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(Circulation Research. 2003;93:464.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Serine Protease Inhibitor Serp-1 Strongly Impairs Atherosclerotic Lesion Formation and Induces a Stable Plaque Phenotype in ApoE^-/- Mice

Ilze Bot, Jan H. von der Thüsen, Marjo M.P.C. Donners, Alexandra Lucas, Madelon L. Fekkes, Saskia C.A. de Jager, Johan Kuiper, Mat J.A.P. Daemen, Theo J.C. van Berkel, Sylvia Heeneman, Erik A.L. Biessen

From the Division of Biopharmaceutics (I.B., J.H.v.d.T., M.L.F., S.C.A.d.J., Th.J.C.v.B., E.A.L.B.), Leiden/Amsterdam Center for Drug Research, Leiden, the Netherlands; John P. Robarts Research Institute (A.L.), Vascular Biology Research Group, University of Western Ontario, London, Ontario, Canada; and the Department of Pathology (M.M.P.C.D., S.H., M.J.A.P.D.), Cardiovascular Research Institute Maastricht, University of Maastricht, the Netherlands.

Correspondence to Ilze Bot, Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, the Netherlands. E-mail i.bot{at}lacdr.leidenuniv.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The myxoma virus protein Serp-1 is a member of the serine protease inhibitor superfamily. Serp-1 potently inhibits human serum proteases including plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA). Serp-1 also displays a high antiinflammatory activity, rendering it a promising candidate for antiatherosclerotic therapy. In this study, we have thus examined the effect of Serp-1 on de novo atherosclerotic plaque formation and on advanced lesions. Perivascular collars were placed around carotid arteries of ApoE^-/- mice to induce atherosclerotic plaques and Serp-1 treatment started at week 1 and week 5 after collar placement. Effects of Serp-1 on de novo atherogenesis were characterized by a significantly lower plaque size than that of control mice (18±5x10³ versus 57±12x10³ µm², respectively; P=0.007). Immunostaining showed a 50% (P=0.004) decrease in the MOMA-2–stained lesion area of Serp-1–treated mice. Treatment of advanced lesions with Serp-1 resulted in a decrease in plaque size and lumen stenosis (P=0.028). -Actin staining of these lesions was significantly increased compared with the control (P=0.017). In both studies, a higher cellularity of the plaque and increased collagen content was observed in Serp-1–treated mice. In vitro studies showed that Serp-1 induces proliferation and migration of vascular smooth muscle cells. In conclusion, Serp-1 inhibits carotid artery plaque growth and progression in ApoE^-/- mice. Equally relevant, it enhances cellularity of the plaque core potentially leading to improved plaque stability. The above results indicate that Serp-1 constitutes a promising lead in antiatherosclerotic therapy.

Key Words: atherosclerosis • carotid arteries • plaque stability • serpin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atherosclerosis is a complex inflammatory disease afflicting medium- and large-sized arteries. In addition to the cellular constituents of the atherosclerotic plaque, which are primarily of smooth muscle cell, monocytic, and lymphocytic origin, a plethora of paracrine and autocrine mediators have been found to be instrumental in atherogenesis.¹ These include components of the coagulation,^2,3 fibrinolytic,⁴ and complement activation^5,6 cascades, which are known to be present in atherosclerotic plaques and to exert a variety of proinflammatory effects.^7,8 Moreover, the serine proteases responsible for the generation of factors belonging to these cascades are themselves believed to exert pleiotropic effects, including the upregulation of cytokine responses, receptor activation, extracellular matrix remodeling, as well as cellular migration and proliferation of smooth muscle cells.^9,10 All of these processes have been implicated in the pathogenesis of atherosclerosis and their inhibition could therefore constitute an attractive strategy for the prevention of atherosclerotic plaque formation and/or progression.

