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(Circulation. 1996;94:2890-2900.)
© 1996 American Heart Association, Inc.

Articles

Virus-Encoded Serine Proteinase Inhibitor SERP-1 Inhibits Atherosclerotic Plaque Development After Balloon Angioplasty

Alexandra Lucas, MD; Li-ying Liu, MD; Joanne Macen, PhD; Piers Nash, BSc; Erbin Dai, MD; Michael Stewart, MSc; Kathryn Graham, PhD; Wai Etches, MD; Lynn Boshkov, MD; Patric N. Nation, MD; Dennis Humen, MD; Marita Lundstrom Hobman, PhD; Grant McFadden, PhD

the Division of Cardiology, Department of Medicine (A.L., L.-y.L., E.D., D.H., M.L.H.); the Department of Biochemistry (J.M., P.N., K.G., G.M.); Health Sciences Laboratory Animal Services (P.N.N.); the Department of Laboratory Medicine and Pathology (M.S., W.E., L.B.), University of Alberta, Edmonton, Alberta, Canada.

Correspondence to Dr Alexandra Lucas, John P. Robarts Research Institute, University of Western Ontario, PO Box 5015, 100 Perth Dr, London, Ontario N6A 5K8, Canada. E-mail arl@rri.uwo.ca.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Recurrent atherosclerotic plaque growth, restenosis, is a significant clinical problem after interventional procedures. Initiation of restenosis involves activation of inflammatory and thrombotic cascades, which are regulated by serine proteinase enzymes and inhibitors. We have investigated the use of a viral serine proteinase inhibitor, SERP-1, to reduce plaque development after primary balloon angioplasty. This is the first experimental report of the use of a viral anti-inflammatory protein for the prevention of atherosclerosis.

Methods and Results Seventy-four cholesterol-fed rabbits were treated with either local or systemic infusions of SERP-1 protein (or control solutions) after balloon-mediated injury. Sites of SERP-1 infusion in rabbits had dramatically reduced plaque compared with control infusions at the 4-week follow-up. At low-dose infusions (30 to 300 pg), only the primary infusion site had a demonstrable decrease in plaque, whereas at higher-dose infusions (>3000 pg), a generalized reduction in plaque development was detected. An associated decrease in mononuclear cell infiltration of the arterial wall was detected after SERP-1 infusion within the first 24 hours. Infusion of an active-site mutant of SERP-1 (P₁-P₁', ala-ala) lacking serine proteinase inhibitory activity failed to prevent plaque growth.

Conclusions Purified SERP-1, a virus-encoded secreted glycoprotein, reduces plaque growth after primary balloon-mediated injury. Plaque development is decreased by inhibition of serine proteinase activity and is associated with a focal reduction in macrophage infiltration immediately after injury. Investigation of serine proteinase inhibitors may provide new insight into the regulation of arterial responses to injury.

Key Words: plaque • serpin • angioplasty • immune system

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
All interventional devices currently used to open occluded arteries have a high associated incidence, 20% to 50%, of recurrent atherosclerotic plaque growth (restenosis).^{1 2} The focal inflammation that inevitably follows the vascular damage produced by techniques such as balloon angioplasty is believed to initiate a chain of events that culminate in restenosis.^{3 4 5 6} Many enzymes in the inflammatory and thrombotic enzyme cascades have serine proteinase activity and are regulated by serine proteinase inhibitors.⁷ Coagulation and fibrinolytic enzymes have been demonstrated to modulate monocyte motility and chemotaxis, tissue invasion, cytokine responses, mononuclear cell proliferation, and apoptosis.^{7 8 9 10} One such serine proteinase inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in atherosclerosis development on the basis of the fact that transgenic mice that specifically lack PAI-1 have evidence of accelerated intimal hyperplasia on histological examination.^{8 9} Similarly, inhibition of plasminogen with tranexamic acid decreased smooth muscle cell migration in a rat model of arterial balloon injury.¹⁰ Urokinase-like plasminogen activator–mediated plasmin formation may aid in the breakdown of fibrin and matrix components during cellular migration, tissue invasion, and vascular remodeling and in regulation of mononuclear cell chemotaxis.^{7 8 9} The precise role of the serine proteinase inhibitors in plaque development and specifically in restenosis remains to be delineated, as does the role of the acute inflammatory response after physical trauma associated with angioplasty procedures. Given the complex nature of the restenosis problem, we have begun to investigate the use of specific therapies such as anti-inflammatory serpins in an attempt to interrupt the injury response cascade at the earliest possible stage after the interventional procedure. To do this, we have exploited DNA viruses that express proteins that directly interfere with elements of the host immune system.

Many of the larger DNA viruses, particularly those that encode more proteins than required for minimal replication of the virus in infected cells (ie, poxviruses, herpesviruses, adenoviruses), have evolved multiple genes whose expressed proteins are designed specifically to inhibit the host immune and inflammatory responses.^{11 12 13 14} Many of these virus-encoded anti-immune proteins are secreted as extracellular glycoproteins from virus-infected cells and are classified as virokines (mimics of cellular cytokines or other soluble immune regulators of the host) or viroceptors (mimics of cellular receptors), depending on their mode of action.¹⁴ The most abundant known source of secreted immunoinhibitory proteins is the poxviruses, which encode several hundred proteins, many of which fall into this class.^{13 14 15} The protein used in the studies described here, SERP-1, is a serine proteinase inhibitor (serpin) encoded by myxoma virus, a poxvirus pathogen of rabbits and the agent of myxomatosis.¹⁶ Myxoma virus encodes a plethora of anti-immune proteins, each targeted to a different aspect of the immune or inflammatory cascades that are normally activated by viral infection.¹⁷ SERP-1 is a secreted glycoprotein that inhibits the early cellular inflammatory response to the virus infection, and deletions of this gene within recombinant virus results in an attenuated virus.^{18 19} Biochemically, SERP-1 is a member of the serpin superfamily of serine proteinase inhibitors, which are important regulators of serine proteinases that modulate a wide variety of immune and inflammatory processes.²⁰ Although the precise biological target of SERP-1 has not been formally defined within infected tissues, in vitro the protein inhibits a variety of cellular proteinases, including plasmin, tissue plasminogen activator, and urokinase.^{21 22}

