(Circulation. 2000;102:III-289.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Gene Transfer of the Serine Elastase Inhibitor Elafin Protects Against Vein Graft Degeneration
From the Program in Cardiovascular Research (S.B.O., S.H.E.Z., A.Y.L.C., B.M., M.R.), The Hospital for Sick Children, and Departments of Pediatrics (M.R.), Laboratory Medicine and Pathobiology (S.B.O., S.H.E.Z., M.R.), Medicine (M.R.), and Cardiovascular Surgery (S.B.O.), University of Toronto, Toronto, Ontario, Canada; and Institute for Molecular and Cellular Biology (Y.K.), Osaka University, Osaka, Japan.
Correspondence to Dr Marlene Rabinovitch, Division of Cardiovascular Research, Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8. E-mail mr{at}sickkids.on.ca
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Abstract |
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Background—Leukocyte infiltration and serine elastase activity lead to smooth muscle cell proliferation in association with posttransplant coronary arteriopathy and may also be involved in vein graft neointimal formation. A number of therapies have targeted cellular proliferation, but the inhibition of serine elastase–mediated extracellular matrix remodeling has not been investigated as a potential strategy to prevent neointimal formation and subsequent atherosclerotic degeneration in vein grafts.
Methods and Results—We studied jugular vein grafts 48 hours after
interposition into the carotid arteries of rabbits and demonstrated
inflammatory cell infiltration and elevated serine elastase
activity, a stimulus for matrix remodeling and deposition of elastin.
Therefore, elastolytic activity in vein grafts was targeted through
transient expression of the selective serine elastase
inhibitor elafin with hemagglutinating virus of Japan
liposome–mediated gene transfer. Elafin transfection reduced
inflammation by 60% at 48 hours and neointimal formation
by 
50% at 4 weeks after implantation. At 3 months, a 74% decrease
in neointimal elastin deposition correlated with protection
against cholesterol-induced macrophage infiltration
and lipid accumulation, which were both reduced by 50% in
elafin-transfected grafts relative to controls.
Conclusions—Gene transfer of the selective serine elastase inhibitor elafin in vein grafts is effective in reducing the early inflammatory response. Although transient expression of elafin delays neointimal formation, it is also sufficient to cause an alteration in elastin content of the extracellular matrix, making it relatively resistant to atherosclerotic degeneration.
Key Words: atherosclerosis • hypercholesterolemia • leukocytes • plaque • remodeling • genes • elafin
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Introduction |
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The long-term effectiveness of vein grafts in the surgical treatment of coronary artery disease is limited by atherosclerotic degeneration.1 The rapidity with which this complication sometimes develops is reminiscent of the neointimal formation that occurs in coronary arteries after heart transplantation that is associated with inflammation and heightened serine elastase activity.2 3 Serine elastases activate4 and release extracellular matrix (ECM)-bound growth factors,5 and elastin degradation products enhance fibronectin expression.6 7 These features are critical for smooth muscle cell (SMC) proliferation and migration, respectively. Elafin is a 6-kDa peptide found in human skin that has potent and specific inhibitory activity against serine elastases.8 The administration of elafin to rabbits after heart transplantation limits neointimal formation in coronary arteries by preventing both the migration and proliferation of vascular SMCs.3
We now show that inflammatory cell infiltration is associated with serine elastase activity in rabbit vein grafts. Gene transfer with elafin before implantation reduces early inflammation, and this limits the later development of neointimal formation at 4 weeks. Moreover, the transient expression of elafin that we have documented for only 1 week after gene transfer induces a sustained modification of the neointimal ECM composition that is evident at 3 months and characterized by reduced deposition of elastin. This is associated with protection against cholesterol-induced infiltration of macrophages and deposition of lipid. The present study is the first to implicate serine elastases in bypass vein graft neointimal formation and the accumulation of neointimal elastin in atherosclerotic degeneration, thereby suggesting a potential therapeutic strategy for the enhancement of graft longevity.
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Methods |
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Animal Model
Sixty-seven New Zealand White rabbits (3.5 to 4 kg) were anesthetized with halothane (Halocardon Laboratories) and heparinized (1000 U IV; Organon Teknika Inc). The right external jugular vein was excised and treated ex vivo as outlined here. With the aid of a dissecting microscope, a 1- to 2-cm segment of right common carotid artery was excised, and the external jugular vein was inserted as an interposition graft in the reversed orientation with interrupted 10-0 nylon sutures. A single vein graft was placed in each animal. Penicillin (15 000 U Penlong XL; Rogar) and buprenorphine (0.05 mg/kg; Reckitt & Colman Pharmaceutical Inc) were administered subcutaneously at the end of the procedure. Animals received standard care in compliance with guidelines formulated by the Canadian National Society for Medical Research. Animals were fed a standard laboratory diet (n=55) or a high-cholesterol diet (n=12) (0.5%; Ren’s Feed & Supplies Ltd) as indicated in Results.
