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Circulation. 1997;96:4333-4342

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(Circulation. 1997;96:4333-4342.)
© 1997 American Heart Association, Inc.

Articles

Remodeling and Neointimal Formation in the Carotid Artery of Normal and P-Selectin–Deficient Mice

Anjali Kumar, PhD; Jennifer L. Hoover, BS; Carol A. Simmons, BS; Volkhard Lindner, MD, PhD; ; Ronald J. Shebuski, PhD

From Cardiovascular Pharmacology, Pharmacia and Upjohn, Inc, Kalamazoo, Mich, and Department of Surgery, Maine Medical Center Research Institute, South Portland (V.L.). Dr. Kumar is currently at Preclinical R & D Genetics Institute, Andover, Me.

Correspondence to Ronald J. Shebuski, PhD, Pharmacia and Upjohn, Inc, 301 Henrietta St, Kalamazoo, MI 49007. E-mail akumar{at}genetics.com


*    Abstract
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 *Abstract
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Background Inflammatory reactions such as leukocyte activation with platelet adherence and release of inflammatory mediators occur after percutaneous transluminal coronary angioplasty and may play a role in restenosis. Vascular remodeling with neointimal formation was studied in normal C57Bl/J6 and P-selectin–deficient mice.

Methods and Results The left common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, left carotids and contralateral controls were snap-frozen. Computer-aided morphometry was performed to measure ratios of neointimal to medial area (NI/M) in 10 sections per animal as a measure of the thickness of the neointimal lesion. For normal mice, NI/M was 1.13±0.2 (n=20), whereas NI/M was reduced by 76% to 0.27±0.1 (n=19) in P-selectin knockout mice. Vascular constriction (as measured by the length of external elastic lamina) was the same in both groups, but the circumference of the lumen in knockout mice was 26% larger. Also, normal and P-selectin–deficient mice were killed at 3 and 7 days after ligation (n=6 for each group per time point). Histological staining and immunostaining for CD45 showed no inflammatory cell presence in P-selectin knockout mice. However, in normal mice, leukocyte infiltration was observed in the adventitia, media, and developing neointima. Also, P-selectin immunostaining was observed in media and developing neointima of normal mice.

Conclusions These data suggest that P-selectin is involved in processes leading to cell migration and proliferation associated with vascular remodeling, presumably by mediating leukocyte recruitment and the interaction between platelets and leukocytes.


Key Words: restenosis • immunohistochemistry • remodeling • cells


*    Introduction
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 *Introduction
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 down arrowResults
 down arrowDiscussion
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Restenosis is one of the major limitations of PTCA and can be thought of as a combination of neointimal formation and arterial remodeling in response to balloon injury. Although intimal hyperplasia has been described as the "pathological hallmark of restenosis after angioplasty,"1 it may not be the primary mechanism by which restenosis occurs. Vascular remodeling has a significant impact on chronic lumen area2,3 and may be responsible for 50% to 90% of late luminal area loss.4–7

Remodeling is an adaptive process that occurs in response to chronic changes in hemodynamic conditions8,9 and involves changes in many processes, such as cell growth, cell death, cell migration, and changes in extracellular matrix composition, that lead to a compensatory adjustment in vessel diameter and lumen area. In the context of restenosis after balloon angioplasty, vascular remodeling refers to loss of lumen area by a combination of reduction in vessel diameter and neointimal thickening. The exact mechanism of arterial remodeling remains to be fully understood and may involve growth factors, vasoactive agents, and matrix modulators. Endothelial cells play an important role in sensing changes in mechanical and biochemical forces9; however, the time to endothelial regrowth after angioplasty in humans is unknown. The blood vessel essentially is thought to remodel itself in response to long-term changes in flow, such that the lumen area is modified to maintain a predetermined level of shear stress.10

P-selectin is a membrane glycoprotein contained within platelet {alpha}-granules11,12 and Weibel-Palade bodies of endothelial cells13,14 that is rapidly mobilized to the plasma membrane on cell activation and granule secretion. It is a member of the selectin family, which also includes E-selectin and L-selectin. Selectins have been implicated in mediating transient interactions between endothelial cells and leukocytes in what is known as leukocyte "rolling," generally believed to be the prerequisite for firm adhesion.15,16 In addition, P-selectin has also been reported to mediate adherence of activated platelets to monocytes and neutrophils17,18 via its carbohydrate ligands sialyl LewisX and P-selectin glycoprotein ligand-1.19

Previous studies suggest that inflammatory reactions and platelet accumulation occur after PTCA. Activation of granulocytes20,21 and neutrophils22 after coronary angioplasty in humans provides evidence that these cells may be important in the restenotic process. Marmur et al1 reported that thrombin is generated in human coronary arteries after PTCA. Thrombin-activated platelets adhere to monocytes and neutrophils via P-selectin.17,18 Recently, Mickelson et al23 reported that leukocyte activation with platelet adherence occurs after coronary angioplasty and that the magnitude of leukocyte activation and platelet adherence was higher in patients experiencing late clinical events. In addition, higher plasma P-selectin levels have been reported in patients with restenosis within 6 months of PTCA.24

Platelet-leukocyte interaction is of considerable pathophysiological interest because it not only targets both cell types to appropriate sites of inflammation and/or hemostasis but also causes functional alteration in these cells. For example, neutrophil-platelet interaction has been associated with neutrophil activation, adherence, and aggregation.25,26 Metabolic cooperation, such as the transcellular metabolism of lipid intermediates from one cell type by the other,27,28 may be important in inflammation, thrombogenesis, and wound healing. P-selectin–mediated binding to monocytes may also induce tissue factor expression29,30 and hence play a role in thrombogenesis.31

We recently established a model for studying vascular remodeling in the mouse.32 Interruption of flow caused by ligating the left common carotid artery just proximal to the carotid bifurcation caused an 80% reduction in lumen area by a combination of intimal hyperplasia together with decreased vessel diameter. This model has the potential to identify molecules contributing to the remodeling process in vivo by studying animals carrying targeted disruption of genes or expressing transgenes. In this study, neointimal lesion formation in normal C57Bl/J6 mice and P-selectin knockout mice was compared to investigate the role of P-selectin in the restenotic process.


