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(Stroke. 1997;28:2214-2218.)
© 1997 American Heart Association, Inc.

Articles

Soluble Adhesion Molecules Reflect Endothelial Cell Activation in Ischemic Stroke and in Carotid Atherosclerosis

C.J.M. Frijns, MD; L.J. Kappelle, MD; J. van Gijn, MD, FRCP; H.K. Nieuwenhuis, MD; J.J. Sixma, MD; R. Fijnheer, MD

From the Departments of Neurology (C.J.M.S., L.J.K., J. van G.) and Hematology (H.K.N., J.J.S., R.F.), University Hospital Utrecht (Netherlands).

Correspondence to C.J.M. Frijns, MD, Department of Neurology, University Hospital Utrecht, PO Box 85500, Heidelberglaan 100, 3584 CX Utrecht, Netherlands. E-mail C.Frijns{at}neuro.azu.nl

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose Activation of endothelial cells and platelets plays an important role in the development of atherosclerosis and thrombotic disorders. Soluble adhesion molecules originating from these cells can be demonstrated in plasma. We hypothesized that elevated plasma concentrations of soluble P-selectin (sP-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-selectin) can reflect activation of endothelial cells and/or platelets in acute ischemic stroke and in previously symptomatic internal carotid artery stenosis.

Methods Plasma was sampled from patients within 2 days of acute ischemic stroke (n=28), from patients with a previous (>1 week) transient or persistent ischemic neurological deficit associated with stenosis of the internal carotid artery (n=34), and from control patients without a history of vascular disease (n=34). Concentrations of sP-selectin, sICAM-1, sVCAM-1, and sE-selectin were measured by means of an enzyme-linked immunosorbent assay.

Results Compared with control subjects, sP-selectin and sE-selectin were significantly elevated in the acute stage of ischemic stroke (P<.0001 and P=.001, respectively) as well as in previously symptomatic carotid stenosis (P<.0001 and P=.0007). sICAM-1 and sVCAM-1 were not increased.

Conclusions The elevated levels of sE-selectin indicate that endothelial cell activation occurs both in the acute stage of ischemic stroke and in previously symptomatic carotid atherosclerosis. Increased sP-selectin concentrations reflect endothelial cell activation as well but may also be caused by platelet activation.

Key Words: carotid stenosis • cell adhesion molecules • cerebral infarction • endothelial

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Adhesion molecules are involved in leukocyte rolling, firm adhesion, and transmigration and as such play a role in inflammatory disorders as well as a variety of other pathophysiological processes.^1–3 ICAM-1 and VCAM-1 are members of the immunoglobulin gene superfamily of adhesion molecules. ICAM-1 is expressed on leukocytes, fibroblasts, epithelial cells, and endothelial cells, whereas VCAM-1 is found only on endothelial cells. P- and E-selectin belong to the selectin family of adhesion molecules. E-selectin is found only on activated endothelium. P-selectin is a membrane molecule of the -granules in platelets and of the endothelial Weibel-Palade bodies. As a result of alternative splicing, two isoforms exist: one with and one without a transmembrane region. The latter soluble molecule is secreted from the -granules or from the Weibel-Palade bodies. The isoform with the transmembrane part as well as the other adhesion molecules are expressed on the cell surface after activation. They are shed into the circulation by proteolysis.

Activation of endothelial cells and platelets plays an important role in the development of atherosclerosis and thrombotic disorders and is accompanied by an increased expression of endothelial adhesion molecules at the site of thrombosis or infarction. In vitro activation of platelets and/or endothelial cells results in shedding of selectins and immunoglobulin gene superfamily molecules in the supernatant.¹ Recently, several soluble isoforms of adhesion molecules were found to circulate in the peripheral blood and to be elevated in diseases such as sepsis and vasculitis.^3,4 P-selectin, ICAM-1, and VCAM-1 are strongly expressed on endothelium overlying human atherosclerotic plaques.^5,6 In animal models of coronary ischemia and reperfusion, as well as in myocardial infarction in humans, a role of endothelial as well as leukocyte adhesion molecules has been reported.^7–12 Adhesion molecules are implicated in peripheral vascular disease as well.^9,13–15 In animal experiments of MCA occlusion and reperfusion, ICAM-1 and P-selectin have been demonstrated to play an important role in the pathogenesis of ischemic infarction.¹⁶ Recently, upregulation of endothelial ICAM-1 in infarcted areas has been found in patients with acute ischemic stroke.¹⁷ Upregulation of E-selectin expression in ischemic rat cortex up to 2 days after MCA occlusion has also been reported,¹⁸ as well as in MCA occlusion and reperfusion in nonhuman primates at 24 hours of reperfusion.¹⁹ In focal central nervous system ischemia, anti-CD18 MoAbs inhibit leukocyte adherence to endothelium,^20,21 increasing postreperfusion microvessel patency, whereas MoAbs against ICAM-1 reduce ischemic cell damage.^22–25

