Published online before print September 25, 2008, doi: 10.1161/CIRCRESAHA.108.180273
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© 2008 American Heart Association, Inc.
Cellular Biology |
Pathophysiological Levels of Soluble P-Selectin Mediate Adhesion of Leukocytes to the Endothelium Through Mac-1 Activation
From the Baker IDI Heart and Diabetes Institute (K.J.W., A.S., E.E.H., S.U.E., K.P., A.M.D., J.P.F.C.-D.) and Australian Centre for Blood Diseases (S.P.J.), Alfred Medical Research & Education Precinct; and Centre for Inflammatory Diseases (M.J.H.), Monash University, Melbourne, Australia; and Department of Plastic and Hand Surgery (S.U.E.), University of Freiburg Medical Centre, Germany.
Correspondence to Dr Kevin Woollard, Baker IDI Heart and Diabetes Institute, 75 Commercial Rd, Melbourne, 3004, Australia. E-mail Kevin.Woollard{at}bakeridi.edu.au
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Abstract |
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Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte–endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1–specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-
–activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin–dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1–mediated rolling and dependent on endothelial P-selectin expression.
Key Words: adhesion molecules • endothelium • monocytes • inflammation • cardiovascular disease
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Introduction |
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The incidence of asymptomatic peripheral artery occlusive disease (PAOD) is steadily increasing among North American adults, with prevalence in people 40 years and older significantly rising from 3.7% in the 1999 to 2000 NHANES survey to 4.2% in the 2001 to 2002 survey and 4.6% in the 2003 to 2004 survey.1 We recently demonstrated that the elevated levels of sP-selectin present in PAOD patients are capable of activating leukocytes and promoting their adhesion to platelet monolayers.2 However, the initial stages of atherogenesis involve interactions of leukocytes with an intact endothelial surface,3,4 and the ability of sP-selectin to promote such interactions remains poorly described.
Recruitment of leukocytes to the endothelium of large arteries (where atherosclerosis predominantly occurs) is thought to take place within an environment of high shear forces and low adhesion molecule expression.5 Although the exact mechanism of how this occurs still remains largely unknown, there is evidence this may be via novel interactions with specific inflammatory antigens.6,7 The majority of studies describe the link between generation of turbulent flow at bifurcation sites and/or after lesion formation of the plaque itself, leading to low shear rates, which facilitate leukocyte–endothelial cell interactions.8–11 Regardless of the exact mechanism, the adhesion cascade involving endothelial adhesion molecule expression, including P-selectin, remains fundamental for leukocyte recruitment within atherogenesis.12,13
P-selectin is a member of the selectin family, localized in the membranes of the -granules of platelets and the Weibel–Palade bodies of endothelial cells and expressed on the surface of activated platelets and endothelial cells.14 It contributes to leukocyte recruitment at sites of vascular injury and inflammation and acts via the engagement of the ligand P-selectin glycoprotein ligand (PSGL)-1.15,16 Previous studies have shown platelet P-selectin engagement of PSGL-1 leads to Mac-1 activation and subsequent firm adhesion, which may also be important for endothelial adhesion.17–19 Deficiency of P-selectin or antibody-mediated inhibition of its adhesive function reduces early atherogenesis in animal models.13,20,21 The soluble form of P-selectin, arising from either proteolytic cleavage or secretion of an alternatively spliced isoform,22 is increased in disease states, prompting its identification as a potentially valuable clinical biomarker of vascular disease risk.23 Previous cellular studies conducted under static conditions have shown that the soluble ectodomain of purified P-selectin competes with the membrane-bound form of P-selectin to reduce leukocyte adhesion to endothelial cells and that sP-selectin reduces superoxide generation in tumor necrosis factor (TNF)-activated neutrophils.24,25 The potential pathophysiological significance of such findings, however, requires further experiments under flow conditions. Furthermore, the concentrations of sP-selectin used in some of these reports are pharmacological rather than pathophysiological.2,13,23 A recent observation that sP-selectin intervention in the P-selectin–deficient mouse can restore leukocyte recruitment, however, does indicate a possible role for sP-selectin in promoting recruitment in vivo.26 Nevertheless, these studies are still limited to observations in rodents and no conclusions can be made regarding vessels prone to atherosclerosis with raised plasma sP-selectin found in a clinical setting.
