Platelet, Not Endothelial, P-Selectin Is Required for Neointimal Formation After Vascular Injury
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动脉硬化血栓血管生物学 2005年第8期
From the Departments of Cardiovascular Medicine (K.W., Z.Z., L.F., A.M.L., E.J.T., M.S.P), Cell Biology (X.Z., N.M., M.Z., M.S.P.), and Molecular Cardiology (E.F.P.), and the Joseph J. Jacobs Center for Thrombosis and Vascular Biology (E.F.P., E.J.T.), Cleveland Clinic Foundation, Cleveland, Ohio.
Correspondence to Kai Wang, MD, PhD, Department of Cardiovascular Medicine/F25, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail wangk@ccf.org
Abstract
Background— P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury.
Methods and Results— Using wild-type (WT) and P-selectin–knockout (Psel–/–) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel–/– mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel–/– mouse group (0.015±0.004 vs 0.004±0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006±0.004 mm2) compared with Plt-Psel mice (0.011±0.005 mm2, P=0.026) and WT mice (0.015±0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel–/– and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury.
Conclusions— Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.
The relative importance of platelet and endothelial P-selectins to neointimal formation after vascular injury was determined by bone marrow transplantation. Significantly less neointima was observed from EC-Psel mice when compared with Plt-Psel mice and WT mice. Decreased neointimal formation was accompanied by reduced inflammation.
Key Words: P-selectin ? arterial injury ? platelets ? endothelial cells ? neointima
Introduction
The process of restenosis has been attributed to the inflammatory response induced by arterial injury.1–3 This local inflammatory response is critically dependent on of the 3 families of cell adhesion molecules: the selectins, the integrins, and the immunoglobulin supergene family. P-selectin is critical for initial leukocyte adhesion to platelets and endothelial cells (ECs)4,5 and is stored in the -granules of platelets and Weibel-Palade bodies of ECs. After platelet or EC activation, P-selectin is rapidly translocated to the cell surface.
P-selectin has an important role in the inflammatory response and development of neointimal formation after vascular injury.6–15 P-selectin upregulates tissue factor in monocytes and leads to leukocyte accumulation in areas of vascular injury associated with thrombosis and inflammation.6,7 P-selectin binding to monocytes through P-selectin glycoprotein ligand-1 promotes local inflammation through the release of tumor necrosis factor (TNF)-, monocyte chemoattractant protein (MCP)-1), interleukin-88–10 and tissue factor microparticles.11,12 A more recent study showed that P-selectin promoted the deposition of RANTES, the platelet-derived CC chemokine, on ECs, subsequent monocyte arrest, and intimal hyperplasia.13 The potential importance of P-selectin in neointimal formation was further demonstrated by a significant decrease in neointimal formation in P-selectin–null mice.14,15 Immunohistochemistry showed that intense P-selectin–positive staining accompanied by leukocyte recruitment was observed in injured wild-type carotid arteries14 but was absent in the P-selectin–null mice.
We have recently demonstrated that specific antagonism of P-selectin binding inhibits neointimal formation in the porcine coronary artery balloon injury model16 and in the diabetic and nondiabetic rat carotid artery injury model.17 Despite this strong evidence of a central role for P-selectin in the initiation of neointimal formation after arterial injury, the relative importance of platelet and EC P-selectin expression on neointimal formation after arterial injury remains unknown. Expression of both platelet and EC P-selectins has recently been shown to be important in the atherosclerotic process,18,19 suggesting that P-selectin expression in each cell type may be important in the response to arterial injury. In the current study, we evaluated the contribution of EC and platelet P-selectins to neointimal growth after arterial injury.
Methods
Animals and Bone Marrow Transplant (BMT)
P-selectin–knockout (Psel–/–; Wyeth/Genetics Institute, Andover, MA) mice were backcrossed 8 times to C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me) to the C57BL/6J background. Then Psel–/– and nonlittermate control C57BL/6J mice were subjected to total-body irradiation from a cesium source (model 143, J.L. Shepherd) to eliminate endogenous bone marrow stem cells. For the preparation of bone marrow, donor mice (C57BL/6J or Psel–/–) were killed, the femur and tibia bones were isolated under sterile conditions, and the bone marrow was harvested by flushing the bones with medium (RPMI, 5% fetal bovine serum [FBS], and 1 mmol/L HEPES). The cells were washed and resuspended in RPMI containing 5% FBS. Recipient mice were lethally irradiated (12.0 Gy) in 2 split doses 3 hours apart. Immediately after the second dose of irradiation, the mice were injected in the lateral tail vein with 4x106 freshly prepared donor bone marrow cells. After the procedure, transplanted mice were transferred to a microisolator unit, fed with sterile chow and water, administered cefozolin (50 mg[middot]kg–1[middot]d–1 for 3 days), and observed daily for 4 weeks.
Determination of Bone Marrow Reconstitution
Flow cytometry was performed as described19 to determine bone marrow reconstitution. In brief, blood from Psel–/– mice receiving bone marrow from C57BL/6J mice was collected into 3.8% sodium citrate (9:1, vol/vol). Platelet-rich plasma was obtained by centrifuging the blood at 150g for 15 minutes at 24°C. Platelets were activated with 10 μmol/L thrombin receptor activating peptide for 20 minutes, incubated for 40 minutes at room temperature with fluorescein isothiocyanate–labeled anti-mouse P-selectin antibody (Pharmingen), and analyzed by fluorescence-activated cell sorting (FACS) with a Becton-Dickinson FACS.
