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Stroke. 1996;27:1386-1392

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(Stroke. 1996;27:1386-1392.)
© 1996 American Heart Association, Inc.

Articles

E-Selectin Appears in Nonischemic Tissue During Experimental Focal Cerebral Ischemia

Hans-Peter Haring, MD; Ellen L. Berg, PhD; Naoya Tsurushita, PhD; Masafumi Tagaya, MD Gregory J. del Zoppo, MD

the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla (H.-P.H., M.T., G.J. del Z.), and Protein Design Labs, Inc, Mountain View (E.L.B., N.T.), Calif.

Correspondence to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, SBR17, The Scripps Research Institute, 10666 N Torrey Pines Rd, La Jolla, CA 92037.


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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 down arrowIntroduction 
 down arrowReferences 
 
Background and Purpose E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia.

Methods The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n=4) and 3 hours of occlusion with reperfusion (1 hour, n=4; 4 hours, n=6; 24 hours, n=6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy.

Results Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin=0.32±0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61±0.17) compared with control and nonischemic tissues (2P<.003). (3) In the contralateral nonischemic basal ganglia and other nonischemic tissues, low but significant E-selectin levels appeared by 24 hours of reperfusion (2P=.0005). The latter were further confirmed by an E:P-selectin immunoprobe.

Conclusions E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.


Key Words: cell adhesion molecules • cerebral ischemia, focal • reperfusion • selectins • microvessels


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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 down arrowIntroduction 
 down arrowReferences 
 
The involvement of polymorphonuclear leukocytes in focal cerebral I/R has directed attention to the responses of the microvascular endothelium to ischemia, which involve sequential expression of adhesion receptors for polymorphonuclear and other leukocytes.1 Upregulation of endothelial intercellular adhesion molecule-1 mRNA and protein and P-selectin protein has been observed within hours after MCAO in experimental focal ischemia and I/R in baboon and rat brains.2 3 E-selectin (endothelial leukocyte adhesion molecule-1) mRNA expression has been seen beginning at 6 hours and peaking at 12 hours after MCAO in ischemic cortex of the spontaneously hypertensive rat.2 In a recent study, transient elevated levels of circulating soluble E-selectin were found in acute ischemic stroke patients.4 Those data imply participation of cerebral endothelial E-selectin in the later inflammatory events after MCAO/R, leading to ischemic cerebral tissue injury.

E-selectin, L-selectin, and P-selectin constitute a distinct group of cell adhesion receptor molecules,5 6 characterized by common sequence and structural features. They are expressed on activated endothelial cells (P-selectin and E-selectin), leukocytes (L-selectin), and activated platelets (P-selectin).5 6 7 As a member of the selectin superfamily of endothelial adhesion receptors, the responses of E-selectin to tissue I/R are unique.3 8 9 10 11 12 13 Both E-selectin and P-selectin mediate leukocyte rolling, which is followed by firm adhesion and transmigration of activated leukocytes into the parenchyma.5 6 7 Upregulation of E-selectin protein has been shown after experimental I/R in myocardium, skin, and kidney.9 11 12 13 Furthermore, evidence exists that cytokine-mediated expression of E-selectin in nonischemic lung microvessels follows local limb ischemia in rats.14 15

Given the biology of E-selectin, it was reasonable to expect its appearance on brain microvessels late during the inflammatory phase of experimental cerebral I/R in the primate. However, in view of previous experimental and clinical studies, the exact expression patterns for E-selectin antigen were likely to be complex.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The procedures used throughout this study were approved by the Institutional Animal Research Committee and performed in accordance with the standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals), the National Institutes of Health Policy on Humane Care and Use of Laboratory Animals, and the US Department of Agriculture Animal Welfare Act. In compliance with these standards, every effort was made to ensure that the subjects were free of pain or discomfort. The principal investigator, veterinarians, and primate handling staff were present for all procedures.

Experimental Procedures
Thirteen adolescent male baboons (Papio anubis/cynocephalus) were used in this study. Before study entry, all animals were seen to lack evidence of disease during a mandated standard quarantine period.