In the present report, we explore the role of pathological concentrations of sP-selectin, examining leukocyte adhesion under shear flow conditions to endothelial cells in vitro, to the intact endothelium in veins and arterial environments ex vivo and to the intact endothelium in vivo. We demonstrate that pathological levels of sP-selectin consistently promote leukocyte–endothelial adhesion through activation of the leukocyte integrin Mac-1. Furthermore, using plasma from PAOD patients exhibiting raised sP-selectin is shown to increase leukocyte recruitment before overt endothelial activation.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Blood Cell Preparation and In Vitro Perfusion
Resting human neutrophils and monocytes were isolated from whole blood using density centrifugation and a commercially available monocyte isolation kit (Dynal). Perfusion studies with human iliac artery endothelial cells (HIAECs) were carried out within a circular parallel plate chamber as previously described.27
Cremaster Intravital Microscopy Experiments
The cremaster was exposed in anesthetized mice and leukocyte adhesion characteristics analyzed as previously described,28,29 from resting controls or treated mice. In some experiments, mice were treated with 500 ng of TNF- IP 4 hours before each experiment. Further experiments were also performed with a blocking monoclonal antibody against CD11b (clone 5C6; 100 µg/mL) 45 minutes before each experiment. Appropriate isotype-matched control (antirat IgG2b) experiments were also performed.
Ex Vivo Treatment of Leukocytes
Leukocytes were isolated from donor mice by density centrifugation and fluorescently labeled with rhodamine-6G (1:200). Leukocytes (1x106/mL) were treated with either wortmannin (0.2 mmol/L), phosphatase (PP)1 (20 µmol/L), or vehicle control for 10 minutes and then washed in PBS. Labeled leukocytes were reinjected into a recipient mouse that was treated with or without sP-selectin and leukocyte adhesion assessed by fluorescence microscopy.
Ex Vivo Vessel Chamber Studies
Isolated aortas were mounted in a vessel chamber primed with Krebs buffer and maintained at physiological pH by infusing carbogen gas through the buffer at 37°C. Anticoagulated (hirudin) human whole blood (from healthy or PAOD donors) or isolated neutrophils were labeled with DiIC18 (1:1000) and perfused through the aortas at 0.12 mL/min using a syringe pump (Harvard Apparatus). We found no changes in cell adhesion to the vasculature comparing blood from different species/donors (Figure I in the online data supplement). Images of vessel wall–cell interactions were observed using an Olympus BX51 fluorescence microscope fitted with a x10 LWD lens (Olympus, Tokyo, Japan) and analyzed using a Hamamatsu camera, coupled to Image ProPlus software. In some experiments, vessels were pretreated with TNF- (4 ng/mL; 4 hours at 37°C) or blood pretreated (10 minutes) with MAN-1 or isotype control antibody (10 µg/mL), or with phorbol 12-myristate 13-acetate (PMA) (1 µmol/L) as a positive control. Vessels were confirmed to be viable (endothelium) pre- and postexperiment by examining endothelial function using pharmacology techniques and PECAM-1 expression by immunohistochemistry (supplemental Figure II).
Human Vessel Ex Vivo Perfusion Studies
Radial veins from patients under going coronary arterial graft surgery were obtained. Immediately after surgery, radial veins were collected, cleaned, and mounted within the vessel chamber and exogenous whole blood from healthy volunteers was supplemented with or without sP-selectin (150 ng/mL) perfused through each vessel, as described under Ex Vivo Vessel Chamber Studies (see above).
Flow Cytometric Analysis
HIAEC intercellular adhesion molecule (ICAM)-1 expression before and after treatment was analyzed using fluorescence-activated cell sorting. All samples were compensated for using appropriate isotype-matched positive control (anti–IgG2a-FITC).
