasty balloon
was introduced and inflated to 6 atm for 1 minute in 2 cycles. The
segment was isolated, and the contents were aspirated before incubation
of 400 µL of saline alone, 400 µL of 5x109
pfu/mL Adv/RSV-ßgal, or 400 µL of 5x109
pfu/mL Adv/RSV-THM (number as detailed for each experiment below).
Local THM Expression Levels
Animals underwent balloon injury and virus delivery as above
(n=3 animals for each group) for PBS, Adv/RSV-ßgal, or Adv/RSV-THM 3
days before vessel harvesting for immunohistochemical evaluation of
sections (1 per segment) with a goat anti-rabbit THM primary antibody
(kind gift of K. Wright, M.D. Anderson Cancer Center, Houston, Tex) at
1:1000 dilution overnight. Paired sections were used as before for CD31
(endothelial staining, Santa Cruz Biotech, at a
dilution of 1:400) or 
-actin (SMC staining, 1A4, DAKO, at a dilution
of 1:75) to identify cell-specific THM distribution after treatment.
Automated immunohistochemistry was performed on a TechMate 500 (Ventana
Medical Systems). A Diagnostic Instruments SPOT true-color
digital camera recorded noninterpolated microscopic images of each
slide at high resolution. The resulting images were analyzed
with the ImagePro Plus analysis system to determine percent THM
expression. Images underwent red channel extraction, thresholding at 4
to 200 (=0), addition of paired CD31 and THM or -actin and THM
images, and counting of areas of interest (double-positive areas
represented by zero), followed by summation. These results
were normalized to total area as determined by area on floating point
conversions of the same image and compared with THM total results
(single stain) to determine nonendothelial/non-SMC
distribution by subtraction. For all experiments, mean and SEM were
determined for each group, with statistical comparisons made with
1-factor repeated-measures ANOVA with significance evaluated at 95%
and with Bonferroni, Tukey-A, and Student-Newman-Keuls post hoc testing
performed in Statview and SPSS 6.1 for the Macintosh (Prentice Hall),
and individual probability values were determined as reported in the
text.
Tissue Harvesting and Preparation and Determination of
Intima-to-Media Ratios
At 7 or 28 days after mechanical dilation and delivery of PBS,
Adv/RSV-ßgal, or Adv/RSV-THM (n=4 per group for 28-day time points
and n=5 for 7-day time points), the treated vessel segment was
harvested. The resulting segment, 
2 cm long, was fixed in 10%
neutral buffered formalin and divided into 3 equal segments. Vessels
from age-matched unmanipulated controls were also harvested for each
time point. Serial cross sections (4 µm) were obtained from the
proximal face of each. Three cross sections per vessel (1 per segment)
were obtained by a blinded observer for each method detailed below.
Routine methods were used for hematoxylin-eosin staining or Verhoeff
elastica staining. Microscopic images were analyzed to
determine the intima-to-media ratio of each on the basis of area
measurements. Negligible neointima formation was confirmed
in contralateral unmanipulated vessels for all groups to validate this
model.
Evaluation of Intra-Arterial Thrombus
Formation
Seven days after mechanical dilation and gene delivery as above,
1 cross section per segment underwent PTAH staining to identify
cross-sectional thrombus area. Total percent cross-sectional thrombus
was determined via image analysis as previously
reported.23 24
Evaluation of Local Extracellular Matrix Elements
Elastic lamina and collagenous connective tissue were
demonstrated with a combination of Verhoeff stain and Masson’s
trichrome stain for collagen (sections from 7-day experiment above).
Total collagen was evaluated after extraction of red channel,
thresholding from 0 to 127, and summation. The results were divided by
the total image area and reported as cross-sectional percent matrix.
Elastin content was determined by floating point conversion,
thresholding at 0 to 35, and summation and confirmed by
single-stained Verhoeff–van Gieson results.
Evaluation of Local Inflammatory Cell Infiltrate
Cross sections at the 7-day time point evaluated above were
obtained (1 per segment) from each vessel and incubated with a primary
monoclonal antibody to rabbit macrophage (RAM-11, 1/800
dilution, DAKO), incubated with a primary monoclonal antibody to rabbit
CD4 (KEN-4, Spring Valley Laboratories), or subjected to chloroacetate
esterase staining to visualize neutrophilic granulocytes as previously
reported.23 Images of each section were acquired to
determine the number of positive cells per cross section. RAM-11 and
esterase images each underwent red channel extraction, thresholding at
0 to 127, and application of a 3x3 circular closing filter once
before counting. The resulting processed images were used to count the
number of positive cells per cross section.
Reproducibility
Neointima formation at 28 days was confirmed in
duplicate experiments with Cordis SAVVY angioplasty balloons and
TEGwire balloons. Across experiments from 2 institutions, 2 different
groups of interventionists, and 2 types of angioplasty balloons, local
delivery of Adv/RSV-THM exhibited statistically significant decreases
in neointima similar to those presented here.