Given the potency of SERP-1 in preventing the early cellular inflammatory response to viral infection, we have examined whether this viral protein could be purified and used as a clinical reagent to ameliorate a syndrome believed to be initiated by excessive inflammation, such as balloon injury–induced atheroma. Accordingly, we have expressed the SERP-1 protein from a recombinant vaccinia virus vector, purified the secreted and fully processed glycoprotein, and tested the protein as an anti-inflammatory reagent in a rabbit angioplasty model.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Expression and Purification of SERP-1 Protein
SERP-1 protein was harvested from the supernatant of BGMK cells infected with a recombinant vaccinia virus (VV-S1) that overexpresses SERP-1¹⁹ and purified by column chromatography. Cells were adsorbed with VV-S1 or VV-601 control virus for 1 to 2 hours at 37°C, washed with PBS, and incubated with Dulbecco's modified Eagle's medium (15 mL per bottle) without serum. After 24 hours, the medium was harvested, clarified at 10 000g for 10 minutes, concentrated x10 with a Centriprep 10 (Amicon) concentrator, dialyzed against 25 mmol/L Tris-HCl, pH 8, and loaded onto a MonoQ (Pharmacia) column. Proteins were eluted with a linear 0 to 1.0 mol/L NaCl gradient; fractions containing SERP-1 were identified by SDS-PAGE and immunoblotting with anti-SERP-1 antiserum and loaded onto a Superdex G75 (Pharmacia) column equilibrated with 25 mmol/L Tris-Cl, pH 8.0, and 150 mmol/L NaCl. Vaccinia vector control proteins secreted from cells infected with vaccinia construct lacking SERP-1 (VV-601) were purified in parallel with recombinant vaccinia that overexpresses SERP-1. The same fractions from the MonoQ column during the purification of the vaccinia vector protein preparation (Fig 1, lane 1) were used for the control infusions in the initial series of pilot experiments in which MonoQ-purified SERP-1 (lane 2) was tested in the rabbit model (Figs 2 and 3). SERP-1 purified to apparent homogeneity by Superdex 75 chromatography (Fig 1, lane 3) was used for the second series of experiments with rabbits (Figs 4 through 9), in which the identically treated control samples from the VV-601 controls showed no detectable protein (Fig 1, lane 5). An active-site mutant of SERP-1 protein deficient in serine proteinase inhibitory activity was prepared by mutating the P₁-P₁' site (arg-asn) with an ala-ala sequence (Fig 1, lane 4).²²

View larger version (63K): [in this window] [in a new window]   Figure 1. Purification of SERP-1 expressed as a secreted glycoprotein from a recombinant vaccinia vector. SERP-1–containing fractions were analyzed by a silver-stained SDS-PAGE. MonoQ column–purified vaccinia vector (VV-601) control proteins secreted from infected cells (lane 1), and the parallel preparation with recombinant vaccinia (VV-S1) that overexpresses SERP-1 (lane 2) were used for the experiments in Figs 2 and 3. The Superdex G75 column–purified SERP-1 preparation (lane 3) was used for the experiments shown in Figs 4 through 9. The purified SERP-1 migrates as a diffuse 50- to 55-kD species (and minor 110-kD dimer) because it is secreted from virus-infected cells as an N-linked glycoprotein.19 Superdex G75 column–purified ala-ala mutant SERP-1 (lane 4) migrates like the wild-type protein. Parallel MonoQ- and Superdex G75–purified VV-601 control proteins secreted from infected cells with minimal protein evident (lane 5). Molecular weight markers indicated as dashes, from top to bottom, are phosphorylase b (106 kD), BSA (80 kD), ovalbumin (49.5 kD), and carbonic anhydrase (32.5 kD).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effect of MonoQ-purified SERP-1 and control MonoQ-purified vaccinia vector proteins on plaque development after balloon angioplasty. The plaque area measurements are expressed as cross-sectional plaque area at the primary infusion site (solid lines) and at the secondary balloon-injured but noninfused upstream (proximal) site above the site of Wolinsky infusion (dotted lines). Rabbits were subjected to balloon-mediated denudation of the aorta above the iliac bifurcation, infused with indicated dosages of MonoQ-purified SERP-1 (A; Fig 1, lane 2) or vaccinia virus vector control proteins (B; Fig 1, lane 1), and killed after 4 weeks on a cholesterol-rich diet (total of 25 rabbits). Plaque area measurements were performed at the primary site of balloon injury and Wolinsky infusion in the distal abdominal aorta and at balloon-injured but noninfused secondary sites in the proximal abdominal aorta, as described in "Methods."

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of infused Superdex G75–purified SERP-1 (Fig 1, lane 3) on plaque area reduction at the primary infusion site (solid line) and the upstream secondary balloon-injured but noninfused site (dotted line). The error bars for several samples are too narrow to be visualized in the graph (19 rabbits).

To examine SERP-1 distribution in the vessel wall, bloodstream, and various organs (liver, heart, kidney and urine) after infusion through a Wolinsky catheter, purified SERP-1 was first labeled with ¹²⁵I by use of IODO-BEADS,²³ according to the manufacturer's protocol. Gel filtration was then performed to remove unincorporated label, and 250 ng (6.3x10⁵ cpm) of ¹²⁵I-labeled SERP-1 was infused through a Wolinsky balloon catheter²⁴ immediately after balloon-mediated endothelial damage in the rabbit aorta as described below. Specimens of blood, urine, abdominal aorta and iliac arteries, kidney, thyroid, and heart were taken, and the radioactivity was quantified by a gamma counter.