Arterial Injury
Eight weeks after BMT, C57BL/6J and Psel–/– mice that had received BMT with cells derived from either Psel–/– or C57BL/6J mice underwent a carotid air-desiccating/pressure double injury.20 In brief, the external carotid artery was ligated, and an incision was made in the external carotid artery. A 30-gauge needle connected to a saline-filled syringe with tubing was introduced into the external carotid artery. After irrigating the isolated common carotid artery with saline to remove blood, the syringe was replaced with an angioplasty inflation device, and the isolated, saline-filled, common carotid artery segment was dilated at a pressure of 1.5 atm for 30 seconds. The inflation device was replaced with an air-filled 60-mL syringe, and a 30-gauge air-exit hole was made at the proximal end of the common carotid artery. The carotid artery was then exposed to air desiccation for 3 minutes at 20 mL for air per minute. After air drying, the artery was refilled with saline, the needle was removed, and anterograde blood flow was reestablished. The wound was irrigated with saline and closed. All experiments conformed to the position of the American Heart Association on research animal use and care and were conducted with the approval of Animal Research Committee of the Cleveland Clinic Foundation.
Tissue Harvest and Preparation
The animals were euthanized at 4 or 21 days after injury. For animals euthanized at day 4, the excised carotid arteries were embedded in paraffin for immunohistochemical analysis. For animals euthanized 21 days after injury, the injured vessel segments were perfusion-fixed with 5% Histochoice (Amresco) for 5 minutes and then harvested. Specimens were stored in 5% Histochoice for at least 24 hours before embedding.
Morphometry
The fixed carotid arteries were embedded in paraffin and cut in serial sections, at 5-mm intervals, from the proximal to the distal end. Slides were stained with hematoxylin-eosin and van Gieson’s elastic stains. An observer blinded to the study groups performed morphometric analysis by using a computerized digital microscopic planimetry software package (Image-Pro Plus, version 4.0 for Windows, Media Cybernetics). Five sections from the injured arterial segment per vessel were evaluated and averaged. The neointimal and medial boundaries were determined; the cross-sectional areas subtended by the luminal border, the areas bounded by the internal elastic lamina, and the external elastic lamina were measured; and the ratio of intimal to medial area was calculated.
Cellular Inflammatory Infiltrates
Immunostaining for neutrophils and monocytes at days 4 and 21 after arterial injury was performed to evaluate the role of P-selectin expression on inflammatory cell infiltrate in the platelet-only P-selectin (Plt-Psel) and endothelial-only P-selectin (EC-Psel) groups. The positive neutrophils and monocytes were identified by immunostaining with monoclonal rat anti-mouse neutrophil IgG2a (Serotec Ltd) antibody and a rat anti-mouse monocyte/macrophage IgG1 anti–MAC-3 (BD Bioscience) antibody. Biotinylated goat anti-rat IgG was used as a secondary antibody. The numbers of neutrophils and monocytes/macrophage were counted within the intima and media from 5 sections from each animal by 2 observers blinded to the identity of individual animals.
MCP-1 and RANTES Expression
Immunostaining for MCP-1 and RANTES was performed to assess the effects of P-selectin expression on chemokine expression after arterial injury. In brief, the expression of MCP-1 and RANTES was semiquantified by measuring the percent positive area of arterial cross section in which the chemokines were expressed. A blinded observer quantified the area of expression by using a computerized digital microscopic planimetry algorithm described earlier. MCP-1– and RANTES-positive cells were immunolocalized by incubation with a mouse monoclonal antibody against MCP-1 (Santa Cruz Biotechnology, Inc) and RANTES (Chemicon International), followed by application of a biotinylated rabbit anti-mouse secondary antibody (1.25 μg/mL) for 30 minutes. Detection of MCP-1 and RANTES was completed with a diaminobenzidine chromogen substrate that produces a brown reaction product in MCP-1– and RANTES-positive tissue. All evaluations were performed on 5 sections from the injured arterial segment per vessel and then averaged.
To identify the source of MCP-1, double immunofluorescence staining was performed. The following cell type–specific antibodies were used: mouse monoclonal anti–-smooth muscle cell (SMC) actin/Cy3-conjugated antibody (Sigma) and rat anti-mouse monocyte/macrophage IgG1 anti–MAC-3 (BD Bioscience) antibody. Goat anti-rat IgG-PE (Santa Cruz Biotechnology, Inc) and donkey anti-goat IgG Alexa Fluor 488 antibody (Molecular Probes) were used as secondary antibodies. Tissue was analyzed with an upright spectral laser scanning confocal microscope (model TCS-SP, Leica Microsystems) equipped with blue argon (for 4'-6-Diamidino-2-phenylindole [DAPI]), green argon (for Alexa Fluor 488), and red krypton (for Alexa Fluor 594) laser. Data were collected by sequential excitation to minimize "bleed-through." Image processing, analysis, and the extent of colocalization were performed with Leica confocal software.
Statistical Analysis
All data were expressed as mean±SD. Statistical analysis was performed with SPSS software (version 10.0 for Windows, SPSS Inc). Continuous variables were compared by ANOVA with Bonferroni correction. A value of P0.05 was considered statistically significant.