Elafin and Chloramphenicol Acetyltransferase Expression
Vectors
The cDNA encoding the amino acid sequence and 3' untranslated
region of human elafin was obtained from Dr J.-M. Sallenave (University
of Edinburgh, Edinburgh, Scotland). The 5' untranslated region was
cloned, and the coding sequence was modified to produce a
carboxyl-terminal FLAG epitope–tagged fusion protein. The preserved
serine elastase inhibitory activity of FLAG-tagged
recombinant elafin has been previously documented.9 The
elafin/FLAG cDNA was ligated into the vector pcDNA3 (Invitrogen Corp)
with expression driven by the cytomegalovirus promoter. A negative
control vector was created through ligation of the cDNA encoding the
bacterial protein chloramphenicol acetyltransferase (CAT) into the same
parent plasmid. Plasmid DNA preparation was performed with a commercial
kit (Maxiprep; Qiagen).
Preparation of Hemagglutinating Virus of Japan Liposomes
The preparation of hemagglutinating virus of Japan (HVJ)
liposomes has been previously described in detail.10
Briefly, plasmid DNA was incubated with the chromosomal protein
high-mobility group 1 (Wako Pure Chemicals Industries), incorporated
into liposomes (cholesterol, phosphatidylcholine, and
phosphatidyl serine sodium; Sigma Chemical Co) and incubated with
inactivated HVJ. Purified HVJ liposomes were resuspended in
balanced salt solution (8 g NaCl, 0.4 g KCl, 1.21 g Tris
base/L H2O, pH 7.6).
Transfection of Rabbit Jugular Vein Grafts
Before implantation, the veins were flushed with the HVJ
liposome solution (0.4 mL, 67 µg DNA/graft) and then incubated in the
effluent for 10 minutes, with both the adventitial and
endothelial surfaces exposed to the transfection
solution. Grafts were either transfected with the elafin expression
vector or the CAT negative control vector or incubated with normal
saline alone.
Tissue Preparation
Rabbits were killed with an anesthetic overdose (Euthanol; MTC
Pharmaceutical). Vein grafts were excised and fixed by perfusion with
2% paraformaldehyde in PBS at 70 mm Hg before
being embedded in paraffin. A segment was also embedded for frozen
sectioning. A group of grafts and normal veins were frozen in liquid
nitrogen for assay of elastase activity.
Assay of Serine Elastase Activity
Tissues were homogenized in buffer (1 mol/L
NaCl with 2 mmol/L N-methylamine), and the supernatants were
collected. Serine elastase activity was assayed with monitoring of
the degradation of a synthetic fluorescent substrate
(Suc-Ala-Ala-Ala-AMC; Bachem Biochemical Inc). A standard curve was
generated with human leukocyte elastase (HLE) (Elastin Products
Company), and elastolytic activity was expressed as nanograms of HLE
per milligram of tissue. Vein graft extracts were also assayed in the
presence of 4 µg recombinant human elafin (1 mg/mL in water; Zeneca
Pharmaceuticals).
Processing and Functional Activity of FLAG-Tagged Elafin
Transgene
Elafin- and CAT-transfected veins were incubated for 48 hours in
M199 with 1% antibiotic-antimycotic solution (GIBCO) and 5% fetal
calf serum. Conditioned media were dialyzed, lyophilized, and
resuspended in H2O. The supernatants were
collected, and samples of equal protein concentration were loaded onto
4% to 20% Tris-glycine gels. SDS–polyacrylamide gel
electrophoresis was performed, and the proteins were transferred to a
nitrocellulose membrane. The membranes were incubated with a monoclonal
anti-FLAG antibody (10 µg/ml; Sigma Chemical Co) and then with
horseradish peroxidase–conjugated secondary antibody and developed
with a chemiluminescence system (ECL kit; Amersham International).
To confirm the functional activity of the elafin transgene, tissues maintained in organ culture as described here were homogenized. The resultant pellets were resuspended in H2O and boiled for 1 minute to release the heat-stable tissue-bound elafin. The ability of the supernatant to inhibit the activity of HLE was assessed with the fluorescent synthetic elastase substrate as described earlier. HLE (20 ng) was incubated with 2 µL fluorescent substrate (4 mmol/L) in 1 mL buffer. Baseline HLE activity was measured, tissue extract was added, and the decrease in the rate of substrate degradation was determined. Inhibitory activity was expressed in terms of units per microgram, where 1 inhibitory unit represents a decrease in the activity of 1 ng of HLE by 50%.