A major subgroup of these inhibitors consists of the so-called serpins, which belong to a conserved superfamily of single-chain proteins that act as irreversible covalent "suicide" protease inhibitors.¹¹ The precise relevance of these inhibitors to atherosclerosis is largely unclear. Thus, some researchers have found the serpin plasminogen activator inhibitor-1 (PAI-1) to have no effect on de novo atherogenesis in hypercholesterolemic mice,¹² whereas others have demonstrated PAI-1 deficiency to be atheroprotective in early atherosclerosis² and to accelerate plaque progression in advanced atherosclerosis.¹³ Its effect in restenosis appears to be more clear-cut, as studies have demonstrated an exacerbating role for PAI-1 in neointima formation by promoting smooth muscle cell migration and proliferation.^14–17

In the quest for an atheroprotective serpin with therapeutic potential, attention has turned to virally encoded protease inhibitors. The myxoma virus serpin, Serp-1, is a secreted glycoprotein that protects virally infected cells from clearance by the host’s immune system by inhibiting the early inflammatory response.^18–21 It has been found to inhibit a range of proinflammatory proteases that are considered to be involved in atherogenesis, including urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, and Xa.^22,23 Infusion of Serp-1 protein has been reported to attenuate postangioplasty plaque formation^24,25 and transplant vasculopathy^25–27 in a variety of animal models. Its effect on atherosclerosis remains uncharted, however, as the experience gained with PAI-1 implies that data obtained from endothelial injury experiments cannot necessarily be extrapolated to a beneficial effect in de novo atherogenesis.

In this study, we therefore aimed to delineate the effect of Serp-1 on de novo carotid atherogenesis and lesion progression in atherosclerosis-prone ApoE knockout (ApoE^-/-) mice. Accelerated atherosclerotic plaque formation was induced by perivascular collar placement as described previously,²⁸ and Serp-1 glycoprotein was administered 1 (effect on de novo plaque formation) or 5 weeks (effect on advanced lesions) after collar placement by continuous subcutaneous infusion. After 4 weeks of Serp-1 treatment, the effect of Serp-1 was determined by morphometric and immunohistochemical analysis of the resultant plaques.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Male ApoE^-/- mice (12 to 14 weeks old) were obtained from TNO-PG (Leiden, The Netherlands). All animal work was approved by the regulatory authority of Leiden and performed in compliance with the Dutch government guidelines. Western type diet and water were provided ad libitum.

Collar Placement and Serp-1 Administration
To determine the effect of Serp-1 on de novo atherosclerotic lesion formation, two groups of male ApoE^-/- mice (control group: n=8, Serp-1 group: n=9) were fed a Western type diet 2 weeks before surgery and throughout the experiment. Carotid atherosclerotic lesions were induced by perivascular collar placement as described by von der Thüsen et al.²⁸ Mice were anesthetized by subcutaneous injection of ketamine (60 mg/kg; Eurovet Animal Health), fentanyl citrate and fluanisone (1.26 mg/kg and 2 mg/kg, respectively; Janssen Animal Health). Sterile osmotic pumps (Alzet, type 2004, Durect Corporation) containing 0.2 µmol/L endotoxin-free Serp-1 in 0.9% saline (2.0 µg/kg per day)²⁴ were placed subcutaneously using isoflurane anesthesia 1 week after collar placement. Two pumps were filled with ¹²⁵I-Serp-1 (Iodogen method, 0.2 pmol/L, 0.34 cpm/ng) in order to measure plasma levels.

To determine the effect of Serp-1 on advanced atherosclerotic lesions, the administration of Serp-1 (2.0 µg/kg per day) was started in 13 male ApoE^-/- (compared with 14 control mice) 5 weeks after collar placement. Identical osmotic minipumps were used and Serp-1 treatment endured for 4 weeks. Control mice received osmotic pumps containing PBS. During treatment, plasma total cholesterol levels were measured spectrophotometrically using enzymatic procedures (Roche Diagnostics). Triglyceride levels were quantified using a commercially available kit (Roche). In both assays, Precipath standardized serum (Boehringer Mannheim) was used as an internal standard.