Rabbit Model
All rabbits (New Zealand White) were fed 2% cholesterol in 10% peanut oil diet for 7 d/wk (Figs 2 through 4) or 4 d/wk (Figs 5 through 9), beginning 2 weeks before balloon injury and continuing for 4 weeks. Although the 7-day regimen produced more profuse plaque, the 4-day regimen had fewer toxic effects. A 3- to 3.5-mm angioplasty balloon catheter (1:1 ratio of balloon to aorta diameter) was introduced through a femoral arterial cutdown after anesthesia (40 mg/kg ketalean, 8 mg/kg xylazene, and 0.5 mg/kg acepromazine IM). The balloon was inflated to 8 bars in the distal abdominal aorta, advanced retrograde to the distal thoracic aorta, and then withdrawn three times under fluoroscopic control in each rabbit. Contrast angiograms were recorded before and after balloon angioplasty–mediated trauma and at the 4-week follow-up. Heparin (400 U) was given immediately after femoral access was obtained. Six rabbits were killed 24 hours after SERP-1 or saline infusion for acute immunohistochemical assessment; all other rabbits (74 in total) were killed 4 weeks after infusion. Rabbits given local Wolinsky infusions that developed minimal plaque (plaque area proximal to the Wolinsky infusion site <0.02 mm²) were not included in the final assessment of data to avoid confounding factors associated with variability in plaque development. Five of the control and SERP-1–infused rabbits tested were therefore excluded from the study. All surgical procedures and care of rabbits were according to the University of Alberta Animal Welfare Committee and national guidelines. In the initial pilot study, MonoQ-purified SERP-1 (Fig 1, lane 2) at protein doses of 50 to 5000 pg per sample was infused immediately after balloon-mediated injury in the distal abdominal aorta (2 rabbits received 0 pg, 4 rabbits received 50 pg, 6 rabbits received 500 pg, and 4 rabbits received 5000 pg). A parallel preparation of MonoQ-purified vaccinia vector control proteins lacking SERP-1 (Fig 1, lane 1) was infused locally into the distal abdominal aorta at comparable doses (3 rabbits received 50 pg, 3 rabbits received 500 pg, and 3 rabbits received 5000 pg). Each infusate was administered through a Wolinsky catheter²⁴ in a total volume of 10 mL sterile 0.9% saline immediately after balloon-mediated injury. In a second series of experiments to test purified SERP-1 (Fig 1, lane 3), 19 rabbits had infusions of 0 (5 rabbits), 30 (5 rabbits), 300 (5 rabbits), or 3000 (4 rabbits) pg. Note that 5000 pg of the partially purified SERP-1 protein (MonoQ) used in the first series was judged to be 60% pure and thus corresponds to 3000 pg of Superdex-purified SERP-1. To examine the specificity of the serine proteinase activity of SERP-1 in this biological system, 8 rabbits were also infused in the same manner with either 20 ng purified SERP-1 (4 rabbits; Fig 1, lane 3) or 20 ng of a purified ala-ala mutant of SERP-1 (4 rabbits; Fig 1, lane 4). All infusions were through a 3.25-mm Wolinsky balloon (inflated to a final pressure of 6±1 bars for 2 minutes) in the abdominal aorta proximal to the iliac bifurcation. The Wolinsky balloon was positioned immediately above the iliac bifurcation under fluoroscopic control so that the perfusion balloon was routinely located as close as possible to 0.5 to 2.5 cm above the bifurcation under fluoroscopic control; this site was designated the primary infusion site. Upstream secondary sites were defined in the region >2.5 cm proximal to the iliac bifurcation.

View larger version (139K): [in this window] [in a new window]   Figure 3. Histological profiles of aortic specimens taken from cholesterol-fed rabbits 4 weeks after balloon angioplasty injury and MonoQ-purified (Fig 1, lane 2) SERP-1 protein (A and D), control VV-601 vaccinia vector (Fig 1, lane 1) protein (B and E), or saline (C and F) infusions. All sections are stained with hematoxylin and eosin (magnification x260). A, Section of aorta at the primary site of SERP-1 infusion (0.05 ng). B, Section at the primary infusion site of 5 ng vaccinia vector control infusion. C, Section from the primary saline infusion site. D, Section taken from a secondary noninfused site of a rabbit that received 5 ng SERP-1 infusion. E, Section taken at a secondary upstream, noninfused site in a rabbit treated with 5 ng vaccinia control protein infusion. F, Section taken at the secondary upstream, noninfused site in a rabbit treated with saline infusion.

View larger version (19K): [in this window] [in a new window]   Figure 5. Titration of dose response to systemic Superdex G75–purified (Fig 1, lane 3) SERP-1 infusion before balloon injury. Twenty-two rabbits had balloon angioplasty injury immediately after single intravenous or intra-arterial injection of purified SERP-1 protein at the indicated dosage (ie, non-Wolinsky). Four weeks after treatment, plaque was analyzed at balloon-damaged sites as in Fig 2. All specimens from individual rabbits taken from the balloon injury sites from the proximal abdominal aorta to the iliac bifurcation are included in mean values for each rabbit and each dose infused.

View larger version (109K): [in this window] [in a new window]   Figure 6. Histological examination of plaque at balloon-damaged sites 4 weeks after injury and systemic injection of purified SERP-1 protein (Fig 1, lane 3) or control. A, Hematoxylin and eosin–stained section taken from the abdominal aorta of a rabbit 4 weeks after balloon injury with systemic, intravenous infusion of 3.0 ng of SERP-1. B, Hematoxylin and eosin–stained section taken from the distal thoracic aorta of a rabbit 4 weeks after balloon injury with systemic, intravenous infusion of SERP-1 (3.0 ng). C, Hematoxylin and eosin–stained section taken from the abdominal aorta of a rabbit 4 weeks after balloon injury with systemic, intravenous infusion of saline. D, Hematoxylin and eosin–stained section taken from the distal thoracic aorta of a rabbit 4 weeks after balloon injury with systemic, intravenous infusion of saline.