*    Methods
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 *Methods
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All animals used in this study were handled in compliance with the Guide for the Care and Use of Laboratory Animals, 1996, a publication of the National Research Council, National Academy Press, Washington, DC. All experiments were performed in an American Association for Accreditation of Laboratory Animal Care–approved laboratory according to protocols that were reviewed and approved by the Institutional Animal Care and Use Committee.

Animal Procedures
Mouse Carotid Ligation Model
Adult male C57Bl/J6 mice (n=20) and P-selectin–deficient mice (n=20) were used in the carotid ligation model. P-selectin knockout mice, generated on the C57Bl/J6 strain, were provided by Transgenics, Pharmacia & Upjohn, Inc. Briefly, the animals were anesthetized with a solution of ketamine (80 mg/kg body wt; Fort Dodge Laboratories, Inc) and xylazine (5 mg/kg; Lloyd Laboratories) injected intraperitoneally. The left common carotid artery was exposed through a small midline incision in the neck. The artery was completely ligated just proximal to the carotid bifurcation to disrupt blood flow. The animals were allowed to recover for 4 weeks. All animals except one P-selectin knockout mouse recovered. The animals were then reanesthetized as described above, and 50 µL of a 5% solution of Evans blue dye (Sigma Chemical Co) in Ringer's solution (Baxter Healthcare Corp) was injected into the renal vein and allowed to circulate for 1 minute. For each animal, after perfusion fixation under physiological pressure with formaldehyde solution (10% vol/vol in aqueous phosphate buffer) as previously described,33 a 5-mm segment of the left carotid just proximal to the suture and a similar segment of the right contralateral control artery were excised.

Perfusion-fixed artery segments were placed (ligated end down for left carotid segments) in Tissue-Tek O.C.T. embedding medium (Miles Inc), snap-frozen at -160°C in liquid nitrogen–cooled isopentane (Baxter Scientific), and stored at -84°C. Fresh-frozen samples were sectioned on a Leitz cryostat and placed on Fro-Pen–coated (Zymed Laboratories Inc) ProbeOn slides (Fisher Scientific) for immunohistochemical analysis and Fro-Pen–coated microscope slides for histological staining. Normal C57Bl/J6 and P-selectin knockout mice were also killed at days 3 and 7 (6 of each at both time points) after carotid artery ligation and tissue samples were analyzed for possible early involvement of inflammatory cells.

Bleeding Time Measurements
Determinations of bleeding times for 25 each of the C57 Bl/J6 and P-selectin knockout mice were made as previously described.34,35 Briefly, conscious mice were held in a restrainer, and a distal 2-mm segment of the tail was transected with a disposable surgical blade. The tail was quickly immersed in a 100-mL beaker of 0.9% isotonic saline at 37°C. Bleeding time was measured from the moment the tip of the tail was severed until bleeding ceased completely and there was no rebleeding within 30 seconds, up to a maximum observation period of 10 minutes. Those animals for which bleeding did not cease in this period were assigned a bleeding time of 10 minutes.

Complete Blood Counts
Normal C57Bl/J6 and P-selectin–deficient mice were anesthetized as above (n=10 each), and blood samples were drawn by cardiac puncture into EDTA-containing Microtainer tubes (Becton-Dickinson). These samples were analyzed by Clinical Research, Pharmacia & Upjohn, Inc, and complete blood counts and differential leukocyte counts were reported. Briefly, complete blood and differential leukocyte counts were performed in a Bayer Technicon H-1 loaded with species-specific software. This apparatus uses fixation followed by flow-cytometric light scattering to determine parameters such as red cell count, size, hemoglobin content, and platelet count and volume in a laser-based optics channel. The peroxidase method was used for leukocyte counts after red cell lysis and fixing of white blood cells and intracellular enzymes. Differential leukocyte counts were performed after cytochemical staining at the sites of peroxidase activity. The final counts were obtained by a combination of light scatter, peroxidase staining, and nuclear complexity. Blood smears were examined to check cell morphology. The automated Technicon system was frequently calibrated by manual hemocytometric counting after red cell lysis.

Immunohistochemistry
P-Selectin Immunostaining
Representative cryo-step sections (50 µm apart/30 per animal) from normal and P-selectin knockout mice at 3, 7, and 28 days after ligation were immunohistochemically assessed for P-selectin antigen expression with an indirect immunoenzymatic ultrastreptavidin detection method (Signet Laboratories). Briefly, tissue cryosections were postfixed in cold 10% formalin (4°C) for 10 minutes. Endogenous peroxidase activity was blocked by 0.3% (vol/vol) H2O2 in methanol for 10 minutes. An avidin/biotin blocking kit (Vector Labs, Inc.) was also applied to block nonspecific binding, followed by preincubation in 5% normal goat serum for 20 minutes. The primary polyclonal P-selectin antibody (PharMingen) or an appropriate nonimmune rabbit IgG control antibody (Sigma ImmunoChemical) was applied for 30 minutes, followed by application of a 1:2 diluent of biotinylated secondary antibody for 20 minutes. An ultrastreptavidin horseradish peroxidase–conjugated labeling complex was applied for 20 minutes. P-selectin antigen expression was visualized with a DAB chromogen substrate. Sections were counterstained with Mayer's hematoxylin (Sigma Chemical Co) and mounted with Crystal Mount (Fisher Biotech) for light microscopic analysis. Confirmation of method specificity was achieved by PBS buffer substitution of primary and secondary antibodies or staining with DAB substrate only.