Serum Levels of Serp-1
Radioactivity in plasma, obtained from mice receiving ¹²⁵I-Serp-1, was measured weekly to quantify plasma concentrations of Serp-1. To determine degradation of Serp-1, 50 µL of plasma containing ¹²⁵I-Serp-1 was loaded onto a Superdex 75 column in a SMART gel filtration system (Pharmacia) and radioactivity in a total of 25 fraction was quantified.

Tissue Harvesting
Animals were anesthetized after 4 weeks of Serp-1 administration. In situ perfusion and tissue embedding occurred as described in von der Thüsen et al.²⁸ Transverse 5 µm cryosections were prepared on a Leica CM 3050S Cryostat (Leica Instruments) in a proximal direction for histology and immunohistochemical analysis.

Histology and Immunohistochemistry
Cryosections were stained with hematoxylin (Sigma Diagnostics) and eosin (Merck Diagnostica). Sections were immunohistochemically stained for the presence of macrophages using a rat monoclonal MOMA-2 antibody, dilution 1:50 (a gift from Dr G. Kraal, VU, Amsterdam, The Netherlands). Rabbit anti-Rat biotin (1:300, Sigma Diagnostics) was used as a secondary antibody, and ABC-horse radish peroxidase (1:100) was used for visualization. To visualize vascular smooth muscle cells (VSMCs), cryosections were stained for -smooth muscle actin (ASMA) using an ASMA-fluorescein-isothiocyanate (FITC) conjugated antibody (1:3000, Sigma Diagnostics). Anti–FITC-horse radish peroxidase (1:300) was used as a secondary antibody.

The sections were incubated with primary and secondary antibody for 2 hours at room temperature and stained using 3,3'-diamino-benzidine (Sigma Diagnostics) as enzyme substrate. Sections were stained for collagen, using Picrosirius Red (Direct red 80) and for elastin, using an Accustain elastin staining kit (both Sigma Diagnostics).

Morphometry
Hematoxylin-eosin–stained sections of the common carotid arteries were used for morphometric analysis. Each vessel was assessed 0.5 mm proximal to the collar and the site of maximal stenosis was used for morphometric assessment. The images were digitized and analyzed as previously described.²⁸

Cell Culture
VSMCs were obtained from thoracic aortas from male C57Bl/6 mice using the collagenase digestion method.²⁹ Cells were cultured in a humidified atmosphere (5% CO₂) at 37°C in DMEM containing 10% Newborn Calf Serum (NCS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from BioWhittaker Europe).

The murine macrophage cell line RAW 264.7 was cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere (5% CO₂) and at 37°C.

Proliferation Assay
VSMCs or RAW cells were detached by short incubation with trypsin-EDTA (BioWhittaker) and seeded at a density of 3.0x10⁴ (VSMCs) or 1.0x10⁴ (RAW) cells per well. The cells were allowed to attach and serum starved in DMEM containing 1% calf serum for 24 hours to synchronize the cell cycle. The cells were then incubated for 72 hours with medium containing 20 pmol/L of Serp-1 or control medium and medium±Serp-1 was refreshed daily. After incubation 0.5 µCi [³H]thymidine (Amersham) per well was added and the cells were incubated at 37°C for 5 hours. [³H]Thymidine incorporation was quantified in a liquid scintillation analyzer (Packard 1500 Tricarb). To verify the validity of the [³H]thymidine incorporation data, also the absolute number of VSMCs and RAW cells was counted using FACS analysis (FACScalibur, BD Biosciences) after 72 hours of exposure to Serp-1 or control medium, starting with 10⁵ cells/well.