Histology and Immunohistochemical Staining
Movat pentachrome staining was performed on cross
sections from paraffin-embedded vessel segments. Lipid accumulation was
evaluated by oil red-O staining of frozen tissue sections. Elafin
expression was documented by immunoperoxidase staining with antibody
against the FLAG epitope tag (1:200; Zymed). Inflammatory cells were
characterized by staining frozen tissue sections with a monoclonal
antibody against neutrophils and T cells (RPN3/57, 1:50; Serotec Ltd);
against neutrophils, monocytes, and macrophages (MAC387, 1:50;
Serotec Ltd); or against macrophages (RAM11, 1:50; DAKO).
Immunostaining for SMC 
-actin was performed with a
monoclonal antibody (1A4, 1:100; DAKO), and elastin was stained with an
antibody against bovine tropoelastin (PR396, 1:150; EPC Inc). Immune
complex formation was visualized with the Vectastain ABC amplification
system (Vector Laboratories). Additional sections were incubated with
nonimmune antibody as a negative control and showed minimal background
staining.
Morphometric Analysis and Quantification of
Immunohistochemical Staining
Inflammatory cells identified through immunohistochemical
staining of vein grafts 48 hours after implantation were quantified in
a blinded fashion. The total number of nuclei clearly associated with
positive immunostaining was counted from each section
and expressed as the number of positive cells per
millimeter.2
Morphometric analysis was performed on vein grafts harvested 4 weeks and 3 months after implantation. Low-power images from Movat-stained sections were analyzed with Image-Pro Plus software. Average intimal thickness was measured between tracings of the vessel lumen and the internal elastic lamina. Medial-plus-adventitial thickness was measured between tracings of the internal elastic lamina and the limit of the compact collagenous outer layer of the vessel. Plaque area in the grafts of cholesterol-fed animals was determined with computerized planimetry and standardized to the total area. All measurements were performed on sections from 3 segments (proximal, mid, and distal) of each graft. The computerized morphometric results were confirmed and calibrated by hand with a microscope equipped with a micrometer.
Computerized techniques were used to quantify oil red-O staining for lipid content, RAM11 immunohistochemical staining for macrophages, elastin staining, and tropoelastin immunostaining. Four images were digitized from the top, bottom, left, and right of each section. Image-Pro Plus software was used to calculate the number of pixels positively stained on each image, and this value was expressed as a percentage of the total area. An average value of the 4 images was calculated for each section.
Determination of Serum Cholesterol Levels
Serum samples from animals fed the high-cholesterol
diet and control animals maintained on a standard laboratory diet were
collected before tissue harvest. Total serum cholesterol
was determined with colorimetric methods (Vitros
Ectachem 950; Johnson & Johnson Clinical Diagnostics).
Statistical Analysis
Data are reported as mean±SEM. Analyses were performed
with 1-way ANOVA with post hoc testing according to Fisher’s protected
least significant difference method. A probability value of <0.05 was
considered statistically significant.
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Results |
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Serine Elastase Activity in Vein Grafts
We first showed that at 48 hours, elastase activity was elevated 5-fold in vein grafts compared with normal veins (Figure 1A
, P<0.02). Recombinant
elafin inhibited this activity by 70% (P<0.05), suggesting
that serine elastases are responsible for most of the elastolytic
activity in vein grafts. Both inflammatory cells that infiltrate the
vein grafts and SMCs represent potential sources of serine
elastase activity.
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Elafin Transgene Expression and Function
Our next goal was to assess transgene expression in veins after
gene transfer with the plasmid containing the cDNA-encoding elafin.
After a 48-hour incubation in organ culture, Western
immunoblotting of conditioned media demonstrated elafin
transgene expression in veins transfected with elafin but not the CAT
negative control vector (Figure 1B
). The functional activity of
elafin was evaluated through assay of the inhibitory
activity of vein tissue extracts against the serine elastase HLE. A
38% increase in HLE inhibitory activity was observed in
the elafin-transfected veins relative to CAT-transfected controls
(Figure 1C, P<0.005).
The kinetics of elafin expression were evaluated with immunoperoxidase
staining for the FLAG epitope tag. Elafin expression was associated
with endothelial, medial, and adventitial cells at 48
hours in elafin-transfected vein grafts (Figure 1D). Staining
was also seen at 1 week (Figure 1E), but not 4 weeks, after
transfection (Figure 1F). No specific staining was seen in
CAT-transfected control grafts (not shown).