CD45 Immunostaining
To further characterize involvement of inflammatory leukocytes at days 3 and 7 after ligation, CD45 immunohistochemistry was conducted on 30 adjacent cryostat step sections. CD45-positive cells were immunolocalized by incubation with a rat monoclonal antibody against CD45 (PharMingen) (1.25 µg/mL) overnight (4°C) followed by application of a biotinylated mouse anti-rat secondary antibody (Jackson ImmunoResearch) (5 µg/mL) for 30 minutes. Detection of CD45 was completed with a DAB chromogen substrate that produces a brown cell surface stain on CD45 positive cells.

Histopathology
Adjacent cryostat step sections (50 µm apart) were also stained with H&E for morphometric and light microscopic evaluation of 3- and 7-day (n=6 for both groups per time point) and 4-week (n=20 for normal and n=19 for knockout mice) lesion formation after ligation. To better identify infiltrating inflammatory cells, additional 3- and 7-day samples were embedded in paraffin, and step sections were stained with H&E to achieve superior morphology.

Morphometry
The extent of neointimal proliferation was quantified by measuring the area (µm2) of the neointima and media for 10 H&E–stained cross sections of each left carotid artery (from 20 normal and 19 P-selectin knockout mice). Typically, 30 to 40 H&E–stained sections, 50 µm apart, were obtained from each animal. To select the 10 sections to be measured, the sections from an animal were divided into five nearly equal groups. All sections were examined under a Jenaval photomicroscope (The Microscope Company), and two sections were picked from each group. This was to ensure that the entire length of the arterial sample could be used in the analysis, especially because the thickness of the lesion varied along the length of the artery. Also, all sections were within 5 mm of the ligation site, because only a 5-mm segment had been excised in the first place. The Optimas analysis software (Bioscan, Inc) and a Microcomp image analysis system (The Microscope Company) was used to measure areas enclosed by the EEL, IEL, and the vessel lumen. The medial area was calculated by subtracting the area defined by the IEL from the area defined by the EEL. The neointimal area was determined by subtracting lumen area from the area defined by the IEL. In addition, the length of the EEL (in micrometers) was determined in both groups as a measure of the vascular constriction. The circumference of the IEL and lumen (in micrometers) were also measured. These comparisons were made on the basis of length measurements, which are less sensitive to changes due to collapse of sections during processing.

Data Analysis
Data from morphometric analyses were reported as an average NI/M ratio and circumference of EEL and lumen (in micrometers) for each animal, which were measures of the thickness of the neointimal lesion, vascular constriction, and lumen area, respectively. Values were averaged for the two groups of mice: normal C57Bl/J6 and P-selectin knockout. Bleeding time was reported in minutes. All data are reported as mean±SEM for the two groups of mice. Hypothesis testing on the means of the two groups was performed with unpaired Student's t tests, and probability values were reported. Statistical significance was judged at P<=.05.


*    Results
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 *Results
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Comparison of Lesion Formation in Normal C57Bl/J6 and P-Selectin–Deficient Mice
For normal mice 4 weeks after ligation, the average NI/M ratio calculated for the ligated left carotid arteries by measurement of 10 histological sections per animal was 1.129±0.15 (n=20, Fig 1aDown). Intimal hyperplasia was maximal closest to the ligation site and decreased in thickness in the direction of the aortic arch. As also shown in Fig 1aDown, for P-selectin–deficient mice, NI/M was 0.270±0.07 (n=19). Compared with normal mice, neointimal thickness in P-selectin knockout mice was decreased by 76% (P<.001). We established earlier that in this model, lumen area is reduced by a combination of neointimal formation and reduction in vessel area.32 Fig 1bDown shows that there was no difference in the average circumference of EEL in normal and knockout mice. Hence, both groups exhibit a similar degree of vascular constriction in response to carotid artery ligation. The length of the IEL was the same in both groups too (data not shown), suggesting that medial area was not different. The circumference of the lumen in P-selectin knockout mice was 26% larger than that of normal mice (P=.003, Fig 1bDown).



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  		Figure 1. Morphometric analysis of normal and P-selectin knockout mouse arterial sections. A, Average NI/M for normal (n=20) and P-selectin knockout (n=19) mice. Average NI/M ratio was calculated for 10 histological sections per animal. Neointimal thickness as measured by NI/M was 76% lower in P-selectin knockout mice than in normal mice (P<.001). B, Circumference (µm) of EEL was not different in the two groups; however, circumference of lumen in P-selectin knockout mice was 26% larger than that of normal mice (P=.003). Values are mean±SEM.

Fig 2aDown shows a histological section from the right control artery of a normal C57Bl/J6 mouse. Fig 2bDown is a representative section from the left ligated artery of the same mouse as in Fig 2aDown. As is clear from Fig 2aDown and 2bDown, there is neointimal formation in response to interruption of flow in this model in normal C57Bl/J6 mice. Similarly, Fig 2cDown is a typical right control artery from a P-selectin knockout mouse. Fig 2dDown is a section from the left artery of the same knockout mouse as in Fig 2cDown. Fig 2cDown and 2dDown, along with Fig 1Up, shows that neointimal formation is clearly reduced in response to flow interruption in P-selectin knockout mice.



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  		Figure 2. Representative H&E–stained frozen sections from ligated left and contralateral right control carotids for normal and P-selectin knockout mouse 4 weeks after ligation. a, Control artery section from normal C57Bl/J6 mouse with typical morphology. b, Ligated left carotid artery of same mouse shows thick concentric neointimal proliferation. Arrows demarcate IEL. c, Control artery section from a P-selectin knockout mouse also had normal tissue morphology as in a. d, However, section from ligated left artery of same knockout mouse had markedly reduced neointimal proliferation. Arrows demarcate IEL. Magnification x125; bar=40 µm.