View larger version (289K): [in this window] [in a new window]   Figure 7. Analysis of the effect of the active site mutant of SERP-1 (Fig 1, lane 4) on plaque area at the primary infusion site. A, Eight rabbits were given Wolinsky infusion (20 ng) of the P1-P1' (ala-ala) mutant of SERP-1 or the wild-type SERP-1 protein (Fig 1, lanes 4 and 3, respectively), and plaque area at the primary infusion site was measured 4 weeks later, as described in "Methods." B, Hematoxylin and eosin–stained section from the (ala-ala) mutant at the primary SERP-1 infusion site. C, Hematoxylin and eosin–stained section from SERP-1–infused sample.

View larger version (114K): [in this window] [in a new window]   Figure 8. Immunohistochemical analysis of vascular cell populations at the primary infusion site. Samples were then taken 24 hours (A through D) and 4 weeks (E through J) after balloon angioplasty and Wolinsky infusion with purified SERP-1 protein (Fig 1, lane 3) or saline control. A, Smooth muscle cell (-actin antibody) distribution at the primary site after 3 ng SERP-1 infusion (magnification x260). B, Smooth muscle cell (-actin antibody) distribution at the primary site after control saline infusion. (x260). C, Macrophage (RAM11 antibody positive) at the primary site of 30 ng SERP-1 infusion (x400). D, Macrophage (RAM11 positive) at the primary site of control saline infusion (x400). E, Smooth muscle cell (-actin antibody) distribution at a site of 0.3 ng primary SERP-1 infusion (x400). F, Smooth muscle cell staining (-actin antibody) distribution at a site of control saline infusion (x400). G, Macrophage staining (RAM11 positive) at a primary site of 0.3 ng SERP-1 infusion (x400). H, Macrophage staining (RAM11 positive) at a primary site of saline infusion (x400). I, Control-treated plaque stained with irrelevant VSV protein G primary antibody and biotinylated goat anti-mouse secondary antibody (x400). J, Control-treated plaque stained with biotinylated goat anti-mouse secondary antibody in the absence of primary antibody (x400).

View larger version (122K): [in this window] [in a new window]   Figure 9. Relative cellular populations detected at primary and secondary sites 24 hours (A) and 4 weeks (B) after balloon injury and Wolinsky infusion. Infusions were with 0.3 ng purified SERP-1 (Fig 1, lane 3) or control saline. The primary (P) site refers to the site of Wolinsky infusion of purified SERP-1 (+) or saline (-); the secondary (S) site is an upstream balloon-damaged but noninfused area in the upper thoracic artery. The cell populations stained were smooth muscle cells (SMC) and macrophages (MC).

To assess the efficacy of SERP-1 for the prevention of plaque development when given systemically, plaque development was examined in 22 rabbits after intravenous (12 rabbits) and intra-arterial (10 rabbits) systemic infusion of purified SERP-1 into the bloodstream through either venous puncture or arterial sheath (ie, no Wolinsky balloon was used). Each rabbit had infusion of SERP-1 at doses ranging from 0 to 300 ng in 10 mL saline (6 rabbits received 0 ng, 4 rabbits received 0.3 ng, 4 rabbits received 3 ng, 4 rabbits received 30 ng, and 4 rabbits received 300 ng) immediately before balloon injury. Contrast angiograms were recorded before SERP-1 infusion and at follow-up.

Analysis of Lipids and Clotting Factors After SERP-1 Infusion
Cholesterol and triglyceride levels were assessed in blood specimens from rabbits 9 days after intravenous SERP-1 infusion. Blood was also taken from 1 rabbit on normal diet and 2 cholesterol-fed rabbits before balloon injury and infusion for analysis of clotting parameters (prothrombin time, partial thromboplastin time, and fibrinogen levels). Purified SERP-1 protein was added directly to aliquots of these samples to 30 or 300 pg/mL before measurements. Rabbit blood (4.5 mL) was added to 0.5 mL of 3.8% sodium citrate and centrifuged at 3000 rpm for 10 minutes at 20°C in a Sorvall Instruments GLC4 centrifuge. The supernatant was collected and centrifuged at 12 000 rpm for 2 minutes at 20°C and then stored at -86°C for later analysis. Cholesterol and triglyceride levels were measured with the Sigma Diagnostics cholesterol 50 and the triglyceride 10 (GPO-Trinder) single reagent systems. Absorption at 492 nm was read on an EAR 340 AT Easy Reader plate reading spectrophotometer (SLT-Labinstruments). The prothrombin time was read on a Medical Laboratory Automation Inc Electra 700 Automatic Coagulation Timer with Dade Actin FSL Activated PII reagent and Dade Innovin recombinant thromboplastin. The partial thromboplastin time and the fibrinogen levels were assessed with a Dade fibrinogen determination test kit by use of a Fibrometer Precision Coagulation Timer (BBL Becton Dickinson). For coagulation parameters, lipemic plasma was centrifuged in a Beckman microfuge (10 500g for 5 minutes at 20°C).

Histology and Morphometric Analysis
Histological analysis was performed at the primary site of Wolinsky infusion in the distal abdominal aorta representing the primary infusion site as defined by the original positioning of the perfusion balloon. Internal control sections were taken from a downstream noninfused site near the iliac bifurcation (0.5 cm above to 0.5 cm below the bifurcation), and an upstream balloon-damaged but not Wolinsky-infused site (the upper abdominal aorta, 2.5 to 3.5 cm above the iliac bifurcation). The area from 1.5 to 2.5 cm above the iliac bifurcation was considered a border zone with potentially variable infusion doses because of balloon placement and therefore was not included in the analysis. Hematoxylin and eosin staining of formalin-fixed specimens was performed as previously described.²⁵ Sections from each specimen (a minimum of 2 sections per site to a maximum of 18 sections per site) were then stained with hematoxylin and eosin and examined by light microscopy.