The nonhuman primate model of MCAO/R has been described in detail elsewhere.16 17 18 After implantation of the MCAO device, a 7-day procedure-free interval was allowed. All primates that underwent the experimental procedures were clinically free of apparent infection or inflammation and showed normal neurological function (score=100).19

The conditions of cerebral I/R chosen for this study were 2 hours of MCAO (n=2) and 3 hours of MCAO with 1 hour (n=2), 4 hours (n=3), and 24 hours of reperfusion (n=3). Three additional subjects did not undergo device implantation surgery and served as controls. Each experiment was terminated by pressure perfusion with chilled isosmotic perfusion flush solution during reperfusion, as previously reported.3

The brain was immediately excised en bloc from the cranium and immersed in ice. It was subdivided into 1-cm coronal slices. Tissue blocks (1.0x1.0x0.2 to 0.5 cm) from stereoanatomically identical sites of the left and right basal ganglia and temporal cortex were embedded in Tissue-Tek OCT compound (Miles, Inc) in individual cryomolds, frozen with 2-methylbutane/dry ice, and stored at -80°C until sectioning.

Antibodies
A murine IgG1 MoAb against human E-selectin, E-1E4, and a cross-reacting MoAb (IgG1) binding human E-selectin or P-selectin, EP-5C7, were used to localize E-selectin antigen. Both MoAbs are described in detail elsewhere.20 WAPS 12.2, a murine MoAb (IgG1) against human P-selectin, and a rabbit polyclonal antibody against human platelet P-selectin (the kind gift of M. Berndt, Baker Medical Research Institute, Prahran, Australia; see Reference 3) were also used.20 Flow cytometric analysis of these antibodies with baboon platelets activated by human thrombin, performed essentially as described for human platelets,20 confirmed the cross-reactivity of EP-5C7, WAPS 12.2, and the rabbit polyclonal antibody, with baboon P-selectin but not E-1E4. LAM 89, a murine MoAb (IgG1) against human laminin antigen (Sigma Chemical Co), served as a marker for cerebral microvessel basal lamina.21 JC/70 A, a murine MoAb (IgG1) against human CD31 (DAKO), was used as a marker for platelet-endothelial cell adhesion molecule-1 on the endothelium.22

Immunohistochemistry
Cryostat sections (10 µm thick) from two matched blocks from each ischemic basal ganglia and nonischemic basal ganglia, parietal cortex, and cerebellum were prepared for immunohistochemistry. The sections were fixed with acetone for 10 minutes at -20°C and immersed in 100 mmol/L glycine in PBS (100 mmol/L Na2HPO4 and 140 mmol/L NaCl, adjusted at pH 7.4) for 10 minutes. After they were rinsed in PBS wash solution, sections were incubated with Blotto to reduce nonspecific binding. The primary antibody (100 µL) was applied to each section, which was then incubated for 2 hours at 37°C under humidified conditions. Biotinylated secondary antibody was applied for 30 minutes at 37°C (Vector Laboratories). Antibody-bound peroxidase activity was developed with 3-amino-9-ethyl carbazole (AEC kit, Biomeda Corp). The sections were then counterstained with Mayer's hematoxylin (Biomeda Corp), blued in saturated sodium bicarbonate, and mounted in crystal mount medium (Biomeda Corp).

Routine controls for each experiment included deletion of the primary and secondary antibodies.23 A murine MoAb (IgG1) against a human {kappa} myeloma protein (Sigma Chemical Co) served as the irrelevant antigen control.

The specificity of the E-selectin findings was confirmed by blockade of E-1E4 with soluble rhE. This ligand was prepared from supernatants of Chinese hamster ovary (CHO) cells transfected with the gene encoding a truncated form of E-selectin. The coding region for the truncated form of human E-selectin (amino acids 1 to 283 of the mature protein) was inserted into the vector pSEC-4 (pDL 104), a derivative of pVg1,24 which contains an SV40 promoter and the mouse glutamine synthetase gene. pSEC-4 is similar to pMEM20 but contains a sequence encoding a c-myc peptide (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) in place of the P-selectin transmembrane and cytoplasmic sequences. The resulting construct was cotransfected into CHO/K1 cells with the pSV2neo vector, and the resulting transfectants were cultured and selected in 1 mg/mL G418 as described.20 Supernatant E-selectin was purified by affinity chromatography with the EP-5C7 antibody conjugated to CNBr-Sepharose (Pharmacia) according to standard procedures. After dialysis into PBS (pH 7.2), the resulting material was found to be greater than 90% pure as estimated by silver staining on SDS-PAGE gels.