Results
Characteristics of Psel–/– Mice Receiving BMTs
Male Psel–/– and C57BL/6J mice were lethally irradiated and injected with bone marrow of either genotype to obtain the following groups of animals: group 1 (n=4), bone marrow from C57BL/6J mice into C57BL/6J recipient mice having both endothelial and platelet P-selectins; group 2 (n=4), bone marrow from Psel–/– mice into Psel–/– recipient mice having neither platelet nor endothelial P-selectin; group 3 (n=5), bone marrow from C57BL/6J mice into Psel–/– recipient mice having normal P-selectin expression in their platelets but not their ECs; and group 4 (n=4), bone marrow from Psel–/– mice into C57BL/6J recipient mice having normal P-selectin expression in their ECs but not in their platelets. Flow cytometry performed 8 weeks after BMT showed that activation of platelets from Psel–/– mice receiving bone marrow from C57BL/6J (group 3) had P-selectin expression similar to that seen in wild-type (WT) mice (group 1), indicating that the bone marrow of Psel–/– mice, which received C57BL/6J mice bone marrow, was successfully reconstituted (Figure 1).
Figure 1. Flow cytometry results. Activation of platelets from P-selectin–/– mice receiving bone marrow from C57BL/6J had a P-selectin expression similar to that in WT mice, indicating that the bone marrow of P-selectin–/– mice was successfully reconstituted. No P-selectin expression was found in P-selectin–/– mice Activation of platelets from C57BL/6J mice receiving bone marrow from P-selectin–/– mice had a P-selectin expression similar to that in P-selectin–/– mice, indicating that the bone marrow of C57BL/6J mice was successfully reconstituted. Light scatterblot indicates the nonstimulated platelet histogram. Abbreviations are as defined in text.
Relative Importance of EC and Platelet P-Selectins on Neointimal Hyperplasia
Morphometric data showed that the absence of platelet P-selectin (EC-Psel, group 4) resulted in a significant reduction in neointimal formation when compared with animals that lacked only endothelial P-selectin expression (Plt-Psel, group 3): 0.006±0.004 versus 0.011±0.005 mm2, P=0.026 and the WT mice (group 1): 0.015±0.004 mm2, P<0.001. This difference in neointimal formation was still observed when the data were normalized to the area of the media [neointima-media ratio, 0.237±0.159 vs 0.539±0.311 from the Plt-Psel (group 3; P=0.017) and 0.636±0.281 from the WT mice (group 1; P=0.002)]. We observed a trend but not a statistically significant difference in neointimal formation when animals lacking endothelial P-selectin expression (Plt-Psel, group 3) were compared with the WT group (group 1): 0.011±0.005 versus 0.015±0.004 mm2, P=0.079 (Table and Figure 2).
Morphometric Data at 21 Days
Figure 2. Photomicrographs of mice carotid arteries 21 days after injury (elastic staining, x20). A, Representative cross section of injured artery from a WT (group 1) mouse, showing extensive neointimal formation. B, Representative cross section of injured artery from a P-selectin–/– (group 2) mouse, showing significantly reduced neointimal formation. C, Representative cross section of injured artery from a Plt-Psel (group 3) mouse, showing extensive neointimal formation. D, Representative cross section of injured artery from an EC-Psel (group 4) mouse, showing significantly reduced neointimal formation. L indicates lumen; N, neointima; and M, media. Other abbreviations are as defined in text.
Cellular Inflammatory Infiltrates
The numbers of neutrophils and monocytes/macrophages were counted by a blinded observer to determine the inflammatory infiltrates. The luminal binding of neutrophils at 4 and 21 days after arterial injury was significantly less in the EC-Psel group (n=4) compared with the Plt-Psel group (n=4; 4 day, 6±3 vs 17±4 cells per cross section, P=0.005; 21 day, 4±2 vs 12±4 cells per cross section, P=0.012; Figure 3). A similar finding was also observed for monocyte/macrophage binding and infiltration into the neointimal and artery wall 4 and 21 days after arterial injury (4 day, 7±2 vs 19±6 cells per cross section, P=0.009; 21 day, 3±1 vs 14±4 cells per cross section, P=0.002; Figure 4).
Figure 3. Representative immunohistochemical photomicrographs of injured carotid arteries for neutrophil infiltration. Significantly less neutrophil infiltration was observed in the EC-Psel group when compared with the Plt-Psel group at days 4 and 21 after injury (x63). Abbreviations are as defined in text.
Figure 4. Representative immunohistochemical photomicrographs of injured carotid arteries for monocyte/macrophage infiltration. Significantly less monocyte/macrophage infiltration was observed in the EC-Psel group when compared with the Plt-Psel group at days 4 and 21 after injury (x63). L indicates lumen. Other abbreviations are as defined in text.