Morphometric analysis of plaque thickness and area was performed as described elsewhere,²⁵ and area measurements were tabulated for each rabbit. For morphometric analysis, cross-sectional areas of plaque detected by light microscopy were outlined for each section by use of a Nikon Optiphot-Labophot drawing device microscope attachment to a Nikon Labophot two light microscope (Nikon, Nippon Kogaku KK). Each drawing was standardized to a 1-mm marker. The areas were then measured with a Jandel Scientific Sigma Scan program and Summagraphics digitizing Summa sketch pad coupled to a Macintosh IIcx computer. The mean plaque area was calculated for all sections measured from one site, eg, the mean for plaque area as measured from four sections taken from a primary Wolinsky infusion site.

Immunohistochemical analysis was performed on adjacent sections taken from primary and secondary infusion sites with purified SERP-1 or saline as has been previously described.²⁶ In brief, formalin-fixed rabbit arterial sections were cut into 5-µm sections and stained with the indirect peroxidase-labeled antibody technique.^{26 27} Paraffin sections were taken from formalin-fixed rabbit aorta and immunostained with Sigma Diagnostics mouse anti–-human smooth muscle actin antibody for smooth muscle cells (which cross-reacts with rabbit smooth muscle cells) and with mouse ascites RAM11 monoclonal antibody (a kind gift from E. Raines, University of Washington, Seattle) for rabbit monocytes/macrophages. Each primary antibody–treated section was then incubated with biotinylated goat anti-mouse or anti-rat antibody for 30 minutes and avidin-biotin-peroxidase complex for 40 minutes and developed with 3'3-diaminobenzidine for 5 minutes. In negative controls, we substituted an irrelevant primary antibody (IgG1 antibody to the cytoplasmic tail of vesicular stomatitis virus G protein from Dr T. Kreis) and a neutral buffered saline for the primary and/or secondary antibody. Positive controls consisted of human arterial smooth muscle cells and rabbit small bowel (for -actin), tonsil, and spleen (for RAM11). Each section was incubated with 20% normal goat serum before primary antibody addition to decrease nonspecific staining. The percentage of positive staining was assessed in three to five high-power areas per slide, with quantification of the number of positive grids divided by the number of squares in the grid covered by the area of intimal hyperplasia or media.

Statistical Analysis
Mean values for plaque area, angiographic measurements, and biochemical parameters were compared by Student's unpaired t test or by ANOVA. Statview, a Macintosh statistics package, was used for calculation of means, SE, and significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Focal SERP-1 Infusion in the Rabbit Model Reduces Plaque Growth
Secreted SERP-1 protein was harvested from the supernatant of cells infected with a recombinant vaccinia virus that overexpresses SERP-1¹⁹ and purified by MonoQ and Superdex G75 column chromatography. The MonoQ peak elution fractions from the vaccinia control vector and from a parallel preparation from a vaccinia vector that overexpresses SERP-1 (Fig 1, lanes 1 and 2, respectively) were used in a pilot study (25 rabbits) to ascertain first whether SERP-1 had anti-inflammatory properties that could ameliorate plaque development in response to balloon-mediated damage and second whether any bioactive proteins expressed from the vaccinia vector itself might have copurified with SERP-1. Subsequently, this SERP-1 protein preparation purified to homogeneity (Fig 1, lane 3) and a parallel purification of vaccinia vector proteins with minimal residual protein evident (Figure 1, lane 5) were used for the second rabbit study to examine the dose response of SERP-1 (19 rabbits), the biological activity of an active-site mutant (Fig 1, lane 4) of SERP-1 (8 rabbits), and the effect of systemic SERP-1 infusions (22 rabbits). Rabbits were examined for intimal hyperplasia 4 weeks after the balloon angioplasty procedure. Arterial changes associated with treatment at the primary site of infusion in the distal abdominal aorta were compared with adjacent upstream and downstream noninfused sites (internal controls) and at primary sites of control infusions (external controls). Representative sites were sectioned and stained with hematoxylin and eosin, and cross-sectional plaque area measurements were quantified in correlation with infusion dose of SERP-1 (Fig 2A) or vaccinia vector control proteins (Fig 2B). Overall, a significant decrease in plaque area was detected at primary sites of SERP-1 infusions in the distal aorta (Fig 2A, solid line) compared with the summated internal and external control samples. In contrast, much less of an effect was observed at secondary upstream noninfused sites in the proximal (upper abdominal) aorta (Fig 2A, dotted line), although a modest reduction was observed at downstream sites near the iliac bifurcation at the higher doses of SERP-1 (data not shown). There was a preferential decrease in relative plaque development in rabbits treated with each of the three doses (50, 500, and 5000 pg) of SERP-1 tested at the primary infused site compared with the secondary upstream site (Fig 2A), whereas for the parallel-purified vaccinia vector secreted proteins, there was, if anything, a comparable level or even an increase in plaque development at both primary and secondary sites (Fig 2B).

Atherosclerotic plaque detectable at primary SERP-1 infusion sites, when present, was usually only a thin layer of fatty intimal hyperplasia (Fig 3A), whereas after vaccinia control or saline infusions, plaque composition was often complex and comprised extensive fatty and fibrous areas (Fig 3B and 3C, respectively). At balloon-damaged secondary sites upstream of the primary Wolinsky infusions sites, significant plaque development was detectable for all three classes of infusates: SERP-1 (Fig 3D), vaccinia control proteins (Fig 3E), and saline (Fig 3F). However, as indicated in Fig 2A, the thickness of plaque at the upstream sites in animals infused with these moderate doses of SERP-1 was generally intermediate between primary SERP-1 infusion sites and all the other controls (Fig 2B). The histological profiles of rabbits treated with saline infusions were indistinguishable from those treated with the purified vaccinia control proteins and dramatically different from the SERP-1 samples, specifically at the primary infusion sites (P<.0001 for primary sites; P=NS for secondary sites). Contrast angiograms recorded at the 4-week follow-up did not show a significant change in lumen diameter after SERP-1 infusion, although the control rabbits had smaller mean lumen diameters (data not shown). On the basis of the promising results of this pilot study, we then initiated a second set of experiments using highly purified SERP-1 protein and an active-site mutant of SERP-1 that is deficient in all serine proteinase inhibitory activities tested.