Role of Inflammatory Cells in Lesion Formation
H&E–stained paraffin-embedded carotid sections from normal and P-selectin knockout mice at 3 and 7 days after ligation (n=6 for each group at each time point) are shown in Fig 3Down. Evidence of inflammatory leukocyte recruitment was visible in normal mouse sections, around the lumen and in the adventitia, at day 3 (Fig 3aDown). By day 7, inflammatory cells were in the developing neointima, media, and adventitia (Fig 3cDown). In P-selectin knockout mice, no inflammatory cells were observed either at day 3 (Fig 3bDown) or day 7 (Fig 3dDown) after ligation. To better identify inflammatory leukocytes, immunostaining for the common leukocyte antigen CD45 was performed on frozen sections from normal and P-selectin knockout mice 3 and 7 days after ligation. CD45-positive leukocytes, indicated by a brown surface stain in Fig 4Down, were observed in the same pattern as that described for Fig 3Down above. Again, P-selectin knockout mice had no evidence of inflammatory cell recruitment either at day 3 (Fig 4bDown) or at day 7 (Fig 4dDown).



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  		Figure 3. H&E–stained paraffin sections from mice killed 3 and 7 days after carotid ligation. a, At 3 days after ligation, inflammatory cells were observed around lumen and in adventitia (arrows). b, No inflammatory cell recruitment was visible in P-selectin knockout mice at day 3. c, Inflammatory cells were observed in developing neointima, media, and adventitia of normal mouse carotid sections by day 7 (arrows). d, At day 7, inflammatory cells were absent in knockout mice. N indicates normal; KO, P-selectin knockout. Magnification x500; bar=20 µm.



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  		Figure 4. CD45 immunohistostaining from normal and P-selectin knockout mice at 3 and 7 days. Positive CD45 stain for inflammatory cells (brown stain) confirmed recruitment and migration of leukocytes in normal mice at days 3 and 7 (a and c, respectively) as seen in previous H&E–stained sections in Fig 3Up. Inflammatory cells were absent in P-selectin knockout mice at both time points (b and d, respectively). Abbreviations and sizes as in Fig 3Up.

P-selectin immunostaining on frozen sections taken from animals killed 3 and 7 days after ligation is shown in Fig 5Down for one each of the normal and P-selectin knockout mice. Fig 5aDown shows a section from a normal mouse at day 3. Some P-selectin staining (indicated in brown) is visible around the lumen, probably derived from endothelial cells and adhering platelets. Staining is absent in the P-selectin knockout mouse section, as shown in Fig 5bDown. By day 7, P-selectin immunostain for the normal mouse was more intense and extended not only around the lumen but also into the developing neointima and media (Fig 5cDown). Again, there was no staining in the P-selectin knockout mouse at day 7, as shown in Fig 5dDown. P-selectin immunostaining for normal animals was absent at 4 weeks after ligation (data not shown).



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  		Figure 5. Immunoperoxidase P-selectin staining (dark brown) on ligated left carotid artery sections taken from one each of the animals killed 3 and 7 days after ligation. a, Section from normal mouse at day 3 showed P-selectin immunostaining around lumen, probably derived from endothelial cells and adhering platelets. b, No P-selectin stain was observed in knockout mouse sections at day 3. c, By day 7, normal mouse sections had more intense P-selectin staining, observed around lumen and extending into media and developing neointima. d, Staining was absent in P-selectin knockout mice at day 7, as shown. Abbreviations and sizes as in Fig 3Up.

Bleeding Time Measurements
The average time for bleeding to cease after the tip of the tail was severed in normal mice was 2.33±0.6 minutes (n=25); for P-selectin knockout mice (n=25), it was 2.06±0.38 minutes. Hence, bleeding times for normal and knockout mice were not different (P=.70). Bleeding time measurements were made according to the same protocol as Subramaniam et al35; however, unlike the findings in the present work, these investigators reported a 40% increase in the bleeding time for P-selectin knockout mice compared with normal mice. This might be related to the fact that in their study, Subramaniam et al used P-selectin knockout mice on a 129Sv/C57BL background, whereas in the present study, knockout mice were C57BL and were confirmed homozygous, as were the normal animals.

Complete Blood Counts
Complete blood counts and differential leukocyte counts were performed on blood samples from normal C57Bl/J6 and P-selectin–deficient mice (n=10 each). A twofold to threefold increase in basal neutrophil counts in P-selectin–deficient animals compared with normal wild-type animals has been reported previously.36 Also, no difference in the total peripheral leukocyte and platelet counts was reported in either group of animals. In this study, however, total leukocyte count in P-selectin knockout mice was found to be 49% higher than in normal mice (4.81±0.5x103 versus 3.22±0.7x103/µL; n=10, P=.05). The percentage of leukocytes that were neutrophils, lymphocytes, and monocytes was the same in both cases (32%, 62%, and 1%, respectively). Therefore, total counts of neutrophils, lymphocytes, and monocytes were nearly 50% higher in the P-selectin knockout mice than in normal mice, probably as a result of increased half-life and/or reduced margination. Also, the platelet count in P-selectin–deficient mice was found to be 18% lower than in normal mice (1084.6±20.8x103 versus 1317.2±103.3x103/µL; P=.04). Again, these differences from previous reports in blood counts may be a result of differences in the genetic background of the mice studied.


*    Discussion
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 *Discussion
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Coronary restenosis remains a complex pathophysiological problem that continues to defy preventive pharmacological therapy. Processes such as smooth muscle cell migration and proliferation, extracellular matrix synthesis, and mural thrombosis, among others, are posited to contribute to restenosis after PTCA. However, a variety of agents targeting these processes have been largely unsuccessful in clinical trials despite positive effects in animal models of neointimal proliferation. In some of the more promising studies, neointimal hyperplasia in rabbit carotid arteries after balloon injury was reduced by the administration of a small peptide antagonist, GpenGRGDSPCA, which blocked the {alpha}Vß3 integrin, implicated in smooth muscle cell migration.37 Also, a monoclonal antibody directed against the platelet GP IIb/IIIa ({alpha}IIbß3) receptor reduced clinical restenosis, as assessed by composite end-point analysis (death, myocardial infarction, or repeat revascularization).38 This was, however, associated with serious bleeding complications.