Immunohistochemical Assay of MCP-1 and RANTES
Immunohistochemical analysis showed that RANTES expression from Psel–/– (group 2, n=4) and EC-Psel (group 4, n=4) animals was significantly less when compared with that in the WT (group 1, n=4) and Plt-Psel (group 3, n=4) mice, as assessed by percent positive area for RANTES-positive cells [14.9±9.6% (Psel–/–, group 2) and 15.0±2.2% (EC-Psel, group 4) vs 39.2±10.7% (WT, group 1) and 34.4±7.1% (Plt-Psel, group 3), P<0.05; Figure 5A and 5B]. A similar pattern of expression was also observed for MCP-1 expression. Significantly less MCP-1 expression was found in the P-sel –/– (group 2) and the EC-Psel (group 4) groups when compared with WT (group 1) and Plt-Psel (group 3) groups [MCP-1, 12.7±8.6% (Psel–/–, group 2) and 12.8±4.8% (EC-Psel, group 4) vs 32.4±8.5% (WT, group 1) and 30.7±11.3%(Plt-Psel, group 3), P<0.05; Figure 5C and 5D]. Double immunofluorescence staining for MCP-1 with SMC -actin and monocytes/macrophages indicated the source of MCP-1 from both SMCs and monocytes/macrophages (Figure Ia and Ib, available online at http://atvb.ahajournals.org).
Figure 5. Immunohistochemical photomicrographs of injured carotid arteries for RANTES expression (x100) and MCP-1 expression (x63). A, Representative cross section of RANTES expression from the Plt-Psel (group 3) group. B, Representative cross section of RANTES expression from the EC-Psel (group 4) group. C, Representative cross section of MCP-1 expression from the Plt-Psel (group 3) group. D, Representative cross section of MCP-1 expression from the EC-Psel (Group 4). Significantly less RANTES and MCP-1 expression was found in the EC-Psel group, indicating that a greater inflammatory response after arterial injury occurred in the Plt-Psel group. L indicates lumen. Other abbreviations are as defined in text.
Discussion
This study demonstrates that platelet P-selectin, but not endothelium-derived P-selectin, plays a crucial role in the development of neointimal formation after arterial injury. These results reinforce the important role of P-selectin in modulating the inflammatory response to vascular injury and highlight the potential therapeutic strategies targeting neutrophil-platelet interactions in preventing restenosis.
P-selectin plays a prominent role in the adhesion of leukocytes to activated platelets.21–25 Platelet–neutrophil binding promotes the release of vasoactive substances that locally amplify inflammatory and thrombotic responses. P-selectin has been shown to be intensively expressed in adhering platelets on the exposed subendothelium that are bound to adherent leukocytes in injured rat carotid arteries.26 Increased platelet-neutrophil and platelet-monocyte aggregates have also been found in patients with coronary artery disease and after vascular injury.27,28 Consistently, P-selectin–deficient mice form less compact platelet layers on the denuded arterial surface in vivo, accumulate fewer leukocytes into the vascular wall, and develop less neointimal formation after arterial injury.14,20
Studies from our group have demonstrated that specific antagonism of P-selectin significantly reduced the inflammatory response and neointimal formation in the porcine coronary artery balloon injury model15 and rat carotid artery injury model.16 It has been shown that specific antagonism of P-selectin inhibits early platelet-leukocyte adhesion on injured arteries and reduces restenosis through a positive impact on vascular remodeling in a porcine carotid artery double injury model.32–34 It has also been shown that specific antagonism of P-selectin inhibits cytokine secretion, such as MCP-1 and TNF-, from arterial ECs and SMCs, and leads to an inhibited inflammatory response.9 Furthermore, P-selectin also mediates binding of platelets to other cells, such as in platelet-platelet and platelet-endothelial interactions,35 which are also involved in the development of neointimal hyperplasia. RANTES, the platelet-derived CC chemokine, is released from activated platelets.36 It has recently been demonstrated that the deposition of RANTES and subsequent monocyte arrest are promoted by platelet P-selectin and are involved in intimal hyperplasia and that blockade of RANTES receptors decreases neointimal formation and the inflammatory response after vascular injury.13 Consistent with the importance of P-selectin–mediated RANTES expression in neointimal formation, our study demonstrates a significant decrease in RANTES expression and neointimal formation in animals that lacked platelet P-selectin. Recently, a study from Ley’s group18 showed that circulating activated platelets promoted monocyte recruitment to atherosclerotic arteries and accelerated the formation of atherosclerosis in apolipoprotein E–deficient mice. These authors found that activated platelets interacted with monocytes and the endothelium of the vessel wall through platelet P-selectin. This interaction resulted in deposition of platelet-derived proinflammatory factors to the vessel wall, such as RANTES, leading to monocyte recruitment that exacerbated atherosclerosis. This finding is consistent with a more recent study from Wagner’s group,19 in which they demonstrated that targeted disruption of platelet P-selectin led to decreased spontaneous atherosclerosis in apolipoprotein E–deficient mice. However, that study also highlighted the importance of EC P-selectin expression in atherosclerosis. In distinct contrast to our study on neointimal hyperplasia, the earlier study on atherosclerosis showed that animals deficient in only endothelial P-selectin had significantly less atherosclerosis than animals deficient in only platelet P-selectin.
The importance of platelet P-selectin in neointimal hyperplasia in response to arterial injury has recently been demonstrated with BMT from P-selectin–null mice into apolipoprotein E–null mice.37 Our study furthers this work by comparing the relative importance of platelet and EC P-selectin expression in neointimal formation after arterial injury. Our results demonstrate that platelet, but not vascular wall, P-selectin expression has a significant role in the upregulation of inflammatory cytokines and neointimal formation after arterial injury. The lack of effect of blocking only endothelial P-selectin expression on neointimal hyperplasia may serve to highlight the differences in biology between neointimal hyperplasia and atherosclerosis and to emphasize the importance of platelet-leukocyte interactions in restenosis.