Cellular Injury
Cellular DNA scission was taken as a measure of tissue injury.25 26 27 28 29 To detect cellular DNA injury/repair after MCAO/R, the terminal deoxynucleotidal transferase-mediated dUTP nick end labeling reaction was applied to the cryosections using the DIG-dUTP method (ApopTag kit, Oncor).30 By this method, DIG-dUTP binds to free 3' OH ends generated by DNA scission/repair from any cause. Typically, control primate and rodent cerebral tissues prepared in the manner described above demonstrate no dUTP uptake (data not shown). Incorporated nuclear DIG-dUTP was detected according to the manufacturer's instructions. The cryosections were fixed with 10% neutral-buffered formalin and immersed in ethanol/acetic acid (2:1). After the sections were washed, they were treated with 2% H2O2 for 5 minutes and incubated with DIG-dUTP in terminal deoxynucleotidal transferase buffer at 37°C for 60 minutes. Color development followed incubation with anti-DIG-peroxidase conjugate for 30 minutes, with 0.025% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2 in 0.05 mol/L Tris buffer (pH 7.6) for 5 minutes. Sections from ischemic and nonischemic tissue at 3 hours of MCAO and 24 hours of reperfusion were used. Positive controls were generated by incubation of nonischemic tissues with DNase I (Sigma) for 10 minutes at 20°C before DIG-dUTP development.

Quantitative Analysis
Quantitation of microvascular numbers and minimum transverse diameters was assisted by computerized video-imaging microscopy.3 In each section, 250 fields (18.3 mm2) were scanned in a meandering pattern. The overall vessel numbers were categorized according to the previously defined microvascular subclasses.1 17

E-selectin or E:P-selectin presentation was assessed in relation to microvascular laminin, ie, E-selectin/laminin and E:P-selectin/laminin. Perivascular laminin antigen decreases during cerebral I/R with time of reperfusion.31 Laminin antigen was chosen because it more accurately reflects the structurally intact microvessels at any time during I/R in this model.31 In this series, the mean numbers of microvessels identified by LAM 89 in basal ganglia specimens subjected to MCAO or MCAO/R at all time points did not differ from those previously reported.31 No significant differences were found between control basal ganglia and nonischemic (ie, right hemispheric) basal ganglia at any time points (data not shown).

Fluorescence microscopy and laser confocal microscopy were used for the immunofluorescence colocalization experiments with E-selectin and P-selectin, and with E-selectin and laminin. The equipment and techniques have been described elsewhere.31

Statistical Analysis
All data are presented as mean±SD. Two-sample t tests were used for comparisons of antigen presentations as noted. Significance was set at 2P<.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Fluorescence colocalization studies demonstrated E-selectin, at 24 hours of reperfusion after 3 hours of MCAO, only in those microvessels displaying laminin antigen in the ischemic basal ganglia. A reduction in the number of microvessels displaying laminin with MCAO/R was confirmed, as previously reported.31

Three distinct and consistent microvascular distribution and immunoperoxidase intensity patterns of E-selectin antigen presentation were observed in relation to I/R (Fig 1Down). Colocalization experiments confirmed that E-selectin was expressed on microvascular endothelial cells defined by the ubiquitous endothelial cell marker CD31.22 The E-selectin specificity of all observations defined by E-1E4 was corroborated by coincubation with soluble rhE. rhE completely blocked E-1E4 binding to ischemic microvascular endothelium during the entire time course (Fig 1Down). The TableDown and Fig 2Down depict the relative microvascular association of E-selectin and E:P-selectin presentation to laminin antigen.