Elafin Transfection and Inflammatory Cell Infiltration
To determine whether inhibition of serine elastases would, by
preventing the generation of elastin and other matrix peptides,
suppress the chemoattraction of inflammatory cells,11 we
examined the vein grafts 48 hours after implantation. Extensive
infiltration of inflammatory cells was observed in saline and CAT
control grafts. Immunohistochemical studies with the antibody RPN3/57
identified 50% of cells as neutrophils and T cells (Figure 2A), whereas MAC387, an antibody against
neutrophils, monocytes, and macrophages but not T cells,
recognized 30% of cells (data not shown). Macrophages
distinguished by the antibody RAM11 made up only a small proportion of
the cells in the graft wall (data not shown). Immunohistochemically
identified inflammatory cells were reduced by 60% in
elafin-transfected grafts relative to controls (Figure 2C, P<0.05), primarily due to a reduction in the number of
cells identified with the antibody against neutrophils and T cells
(RPN3/57, Figures 2B and 2D, P<0.05).
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Morphometric Analysis of Vein Graft Remodeling
We next investigated the effect of elafin gene transfer on vein
graft remodeling at 4 weeks by examining the extent of
neointimal formation and medial-plus-adventitial
thickening. Computerized morphometric analysis demonstrated
that intimal thickness was reduced by 50% in elafin-transfected
grafts compared with controls (Figures 2E to 2G,
P<0.01). Furthermore, combined medial-plus-adventitial
thickness was also reduced in elafin-transfected grafts (Figure 2H, P<0.001). Vessel radius/wall thickness, a
parameter that is proportional to wall stress, was elevated
by 25% in elafin-transfected grafts relative to controls (10.5±0.8
for elafin versus 8.4±0.4 for saline and 8.6±0.5 for CAT,
P<0.05, n=9).
Late Remodeling and Atherosclerosis
To assess the impact of transient elafin expression on late graft
remodeling and atherosclerosis, elafin-transfected
grafts and CAT-transfected controls were harvested 3 months after
implantation in animals fed either a normal or a
cholesterol-enriched diet (0.5%). Elevated serum
cholesterol levels were found to be similar in the elafin
(15.3±1.3 mmol/L) and CAT (15.8±1.9 mmol/L) groups (normal
<1.29 mmol/L). Intimal thickness in elafin-transfected grafts was
no longer different from CAT-transfected controls at 3 months when
animals were maintained on a normal diet (Figure 2K). However,
accelerated neointimal formation in
cholesterol-fed animals remained somewhat reduced in the
elafin-transfected grafts (Figures 2I to 2K,
P<0.05).
The most striking observation was that atherosclerotic plaque formation
was reduced by 40% in elafin-transfected grafts relative to
CAT-transfected controls (Figures 2I, 2J, and 2L,
P<0.05). Lipid content in vein grafts from
cholesterol-fed animals evaluated with computerized
morphometric analysis of oil red-O–stained sections was
reduced by 50% with elafin transfection compared with
CAT-transfected controls (Figures 3A to
3C, P<0.002). Immunohistochemical studies with the antibody
RAM11 revealed that atherosclerotic plaques in the graft wall were in
large part composed of macrophages (Figure 3D). The area
occupied by positively stained macrophages was reduced by
>50% in elafin-transfected grafts compared with controls (Figures 3D to 3F, P<0.05).
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Neointimal Elastin Deposition
We next addressed whether a difference in the composition of the
ECM induced by the early expression of elafin could have accounted for
the suppression of atherosclerotic degeneration despite only a modest
reduction in neointimal thickening in vein grafts at 3
months. We have previously linked fibronectin deposition with
elastase activity and inflammatory cell infiltration, but
immunohistochemical studies did not reveal a difference in
neointimal fibronectin in elafin-transfected grafts
relative to controls (data not shown). Elastase activity can induce
elastin deposition,12 and elastin fragments are
chemoattractants for and bind to inflammatory cells, including
monocytes.11 13 We therefore evaluated elastin deposition
in the neointima of elafin-transfected grafts with
computerized analysis of Movat-stained sections and found it to
be reduced by 74% relative to CAT-transfected controls (Figures 3G to 3I, P<0.03). A similar reduction in elastin
deposition was also detected with tropoelastin immunoperoxidase
staining (Figures 3J to 3L, P<0.001).