Conclusions
Platelet-derived P-selectin plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting neutrophil-platelet interactions could be effective in inhibiting restenosis.
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Correspondence to Kai Wang, MD, PhD, Department of Cardiovascular Medicine/F25, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail wangk@ccf.org
Abstract
Background— P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury.
Methods and Results— Using wild-type (WT) and P-selectin–knockout (Psel–/–) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel–/– mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel–/– mouse group (0.015±0.004 vs 0.004±0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006±0.004 mm2) compared with Plt-Psel mice (0.011±0.005 mm2, P=0.026) and WT mice (0.015±0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel–/– and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury.
Conclusions— Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.
The relative importance of platelet and endothelial P-selectins to neointimal formation after vascular injury was determined by bone marrow transplantation. Significantly less neointima was observed from EC-Psel mice when compared with Plt-Psel mice and WT mice. Decreased neointimal formation was accompanied by reduced inflammation.
Key Words: P-selectin ? arterial injury ? platelets ? endothelial cells ? neointima
Introduction
The process of restenosis has been attributed to the inflammatory response induced by arterial injury.1–3 This local inflammatory response is critically dependent on of the 3 families of cell adhesion molecules: the selectins, the integrins, and the immunoglobulin supergene family. P-selectin is critical for initial leukocyte adhesion to platelets and endothelial cells (ECs)4,5 and is stored in the -granules of platelets and Weibel-Palade bodies of ECs. After platelet or EC activation, P-selectin is rapidly translocated to the cell surface.
P-selectin has an important role in the inflammatory response and development of neointimal formation after vascular injury.6–15 P-selectin upregulates tissue factor in monocytes and leads to leukocyte accumulation in areas of vascular injury associated with thrombosis and inflammation.6,7 P-selectin binding to monocytes through P-selectin glycoprotein ligand-1 promotes local inflammation through the release of tumor necrosis factor (TNF)-, monocyte chemoattractant protein (MCP)-1), interleukin-88–10 and tissue factor microparticles.11,12 A more recent study showed that P-selectin promoted the deposition of RANTES, the platelet-derived CC chemokine, on ECs, subsequent monocyte arrest, and intimal hyperplasia.13 The potential importance of P-selectin in neointimal formation was further demonstrated by a significant decrease in neointimal formation in P-selectin–null mice.14,15 Immunohistochemistry showed that intense P-selectin–positive staining accompanied by leukocyte recruitment was observed in injured wild-type carotid arteries14 but was absent in the P-selectin–null mice.
We have recently demonstrated that specific antagonism of P-selectin binding inhibits neointimal formation in the porcine coronary artery balloon injury model16 and in the diabetic and nondiabetic rat carotid artery injury model.17 Despite this strong evidence of a central role for P-selectin in the initiation of neointimal formation after arterial injury, the relative importance of platelet and EC P-selectin expression on neointimal formation after arterial injury remains unknown. Expression of both platelet and EC P-selectins has recently been shown to be important in the atherosclerotic process,18,19 suggesting that P-selectin expression in each cell type may be important in the response to arterial injury. In the current study, we evaluated the contribution of EC and platelet P-selectins to neointimal growth after arterial injury.
Methods
Animals and Bone Marrow Transplant (BMT)
P-selectin–knockout (Psel–/–; Wyeth/Genetics Institute, Andover, MA) mice were backcrossed 8 times to C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me) to the C57BL/6J background. Then Psel–/– and nonlittermate control C57BL/6J mice were subjected to total-body irradiation from a cesium source (model 143, J.L. Shepherd) to eliminate endogenous bone marrow stem cells. For the preparation of bone marrow, donor mice (C57BL/6J or Psel–/–) were killed, the femur and tibia bones were isolated under sterile conditions, and the bone marrow was harvested by flushing the bones with medium (RPMI, 5% fetal bovine serum [FBS], and 1 mmol/L HEPES). The cells were washed and resuspended in RPMI containing 5% FBS. Recipient mice were lethally irradiated (12.0 Gy) in 2 split doses 3 hours apart. Immediately after the second dose of irradiation, the mice were injected in the lateral tail vein with 4x106 freshly prepared donor bone marrow cells. After the procedure, transplanted mice were transferred to a microisolator unit, fed with sterile chow and water, administered cefozolin (50 mg[middot]kg–1[middot]d–1 for 3 days), and observed daily for 4 weeks.
Determination of Bone Marrow Reconstitution
Flow cytometry was performed as described19 to determine bone marrow reconstitution. In brief, blood from Psel–/– mice receiving bone marrow from C57BL/6J mice was collected into 3.8% sodium citrate (9:1, vol/vol). Platelet-rich plasma was obtained by centrifuging the blood at 150g for 15 minutes at 24°C. Platelets were activated with 10 μmol/L thrombin receptor activating peptide for 20 minutes, incubated for 40 minutes at room temperature with fluorescein isothiocyanate–labeled anti-mouse P-selectin antibody (Pharmingen), and analyzed by fluorescence-activated cell sorting (FACS) with a Becton-Dickinson FACS.