Inclusion/Exclusion Criteria for Patient Recruitment and sP-Selectin Measurement
Volunteer patients (<75 years old) were recruited with a high atherosclerotic burden were identified by the presence of extensive peripheral arterial occlusive disease (PAOD), and plasma sP-selectin levels were measured as previously described.2
PAOD Plasma Static Adhesion Experiments and Immunodepeltion of sP-Selectin
Neutrophil adhesion to HIAECs was determined using a modified method from Woollard et al30 using fluorescently labeled leukocytes. Plasma from PAOD patients or matched controls was immunodepleted of sP-selectin using protein A–sepharose beads conjugated to WAPS 12.2 or isotype-matched conjugated antibody, as previously described.2
Statistical Analysis
Groups of data were evaluated statistically by ANOVA, followed by Tukey post test for multiple comparisons at each time point, Mann–Whitney test for grouped data, and Spearman’s rank test for identifying correlations.
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Results |
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sP-Selectin Increases Leukocyte Adhesion to Endothelial Cells In Vitro, In Vivo, and in Ex Vivo Animal and Human Vessels
In Vitro Adhesion
Given our focus on PAOD, in vitro perfusion assays using endothelial cells from human iliac arterial vascular beds (HIAECs) were used. Figure 1A shows that sP-selectin significantly increased neutrophil adhesion to resting HIAECs over 10 minutes under shear flow (150 sec–1). This increase in adhesion was dose-dependent, with significant increases in adhesion from 75 to 150 ng/mL of sP-selectin (Figure 1B). There was no increase in adhesion with sE-selectin (Figure 1B).
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) 150 ng/mL sP-selectin–Fc or (
) control. B, Neutrophils were incubated with sP-selectin–Fc (0 to 250 ng/mL) and perfused over resting HIAECs. sE-selectin–Fc (150 ng/mL) was used as a negative control (n=6 in triplicate). *P<0.05 compared with control, as analyzed by 2-way or 1-way ANOVA, followed by Bonferroni and Tukey post hoc test, respectively.
In Vivo Adhesion
The effect of sP-selectin in cremaster postcapillary venules was examined via intravital microscopy. Figure 2 and supplemental Video 1B show that intravenous injection of murine recombinant sP-selectin (150 ng/mL) led to a significant acute increase (15 minutes) in the number of rolling leukocytes relative to vehicle controls (supplemental Video 1A). This increase in the number of rolling cells was associated with a decrease in velocity and a significant increase in the number of adherent leukocytes (Figure 2). This finding was specific to sP-selectin because neither murine recombinant sE-selectin IV (150 ng/mL) (Figure 2) nor control IgG (150 ng/mL) (supplemental Figure VI) had any effect.
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), or saline (100 µL) (
Ex Vivo Leukocyte Adhesion in Aortic Vessels
Figure 3A and representative video images (Figure 3B and supplemental Video 2B) show that sP-selectin (150 ng/mL) led to an acceleration in human neutrophil recruitment to the endothelium of resting aortae compared to vehicle control (supplemental Video 2C). Further experiments were then performed using human whole blood perfusions. Figure 3C and representative video images (Figure 3D and supplemental Video 2B) show that sP-selectin led to significant increase in adhesion from vehicle controls after 5 minutes of perfusion (supplemental Video 2A). In contrast, 150 ng/mL sE-selectin and IgG failed to increase adhesion above that in vehicle-treated controls (Figure 3C and supplemental Figure VI).