Although the use of animal models is not without limitations, it has contributed immensely to the understanding of the process of restenosis. It has now become possible to effectively define appropriate molecular targets by studying animals (usually mice) carrying targeted disruption of genes (knockout mice) or expressing transgenes (transgenic mice). We developed a murine model of vascular remodeling in which a disruption of the flow field was created in the left common carotid artery.32 During the 4 weeks that these animals were allowed to recover, these altered shear stress conditions caused the vessel to "remodel" and shrink in luminal area because of reduction in vessel diameter and neointima formation. The proliferative response of smooth muscle cells may also be stimulated by the increase in arterial wall tension39–41 that occurs proximal to the ligating suture. In this work, this model was used in normal C57Bl/J6 and P-selectin knockout mice to investigate the role of P-selectin in the restenotic process.

The NI/M was reduced by 76% in P-selectin knockout mice compared with normal mice, and the circumference of the lumen was found to be 26% larger (Fig 1Up). Fig 2Up shows the dramatic difference in lesion thickness between the two groups. Clearly, P-selectin is involved in the processes leading to cell proliferation associated with vascular remodeling, much like that which occurs after balloon injury. These data also strongly suggest the early involvement of inflammatory cells in mediating this effect, because leukocytes were observed in the adventitia, media, and developing neointima of normal mouse carotid sections at 3 and 7 days after ligation, but not in P-selectin knockout mice. In other murine models, such as thioglycollate-induced neutrophil influx into the peritoneal cavity,42 a 1- to 2-hour lag in neutrophil recruitment after thioglycollate injection was observed. The recovery of neutrophil recruitment at later time points (>2 hours) was attributed to E-selectin. In the present study, normal C57Bl/J6 and P-selectin–deficient mice were examined 3, 7, 14 (data not shown), and 28 days after carotid ligation. The complete absence of inflammatory cell infiltrate in knockout mouse carotid sections at 3 and 7 days is presented (Figs 3Up and 4Up). Inflammatory cells were not detected within the arterial wall at 14 or 28 days in either group (data not shown). The lesion thickness at 28 days, however, was significantly reduced in P-selectin knockout mice, as shown in Figs 1Up and 2Up. It appears that E-selectin is most likely not involved in this injury model. Although the endothelial cells remain intact, the changes in the local environment on ligation may not be sufficient to induce E-selectin expression, which requires robust cytokine or LPS stimulation over a period of 4 to 6 hours.43 E-selectin immunostaining was not performed in this work, but endothelial cells were found to stain positive for P-selectin. In addition, P-selectin immunostaining in the media and developing neointima reveals that platelets may have a role in contributing to smooth muscle cell migration and proliferation.

In this model, secondary effects associated with no net flow in the left common carotid artery together with turbulence just proximal to the ligating suture may lead to activation of platelets and leukocytes. Both platelets and leukocytes may gain access to the media at sites at which the integrity of the endothelial monolayer is compromised, in the region of turbulence proximal to the ligating suture. Alternatively, platelets and leukocytes adhering to the endothelium may simply be covered by migratory, hyperplastic vascular smooth muscle cells. However, because these processes could just as easily occur in the P-selectin knockout mice, but did not, a third possibility seems more likely. The transmigration of leukocytes into the vessel wall has been extensively characterized.16 Also, the adherence of platelets via P-selectin to its carbohydrate ligands on monocytes and neutrophils has been well documented.17,18 Therefore, it is possible that platelets may be transported to the media through heterotypic aggregation with migratory leukocytes mediated by P-selectin. This would create a more direct spatial association between vascular smooth muscle cells and platelet-derived mitogenic and chemotactic factors, thus enhancing neointimal formation. Conversely, absence of P-selectin may have a two-pronged effect in P-selectin knockout mice. First, the process of leukocyte recruitment is impaired in these mice36 because of the absence of P-selectin, which mediates leukocyte rolling on activated endothelium. In addition, heterotypic aggregation between platelets and leukocytes mediated by P-selectin is not likely to occur in P-selectin–deficient mice. This would explain why inflammatory cells were not observed in P-selectin knockout mouse carotid arteries at 3 and 7 days after ligation, even though it was not surprising to find little or no P-selectin immunostaining in P-selectin–deficient mouse sections. However, P-selectin immunostaining in normal mice, especially in the media at day 7, which is probably platelet derived, suggests that in this model, platelets migrate into the vessel wall either directly or via P-selectin–mediated adherence to leukocytes, potentially influencing smooth muscle cell migration and proliferation.

A recent report suggested a mechanism for platelet-dependent lymphocyte recruitment to high endothelial venules in which activated platelets bound to peripheral node addressin on the endothelium in L-selectin–deficient mice can capture circulating lymphocytes through high-density expression of P-selectin.44 Adherent platelets at sites of vascular damage may recruit circulating neutrophils through P-selectin–mediated adhesion and ß2-integrin–dependent transmigration.45 Interactions such as these are probably responsible for the colocalization of platelets and neutrophils in acute inflammation,46 myocardial infarction,47 and atherosclerosis.48,49 In addition, platelet-leukocyte adherence has been reported in patients undergoing cardiopulmonary bypass50 and after coronary angioplasty,23 the magnitude of leukocyte activation and platelet adherence being higher in patients experiencing late clinical events. Diacovo et al44 suggested that platelets may have the capacity to deliver leukocytes to vascular beds that may not express selectins or selectin ligands but do have receptors for other platelet adhesion molecules. These investigators also report a "dynamic interplay between activated platelets and vascular endothelium" that is characterized by the occurrence of reversible but continuous interactions between endothelial cells and deposited platelets. In this scenario, it is tempting to speculate that migratory leukocytes may also have the capacity to deliver platelets to the media via a P-selectin–dependent interaction in which platelet-derived growth factors and chemotactic factors may influence smooth muscle cell migration and proliferation leading to neointimal formation in such pathological situations as restenosis.