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  		Figure 1. E-selectin presentation in nonischemic (non-I/R; A, C, E) and ischemic (I/R; B, D, F) basal ganglia (see text). No E-selectin was apparent in non-I/R tissue at 3 hours of MCAO and 1 hour of reperfusion (A) but was observed in I/R tissue (B). Presentation was significant at 24 hours of reperfusion (D). Microvessels of the contralateral nonischemic basal ganglia were less intensely stained at 24 hours of reperfusion (C). rhE completely blocked E-1E4 in both nonischemic (E) and ischemic (F) basal ganglia at all time points, demonstrating the specificity of the E-selectin observations.


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  		Table 1. Number of Microvessels Presenting E-Selectin or E:P-Selectin Antigen



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  		Figure 2. Microvascular endothelial E-selectin presentation defined by E-1E4 (A, B) and E:P-selectin expression defined by EP-5C7 (C, D), relative to laminin antigen. Open bars designate low peroxidase signal intensity; closed bars indicate high peroxidase signal intensity (see Fig 1Up). C indicates control; -1, 2 hours of MCAO; 1, 4, 24, 3 hours of MCAO with 1, 4, and 24 hours of reperfusion, respectively. At 24 hours of reperfusion, a significant increase of microvascular E-selectin presentation was observed in the ischemic tissue compared with controls (2P=.0005) (E-selectin, I/R) (B) and with the non-I/R basal ganglia (2P=.0023) (A). E-selectin (A) and E:P-selectin (C) expression at 24 hours of reperfusion were not different in the non-I/R basal ganglia (2P=.350) or the I/R basal ganglia (2P=.535). A direct comparison between the fractions of vessels presenting E-selectin and E:P-selectin is not possible.

(1) E-selectin immunoreactivity, characterized by a low-intensity peroxidase signal, appeared by 2 hours of MCAO in the ischemic basal ganglia (E-selectin/laminin=0.28±0.11) and did not change by 4 hours of reperfusion after 3 hours of MCAO (E-selectin/laminin=0.32±0.10) (Fig 2Up).

(2) By 24 hours of reperfusion, E-selectin expression in the ischemic territory increased. This was evidenced by robust signal intensities (Fig 1Up) and a significant increase in the number of microvessels expressing E-selectin antigen (E-selectin/laminin=0.61±0.17) compared with control (2P=.0005) and with the contralateral nonischemic basal ganglia (2P=.0023) (Fig 2Up). E-selectin presented primarily on precapillary and postcapillary microvessels (80.2±15.3%) but also on capillaries (17.6±15%) and on few microvessels greater than 30 µm (2.2±0.6%) (Fig 3Down). In contrast, no E-selectin antigen was detected in any control subject or in the nonischemic basal ganglia, parietal cortex, or cerebellum before 4 hours of reperfusion (Fig 1Up). The absence of E-selectin antigen (and P-selectin antigen) outside of the ischemic basal ganglia in this interval was confirmed in each subject by studies with EP-5C7 (Fig 2Up).




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  		Figure 3. Appearance of E-selectin antigen in relation to changes in laminin antigen and microvascular diameter (n=6 each). A, Ischemic zone (I/R); B, nonischemic zone (non-I/R). Microvascular diameter distributions of laminin antigen (thin solid line) and E-selectin antigen (thick solid line) in control cohort were identical in both basal ganglia. Note no E-selectin expression in control group. In the I/R zone at 24 hours of reperfusion, the total number of vessels displaying laminin antigen (broken line) decreased and E-selectin antigen (hatched line) increased, while the diameter distributions did not change. In the non-I/R zone, E-selectin (hatched line) was weakly expressed (see text).