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Ex Vivo Leukocyte Adhesion to Human Radial Vein Endothelium
To examine whether a similar pattern of adhesion is mediated by sP-selectin within a human vessel, ex vivo perfusion assays using radial veins from patients undergoing coronary arterial graft surgery were performed. Figure 3E shows that supplementing blood with exogenous sP-selectin (150 ng/mL) led to a significant increase in adhesion, compared to vehicle-supplemented controls.
sP-Selectin Has No Further Effect on Leukocyte Adhesion to Endothelium Activated With TNF-
Preactivating HIAECs with 4 ng/mL of TNF- significantly increased neutrophil adhesion in the absence of sP-selectin. Addition of sP-selectin had no further effect (Figure 4A). Similarly, pretreatment of mice systemically with TNF- (500 ng) induced marked increases in leukocyte rolling flux (
150 cells per min) and adhesion (25 cells per 100 µm) and a decrease in leukocyte rolling velocity, relative to saline controls (Figure 4B and supplemental Video 1C). Administration of sP-selectin did not further increase the number of leukocyte–endothelial cell interactions beyond already elevated levels (Figure 4B). A similar pattern was also observed in the ex vivo arterial vessel chamber model (Figure 4C and supplemental Video 2C).
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) in vivo. C, Isolated aortas were preincubated with (
Mechanism of Action
sP-Selectin Does Not Activate Endothelial Cells
Endothelial cells have been reported to express putative P-selectin receptors GPIb or PSGL-1.31,32 To determine whether sP-selectin mediates adhesion through endothelial activation directly, we analyzed the effects of sP-selectin on modulating ICAM-1 expression on HIAECs. sP-selectin (150 ng/mL) had no effect on ICAM-1 expression, in contrast to both TNF- and lipopolysaccharide (Figure 5). Furthermore, expression of either receptor was undetectable by flow cytometry in our hands (data not shown).
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sP-Selectin Induces Mac-1–Dependent Rolling and Stationary Adhesion
The activation of the leukocyte integrin Mac-1 has been shown to be important in mediating stationary adhesion to endothelial cells.17 Previous work has shown that sP-selectin binding to PSGL-1 on leukocytes leads to outside-in signaling via the non–receptor tyrosine kinase (NRTK) Src.2,26,33 To examine whether a similar mechanism of leukocyte integrin activation occurs in mediating adhesion to endothelial cells, vessel chamber experiments were performed using a novel single-chain antibody (MAN-1) that only blocks the activated epitope of Mac-1.34 We first demonstrated that neutrophil binding of this antibody is increased in response to PMA or sP-selectin (supplemental Figure IIIB), supporting its recognition of the activated form of Mac-1. Preincubation with MAN-1 led to a significant reduction in stationary adhesion induced by PMA (used as a positive control) and sP-selectin (Figure 6A). However, MAN-1 did not inhibit the basal level of adhesion seen with nonactivated neutrophils (Figure 6A). A nonspecific single-chain antibody raised against a scrambled sequence had no effect on adhesion of either resting or activated cells (data not shown). Mac-1–mediated sP-selectin adhesion was confirmed using a standard Mac-1 blocking antibody (clone M1/70; supplemental Figure IIIA).
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To determine the contribution of Mac-1 on sP-selectin–mediated recruitment in vivo, we performed blocking experiments against CD11b within cremaster intravital experiments. Figure 6B shows that blocking Mac-1 led to complete inhibition of stationary adhesion mediated by sP-selectin. No significant change in adhesion was noted using the isotype-matched control (data not shown). Surprisingly, blocking Mac-1 led to a significant reduction in the number of rolling leukocytes mediated by sP-selectin; however, basal rolling was not blocked (Figure 6C), describing a novel role for sP-selectin mediated rolling via Mac-1.
sP-Selectin–Mediated Adhesion Is Dependent on Endothelial P-Selectin
To further examine the contribution of sP-selectin on tethering of leukocytes, we examined sP-selectin–mediated adhesion in the P-selectin knockout (KO) mouse. Figure 6D shows that regardless of treatment with sP-selectin, no adhesion was seen in the venule microcirculation, nor was there any notable rolling of cells (data not shown). However, sP-selectin (150 ng/mL) still mediated similar levels of Mac-1 expression on both leukocytes (in whole blood) from wild-type mice and P-selectin knockout mice (supplemental Figure IV). Interestingly, when analyzing arterial adhesion using aorta from the P-selectin KO mouse, there were low levels of basal adhesion; however, there was no significant increase in adhesion after sP-selectin treatment (150 ng/mL) (Figure 6D).