Migration Assay
VSMCs were serum starved for 24 hours in serum containing 1% NCS before the experiment. Cells were detached using trypsin-EDTA and 3.4x10⁴ cells were seeded into Transwell plates (12-µm filter pores, 12-mm diameter, Costar). On the basolateral side of the Transwell chamber, medium containing 10% NCS (negative control) or medium containing 10% NCS with 20 pmol/L Serp-1 was added. The chemotactic peptide n-formyl-methionine-leucine-phenylalanine (fMLP, Sigma) was used as a positive control (1 nmol/L). After 4 hours of incubation at 37°C and 5%CO₂, VSMCs were fixed and stained with Mayer’s Hematoxylin. The number of cells that has migrated to the basolateral side of the chamber was scored manually.

Apoptosis Assay
VSMCs and RAW 264.7 were seeded at a density of 10⁵ cells/cm² and allowed to attach for 24 hours. The cells were exposed to 20 pmol/L of Serp-1 in medium for 24 hours after which DNA of the cells was stained with propidium iodide (Sigma). RNase H (Boehringer Mannheim) was added to avoid RNA contamination in the measurements. DNA fragmentation was measured using FACS analysis (FACScalibur, BD Biosciences).

Statistical Analysis
Values are expressed as mean±SEM. A two-tailed Student’s t test was used to compare individual groups of animals. A level of P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Because the serum half-life of ¹²⁵I-Serp-1 after intravenous injection into mice was 3.2 minutes, we chose to administer Serp-1 subcutaneously by continuous infusion through osmotic minipumps (infusion rate 0.25 µL/hr per day for 4 weeks, 2.0 µg/kg per day).

Serp-1 infusion did not affect body weight, plasma total cholesterol, and triglyceride levels of the Western-type diet fed ApoE^-/- mice (data not shown). After subcutaneous infusion of Serp-1 by means of the osmotic minipumps, Serp-1 plasma concentration was 14±3 pmol/L (Figure 1A) throughout the duration of the treatment. As a measure of the stability of Serp-1 in plasma, plasma of the ¹²⁵I-Serp-1–treated mice was analyzed using gel filtration (SMART system, Pharmacia Biotech). The radioactivity profile of each plasma sample essentially coincided with that of intact Serp-1 (Figure 1B), substantiating the integrity of the infused Serp-1. Only in the plasma sampled at week 4, some residual radioactivity was recovered at higher elution volumes, which may point to minor degradation of Serp-1 at that time point.

View larger version (14K): [in this window] [in a new window]   Figure 1. A, Plasma levels of Serp-1 after continuous subcutaneous infusion of 125I-Serp-1 via an osmotic minipump into ApoE-/- mice during 4 weeks of treatment. B, Gel filtration analysis of plasma samples obtained from 125I-Serp-1–infused ApoE-/- mice at 2 (), 3 (), 4 (, and 5 () weeks after surgery. Bar indicates the fractions in which intact Serp-1 elutes.

Plaque Size
De Novo Atherogenesis
Plaque size and cell numbers were measured to determine the effect of Serp-1 on de novo atherogenesis. Morphometric analysis of lesions proximal to the collar showed that the plaque sizes of Serp-1–treated mice were significantly lower (67.7%) than that of control mice (18±5x10³ versus 57±12x10³ µm², respectively; P=0.007; Figure 2A). Also, the intima/lumen ratio of Serp-1–treated mice was significantly reduced as compared with the control mice (-44%; 0.29±0.06 versus 0.66±0.11; Table 1). Total vessel areas of the carotid artery at the site of maximal stenosis were 79±9x10³ µm² in control animals versus 61±5x10³ µm² on Serp-1–treated mice (P=NS).

View larger version (23K): [in this window] [in a new window]   Figure 2. Plaque size analysis (A and D), lumen size (B and E), and cellularity (C and F) of cross sections of carotid artery specimens obtained from control and Serp-1–treated ApoE-/- mice (de novo atherosclerosis: A, B, and C; advanced lesions: D, E, and F). Error bars represent SEM. A, Plaque sizes of Serp-1–treated mice compared with that of control mice are significantly reduced (**P=0.007). B, An increase in lumen size was found in Serp-1–treated ApoE-/- mice (*P=0.017). C, Cellularity of lesions of control mice and Serp-1–treated mice. A significant increase of cellularity was found in the Serp-1–treated group (*P=0.014). D, Plaque size of Serp-1–treated mice after 5 weeks of collar (P=0.058). E, Lumen size of Serp-1–treated plaques were found to be increased compared with the controls (*P=0.028). F, Serp-1–treated lesions also display a significant increase in cellularity (*P=0.034).