(3) At 24 hours of reperfusion in the nonischemic basal ganglia, E-selectin antigen appeared in 0.23±0.14 microvessels (Figs 1 and 2UpUp). These findings were confirmed by complete blockade of the E-selectin signal by rhE. Noteworthy is that E-selectin antigen was also detected in nonischemic parietal cortex and cerebellum and their counterpart contralateral tissues only at 24 hours of reperfusion. However, signal intensities were consistently weaker in the contralateral nonischemic tissues (Fig 1Up). The microvascular E-selectin distribution was nearly identical to that seen on ischemic basal ganglia, comprising 80.6±17.8% precapillary arterioles and postcapillary venules, 18.5±17.9% capillaries, and 0.9±0.2% microvessels greater than 30 µm in diameter (Fig 3Up). Parallel experiments with EP-5C7 and fluorescence colocalization of E- and P-selectin confirmed that only E-selectin antigen was associated with microvessels in nonischemic tissues at 24 hours of reperfusion (Fig 2Up).

As shown recently3 and confirmed with WAPS 12.2, no P-selectin antigen was found in any nonischemic brain tissue. However, in ischemic basal ganglia at 24 hours of reperfusion, colocalization experiments (n=6) defined the number of microvessels per square millimeter, which presented E-selectin alone as 92.6±15.5, P-selectin alone as 1.3±1.3, and both E- and P-selectin as 13.9±12.7. Hence, P-selectin colocalized with E-selectin in only 16.2±12.6% of microvessels. At earlier times, P-selectin antigen was also observed; however, the relatively low E-selectin fluorescence intensities precluded quantitation.

Before 24 hours of reperfusion, all E-selectin immunoreactivities in ischemic tissues were confined entirely to the regions of dUTP uptake. No dUTP signal was observed at 24 hours of reperfusion in the nonischemic basal ganglia in the presence of microvascular E-selectin presentation.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
This report characterizes the microvascular appearance of E-selectin antigen during focal cerebral I/R. Its cerebral microvascular presentation in response to MCAO/R in the nonhuman primate occurred in three distinct patterns: (1) significant E-selectin in the ischemic zone by 24 hours of reperfusion; (2) lesser amounts of E-selectin antigen in the ischemic zone at 2 hours of MCAO and up to 4 hours of reperfusion after 3 hours of MCAO; and (3) consistent low levels of E-selectin in the (contralateral) nonischemic basal ganglia, parietal cortex, and cerebellum by 24 hours of reperfusion but not before (Figs 1 and 2UpUp).

All selectins show common structural features: an N-terminal lectin domain, an epidermal growth factor-like domain, a variable number of complement regulatory repeat sequences, a transmembrane domain, and a cytoplasmic domain.5 6 E-selectin, P-selectin, and L-selectin differ in the number of repeat sequences but otherwise share a high degree of sequence homology.5 For instance, E-selectin and P-selectin are 43% to 62% identical in their lectin, epidermal growth factor, and complement regulatory repeat sequences.5 20 The anti-selectin MoAbs used in this study were directed either against a specific E-selectin epitope (E-1E4) or a novel epitope found within the lectin domains common to E- and P-selectin (EP-5C7).20 Whereas P-selectin, on endothelial cell activation, is rapidly recruited by translocation to the cellular surface from intracellular storage vesicles (Weibel-Palade bodies), the expression of E-selectin requires de novo synthesis of both mRNA and protein.5 6 Interleukin-1, tumor necrosis factor-{alpha}, lymphotoxin, and lipopolysaccharides induce E-selectin expression.5 6 8 14 15 E-selectin mediates polymorphonuclear leukocyte rolling in close functional relationship with P-selectin, followed by firm endothelial attachment and transmigration.5 6 7 It thereby appears to play a role in later inflammatory processes of I/R injury. Data from knockout mice, deficient for either the E-selectin or P-selectin gene, indicate that loss of one selectin might be fully compensated by the other,32 33 consistent with the concept that endothelial E-selectin and P-selectin are functionally redundant.32 33

The highest levels of E-selectin upregulation (ie, maximal chromogen intensity and frequency of microvascular E-selectin presentation) were consistently found in the ischemic territory by 24 hours of reperfusion after MCAO (Figs 1 and 2UpUp). This is in agreement with the descriptions of endothelial E-selectin expression in response to I/R in vessels of myocardium, lung, kidney, skin, and skeletal muscle of selected animal models.8 10 11 12 13 14 15 However, only very limited information describing the dynamics of E-selectin antigen regulation during focal cerebral I/R is available.2 4 P-selectin antigen presentation occurred independent of E-selectin, as demonstrated by P-selectin appearance in the time course fluorescence colocalization experiments (and Reference 3) in only 1.3±1.2% of microvessels at 24 hours of reperfusion. Coincident expression of both antigens was seen in 14.9±13.0% of E-selectin immunoreactive microvessels at 24 hours.