Src and Phosphoinositide 3-Kinase Signaling
sP-selectin can mediate downstream signaling events via PI(3)K (phosphoinositide 3-kinase) and the nonreceptor tyrosine kinase Src, contributing to leukocyte integrin activation and adhesion.2,26 By using fluorescently labeled leukocytes from donor mice and ex vivo treatment with either wortmannin or PP1, we examined the effect of inhibiting PI(3)K and Src (respectively) on sP-selectin mediated adhesion in vivo. Figure 6E shows that inhibiting either PI(3)K or Src signaling abrogated any increases in adhesion mediated by sP-selectin.
Plasma sP-Selectin Levels and Function in PAOD
To examine whether the in vivo and ex vivo increases in adhesion noted with pathophysiological concentrations of sP-selectin are also present within a clinical setting, static adhesion experiments to confluent HIAECs were performed using plasma from PAOD patients. PAOD patients exhibit significantly increased plasma levels of sP-selectin up to 150 ng/mL.2 Treating isolated neutrophils with plasma from PAOD patients led to a positive correlation (P<0.001) between neutrophil adhesion and increasing concentrations of sP-selectin (Figure 7).
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To further confirm the role of raised sP-selectin in PAOD-mediating leukocyte–endothelial adhesion, ex vivo perfusions were performed using plasma from PAOD patients, before and after immunodepleting pathological increases in plasma sP-selectin. Figure 8 shows that immunodepleting sP-selectin from PAOD plasma led to a significant reduction in neutrophil adhesion within radial vessels. An ELISA was performed after immunodepleting sP-selectin from PAOD plasma and compared to healthy control plasma. Immunodepletion reduced sP-selectin in PAOD plasma to 6.91±4.11 ng/mL, similar to the control value of 7.29±3.64 ng/mL. Immunodepleting sP-selectin from healthy control plasma or using an isotype-matched antibody in PAOD plasma had no effect on adhesion (Figure 8).
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Discussion |
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We demonstrate, for the first time, a functionally important role for sP-selectin in regulating leukocyte adhesion to resting endothelial cells (in vitro) and to the intact venous and arterial endothelium (in vivo and ex vivo). Furthermore, we demonstrate that pathophysiologically relevant concentrations of sP-selectin are biologically active and induce leukocyte activation, resulting in upregulation of Mac-1–mediated rolling dependent on endothelial P-selectin expression. We show that raised plasma sP-selectin in PAOD patients may increase leukocyte recruitment using a novel model of assessing leukocyte adhesion within a human vessel. Overall, our studies raise the possibility that sP-selectin promotes leukocyte recruitment at sites of vessel wall before endothelial activation or dysfunction, giving it the potential to promote the early stages of atherogenesis in patients at risk of vascular disease.
sP-selectin dose-dependently increased leukocyte adhesion to resting, nonactivated HIAECs. The maximal increase in adhesion was seen at pathophysiological concentrations reported in numerous studies associated with vascular diseases, including risk factors for both peripheral and coronary disease.2,13,23 The lack of any further increase in sP-selectin–induced adhesion in activated endothelial cells and blood vessels may reflect the finding that these endothelial cells are already supporting large numbers of interacting leukocytes. Furthermore, this suggests that the effect induced on leukocyte activation via sP-selectin can also be induced in the absence of sP-selectin by activation of the endothelium with TNF. This may suggest that when endothelial adhesion molecule expression is low, subtle changes in leukocyte integrin adhesiveness can mediate rapid increases in recruitment. In contrast, if endothelial adhesion molecule expression is maximal, recruitment occurs more efficiently and is not affected by sP-selectin–induced changes in leukocyte integrin function.