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Results |
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Local THM Expression Levels After Gene Delivery
At virus doses that yielded 20% marker expression in preliminary ß-galactosidase experiments, total cross-sectional THM antigen expression rose to 320% of buffer controls (P=0.0012) and 233% of viral controls (P=0.0026), whereas both controls were statistically similar to one another (P>0.05), as presented in Table 1
.
Significant increases in THM expression were observed
(P<0.05 for all comparisons except THM versus ßgal
endothelial staining) in endothelial
cells, SMCs, and "other" (endothelial cells
or SMCs with weak antigen levels below threshold or other cell types)
relative to control groups that were comparable to one another
(P>0.05 for all comparisons).
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Intima-to-Media Ratios After Overexpression of THM
As presented in Figure 1, right, local overexpression of THM in an in vivo rabbit model decreases
degree of neointima formation after mechanical overdilation
by 37% relative to balloon-injured controls after 28 days
(P=0.003 and P=0.010, respectively, relative to
buffer and viral controls, which were comparable to one another,
P>0.05). This treatment group also exhibits decreases in
neointimal hyperplasia by 43% of buffer control values
after only 7 days in these same experiments, as presented in
Figure 1, left (P=0.009 and P=0.048,
respectively, relative to buffer and viral controls, which were
comparable to one another, P>0.05). Figure 2 presents
representative photomicrographs of Verhoeff–van
Gieson/Masson–stained sections depicting the predominantly cellular
neointima observed at 28 days.
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Intra-Arterial Thrombus Formation After Local
Overexpression of THM
The degree of intra-arterial thrombus formation was
quantified 7 days after mechanical balloon dilation and gene
delivery as described above and summarized as Figure 3. Local overexpression of THM leads to
statistically significant decreases in intra-arterial
thrombus formation relative to both viral and buffer controls
(P=0.0134 relative to buffer controls and
P=0.0324 relative to viral controls).
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Evaluation of Vessel Wall Matrix After Overexpression of
THM
Quantification of elastin and collagenous extracellular matrix was
undertaken at the 7-day time point (Table 2). Immunohistochemical evaluation was
avoided to evaluate class-wide effects on collagenous extracellular
matrix and elastin content via histochemical analyses. Elastin
content in the Ad/RSV-THM group increased by 65% relative to viral
controls (P=0.001) and was statistically similar to buffer
control levels (P>0.05). Collagenous extracellular matrix
was significantly preserved relative to both buffer (66% increase,
P=0.005) and viral controls (54% increase,
P=0.034). Significant matrix preservation occurred in the
Adv/RSV-THM group relative to controls. These effects could be due to
decreases in local inflammatory infiltrate in the THM group or
secondary to decreased cellular activation of
endothelial cells, SMCs, and macrophages via
thrombin receptor. To evaluate the relative contribution of
inflammation, local infiltrate was examined by cellular subtype.
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Local Inflammatory Responses After Overexpression of THM
Infiltration of granulocytes, CD4+ T cells, and
macrophages was evaluated at 7 days, with the results
presented in Table 3. Adv/RSV-THM
vessels did not exhibit statistically significant alterations in
granulocyte infiltration relative to buffer controls
(P>0.05). Viral controls revealed increased infiltration of
potentially elastase-degrading granulocytes relative to both
Adv/RSV-THM (P=0.040) and buffer controls
(P=0.049). THM prevented the ADV-mediated increase in
granulocyte infiltration observed in viral controls. No statistically
significant differences in number of CD4+ T cells occurred between
groups (P>0.05 for all comparisons). Interestingly,
substantial decreases in local macrophage infiltration occurred
with Adv/RSV-THM treatment relative to both viral and buffer controls
(P=0.001 for each).
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Discussion |
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A great deal of recent evidence suggests that thrombin-mediated proteolytically activated receptor (PAR) activation may play roles in thrombosis, atheroma development and progression, and neointima formation. Thrombin activates these receptors on platelets, SMCs, and endothelial cells and results in phenotypic cellular activation, growth factor release, and proliferation/migration of each cell type.6 7 8 In normal arteries, THM binds thrombin and prevents PAR activation while also catalytically activating protein C, which adds additional antithrombotic and anti-inflammatory effects. In this work, we demonstrate that local overexpression of THM limits neointima formation at 7 and 28 days. Potential contributions to these effects include significant reductions in local thrombus, preserved local extracellular matrix, and decreased local inflammation after THM treatment.
Previous work has validated the use of low-dose adenovirus as a delivery system that can be used with minimal adverse impact on THM expression levels.23 Although the virus doses used here yielded 20% marker expression in preliminary ß-galactosidase experiments, total THM antigen expression rose to 320% of buffer controls and 233% of viral controls with significant percentage of expression that is non–endothelium-based, as described in previous adenovirus-mediated gene transfer experiments after angioplasty. Thus, high levels of Adv/RSV-THM–produced THM expression were achieved throughout these injured arteries. Differences between levels of THM expression and the level of marker expression are most likely due to decreases in control THM expression after injury, as well as to high-level expression achieved per transfected cell in THM treatments. However, because the antibody used recognizes both the human and rabbit antigens, the differences between marker expression levels and THM epitope availability could be due in part to differential antibody reactivity.