Arterial Injury
Eight weeks after BMT, C57BL/6J and Psel–/– mice that had received BMT with cells derived from either Psel–/– or C57BL/6J mice underwent a carotid air-desiccating/pressure double injury.20 In brief, the external carotid artery was ligated, and an incision was made in the external carotid artery. A 30-gauge needle connected to a saline-filled syringe with tubing was introduced into the external carotid artery. After irrigating the isolated common carotid artery with saline to remove blood, the syringe was replaced with an angioplasty inflation device, and the isolated, saline-filled, common carotid artery segment was dilated at a pressure of 1.5 atm for 30 seconds. The inflation device was replaced with an air-filled 60-mL syringe, and a 30-gauge air-exit hole was made at the proximal end of the common carotid artery. The carotid artery was then exposed to air desiccation for 3 minutes at 20 mL for air per minute. After air drying, the artery was refilled with saline, the needle was removed, and anterograde blood flow was reestablished. The wound was irrigated with saline and closed. All experiments conformed to the position of the American Heart Association on research animal use and care and were conducted with the approval of Animal Research Committee of the Cleveland Clinic Foundation.
Tissue Harvest and Preparation
The animals were euthanized at 4 or 21 days after injury. For animals euthanized at day 4, the excised carotid arteries were embedded in paraffin for immunohistochemical analysis. For animals euthanized 21 days after injury, the injured vessel segments were perfusion-fixed with 5% Histochoice (Amresco) for 5 minutes and then harvested. Specimens were stored in 5% Histochoice for at least 24 hours before embedding.
Morphometry
The fixed carotid arteries were embedded in paraffin and cut in serial sections, at 5-mm intervals, from the proximal to the distal end. Slides were stained with hematoxylin-eosin and van Gieson’s elastic stains. An observer blinded to the study groups performed morphometric analysis by using a computerized digital microscopic planimetry software package (Image-Pro Plus, version 4.0 for Windows, Media Cybernetics). Five sections from the injured arterial segment per vessel were evaluated and averaged. The neointimal and medial boundaries were determined; the cross-sectional areas subtended by the luminal border, the areas bounded by the internal elastic lamina, and the external elastic lamina were measured; and the ratio of intimal to medial area was calculated.
Cellular Inflammatory Infiltrates
Immunostaining for neutrophils and monocytes at days 4 and 21 after arterial injury was performed to evaluate the role of P-selectin expression on inflammatory cell infiltrate in the platelet-only P-selectin (Plt-Psel) and endothelial-only P-selectin (EC-Psel) groups. The positive neutrophils and monocytes were identified by immunostaining with monoclonal rat anti-mouse neutrophil IgG2a (Serotec Ltd) antibody and a rat anti-mouse monocyte/macrophage IgG1 anti–MAC-3 (BD Bioscience) antibody. Biotinylated goat anti-rat IgG was used as a secondary antibody. The numbers of neutrophils and monocytes/macrophage were counted within the intima and media from 5 sections from each animal by 2 observers blinded to the identity of individual animals.
MCP-1 and RANTES Expression
Immunostaining for MCP-1 and RANTES was performed to assess the effects of P-selectin expression on chemokine expression after arterial injury. In brief, the expression of MCP-1 and RANTES was semiquantified by measuring the percent positive area of arterial cross section in which the chemokines were expressed. A blinded observer quantified the area of expression by using a computerized digital microscopic planimetry algorithm described earlier. MCP-1– and RANTES-positive cells were immunolocalized by incubation with a mouse monoclonal antibody against MCP-1 (Santa Cruz Biotechnology, Inc) and RANTES (Chemicon International), followed by application of a biotinylated rabbit anti-mouse secondary antibody (1.25 μg/mL) for 30 minutes. Detection of MCP-1 and RANTES was completed with a diaminobenzidine chromogen substrate that produces a brown reaction product in MCP-1– and RANTES-positive tissue. All evaluations were performed on 5 sections from the injured arterial segment per vessel and then averaged.
To identify the source of MCP-1, double immunofluorescence staining was performed. The following cell type–specific antibodies were used: mouse monoclonal anti–-smooth muscle cell (SMC) actin/Cy3-conjugated antibody (Sigma) and rat anti-mouse monocyte/macrophage IgG1 anti–MAC-3 (BD Bioscience) antibody. Goat anti-rat IgG-PE (Santa Cruz Biotechnology, Inc) and donkey anti-goat IgG Alexa Fluor 488 antibody (Molecular Probes) were used as secondary antibodies. Tissue was analyzed with an upright spectral laser scanning confocal microscope (model TCS-SP, Leica Microsystems) equipped with blue argon (for 4'-6-Diamidino-2-phenylindole [DAPI]), green argon (for Alexa Fluor 488), and red krypton (for Alexa Fluor 594) laser. Data were collected by sequential excitation to minimize "bleed-through." Image processing, analysis, and the extent of colocalization were performed with Leica confocal software.
Statistical Analysis
All data were expressed as mean±SD. Statistical analysis was performed with SPSS software (version 10.0 for Windows, SPSS Inc). Continuous variables were compared by ANOVA with Bonferroni correction. A value of P0.05 was considered statistically significant.