Previously described animal models of atherogenesis have also reported neointimal thickening accompanied by leukocyte infiltration.51,52 In the present model, vascular constriction with accompanying neointimal proliferation was studied, because it relates to loss of lumen area in restenosis after angioplasty. No endothelial denudation or thrombus formation was observed. The results obtained suggest early involvement of leukocytes and implicate P-selectin in this process. There is evidence in the literature that agents that antagonize selectins/P-selectin help protect the host from damage occurring during inflammation,53 myocardial infarction, and reperfusion injury.54–57 In addition, antibody inhibition of P-selectin blocked leukocyte accumulation and fibrin deposition within Dacron grafts.31 Also, pretreatment with an anti–P-selectin antibody accelerated streptokinase-induced thrombolysis in a primate model of arterial thrombosis.58 Recently, it was reported that in a rabbit balloon injury model, intimal hyperplasia was reduced by blocking selectins with a sialyl LewisX analogue.59 These data, together with the results from the present study, suggest that P-selectin may be an important therapeutic target for cardiovascular disorders.

P-selectin–deficient mice were found to be grossly normal and fertile. P-selectin immunostaining on blood smears was performed to confirm that these mice in fact did not express platelet P-selectin (data not shown). In addition, the same P-selectin knockout mice were also supplied to another group, which showed that there was no histamine- or LPS-induced expression of P-selectin in the tissues of these mice compared with the wild-type (C57Bl) mice.60 In the present work, tail bleeding times for P-selectin knockout mice were not found to be significantly different from those for normal C57Bl/J6 mice, unlike previously reported data.35 This indicates that P-selectin–deficient mice do not suffer from abnormal intravascular coagulation activity, despite an 18% reduction in platelet count. These mice did exhibit moderate leukocytosis, probably due to reduced margination and/or increased half-life of the cells. It therefore appears that P-selectin antagonism will be appropriate and effective as a therapeutic strategy and will probably not be associated with bleeding complications.

This work provides compelling evidence for a role of P-selectin in a mouse model of arterial remodeling and neointimal formation. This may have implications in the treatment of restenosis, especially because it is now recognized that remodeling has a significant impact on chronic lumen area and is largely responsible for late lumen area loss. Administration of various types of P-selectin inhibitors, such as neutralizing antibodies and competing oligosaccharide ligands, have thus far been successful in animal models for various related pathological conditions, as outlined above. Given the limitations of currently available preventive therapy for restenosis, P-selectin antagonism may be a highly attractive strategy for further clinical investigation.


*    Selected Abbreviations and Acronyms
 
EEL = external elastic lamina
IEL = internal elastic lamina
NI/M = ratio of neointimal to medial area
PTCA = percutaneous transluminal coronary angioplasty
DAB = 3,3'-diaminobenzidine tetrahydrochloride
H&E = hematoxylin and eosin


*    Acknowledgments
 
The authors wish to acknowledge Timothy P. Boyle and Susan L. Kuiper of Transgenics (Unit 7241), Pharmacia and Upjohn, Inc, for providing P-selectin knockout mice and Clinical Research (Unit 7262) for analyzing blood samples. The expert advice of Carol W. Johnson (Unit 7227) on immunohistochemistry is gratefully acknowledged. The completion of these experiments was also greatly facilitated by the assistance of the staff of Laboratory Animal Medicine and Care (Unit 7273), Pharmacia and Upjohn, Inc.

Received July 10, 1997; revision received September 5, 1997; accepted September 11, 1997.