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Discussion |
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Early vein graft remodeling is characterized by inflammation and elevated serine elastase activity. By using gene transfer with the serine elastase inhibitor elafin, we were able to successfully reduce the inflammatory response in vein grafts and limit neointimal formation at 4 weeks. Intimal thickening did continue beyond 4 weeks in elafin-transfected grafts. However, accelerated neointimal formation induced by cholesterol feeding was significantly decreased by elafin transfection. Furthermore, transient elafin expression resulted in a persistent decrease in neointimal elastin deposition associated with a
50% reduction in cholesterol-induced
atherosclerotic plaque formation, lipid content, and macrophage
accumulation. This is the first study to provide evidence for the
important role of serine elastases in early vein graft remodeling
and for the relevance of ECM remodeling and elastin deposition to late
atherosclerotic degeneration. Serine elastase activity is elevated in the coronary arteries of rabbits after heart transplantation, and elafin prevents neointimal formation through inhibition of migration and proliferation of vascular SMCs.3 We have similarly documented serine elastase activity in vein grafts and limited neointimal formation for up to 4 weeks by transient expression of elafin, thus implicating serine elastases in this process. Sources of serine elastases include SMCs, which produce an endogenous elastase in response to endothelial injury or activation.2 14 15 However, infiltrating leukocytes may also contribute to elevated serine elastase activity in vein grafts.16
A critical role for inflammatory cells in neointimal formation has been suggested by the observation that neointimal formation is reduced in vein grafts from rats that lack T cells or are administered cyclosporin A.17 It has been speculated that leukocytes mediate neointimal formation through the release of mitogenic factors.17 However, proteolytic enzymes released from inflammatory cells may also promote SMC proliferation and migration.
We have demonstrated that elafin is effective in markedly reducing inflammation in vein grafts at 48 hours. Elastases activate proforms of inflammatory cytokines18 and degrade components of the endothelial basement membrane, perhaps facilitating the invasion of inflammatory cells into the vessel wall.19 The release of peptides by serine elastase–mediated degradation of elastin promotes leukocyte chemotaxis11 and is prevented by elafin in cultured cells.20 The anti-inflammatory effect of elafin could reflect its ability to inhibit all of these mechanisms.
In addition to the anti-inflammatory properties of elafin transfection, reduced neointimal formation may reflect a more direct effect on SMC migration and proliferation. By releasing and activating growth factors that are bound to the ECM, serine elastases can induce the proliferation of vascular SMCs.4 5 The proliferative aspect of vein graft remodeling, evident in the extensive neointima, was attenuated by elafin; however, this was not sustained beyond 1 month. This finding is consistent with the reduction in elafin expression seen in vein grafts at 4 weeks. The stimulus for ongoing remodeling may be elevated wall stress due to limited thickening of the wall of elafin-transfected grafts at 1 month.
Although neointimal formation in response to elevated cholesterol was only modestly reduced by elafin transfection at 3 months, relative atherosclerotic plaque area, lipid content, and macrophage accumulation were markedly decreased. Protection against lipid deposition and macrophage infiltration appears to be related to a reduction in neointimal elastin induced by transient elafin expression. Elastin is a critical component of atherosclerotic plaques, because it is a preferred ECM component to which monocytes adhere13 and a nidus for both cholesterol and lipid accumulation and subsequent calcification.21
Elastases stimulate elastin synthesis in cultured cells through the cooperative influence of proteolytically degraded ECM and elastin peptides.12 We propose that a similar mechanism is active in the neointima of vein grafts and that transient modulation of proteolytic activity by elafin transfection abrogates the initial stimulus for elastin production and deposition, thereby protecting against later atherosclerotic degeneration.
In summary, we have shown that the transfection of vein grafts to produce a specific serine elastase inhibitor is effective in delaying neointimal formation, thereby establishing a role for elastolytic activity in vein graft remodeling. Although ongoing remodeling in transfected grafts eventually leads to development of a significant neointima, it has a compositionally altered ECM that is relatively resistant to atherosclerotic degeneration. These data provide a rationale for the protection of vein grafts through serine elastase inhibition delivered via gene transfer or even via the administration of an orally bioavailable elastase inhibitor during the acute phase of remodeling to reduce late atherosclerotic complications.
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Acknowledgments |
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This work was supported by Heart and Stroke Foundation of Canada grant HSFO 3170 and by the Primary Pulmonary Hypertension Cure Foundation. Dr Rabinovitch is a Research Endowed Chair and Dr O’Blenes is the Evelyn McGloin Fellow of the Heart and Stroke Foundation of Ontario. We wish to thank the staff of the Animal Facility and Pathology Department at The Hospital for Sick Children, Toronto, for technical assistance, and Joan Jowlabar, Judy Edwards, and Judy Matthews for their help in preparation of the manuscript.
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