Results
Characteristics of Psel–/– Mice Receiving BMTs
Male Psel–/– and C57BL/6J mice were lethally irradiated and injected with bone marrow of either genotype to obtain the following groups of animals: group 1 (n=4), bone marrow from C57BL/6J mice into C57BL/6J recipient mice having both endothelial and platelet P-selectins; group 2 (n=4), bone marrow from Psel–/– mice into Psel–/– recipient mice having neither platelet nor endothelial P-selectin; group 3 (n=5), bone marrow from C57BL/6J mice into Psel–/– recipient mice having normal P-selectin expression in their platelets but not their ECs; and group 4 (n=4), bone marrow from Psel–/– mice into C57BL/6J recipient mice having normal P-selectin expression in their ECs but not in their platelets. Flow cytometry performed 8 weeks after BMT showed that activation of platelets from Psel–/– mice receiving bone marrow from C57BL/6J (group 3) had P-selectin expression similar to that seen in wild-type (WT) mice (group 1), indicating that the bone marrow of Psel–/– mice, which received C57BL/6J mice bone marrow, was successfully reconstituted (Figure 1).
Figure 1. Flow cytometry results. Activation of platelets from P-selectin–/– mice receiving bone marrow from C57BL/6J had a P-selectin expression similar to that in WT mice, indicating that the bone marrow of P-selectin–/– mice was successfully reconstituted. No P-selectin expression was found in P-selectin–/– mice Activation of platelets from C57BL/6J mice receiving bone marrow from P-selectin–/– mice had a P-selectin expression similar to that in P-selectin–/– mice, indicating that the bone marrow of C57BL/6J mice was successfully reconstituted. Light scatterblot indicates the nonstimulated platelet histogram. Abbreviations are as defined in text.
Relative Importance of EC and Platelet P-Selectins on Neointimal Hyperplasia
Morphometric data showed that the absence of platelet P-selectin (EC-Psel, group 4) resulted in a significant reduction in neointimal formation when compared with animals that lacked only endothelial P-selectin expression (Plt-Psel, group 3): 0.006±0.004 versus 0.011±0.005 mm2, P=0.026 and the WT mice (group 1): 0.015±0.004 mm2, P<0.001. This difference in neointimal formation was still observed when the data were normalized to the area of the media [neointima-media ratio, 0.237±0.159 vs 0.539±0.311 from the Plt-Psel (group 3; P=0.017) and 0.636±0.281 from the WT mice (group 1; P=0.002)]. We observed a trend but not a statistically significant difference in neointimal formation when animals lacking endothelial P-selectin expression (Plt-Psel, group 3) were compared with the WT group (group 1): 0.011±0.005 versus 0.015±0.004 mm2, P=0.079 (Table and Figure 2).
Morphometric Data at 21 Days
Figure 2. Photomicrographs of mice carotid arteries 21 days after injury (elastic staining, x20). A, Representative cross section of injured artery from a WT (group 1) mouse, showing extensive neointimal formation. B, Representative cross section of injured artery from a P-selectin–/– (group 2) mouse, showing significantly reduced neointimal formation. C, Representative cross section of injured artery from a Plt-Psel (group 3) mouse, showing extensive neointimal formation. D, Representative cross section of injured artery from an EC-Psel (group 4) mouse, showing significantly reduced neointimal formation. L indicates lumen; N, neointima; and M, media. Other abbreviations are as defined in text.
Cellular Inflammatory Infiltrates
The numbers of neutrophils and monocytes/macrophages were counted by a blinded observer to determine the inflammatory infiltrates. The luminal binding of neutrophils at 4 and 21 days after arterial injury was significantly less in the EC-Psel group (n=4) compared with the Plt-Psel group (n=4; 4 day, 6±3 vs 17±4 cells per cross section, P=0.005; 21 day, 4±2 vs 12±4 cells per cross section, P=0.012; Figure 3). A similar finding was also observed for monocyte/macrophage binding and infiltration into the neointimal and artery wall 4 and 21 days after arterial injury (4 day, 7±2 vs 19±6 cells per cross section, P=0.009; 21 day, 3±1 vs 14±4 cells per cross section, P=0.002; Figure 4).
Figure 3. Representative immunohistochemical photomicrographs of injured carotid arteries for neutrophil infiltration. Significantly less neutrophil infiltration was observed in the EC-Psel group when compared with the Plt-Psel group at days 4 and 21 after injury (x63). Abbreviations are as defined in text.
Figure 4. Representative immunohistochemical photomicrographs of injured carotid arteries for monocyte/macrophage infiltration. Significantly less monocyte/macrophage infiltration was observed in the EC-Psel group when compared with the Plt-Psel group at days 4 and 21 after injury (x63). L indicates lumen. Other abbreviations are as defined in text.
Immunohistochemical Assay of MCP-1 and RANTES
Immunohistochemical analysis showed that RANTES expression from Psel–/– (group 2, n=4) and EC-Psel (group 4, n=4) animals was significantly less when compared with that in the WT (group 1, n=4) and Plt-Psel (group 3, n=4) mice, as assessed by percent positive area for RANTES-positive cells [14.9±9.6% (Psel–/–, group 2) and 15.0±2.2% (EC-Psel, group 4) vs 39.2±10.7% (WT, group 1) and 34.4±7.1% (Plt-Psel, group 3), P<0.05; Figure 5A and 5B]. A similar pattern of expression was also observed for MCP-1 expression. Significantly less MCP-1 expression was found in the P-sel –/– (group 2) and the EC-Psel (group 4) groups when compared with WT (group 1) and Plt-Psel (group 3) groups [MCP-1, 12.7±8.6% (Psel–/–, group 2) and 12.8±4.8% (EC-Psel, group 4) vs 32.4±8.5% (WT, group 1) and 30.7±11.3%(Plt-Psel, group 3), P<0.05; Figure 5C and 5D]. Double immunofluorescence staining for MCP-1 with SMC -actin and monocytes/macrophages indicated the source of MCP-1 from both SMCs and monocytes/macrophages (Figure Ia and Ib, available online at http://atvb.ahajournals.org).