*    References
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  1. Marmur JD, Merlini PA, Sharma SK, Khaghan N, Torre SR, Israel DH, Ardissino D, Ambrose JA. Thrombin generation in human coronary arteries after percutaneous transluminal balloon angioplasty. J Am Coll Cardiol. 1994;24:1484–1491.[Abstract]
  2. Kakuta T, Currier JW, Haudenschild CC, Ryan TJ, Faxon DP. Differences in compensatory vessel enlargement, not intimal formation, account for restenosis after angioplasty in the hypercholesterolemic rabbit model. Circulation. 1994;89:2809–2815.[Abstract/Free Full Text]
  3. Currier JW, Faxon DP. Restenosis after percutaneous transluminal coronary angioplasty: have we been aiming at the wrong target? J Am Coll Cardiol. 1995;25:516–520.[Abstract]
  4. Post MJ, Borst C, Kuntz RE. The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty: a study in the normal rabbit and the hypercholesterolemic Yucatan micropig. Circulation. 1994;89:2816–2821.[Abstract/Free Full Text]
  5. Mintz GS, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. Intravascular ultrasound comparison of restenotic and de novo coronary artery narrowings. Am J Cardiol. 1994;74:1278–1280.[Medline] [Order article via Infotrieve]
  6. Wilensky RL, March KL, Gradus-Pizlo I, Sandusky G, Fineberg N, Hathaway DR. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation. 1995;92:2995–3005.[Abstract/Free Full Text]
  7. Andersen HR, Mæng M, Thorwest M, Falk E. Remodeling rather than neointimal formation explains luminal narrowing after deep vessel wall injury: insights from a porcine coronary (re)stenosis model. Circulation. 1996;93:1716–1724.[Abstract/Free Full Text]
  8. Guyton JR, Hartley CJ. Flow restriction of one carotid artery in juvenile rats inhibits growth of arterial diameter. Am J Physiol. 1985;248:H540–H546.[Abstract/Free Full Text]
  9. Langille BL, O'Donnell F. Reductions in arterial diameter produced by chronic decreases in flow are endothelium-dependent. Science. 1986;231:405–407.[Abstract/Free Full Text]
  10. Kamiya A, Togawa T. Adaptive regulation of wall shear stress to flow change in the canine carotid artery. Am J Physiol. 1980;239:H14–H21.[Abstract/Free Full Text]
  11. Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol. 1985;101:880–886.[Abstract/Free Full Text]
  12. Berman CL, Yeo EL, Wencel-Drake JD, Furie BC, Ginsberg MH, Furie B. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. J Clin Invest. 1986;78:130–137.
  13. McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF. GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in the Weibel-Palade bodies. J Clin Invest. 1989;84:92–99.
  14. Bonfanti R, Furie BC, Furie B, Wagner DD. PADGEM (GMP 140) is a component of Weibel-Palade bodies of human endothelial cells. Blood. 1989;73:1109–1112.[Abstract/Free Full Text]
  15. Lasky LA. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science. 1992;258:964–969.[Abstract/Free Full Text]
  16. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76:301–314.[Medline] [Order article via Infotrieve]
  17. Larsen E, Celi A, Gilbert GE, Furie BC, Erban JK, Bonfanti R, Wagner DD, Furie B. PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell. 1989;59:305–312.[Medline] [Order article via Infotrieve]
  18. Hamburger SA, McEver RP. GMP-140 mediates adhesion of stimulated platelets to neutrophils. Blood. 1990;75:550–554.[Abstract/Free Full Text]
  19. McEver RP. Regulation of function and expression of P-selectin. Agents Actions Suppl. 1995;47:117–119.[Medline] [Order article via Infotrieve]
  20. De Servi S, Mazzone A, Ricevuti G, Fioravanti A, Bramucci E, Angoli L, Stefano G, Specchia G. Granulocyte activation after coronary angioplasty in humans. Circulation. 1990;82:140–146.[Abstract/Free Full Text]
  21. Tsutsui M, Shimokawa H, Tanaka S, Yoshihara S, Higuchi S, Matsuguchi T, Okamatsu S. Granulocyte activation in restenosis after percutaneous transluminal coronary angioplasty. Jpn Circ J. 1996;60:27–34.[Medline] [Order article via Infotrieve]
  22. Ikeda H, Nakayama H, Oda T, Kuwano K, Yamaga A, Ueno T, Yoh M, Hiyamuta K, Koga Y, Toshima H. Neutrophil activation after percutaneous transluminal coronary angioplasty. Am Heart J. 1994;128:1091–1098.[Medline] [Order article via Infotrieve]
  23. Mickelson JK, Lakkis NM, Villarreal-Levy G, Hughes BJ, Smith CW. Leukocyte activation with platelet adhesion after coronary angioplasty: a mechanism for recurrent disease? J Am Coll Cardiol. 1996;28:345–353.[Abstract]
  24. Ishiwata S, Tukada T, Nakanishi S, Nishiyama S, Seki A. Postangioplasty restenosis: platelet activation and the coagulation-fibrinolysis system as possible factors in the pathogenesis of restenosis. Am Heart J. 1997;133:387–392.[Medline] [Order article via Infotrieve]
  25. Redl H, Hammerschmidt DE, Schlag G. Augmentation by platelets of granulocyte aggregation in response to chemotaxins: studies utilizing an improved cell preparation technique. Blood. 1983;61:125–131.[Abstract/Free Full Text]
  26. Tzeng DY, Deuel TF, Huang JS, Senior RM, Boxer LA, Baehner RL. Platelet-derived growth factor promotes polymorphonuclear leukocyte activation. Blood. 1984;64:1123–1128.[Abstract/Free Full Text]
  27. Marcus AJ, Safier LB, Ullman HL, Broekman MJ, Islam N, Oglesby TD, Gorman RR. 12S,20-Dihydroxyicosatetraenoic acid: a new icosanoid synthesized by neutrophils from 12S-hydroxyicosatetraenoic acid produced by thrombin- or collagen-stimulated platelets. Proc Natl Acad Sci U S A. 1984;81:903–907.[Abstract/Free Full Text]
  28. Maclouf JA, Murphy RC. Transcellular metabolism of neutrophil-derived leukotriene A4 by human platelets: a potential cellular source of leukotriene C4. J Biol Chem. 1988;263:2223–2229.[Abstract/Free Full Text]
  29. Celi A, Pellegrini G, Lorenzo R, De Blasi A, Ready N, Furie BC, Furie B. P-selectin induces the expression of tissue factor on monocytes. Proc Natl Acad Sci U S A. 1994;91:8767–8771.[Abstract/Free Full Text]