View this table: [in this window] [in a new window]   Table 1. Effect of Serp-1 on De Novo Atherogenesis

Advanced Atherosclerosis
Analysis of the carotid arteries of the ApoE^-/- mice with preexisting lesions showed a 30% decrease in lesion size after Serp-1 treatment (P=0.058; Figure 2D, Table 2). The lesion size of these Serp-1–treated animals showed a 10% increase in further plaque growth compared with the control mice after 5 weeks of lesion development (P=0.7). Also, the lumen size was significantly increased in treated animals (P=0.028; Figure 2E). Total vessel areas at the site of maximal stenosis was 95±11x10³ µm² in control animals versus 80±7x10³ µm² on Serp-1–treated mice (P=NS).

View this table: [in this window] [in a new window]   Table 2. Effect of Serp-1 on Preexisting Atherosclerotic Lesions

Plaque Morphology
De Novo Atherogenesis
A detailed analysis of the plaque morphology revealed striking differences in lesion composition between Serp-1–treated and control mice. When mice were treated with Serp-1 1 week after collar placement (de novo atherogenesis), lesions were found to differ markedly from that of control mice in terms of core cellularity (6.0±1.0x10³ versus 2.0±0.7x10³ cells/mm²; P=0.014; Figure 2C). To address the actual cell type responsible for the increased cellularity, lesions were stained for the presence of macrophages (MOMA-2) and differentiated SMCs (ASMA).

Immunostaining showed a significant decrease in MOMA-2–stained lesion area of Serp-1–treated mice. The area stained for MOMA-2 as a percentage of the total lesion size was 39±5% in control mice versus 20±3% in Serp-1–treated mice (-50%; P=0.004; Figures 3A through 3C). Lesions of Serp-1–treated mice tended to be enriched in -smooth muscle-actin, calculated as a percentage of total plaque area (control: 9±2%; Serp-1: 14±5%, Figures 3D through 3F). The contribution of extracellular matrix to the plaque structure was determined by measuring collagen content of the lesions using a Picrosirius Red staining, which showed a significant increase in collagen in Serp-1–treated lesions (38±9% compared with 12±3% in control lesions; P=0.012; Figures 3G through 3I).

View larger version (82K): [in this window] [in a new window]   Figure 3. Immunohistochemical analysis of Serp-1–treated and control plaques on the effect of Serp-1 on de novo atherogenesis. MOMA-2 staining (x100, A and B), ASMA staining (x100, D and E), and Picrosirius Red staining (x200, G and H) of control (A, D, and G) and Serp-1–treated (B, E, and H) lesions. Content was calculated as percentage of staining area relative to the total plaque size. A significant decrease in MOMA-2–positive macrophage content was observed in the Serp-1–treated mice (C; **P=0.004). F, Amount of -smooth muscle actin staining. No significant difference between the relative area stained for VSMCs between control and Serp-1 was observed. I, Picrosirius Red–stained collagen was significantly increased in the Serp-1–treated mice (*P=0.012).

Advanced Atherosclerosis
The plaque cellularity of the preexisting lesions treated with Serp-1 was found to be increased in Serp-1–treated mice compared with control mice (3.1±0.2x10³ and 2.1±0.4x10³ cells/mm² respectively; P=0.034; Figure 2F). Immunostaining in these Serp-1–treated lesions showed a 3-fold increase in -smooth muscle-actin staining as percentage of the total plaque area compared with the controls (15±5% and 5±1%, respectively; P=0.017; Figures 4D through 4F). These data show that in control animals the amount of -actin is diminishing in time, whereas this is not observed in Serp-1–treated animals. In the lesions, the macrophage-specific MOMA-2 staining showed no differences between control and Serp-1–treated mice (15±2% versus 15±2%; Figures 4A through 4C). In control animals, the macrophage content of the plaques decreases in time from 38% to only 15% of the total plaque area. In Serp-1–treated mice, the macrophage content is not increasing during lesion development at the expense of necrotic core formation.