The observations of Wang et al2 in the cortex of spontaneously hypertensive rats are consistent with the expected late expression of the intact protein after endothelial cell activation.6 However, the cellular source of E-selectin antigen has not been described. In unrelated studies, the expression of E-selectin was primarily restricted to postcapillary venules but was also found on capillaries and larger vessels.34 35 In the present study, E-selectin antigen was predominantly found on microvascular endothelium within the range of 7.5 to 30.0 µm (precapillary arterioles and postcapillary venules).

Low levels of E-selectin protein could be detected as early as 2 hours after MCAO in ischemic cerebral tissue of the primate, although almost no E-selectin mRNA was found in rat cortex earlier than 6 hours after MCAO.2 While it cannot be excluded that low levels of mRNA may be responsible for the early E-selectin presentation noted here, different sensitivities of techniques used to detect mRNA and protein species-related differences between rodents and primates, varying experimental settings with respect to MCAO/R, and other features may underlie the early presentation in the primate.2 A more intriguing possibility is that soluble E-selectin may adhere to receptors exposed on ischemic microvascular endothelium during ischemia. The recent demonstration that E-selectin can induce chemotaxis of human endothelial cells in culture and angiogenesis in the rat cornea suggests the existence of inducible E-selectin receptors on endothelium.36

E-selectin may also play a role in leukocyte recruitment at early times (1 to 2 hours) after an inflammatory insult. Bosse and Vestweber37 have demonstrated that an anti-E-selectin MoAb inhibited recruitment of granulocytes into the peritoneal cavity of thioglycolate-treated mice at 1 to 2 hours as well as or better than at 4 hours after stimulation. Various strategies aimed at selectin-mediated granulocyte-endothelial interactions have been effective in reducing tissue damage in selected animal models of myocardial infarction and lung injury.10 38 39 40

The observation of E-selectin antigen in nonischemic brain tissue by 24 hours of MCAO but not in control subjects or in nonischemic brain tissues up to 7 hours after MCAO is consistent with humoral control. It is likely that cytokines released from ischemic tissue are the key mediators in this process.5 6 8 14 15 These transferable factors may support inflammatory responses in otherwise unaffected tissue distant to the local acute injury, as suggested by the finding of E-selectin in the absence of nonvascular cellular DNA injury (dUTP uptake). Whether these findings reflect a remote endothelial response to the tissue injury after focal I/R must be confirmed by discrete cellular mRNA expression studies. In support, E-selectin expression in lung microvessels followed I/R injury to skeletal muscle of the isolated rat hindlimb,14 15 which was completely abrogated by tumor necrosis factor-{alpha} and interleukin-1 antagonists.14 In the brain, alterations in cerebral blood flow in the nonischemic hemisphere after contralateral ischemia may be explained in part by these remote cellular phenomena.41 42

Fassbender et al4 reported elevated circulating levels of soluble E-selectin in serum 24 hours after stroke onset in humans. The extracellular fragment of the E-selectin molecule can be shed from the transmembrane domain.4 5 It is expected that increasing soluble E-selectin levels in plasma would accompany endothelial cell E-selectin upregulation.4 This raises the possibility of cross-reactions between the anti-E-selectin or anti-E:P-selectin MoAbs with the soluble ligand bound to endothelial receptors. In the primate, E-selectin presentation early after MCAO did not represent retained intravascular plasma (data not shown).43

The appearance of E-selectin immunoreactivity in microvascular endothelium in nonischemic tissue late during reperfusion after MCAO implies a remote E-selectin response to the initial ischemic injury. The full relevance of these findings must await characterization of mRNA regulation in both ischemic and nonischemic territories.