  30. Osterud B. Cellular interactions in tissue factor expression by blood monocytes. Blood Coagul Fibrinolysis. 1995;6(suppl 1):S20–S25.
  31. Palabrica T, Lobb R, Furie BC, Aronovitz M, Benjamin C, Hsu YM, Sajer SA, Furie B. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature. 1992;359:848–851.[Medline] [Order article via Infotrieve]
  32. Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of flow. Arterioscler Thromb Vasc Biol. 1997:17:2238–2244.
  33. Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ Res. 1993;73:792–796.[Abstract/Free Full Text]
  34. Dejana E, Callioni A, Quintana A, de Gaetano G. Bleeding time in laboratory animals, II: a comparison of different assay conditions in rats. Thromb Res. 1979;15:191–197.[Medline] [Order article via Infotrieve]
  35. Subramaniam M, Frenette PS, Saffaripour S, Johnson RC, Hynes RO, Wagner DD. Defects in hemostasis in P-selectin-deficient mice. Blood. 1996;87:1238–1242.[Abstract/Free Full Text]
  36. Wagner DD. P-selectin knockout: a mouse model for various human diseases. CIBA Found Symp. 1995;189:2–16.[Medline] [Order article via Infotrieve]
  37. Choi ET, Engel L, Callow AD, Sun S, Trachtenberg J, Santoro S, Ryan US. Inhibition of neointimal hyperplasia by blocking {alpha}Vß3 integrin with a small peptide antagonist GpenGRGDSPCA. J Vasc Surg. 1994;19:125–134.[Medline] [Order article via Infotrieve]
  38. Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT, on behalf of the EPIC Investigators. Randomised trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet. 1994;343:881–886.[Medline] [Order article via Infotrieve]
  39. Zarins CK, Zatina MA, Giddens DP, Ku DN, Glagov S. Shear stress regulation of artery lumen diameter in experimental atherogenesis. J Vasc Surg. 1987;5:393–400.[Medline] [Order article via Infotrieve]
  40. Langille BL, Bendeck MP, Keeley FW. Adaptations of carotid arteries of young and mature rabbits to reduced carotid blood flow. Am J Physiol. 1989;256:H931–H939.[Abstract/Free Full Text]
  41. Glagov S. Intimal hyperplasia, vascular remodeling, and the restenosis model. Circulation. 1994;89:2888–2891.[Free Full Text]
  42. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell. 1993;74:541–554.[Medline] [Order article via Infotrieve]
  43. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone MA Jr. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci U S A. 1987;84:9238–9242.[Abstract/Free Full Text]
  44. Diacovo TG, Puri KD, Warnock RA, Springer TA, von Adrian UH. Platelet-mediated lymphocyte delivery to high endothelial venules. Science. 1996;273:252–255.[Abstract]
  45. Diacovo TG, Roth SJ, Buccola JM, Bainton DF, Springer TA. Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the ß2-integrin CD11b/CD18. Blood. 1996;88:146–157.[Abstract/Free Full Text]
  46. Issekutz AC, Ripley M, Jackson JR. Role of neutrophils in the deposition of platelets during acute inflammation. Lab Invest. 1983;49:716–724.[Medline] [Order article via Infotrieve]
  47. Bednar M, Smith B, Pinto A, Mullane KM. Neutrophil depletion suppresses 111In-labelled platelet accumulation in infarcted myocardium. J Cardiovasc Pharmacol. 1985;7:906–912.[Medline] [Order article via Infotrieve]
  48. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488–500.[Medline] [Order article via Infotrieve]
  49. Lehr HA, Olofsson AM, Carew TE, Vajkoczy P, von Adrian UH, Hübner C, Berndt MC, Steinberg D, Messmer K, Arfors KE. P-selectin mediates the interaction of circulating leukocytes with platelets and microvascular endothelium in response to oxidized lipoprotein in vivo. Lab Invest. 1994;71:380–386.[Medline] [Order article via Infotrieve]
  50. Rinder CS, Bonan JL, Rinder HM, Mathew J, Hines R, Smith BR. Cardiopulmonary bypass induces leukocyte-platelet adhesion. Blood. 1992;79:1201–1205.[Abstract/Free Full Text]
  51. Hirosumi J, Nomoto A, Ohkubo Y, Sekiguchi C, Mutoh S, Yamaguchi I, Aoki H. Inflammatory responses in cuff-induced atherosclerosis in rabbits. Atherosclerosis. 1987;64:243–254.[Medline] [Order article via Infotrieve]
  52. Kling D, Holzschuh T, Betz E. Recruitment and dynamics of leukocytes in the formation of arterial intimal thickening: a comparative study with normo- and hypercholesterolemic rabbits. Atherosclerosis. 1993;101:79–96.[Medline] [Order article via Infotrieve]
  53. Welply JK, Keene JL, Schmuke JJ, Howard SC. Selectins as potential targets of therapeutic intervention in inflammatory diseases. Biochim Biophys Acta. 1994;1197:215–226.[Medline] [Order article via Infotrieve]
  54. Weyrich AS, Ma X, Lefer DJ, Albertine KH, Lefer AM. In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest. 1993;91:2620–2629.
  55. Winn RK, Liggitt D, Vedder NB, Paulson JC, Harlan JM. Anti-P-selectin monoclonal antibody attenuates reperfusion injury to the rabbit ear. J Clin Invest. 1993;92:2042–2047.
  56. Seekamp A, Till GO, Mulligan MS, Paulson JC, Anderson DC, Miyasaka M, Ward PA. Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am J Pathol. 1994;144:592–598.[Abstract]
  57. Silver MJ, Sutton JM, Hook S, Lee P, Malycky JL, Phillips L, Ellis SG, Topol EJ, Nicolini FA. Adjunctive selectin blockade successfully reduces infarct size beyond thrombolysis in the electrolytic canine coronary artery model. Circulation. 1995;92:492–499.[Abstract/Free Full Text]
  58. Toombs CF, Degraaf GL, Martin JP, Geng JG, Anderson DC, Shebuski RJ. Pretreatment with a blocking monoclonal antibody to P-selectin accelerates pharmacological thrombolysis in a primate model of arterial thrombosis. J Pharmacol Exp Ther. 1995;275:941–949.[Abstract/Free Full Text]
  59. Barron MK, Lefer DJ, Lake RS, Buda AJ, Tenaglia AN. Intimal hyperplasia following balloon injury is reduced by blocking selectins: a possible role for cell adhesion molecules in restenosis. Circulation. 1996;949(suppl I):I-42. Abstract.
  60. Eppihimer MJ, Wolitzky B, Anderson DC, Labow MA, Granger DN. Heterogeneity of expression of E- and P-selectins in vivo. Circ Res. 1996;79:560–569.[Abstract/Free Full Text]



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