SERP-1 protein was purified to homogeneity (Fig 1, lane 3) and used for a second study to quantify SERP-1 inhibition of plaque development. Plaque area was again measured 4 weeks after balloon angioplasty and Wolinsky infusion into the distal abdominal aorta. Purified SERP-1 was infused at a series of concentrations to titrate the dose response for the inhibition of plaque development. At low doses (30 to 300 pg), as was demonstrated with MonoQ-purified SERP-1, there was a significant reduction of plaque growth after SERP-1 at the primary infusion site (Fig 4, solid line) compared with the secondary damaged but noninfused sites (Fig 4, dotted line). Plaque, when present at an SERP-1–treated site, was again a thin layer of fatty plaque compared with the larger, more complex plaque development after control infusions (data not shown). When the plaque area measurements were plotted against the infusion dose, there was a clear association between the SERP-1 infusion dose and the relative extent of subsequent plaque development at the primary infused site (P<.0001 at the primary infusion site by unpaired t test and ANOVA analysis for each infusion dose compared with saline). We also observed that at progressively higher concentrations of SERP-1 (0.3 to 3.0 ng), plaque development at the upstream secondary damaged but noninfused site also became significantly reduced (Fig 4, dotted line). At the 3.0-ng infusion dose, a systemic effect with reduced plaque development at both primary infused sites and secondary noninfused sites became especially apparent, and this effect was investigated further (next section).

A second blinded investigator confirmed significant differences between SERP-1–treated and control samples at the primary infusion site using morphological and histological criteria of plaque parameters (P<.0017 for plaque thickness; P<.0042 for plaque extent; and P<.0025 for plaque composition). Taken together, these results demonstrate that SERP-1 is an extremely potent inhibitor of atheroma development at sites of balloon-mediated vascular injury.

Other investigators have reported that generally with Wolinsky infusion of a variety of reagents with molecular weights significantly larger than the SERP-1 protein (55 kD), the infused test reagent can be detected throughout the arterial wall extending to the adventitia.^{24 28 29} To assess the distribution of SERP-1 in both the arterial wall and the bloodstream, we examined ¹²⁵I-radiolabeled SERP-1 distribution 2 hours after infusion through a Wolinsky catheter. Of the radiolabeled SERP-1 protein detected in the arterial wall at the primary site of ¹²⁵I SERP-1 infusion, 65% was detected in the media and intima, and 35% had penetrated to the surrounding adventitia. There was considerable release of SERP-1 into the bloodstream, with a 147-fold excess of the injected radiolabeled SERP-1 in the circulating blood, but almost no SERP-1 was detected in the organ specimens or secondary arterial wall samples.

Systemic Infusion of Higher Doses of SERP-1 Reduces Plaque Growth
Because Wolinsky infusions at higher concentrations (0.3 to 3.0 ng) of SERP-1 protein indicated a reduction in plaque at secondary noninfused, balloon-damaged sites (Fig 4, dotted line), efficacy of plaque growth reduction after intravenous and intra-arterial systemic infusion of SERP-1 was also assessed in cholesterol-fed rabbits after balloon angioplasty. At each infusion dose, 2 rabbits received intravenous infusion and 2 rabbits received intra-arterial infusion immediately after balloon injury for a total of 4 rabbits at each SERP-1 dose. A generalized and progressive reduction in plaque development at balloon-damaged sites was seen at doses of SERP-1 >3 ng (Fig 5). Histological sections had reduced plaque growth throughout the balloon-damaged sites of the entire abdominal aorta (Fig 6A and 6B) compared with the same sites after control infusions (Fig 6C and 6D). A clear dose response in the reduction in plaque area was seen, with the greatest reduction at SERP-1 doses >30 ng (Fig 5). Contrast angiograms recorded at the 4-week follow-up did not show a significant change in lumen diameter at these SERP-1 doses, but control rabbits had a smaller mean lumen diameter (mean diameter for SERP-1 infusion, 4.1±0.2 mm; for saline infusion, 3.4±0.1 mm, P<.03).

Focal Infusion of an Active-Site Mutant of SERP-1
To determine whether the reduction of plaque growth was the result of the actual serine proteinase inhibitory activity of the SERP-1 protein, studies were conducted with an active-site SERP-1 mutant in which the P₁-P₁' site of the serpin reactive loop was changed from (arg-asn) to (ala-ala) to ablate the ability of the protein to inhibit all serine proteinases tested.²² Wolinsky catheter infusion of the (ala-ala) mutant of SERP-1 in cholesterol-fed rabbits after balloon angioplasty had no effect on plaque growth, whereas a parallel series of rabbits infused with wild-type SERP-1 reduced plaque growth significantly at the primary infusion site (Fig 7A). This loss of plaque inhibitory activity was significant (P<.0001) and illustrates that the plaque inhibitory activities of SERP-1 are due to its biochemical properties as an inhibitory serpin and not to an unrelated property of the SERP-1 protein. This conclusion is underscored by the histological analysis of primary site lesions from the rabbits infused with the (ala-ala) mutant (Fig 7B) compared with the wild-type SERP-1 (Fig 7C).