*    Selected Abbreviations and Acronyms
 
DIG = digoxigenin
E:P-selectin = common epitope of E- and P-selectin
I/R = ischemia/reperfusion
MCA = middle cerebral artery
MCAO = middle cerebral artery occlusion
MCAO/R = middle cerebral artery occlusion and reperfusion
MoAb = monoclonal antibody
rhE = recombinant human E-selectin


*    Acknowledgments
 
This study was supported by grant NS 26945 (Dr del Zoppo) from the National Institutes of Health and the Schrodinger Fellowship (Dr Haring) from the Fonds zur Forderung der wissenschaftlichen Forschung (Austria).


*    Footnotes
 
This is publication No. 9652-MEM from The Scripps Research Institute, La Jolla, Calif.

Received February 16, 1996; revision received March 28, 1996; accepted April 1, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

Michael J Eppihimer, PhD, Guest Editor

Department of Physiology, Louisiana State University Medical Center, Shreveport, La


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
In the accompanying article, Haring and colleagues report that MCAO/R alters the surface expression of E-selectin on microvessel endothelium in ischemic and nonischemic regions of the brain. The expression of E-selectin was detected primarily (>80%) on precapillary and postcapillary microvessels with diameters less than 30 µm. While the type of microvessel expressing E-selectin was not delineated, the fact that leukocyte-endothelium adhesion occurs preferentially in postcapillary venules after MCAO1 implies that E-selectin is expressed in part on the postcapillary venular endothelium.

A distinguishing observation of the study by Haring et al is the time course for E-selectin expression in ischemic and nonischemic regions after MCAO/R. Under normal conditions, E-selectin is not expressed on the endothelium of postcapillary microvessels. However, agents such as tumor necrosis factor-{alpha}, interleukin-1, and lipopolysaccharide have been shown to induce the expression of E-selectin to the endothelial cell surface during a time period of 12 hours (maximum at 3 to 6 hours), which returns to near basal levels within 24 hours.2 3 The expression of E-selectin after stimulation with these cytokines requires protein synthesis, which suggests that E-selectin is regulated at the mRNA level. While E-selectin was observed in microvessels of ischemic regions after 1 and 4 hours of reperfusion, its expression increased approximately twofold after 24 hours of reperfusion. One explanation for the sustained and elevated expression of E-selectin after 24 hours may be that oxidants and cytokines produced by reperfusion act on endothelial cells to alter the transcriptional rate of E-selectin mRNA and its subsequent translation into protein. Another possible explanation for the persistent expression of E-selectin at 24 hours is that the mechanism responsible for endocytosis, through the endosomes and lysosomes, is rendered inoperative by cellular changes induced by I/R. Inhibition of the lysosomes with chloroquine resulted in persistent E-selectin expression lasting longer than 18 hours.4 While an absence of E-selectin was observed in microvessels in nonischemic regions at early time points, there was a significant elevation in its expression after 24 hours of reperfusion. The presence of E-selectin in nonischemic regions of the brain indicates that distant sites may be at risk of an inflammatory response after reperfusion of an ischemic region. While the expression of E-selectin in nonischemic regions is attenuated compared with ischemic regions, the present study suggests that inflammatory stimuli, such as oxidants and cytokines, generated from the initial ischemic injury may act remotely through their circulation to induce E-selectin expression on vascular endothelium of nonischemic tissues. However. the relevance of this finding to leukocyte recruitment and cellular injury in nonischemic tissues remains to be explored.

Another interesting observation of this study was the colocalization of P-selectin with E-selectin in a small fraction of microvessels. Recent studies indicate that, in addition to a rapid translocation of P-selectin from storage granules (within minutes). P-selectin may also be transcriptionally regulated.5 The presence of P-selectin in approximately 15% of the microvessels agrees favorably with observations from Okada et al6 (1994), in which 10% of the microvessels from the ischemic region displayed P-selectin. The considerably lower number of microvessels expressing P-selectin compared with E-selectin may presumably be due to differences between their kinetics of expression within a given microvessel in response to I/R. For example, P-selectin expression induced by I/R may be short-lived on the endothelial cell surface such that at any given time throughout the period of reperfusion only a small number of microvessels will display P-selectin.