Experiments examining endothelial ICAM-1 expression suggested that the increase in in vitro adhesion by sP-selectin was independent of effects on endothelial cells directly. This finding is potentially explained by a lack of PSGL-1 and GPIb expression on HIAECs. The expression of these receptors remains controversial and has only been described on other endothelial phenotypes.31,32,35 Whether other endothelial cell types may indeed mediate responses to sP-selectin remains to be investigated.
The evidence that human pathophysiological concentrations of sP-selectin can induce increases in adhesion only to nonactivated resting endothelium was confirmed in vivo by analyzing adhesion to venules within the cremaster microcirculation. This is consistent with previous reports using a dissimilar dose of sP-selectin.26 These injections led to a striking increase in acute adhesion within 15 minutes, which was abrogated if the vasculature had been prestimulated with TNF-. The increase in adhesion within resting venules was correlated with a decrease in rolling velocity, evidence for sP-selectin mediating leukocyte activation in vivo.26 The rapid increase and subsequent reduction in adhesion was not attributable to Mac-1 recycling or decreased expression over time (supplemental Figure V); however, it may be through the elimination of exogenous circulating plasma sP-selectin and requires further investigation. These acute effects in mediating changes in adhesion were not caused by endotoxin contamination and were further confirmed to be specific to sP-selectin through using sE-selectin as a negative control. Preliminary reports have described an ability of sE-selectin to engage PSGL-1 and modulate adhesion.36 However, further studies also show alternative PSGL-1–independent sE-selectin ligands including ESL-1 and CD44, especially within the models used here.37 The lack of adhesion mediated by sE-selectin is also consistent with previous data.2
We have shown for the first time that increases in leukocyte rolling mediated by sP-selectin to resting endothelium is through Mac-1. Previous work has shown that leukocyte integrin activation is able to mediate leukocyte rolling to a certain extent on activated endothelium;26,38,39 however, to our knowledge, this is the first clear demonstration of Mac-1–dependent rolling to the resting endothelium. Furthermore, we demonstrate that this in vivo mechanism of increased leukocyte rolling and stationary adhesion mediated by sP-selectin is dependent on endothelial P-selectin expression, as determined in the P-selectin KO mouse. This is contrary to previous published work showing that sP-selectin can restore leukocyte adhesion in the P-selectin KO mouse.26 The difference between the 2 observations requires more work; however, it is interesting to note that the authors26 failed to note any rolling leukocytes (from any treatment) but reported significant increases in adherent cells, suggesting instantaneous leukocyte arrest, which was not seen within our results. Moreover, we have described the partial inside-out signaling mechanism responsible for sP-selectin–mediated adhesion in vivo. Using ex vivo–treated leukocytes from a donor mouse, treatment with PI(3)K and Src inhibitors wortmannin and PP1, completely inhibited increases in adhesion mediated by sP-selectin.
To identify whether sP-selectin may exhibit increases in adhesion to vascular beds prone to atherosclerosis, we designed a novel ex vivo perfusion model. sP-selectin led to a significant increase in leukocyte adhesion both with purified neutrophils and whole blood. To examine the mechanism of how sP-selectin increases leukocyte recruitment and the in vivo observation that Mac-1 was responsible for sP-selectin mediated rolling, we investigated the effect on Mac-1 activation using a novel single-chain antibody (MAN-1) that only blocks the activated epitope of Mac-1.34 MAN-1 significantly inhibited stationary adhesion induced by sP-selectin, whereas it had no effect on resting adhesion. This is consistent with previous data describing sP-selectin–mediated signaling via PSGL-1, leading to inside-out Src signaling and Mac-1 activation.2 These results show that the antiadhesive effect of MAN-1 is restricted to adhesion induced following Mac-1 activation, as previously described,34,40 and confirms data describing a reduction in PMA activated monocyte adhesion to fibrinogen-coated microslides under flow.34 This evidence points toward the use of MAN-1 as a possible therapeutic tool to reduce Mac-1–activated mediated adhesion. Furthermore, the design of a single-chain antibody reduces the Fc-mediated immune response associated with traditional antibody preparations.