Effect of SERP-1 on Clotting Factors and Serum Cholesterol Levels
No significant correlation between the mean cholesterol and triglyceride concentrations in blood taken from rabbits at the 4-week follow-up was detected, indicating that SERP-1 does not act as a chronic systemic cholesterol-lowering agent. The mean cholesterol and triglyceride levels were 59.8±5.4 and 1.05±0.49 mmol/L after SERP-1 infusion, whereas the mean cholesterol and triglyceride levels were 54.8±10.1 and 4.12±2.56 mmol/L, respectively in the controls (P=NS). There was also no significant acute effect of SERP-1 on the prothrombin time, partial thromboplastin time, and fibrinogen levels when SERP-1 was mixed with the blood from normal or lipemic rabbits. In the blood taken from 3 rabbits, the baseline partial thromboplastin time before SERP-1 was added varied from 32.4 to 52.75 seconds. After addition of SERP-1, the partial thromboplastin time remained in the same range from 31.4 to 49.65 seconds (P=NS). These data indicate that SERP-1 has no measurable short- or long-term effect on standard clotting parameters.

Effect of SERP-1 on the Influx of Rabbit Macrophage Into Damaged Vascular Sites
Two time points were chosen for analysis: at 24 hours after infusion, samples were used to assess the level of acute cellular infiltration in response to the Wolinsky infusion at balloon-damaged sites, and at 4 weeks after infusion, samples were used to evaluate long-term responses. The rabbit cell populations measured were vascular smooth muscle cells and monocytes/macrophages. The sections that displayed short-term responses 24 hours after infusion showed no significant differences in smooth muscle cell numbers at primary sites of SERP-1 infusion (Fig 8A) versus saline control (Fig 8B), but significant reductions were observed in the infiltration of macrophages (Fig 8C) into primary sites perfused by purified SERP-1 protein compared with the saline controls (Fig 8D). When comparable sections from infusion sites were stained for these same cell populations 4 weeks after infusion with either SERP-1 or control, similar profiles were observed for smooth muscle cells (Fig 8E and 8F), but again marked reductions at SERP-1–treated sites were observed for macrophages (Fig 8G) compared with saline controls (Fig 8H). The immunohistochemical controls (Fig 8I and 8J) illustrate the specificity of staining.

To quantify the effect of SERP-1 infusion on cellular infiltration at primary infused sites in the same rabbit, the percent of cells staining positive for each of the two antibodies was determined for primary and secondary sites (Fig 9A). As Fig 9A shows, at 24 hours after infusion, there were no significant differences in smooth muscle cell populations at either primary or secondary sites, whereas reductions in the infiltration of macrophages were restricted to the primary sites of SERP-1 infusion but not saline controls and were not observed at secondary sites in either case at this SERP-1 infusion dose (0.3 ng). When the same analysis was performed on 4-week samples (Fig 9B), again no major effect of SERP-1 was observed on smooth muscle cell proliferation at primary or secondary sites, but the chronic levels of infiltration of RAM11-positive macrophages remained low at the sites of primary SERP-1 infusion compared with any of the secondary sites. Thus, we conclude that by 24 hours after SERP-1 infusion, there was a significant decrease in the influx of RAM11-positive macrophages (P<.01) compared with saline infusion at sites of primary Wolinsky infusion or any of the secondary sites. Similarly, in the 4-week samples, macrophage staining remained significantly decreased at sites of primary SERP-1 infusion both in the body of the intimal plaque and in the deeper medial layers of the vessel wall (P<.0001).

Assessment of Potential Adverse Effects of SERP-1 Infusion
Occasional complications were observed in the first series of experiments with partially purified proteins. Foot-pad necrosis was observed in 4 rabbits with the MonoQ-purified SERP-1 and VV-601 preparations that contained control MonoQ-purified vaccinia proteins. Jaundice was seen in 2 rabbits (1 treated with partially purified SERP-1 and 2 treated with VV-601 control proteins). Pulmonary edema developed in 2 rabbits given purified SERP-1 (30 ng) through a Wolinsky catheter but was not seen in any later studies at higher systemic concentrations of SERP-1. Foot-pad necrosis was seen in 2 rabbits infused through a Wolinsky catheter with purified SERP-1. Postoperative paralysis (2 rabbits after VV-601 infusion, 1 rabbit after SERP-1 infusion) and dissection (2 rabbits after VV-601 or saline infusion, 1 rabbit after SERP-1 infusion) related to the surgical procedures were observed in equal numbers of rabbits treated with VV-601 or SERP-1.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The accelerated development of plaque at sites where interventional devices have been previously used to open occluded arteries is a serious medical complication, necessitating repeated angioplasty procedures or surgery. High restenosis rates (20% to 50%) are observed with all devices (laser and balloon angioplasty, atherectomy, and stent implant) used to clear narrowed or occluded segments.^{1 2 5} Multiple theories have been advanced to rationalize the chain of events that lead to accelerated plaque growth after angioplasty. The stimulus for smooth muscle cell or monocyte proliferation^{3 4 5 6} has been variously attributed to metabolic disorders (hyperlipidemia, diabetes, cigarettes, or homocystinuria),^{3 4 5 6 30} autoimmune reactions,³¹ viral or autocrine induction of benign smooth muscle tumors,^{32 33} and injury produced iatrogenically by intervention itself.^{1 2} The inflammatory response, initiated when areas of obstruction are mechanically cleared, is believed to be responsible for the initiation of a cascade that involves multiple classes of immune cells and inflammatory regulators.^{34 35 36} Multiple protease enzymes are activated during the inflammatory responses that accompany vascular injury and unstable plaque rupture. The thrombotic and inflammatory cascades in particular are regulated by cascades of serine proteinase enzymes and their inhibitors. We demonstrate here that the infusion of a purified viral serine proteinase inhibitor with documented anti-inflammatory properties¹⁹ reduces plaque growth at sites of balloon injury when infused locally at low (picogram) doses or infused systemically at higher (nanogram) doses at the time of balloon angioplasty arterial injury.