View larger version (91K): [in this window] [in a new window]   Figure 4. Immunohistochemical analysis of Serp-1–treated and control plaques on the effect of Serp-1 on advanced lesions. MOMA-2 staining (x200, A and B), ASMA staining (x200, D and E), and Picrosirius Red staining (x200, G and H) of control (A, D, and G) and Serp-1–treated (B, E, and H) lesions. Content was calculated as percentage of staining area relative to the total plaque size. C, Extent of MOMA-2 staining. No significant difference between the relative area stained for macrophages between control and Serp-1 was observed. F, Amount of ASMA staining: -actin content was found to be significantly increased in Serp-1–treated mice (*P=0.017). I, Significant increase in collagen in Serp-1–treated mice was measured (**P=0.001).

The Serp-1–treated plaques contained a significantly higher amount of collagen: 49±3% compared with 27±4% in control plaques (P=0.001; Figures 4G through 4I). In control lesions, collagen was mainly present in the cap of the plaque, in contrast with the treated plaques, which show collagen staining in both cap and core. Elastin staining of the cryosections showed no difference in lesional elastin content (4.8±1.1% in the control group versus 4.7±0.9% in the Serp-1–treated group). Likewise, fibrin staining did not reveal any differences in fibrin content of the control lesions (19±6%) versus Serp-1–treated arteries (16±4%, P=0.7).

In Vitro Studies
In order to explain the hypercellularity of the Serp-1–treated lesions, in vitro experiment were performed using VSMCs. Proliferation studies with VSMCs showed an increased [³H]thymidine incorporation in VSMCs exposed to 20 pmol/L of Serp-1, corresponding with the plasma concentration used in the in vivo experiments, compared with control VSMCs (170%±12% versus 100%±20% in control cells; P=0.003; Figure 5A). FACS analysis confirmed that Serp-1 incubation (20 pmol/L) led to an increase in VSMC count (62 485±5023 cells compared with 47 031±3574 control cells; P=0.04; Figure 5B). Serp-1 incubation also resulted in a small but significant increase (35%) in [³H]thymidine incorporation (P<0.05) in RAW 264.7 cells. However, the absolute number of RAW cells was not significantly altered after Serp-1 exposure (40 626±4212 versus 53 311±4750 cells for the control). This indicates that the increased incorporation of [³H]thymidine was due to increased expansion rather than to increased proliferation of the RAW cells.

View larger version (17K): [in this window] [in a new window]   Figure 5. In vitro studies performed with VSMCs. A, Proliferation assay of VSMCs exposed to 20 pmol/L of Serp-1; [3H]thymidine incorporation was used to measure proliferation. Exposed to Serp-1, VSMCs display a significantly higher tendency to proliferate (**P=0.003). B, Amount of VSMCs after exposure to 20 pmol/L of Serp-1 was significantly increased compared with the number of control cells (*P=0.04). Thus, increase in proliferation shown by [3H]thymidine incorporation was mainly due to an increased number of cells rather than expansion of the VSMCs. C, Migration assay of VSMCs exposed to 20 pmol/L of Serp-1 at the basolateral side of a Transwell filter. A significantly higher number of VSMCs migrated in the presence of Serp-1 compared with the negative control (**P=0.003). Serp-1 appeared to be an even more potent chemoattractant for VSMCs than the established chemotactic peptide fMPL (positive control: 1 nmol/L).