To determine whether the raised circulating sP-selectin levels in PAOD were capable of stimulating increased leukocyte adhesion, we compared the effects of blood from patients and of blood from disease-free controls. In addition, we determined the consequences of reducing sP-selectin levels in PAOD blood by immunodepletion. These experiments showed a clear correlation between leukocyte adhesion and the prevailing sP-selectin concentration. These data shows for the first time that an increase in this plasma biomarker of disease mediates direct changes to leukocyte adhesion on the intact endothelium. Although our studies do not exclude a functionally important role for other raised circulating plasma biomarkers/proinflammatory molecules, such as C-reactive protein or sCD40L, in regulating leukocyte adhesion,30,41 they nonetheless point to a potentially important role for sP-selectin in this process. Future studies will be required to fully delineate the role of these various inflammatory markers in combination or separately in regulating leukocyte adhesive function.
To extend these translational studies into a functional assay and reduce the effect of cross-species observations related to a mouse specific phenotype (mouse aorta), we designed an ex vivo perfusion assay using isolated human radial veins. To the authors knowledge, this is the first time live cell imaging has been used to analyze leukocyte recruitment and adhesion to the endothelium within a peripheral human radial vessel. We have shown that like the assays performed in vitro with human isolated endothelial cells and ex vivo with mouse aorta, sP-selectin significantly increased leukocyte adhesion to resting endothelium under flow. To further confirm the role of sP-selectin within a clinical setting, immunodepletion experiments were performed with plasma isolated from PAOD patients. Strikingly immunodepleting raised plasma sP-selectin in PAOD samples led to a significant reduction in adhesion under flow within the isolated human vessel. This is consistent with previous data analyzing PAOD plasma adhesion in vitro.2
Clinical Implications
Our results demonstrate a potentially important mechanism for sP-selectin in promoting leukocyte recruitment to the intact endothelium before the endothelium has become activated. Many atherosclerosis risk factors, such as diabetes, hypertension, and obesity, are associated with raised plasma sP-selectin.13,22 Therefore, before these patients exhibit endothelial dysfunction, raised plasma sP-selectin may contribute to the initiation of atherosclerosis through increased recruitment of leukocytes. The source of raised plasma sP-selectin remains to be fully explored. Endothelial sources may arise from venule inflammation within the microcirculation, which is protected from atherosclerosis. Why venules are protected from atherosclerosis even in the presence of high sP-selectin levels remains to be explored; however, this may arise from differences in shear rates, contractility, and endothelial adhesion molecule expression.42 Platelet sources of sP-selectin may arise in patients at risk of cardiovascular disease who exhibit evidence of circulating activated platelets, including increased leukocyte–platelet aggregates and P-selectin expression.22 However, it is interesting to note that reports describe that platelet P-selectin expression appears to be predominant in contributing to the circulating sP-selectin plasma pool.43
Summary
We describe a new model for analyzing both leukocyte adhesion to the murine arterial vascular bed and human radial vasculature. Our studies with these models demonstrate a potentially important role for elevated levels of sP-selectin in regulating leukocyte adhesive function in patients at risk of atherosclerotic disease. Although it remains to be established whether such effects enhance leukocyte recruitment, leading to increased risk of accelerated atherosclerosis in vivo, they do nonetheless indicate that raised plasma sP-selectin may be important for increasing leukocyte recruitment to the endothelium before endothelial activation or dysfunction.
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Acknowledgments |
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Sources of Funding
This work was supported by National Heart Foundation Australia grant G06M2605. K.J.W. was supported by a National Heart Foundation Australia postdoctoral fellowship. J.C.D., A.M.D., K.P., M.J.H., and S.P.J. are National Health and Medical Research Council senior research fellows.
Disclosures
S.U.E. and K.P. are inventors in a patent application filed to protect the intellectual property of human activation-specific anti–Mac-1 antibodies and their derivatives.
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Footnotes |
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Original received May 28, 2008; revision received September 11, 2008; accepted September 16, 2008.
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References |
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