Restenosis is characterized by rapid cellular proliferation and leukocyte infiltration over 2 to 4 weeks that is followed by a secondary wave of connective tissue deposition.^{3 4 37} After an obstructed artery is opened by interventional approaches, circulating leukocytes involved in the acute inflammatory response respond to the induction of proinflammatory signals, particularly from damaged endothelial cells at the area of injury, and undergo directed adhesion and chemotaxis into the damaged vasculature.^{3 4 34 35 36 37 38} If the cellular inflammatory healing response to injury is excessive, then the balance of healing is lost, resulting in recurrent plaque growth that again occludes blood flow in the artery. Similar acute inflammatory reactions may also be involved in the necrosis and rupture that induce acute thrombosis and vascular occlusion in unstable coronary syndromes. The border zones at the margins of the fibrous cap in unstable lesions, where plaque rupture occurs, frequently have monocyte infiltrates, signifying local acute inflammatory reactions.³⁸ Effective therapy for the prevention of plaque development, whether de novo or recurrent, remains limited.^{39 40 41} In this study, we have therefore taken a novel approach to therapeutic intervention that involves the delivery of naturally occurring anti-inflammatory proteins encoded by DNA viruses to dampen the acute cellular inflammatory response to injury at the earliest possible stage after intervention.

Many DNA viruses, particularly those with large genomes, have acquired viral genes whose encoded proteins protect the virus from host responses.^{11 12 13} These viral anti-immune proteins have coevolved with the vertebrate immune system after millions of years of selection pressure and constitute a valuable repository for potent immunomodulators and inflammatory inhibitors.¹⁴ The SERP-1 protein is a virus-encoded serine proteinase inhibitor reported to be secreted from infected cells and to repress effective leukocyte cellular infiltration into the tissues harboring myxoma virus.¹⁹ Serine proteinase enzymes have been implicated in leukocyte chemotaxis, migration, connective tissue breakdown, tissue invasion, activation of platelets, and clot formation.⁷ The results in this report demonstrate that the purified SERP-1 protein, in the absence of any other viral factors, can dramatically reduce plaque growth at sites of balloon-mediated vascular injury in this rabbit model. Whether SERP-1 might be effective in humans depends critically on the conservation of the individual serine proteinase(s) that this particular viral serpin is targeted to inhibit. In this regard, it is important to note that SERP-1 substantially reduces balloon injury–induced atheroma in the cholesterol-fed rabbit model at doses that range from picograms (Wolinsky infusions) to nanograms (systemic infusions), suggesting that further investigation of this protein as a clinical reagent are warranted.

We propose that the dramatic decrease in plaque development after SERP-1 infusion in rabbits is the result of the direct inhibition of cellular proteinases, such as plasmin or urokinase-like plasminogen activator, that mediate the induction of proinflammatory signaling molecules in response to balloon damage. The fact that a reactive-site mutant of SERP-1 protein, in which the P₁-P₁' scissile bond that serves as a substrate for the target serine proteinases has been mutated from (arg-asn) to (ala-ala), is unable to protect against plaque development after balloon injury (Fig 7) demonstrates that it is the actual serpin activity of the SERP-1 protein that inhibits atheroma growth. In this article, we have also demonstrated that there is a dramatic decrease in the local infiltration of reactive macrophage cells at primary sites of SERP-1 infusion within the first 24 hours and that this is followed by decreases in both plaque growth and chronic macrophage infiltration that persists until at least 4 weeks after SERP-1 infusion. Biochemically, SERP-1 protein is known to form 1:1 inhibitory complexes in vitro with a variety of serine proteinases, including plasmin, urokinase, and tissue plasminogen activator,^{21 22} but more information is needed to ascertain whether any of these proteinases are specifically targeted in vivo by SERP-1 in this model. Other lines of evidence suggest that regulatory serpins produced by endothelial cells may act to protect vascular sites from inflammation,^{9 42} and it is conceivable that the viral SERP-1 protein was originally derived by genetic capture from a host progenitor serpin that naturally regulates inflammation. Thus, the acquisition of such an anti-inflammatory strategy could very well have provided a selective advantage for the virus within infected tissues. All the data presented here are consistent with the hypothesis that damaged vascular endothelial cells and/or activated inflammatory leukocytes recruited to the injured site are targets for SERP-1 inhibition. Although the time window in which SERP-1 actively suppresses the influx of reactive inflammatory cells into the damaged site after balloon angioplasty currently is unknown, we hypothesize that once healing of the endothelial layer is complete, the continued inhibitory effect of SERP-1 may no longer be required. This model of SERP-1 action postulates that cellular serine proteinases, possibly at the surface of luminal endothelial cells or incoming monocytes, are key mediators of the inflammatory cascade that signals reactive leukocyte influx into the traumatized vasculature.

This is the first use of a purified virus-encoded serine proteinase inhibitor for the prevention of inflammation and immune reactions associated with a nonviral disease progression, borrowing from the same strategy that DNA viruses use to evade their own immune destruction.¹⁴ Such virus-encoded anti-inflammatory proteins may also have future applications to other diverse immune and inflammation-based disorders.

	Acknowledgments

 
This work was funded by grants from the National Cancer Institute of Canada (Dr McFadden), the Heart and Stroke Foundation of Canada (Dr Lucas), and Biogen, Inc (Drs Lucas and McFadden). Dr Lucas is a clinical investigator at the Alberta Heritage Foundation for Medical Research and a scientist at the Robarts Research Institute. Dr McFadden is a medical scientist at the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada. We thank S. Kasinec for help with the manuscript and W.D. Yan, C. Bleackley, M. Michalak, F. Rachubinski, R. Rachubinski, and P. Armstrong for helpful discussions.

Received December 5, 1995; revision received June 25, 1996; accepted July 8, 1996.

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