Figure 5. Immunohistochemical photomicrographs of injured carotid arteries for RANTES expression (x100) and MCP-1 expression (x63). A, Representative cross section of RANTES expression from the Plt-Psel (group 3) group. B, Representative cross section of RANTES expression from the EC-Psel (group 4) group. C, Representative cross section of MCP-1 expression from the Plt-Psel (group 3) group. D, Representative cross section of MCP-1 expression from the EC-Psel (Group 4). Significantly less RANTES and MCP-1 expression was found in the EC-Psel group, indicating that a greater inflammatory response after arterial injury occurred in the Plt-Psel group. L indicates lumen. Other abbreviations are as defined in text.
Discussion
This study demonstrates that platelet P-selectin, but not endothelium-derived P-selectin, plays a crucial role in the development of neointimal formation after arterial injury. These results reinforce the important role of P-selectin in modulating the inflammatory response to vascular injury and highlight the potential therapeutic strategies targeting neutrophil-platelet interactions in preventing restenosis.
P-selectin plays a prominent role in the adhesion of leukocytes to activated platelets.21–25 Platelet–neutrophil binding promotes the release of vasoactive substances that locally amplify inflammatory and thrombotic responses. P-selectin has been shown to be intensively expressed in adhering platelets on the exposed subendothelium that are bound to adherent leukocytes in injured rat carotid arteries.26 Increased platelet-neutrophil and platelet-monocyte aggregates have also been found in patients with coronary artery disease and after vascular injury.27,28 Consistently, P-selectin–deficient mice form less compact platelet layers on the denuded arterial surface in vivo, accumulate fewer leukocytes into the vascular wall, and develop less neointimal formation after arterial injury.14,20
Studies from our group have demonstrated that specific antagonism of P-selectin significantly reduced the inflammatory response and neointimal formation in the porcine coronary artery balloon injury model15 and rat carotid artery injury model.16 It has been shown that specific antagonism of P-selectin inhibits early platelet-leukocyte adhesion on injured arteries and reduces restenosis through a positive impact on vascular remodeling in a porcine carotid artery double injury model.32–34 It has also been shown that specific antagonism of P-selectin inhibits cytokine secretion, such as MCP-1 and TNF-, from arterial ECs and SMCs, and leads to an inhibited inflammatory response.9 Furthermore, P-selectin also mediates binding of platelets to other cells, such as in platelet-platelet and platelet-endothelial interactions,35 which are also involved in the development of neointimal hyperplasia. RANTES, the platelet-derived CC chemokine, is released from activated platelets.36 It has recently been demonstrated that the deposition of RANTES and subsequent monocyte arrest are promoted by platelet P-selectin and are involved in intimal hyperplasia and that blockade of RANTES receptors decreases neointimal formation and the inflammatory response after vascular injury.13 Consistent with the importance of P-selectin–mediated RANTES expression in neointimal formation, our study demonstrates a significant decrease in RANTES expression and neointimal formation in animals that lacked platelet P-selectin. Recently, a study from Ley’s group18 showed that circulating activated platelets promoted monocyte recruitment to atherosclerotic arteries and accelerated the formation of atherosclerosis in apolipoprotein E–deficient mice. These authors found that activated platelets interacted with monocytes and the endothelium of the vessel wall through platelet P-selectin. This interaction resulted in deposition of platelet-derived proinflammatory factors to the vessel wall, such as RANTES, leading to monocyte recruitment that exacerbated atherosclerosis. This finding is consistent with a more recent study from Wagner’s group,19 in which they demonstrated that targeted disruption of platelet P-selectin led to decreased spontaneous atherosclerosis in apolipoprotein E–deficient mice. However, that study also highlighted the importance of EC P-selectin expression in atherosclerosis. In distinct contrast to our study on neointimal hyperplasia, the earlier study on atherosclerosis showed that animals deficient in only endothelial P-selectin had significantly less atherosclerosis than animals deficient in only platelet P-selectin.
The importance of platelet P-selectin in neointimal hyperplasia in response to arterial injury has recently been demonstrated with BMT from P-selectin–null mice into apolipoprotein E–null mice.37 Our study furthers this work by comparing the relative importance of platelet and EC P-selectin expression in neointimal formation after arterial injury. Our results demonstrate that platelet, but not vascular wall, P-selectin expression has a significant role in the upregulation of inflammatory cytokines and neointimal formation after arterial injury. The lack of effect of blocking only endothelial P-selectin expression on neointimal hyperplasia may serve to highlight the differences in biology between neointimal hyperplasia and atherosclerosis and to emphasize the importance of platelet-leukocyte interactions in restenosis.
Conclusions
Platelet-derived P-selectin plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting neutrophil-platelet interactions could be effective in inhibiting restenosis.
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