Because thrombus formation has been shown to enhance both neointima formation and clinical restenosis after angioplasty, degree of intra-arterial thrombus formation was evaluated histologically. In the present model, local overexpression of THM results in decreased local thrombus bulk. Given that thrombosis represents a significant clinical complication in up to 20% of angioplasties, this therapy may eventually offer survival benefits beyond even the observed reduction in neointima formation.25
Significant early collagen and elastin preservation were also observed in the THM group relative to controls. Normal extracellular matrix provides a primary functional and architectural barrier to the smooth muscle and inflammatory cell migration that, in part, characterize neointima formation. Moreover, several elements of normal extracellular matrix have been shown to prevent smooth muscle mitogenesis directly, even blunting responses to proliferative stimuli as strong as platelet-derived growth factor (PDGF)-BB.26 27 28 Degraded matrix elements, in turn, actually directly stimulate smooth muscle cell migration and proliferation under certain conditions.26 27 28 29 Matrix-degrading enzymes, such as matrix metalloproteinases and elastase, have been implicated in neointima formation, and therapies targeting each of these enzymes have significantly decreased neointima formation after mechanical overdilation injury.30 31 Local overexpression of THM thus not only offers antithrombotic effects but also provides significant matrix preservation, which may blunt neointima formation.
Previous work has demonstrated that adenovirus doses substantially
higher than those presented here result in vigorous local
inflammatory responses,32 33 whereas our relatively
low-dose strategies achieve therapeutic effects without these high
rates of inflammation.23 Consistent with these
conclusions, no increases in local neutrophil or CD4+ T-cell infiltrate
were observed in Adv/RSV-THM animals relative to controls.
Interestingly, significant decreases in local macrophage
infiltration occurred in the THM group relative to both controls. Both
THM-mediated binding of thrombin (to prevent PAR activation) and
protein C activation probably contribute to decreased local
inflammatory infiltrate and decreased inflammation-mediated damage,
because thrombin binding to thrombin receptors (inhibited by THM
binding) has been shown to enhance local inflammation through
alteration of endothelial adhesion molecule
expression,34 35 and protein C itself has been shown to
possess some direct anti-inflammatory properties.36 Recent
views of atherosclerosis as an inflammatory disease
have also emphasized the role of inflammation in neointima
formation and clinical restenosis.26
Macrophages and T cells have both been associated with
restenosis and neointima formation and underlying
processes.37 38 39 Macrophages increase local matrix
proteolytic enzymes, PDGF-BB, monocyte chemotactic protein (MCP)-1,
tumor necrosis factor- (which, in turn, can induce
macrophage colony–stimulating factor, MCP-1, tissue factor,
epidermal growth factor, and other growth regulators),39
and local tissue factor levels, each of which also affects smooth
muscle mitogenesis.40 T cells add 
-interferon and
transforming growth factor-ß to further increase the complex local
growth factor and cytokine/lymphokine profile with broad
downstream effects.39 41 42 43 Blockade of local
inflammatory infiltration and function each have been shown to limit
neointima formation after balloon
injury.44 45 46 Thus, the decreases in neointima
formation observed in THM-treatment animals may also be due, in part,
to decreased local macrophage infiltration.
At Adv/RSV-THM doses that afford only modest increases in THM expression, we have previously reported substantial decreases in thrombosis in response to complex injuries.23 Here, we find that the same doses afford protection against neointima formation after mechanical overdilation injury via multiple pathways. In broader terms, local overexpression of THM reverses the decreases in THM expression that accompany local perturbation and, in turn, preserves more normal phenotypic arterial resistance to local inflammation, thrombosis, and matrix remodeling.
Given that therapies that individually target inflammation, thrombosis, and matrix remodeling have met with success in limiting neointima formation, such multitarget strategies as THM may offer significant improvements. Realistically, all these processes probably play roles in patients undergoing balloon angioplasty. However, one process may prove to be central in some patients, whereas another becomes more important in other patients. Because it will be difficult to identify each group a priori, therapies that target only one may be relatively limited in a broad cross section of patients. In contrast, such therapies as THM may prove effective in limiting neointima formation in each of these environments and may thus be applied more broadly.
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Acknowledgments |
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This work was supported in part through NIH HL-50422 (Dr Woo), Swiss National Science Foundation Fellowship (Dr Hilfiker), and the Holderbank Foundation (Dr Hilfiker).
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Footnotes |
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Guest Editor for this article was Ralph N. Nachman, MD, Cornell University, New York, NY.
Received September 21, 1999; revision received January 21, 2000; accepted February 10, 2000.
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