The finding that MCAO/R increases microvessel E-selectin expression suggests that immunoneutralization of E-selectin may attenuate cerebrovascular injury induced by I/R. Studies have demonstrated that administration of antibodies directed against functional epitopes of E-selectin attenuates neutrophil rolling in cytokine-treated venules7 and leukocyte accumulation in the lung.8 In addition, previous studies have demonstrated that administration of antibodies directed against P-selectin and intercellular adhesion molecule-1 attenuates microvascular permeability and cellular dysfunction attendant to I/R.9 10 Both P-selectin and intercellular adhesion molecule-1 expression have been shown to increase in response to MCAO/R.6 These observations indicate that receptor-mediated leukocyte adhesion to microvascular endothelium plays an essential role in the degradation of vascular and cellular function. Given the present study's finding that compared with P-selectin, E-selectin is predominantly expressed in microvessels in response to I/R suggests that blocking E-selectin function may attenuate leukocyte-induced microvascular dysfunction induced by focal cerebral ischemia. However, studies designed to address the role of leukocyte–endothelial cell adhesion molecules of postischemic and nonischemic cerebral microvessels in the development of microvascular injury are warranted.


*    Selected Abbreviations and Acronyms
 
DIG = digoxigenin
E:P-selectin = common epitope of E- and P-selectin
I/R = ischemia/reperfusion
MCA = middle cerebral artery
MCAO = middle cerebral artery occlusion
MCAO/R = middle cerebral artery occlusion and reperfusion
MoAb = monoclonal antibody
rhE = recombinant human E-selectin

n indicates number of samples; non-I/R, non-ischemia/reperfusion zone; I/R, post-ischemia/reperfusion zone; MCAO, 2-hour middle cerebral artery occlusion; and MCAO/R, 3-hour middle cerebral artery occlusion with indicated periods of reperfusion. Values are mean±SD per 250 fields.


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. del Zoppo GJ, Schmid-Schonbein GW, Copeland BR, Chang C-M. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. 1991;22:1276-1283.
  2. Fries JWU, Williams AJ, Atkins RC, Newman W, Lipscomb MF, Collins T. Expression of VCAM-1 and E-selectin in an in vivo model of endothelial activation. Am J Pathol. 1993;143:725-737.[Abstract]
  3. Gotsch U, Jager U, Dominis M, Vestweber D. Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-{alpha} in vivo. Cell Adhes Commun. 1994;2:7-14.[Medline] [Order article via Infotrieve]
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  5. Hahne M, Jager U, Isenmann S, Hallmann R, Vestweber D. Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J Cell Biol. 1993;121:655-664.[Abstract/Free Full Text]
  6. Okada Y, Copeland BR, Mori E, Tung M-M, Thomas WS, del Zoppo GJ. P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke.. 1994;25:202-211.[Abstract]
  7. Olofsson AM, Arfors K-E, Ramezani JD, Chambers HD, Ochs JM, Harlan JM, Paulson JC, Etzioni A, von Adrian UH. E-selectin mediates leukocyte rolling in interleukin-1 treated rabbit mesentery venules. Blood.. 1994;84:2749-2758.[Abstract/Free Full Text]
  8. Mulligan MS, Varani J, Dame MK, Lane C, Smith CW, Anderson DC, Ward PA. Role of endothelial-leukocyte adhesion molecule-1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin Invest.. 1991;88:1396-1406.
  9. Cardon DL, Young JA, Granger DN. Pulmonary microvascular injury following intestinal ischemia/reperfusion: role of P-selectin. J Appl Physiol.. 1993;75:2529-2534.[Abstract/Free Full Text]
  10. Horgan MJ, Ge M, Gu J, Malik AB. Role of ICAM-1 in neutrophil-mediated lung vascular injury after occlusion and reperfusion. Am J Physiol.. 1991;261:H1578-H1584.[Abstract/Free Full Text]



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