In the apoptosis assay, incubation of VSMCs and RAW 264.7 murine macrophage cells with 20 pmol/L of Serp-1 did not lead to DNA fragmentation, indicating that Serp-1 did not induce apoptosis in either cell type (data not shown) at concentrations used in the in vivo situation.

In analogy to PAI-1, Serp-1 may promote VSMC migration. Indeed, our VSMC migration assay data clearly demonstrate a highly significant increase of VSMC migration across the filter, when cells were exposed to 20 pmol/L of Serp-1 on the basolateral side of the Transwell chamber. Incubation of VSMCs with Serp-1 resulted in a transmigration of 386±17 cells per microscopic field compared with 63±9 cells per field in control VSMCs (P=0.003; Figure 5C). Incubation of VSMCs with the chemotactic peptide fMPL, which was used as a positive control, resulted in a transmigration of 290±10 cells per microscopic field.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, the effect of the myxoma virus–derived serine protease inhibitor Serp-1 was investigated on de novo atherosclerosis and on advanced atherosclerotic lesions in apoE^-/- mice equipped with a perivascular collar to induce carotid artery lesions.²⁸ Serp-1 treatment led to a striking reduction in de novo lesion formation, whereas Serp-1 did not affect plasma total cholesterol and triglyceride levels as well as the total body weight of the animals or promote outward remodeling of the carotid artery. Plasma levels of Serp-1 after subcutaneous infusion remained constant during treatment at approximately 13.5 pmol/L. These low picomolar Serp-1 levels have been reported to suffice for effective serine protease inhibition and for blocking the host’s immune response to myxoma infection.^20,21 Further gel filtration analysis revealed that Serp-1 was not degraded during the timeframe of the experiment. Blood cell levels did not differ between control and Serp-1 treatment groups, suggesting that Serp-1 did not act atheroprotective by inducing a systemic antiinflammatory response.

Interestingly, a 3-fold increase in plaque cellularity was observed in the Serp-1–treated mice. The nature of the cells responsible for the hypercellularity of Serp-1 lesions remains to be elucidated. The plaque macrophage content was shown to be reduced by 50%. Moreover, we did not observe a significant difference in ASMA-positive VSMC content between control and treated mice. Conceivably, the vascular wall may be enriched in VSMCs that are redifferentiated toward an ASMA negative phenotype, previously reported to be present in atherosclerotic lesions.^30,31 This is in keeping with the fact that plaque collagen content was found to be increased by Serp-1 treatment. Furthermore, our in vitro studies showed that VSMCs proliferate and display an enhanced tendency to migrate when exposed to Serp-1 at concentrations used in in vivo experiment, indicating that the hypercellularity of the lesions may indeed be explained by an increased VSMC content.

In line with the antiinflammatory effect of Serp-1 as proposed by Macen et al,²⁰ the observed 50.4% reduction in macrophage content of de novo formed lesions suggests that Serp-1 acts partly by impairing monocyte recruitment when administered while the atherosclerotic plaque is formed. The murine macrophage cell line RAW 264.7 showed no increase in apoptosis of these cells on exposure to Serp-1, indicating that the decrease in macrophage content in the lesions cannot be explained by apoptosis of macrophages in the plaque, but may be caused by an impaired monocyte influx. A similar inhibitory effect of Serp-1 on monocyte infiltration was reported by Lucas et al.²⁴

To elucidate the effect of Serp-1 on lesion progression and development, the effect of Serp-1 on advanced lesions was determined by initiating Serp-1 treatment 5 weeks after collar placement. Plaque size and morphology analysis showed an almost 30% decrease in plaque size and a significant 3-fold increase in lumen size. Importantly, total vessel area was not affected by Serp-1 treatment. This indicates that Serp-1 did not promote outward remodeling of the arteries and that the risk of aneurysm formation after Serp-1 treatment is minor. The plaque size of these Serp-1–treated arteries (57x10³ µm²) is similar to the plaque size of control arteries (63x10³ µm²) in the study on de novo atherogenesis, suggesting that plaque progression is almost completely blunted by Serp-1 administration. Importantly, a significant increase in cellularity of the plaque was again observed in Serp-1–treated mice, which in this study can be explained by an increase in amount of -actin positive VSMCs. The lesional VSMC content in control animals decreased in time, suggesting apoptosis of VSMCs in the cap,³² which may be inhibited by Serp-1.

The Serp-1–treated lesions were found to be was enriched in collagen, which was not only present in the cap, but also in the core, as was also observed in the study on de novo atherogenesis. Quantitative gene expression analysis of VSMCs after exposure to 20 pmol/L of Serp-1 did not demonstrate an effect on procollagen-I mRNA levels (data not shown), pointing to an effect of Serp-1 on collagen degradation in the lesion rather than to an increase in production of collagen. Plasmin, activated by uPA and tPA, is a potent activator of collagenase (MMP-1), stromelysin (MMP-3), and gelatinase (MMP-2 and -9), which are matrix metalloproteinases (MMPs),³³ commonly present in atherosclerotic lesions. Conceivably, an impaired activation of MMPs via inhibition of uPA by Serp-1 can lead to increased collagen content in the lesions. Therefore, Serp-1 may promote a stable plaque phenotype by increasing collagen and -actin–positive smooth muscle cell content. Macrophage-specific MOMA-2 staining did not reveal differences in macrophage content between the groups of mice. It should be realized, however, that macrophage infiltration is a process mainly occurring in the early stage of atherogenesis rather than in later stages of lesion development,¹ thus proceeding before administration of Serp-1. We did show a decrease in macrophage content of the lesions in control mice in time from 40% to 15% at the expense of enhanced necrotic core formation, which was not observed in Serp-1–treated animals.

The activity profile of Serp-1 considerably overlaps with that of PAI-1 in that both are potent inhibitors of uPA, tPA, and plasmin. Comparison of the effects of Serp-1 to that of PAI-1 may thus hold a key to elucidation of the actual mechanism by which Serp-1 exerts its atheroprotective effect. Unfortunately, conflicting data on PAI-1^2,12,13 emphasize that the effects of modulation of serine protease activity are intricately dependent on the local protease milieu and the disease progression and leave unanswered to what extent antiinflammatory effects of Serp-1 may contribute to its beneficial effect as well. Other studies on the role of tPA and uPA in atherogenesis have shown that overexpression of uPA in carotid arteries in rabbits results in an acceleration of plaque growth, although no difference in plaque morphology was observed.³⁴ Carmeliet et al³⁵ have shown that in uPA^-/- mice neointima formation after electric injury is reduced. However, in uPA^-/- mice, VSMC migration was found to be reduced, suggesting that additional effects are involved in the Serp-1 effect as well. Just recently, data by Dai et al³⁶ indicate that Serp-1 acts through an uPA receptor–dependent mechanism, as lesion formation after allograft transplantation of uPAR^-/- aortas to uPAR^+/+ animals was not affected by Serp-1 treatment.

In conclusion, the viral glycoprotein Serp-1 inhibited de novo carotid artery plaque growth in ApoE^-/- mice by a significant 68%. This inhibition was mainly caused by a striking reduction in macrophage infiltration. On advanced lesions, Serp-1 inhibited plaque growth with almost 30%. Serp-1 was found to induce hypercellularity and increased the collagen content of the lesion core suggestive of improved plaque stability. The above results thus indicate that Serp-1 or serpins as a general class of antiinflammatory proteases constitute an attractive lead in antiatherosclerotic therapy.

	Acknowledgments

 
This study was supported by grant 016.026.019 from the Netherlands Organization for Scientific Research (I.B., E.B.) and grant M93.001 from the Netherlands Heart Foundation (J.v.d.T).

	Footnotes

 
Original received December 23, 2002; resubmission received June 26, 2003; revised resubmission received August 1, 2003; accepted August 1, 2003.

	References

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