We investigated plasma samples of patients with a recent cerebral infarct or with (transient) neurological deficit associated with stenosis of the ICA to determine whether in these conditions activation of endothelial cells and platelets can be detected by measuring soluble adhesion molecules. Values were compared with those obtained in patients without evidence of vascular disease.

	Subjects and Methods

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Subjects
Blood samples were collected from patients admitted to the Department of Neurology. The first group consisted of 28 patients with a diagnosis of recent ischemic stroke. The diagnosis was established by history, clinical examination, and cerebral CT or MRI scans. Plasma samples were taken within 48 hours after onset of symptoms. The infarcts were supratentorial in 23 of the 28 patients with a recent ischemic stroke; of the other 5 patients, 3 had an infarct in the brain stem and 2 in the cerebellum. Duplex ultrasonography of the ICA was performed in 23 of these 28 patients. Less than 30% stenosis on either side was found in 16, moderate stenosis (30% to 70%) of at least one carotid artery was present in 2, and 5 others showed severe stenosis of more than 70% unilaterally or bilaterally. Relevant concomitant disorders at the time of blood sampling in the acute stroke group were fever of unknown origin and diabetes complicated by renal failure, each in 1 patient. The second group consisted of 34 patients with a history of a TIA or minor ischemic stroke who also had a moderate to severe symptomatic ICA stenosis. Amaurosis fugax was the presenting symptom in 4, hemispheric TIA in 14, and a nondisabling ischemic stroke in 16. Blood sampling took place at least 7 days after the last cerebral ischemic attack (median, 44 days). The degree of carotid stenosis was assessed by carotid angiography according to the North American Symptomatic Carotid Endarterectomy Trial method^26,27 in 30 of these 34 patients, by CT or MR angiography in 3, and by duplex ultrasonography alone in 1 patient. Carotid stenosis was moderate (30% to 70%) in 2 patients and severe in 32; 16 had a unilateral and 5 a bilateral occlusion of the ICA. Control subjects without evidence of cardiovascular, malignant, inflammatory or autoimmune diseases were recruited from the Department of Neurology (n=18) and from the Outpatient Clinic of Internal Medicine (n=16). Relevant data, such as risk factors for vascular disorders, were obtained from hospital charts.

Blood Sampling and Laboratory Investigations
Blood was drawn by the evacuated tube system in all patients in the same way and collected in citrate anticoagulant (13 mmol/L). It was immediately centrifuged at 2000g for 15 minutes at 4°C. The supernatant was removed and centrifuged a second time. Plasma samples were then stored at -70°C. Levels of sP-selectin were measured by means of an enzyme-linked immunosorbent assay developed at our department. Microtiter plates were coated overnight at 4°C with IgG MoAb 2.15. In each plate, a standard of at least six dilutions of platelet lysate, with a known amount of sP-selectin, was included. The sP-selectin concentration in the platelet lysate was established by means of immunopurified P-selectin (R&D Systems Ltd). The second MoAb was biotinylated IgG 1.18.²⁸ Binding of this MoAb was detected by addition of streptavidin–horseradish peroxidase. Staining was done with o-phenylenediamine dihydrochloride. The optical density at 490 nm (OD₄₉₀) was read in a VMAX plate reader (Molecular Devices Corp), and P-selectin levels of test samples were calculated by comparison with the OD₄₉₀ of the standard samples containing predetermined levels of P-selectin. The median intra-assay coefficient of variation was 3%.

sVCAM-1, sICAM-1, and E-selectin were measured with commercially available kits (R&D Systems). Median intra-assay coefficients of variation were 1.7%, 4.6%, and 2.5%, respectively. Statistical evaluation was performed with the Mann-Whitney U test. Data are presented as mean±SD values. Significance levels were set at P<.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Baseline characteristics of the patients are presented in Table 1. The concentrations of adhesion molecules (mean±SD; range) are summarized in Table 2 and in the Figure . Control values are in accordance with those mentioned in previous studies.³ sP-selectin concentration was significantly elevated in both acute ischemic stroke (238±92 ng/mL; P<.0001) and carotid atherosclerosis (200±73 ng/mL; P<.0001) compared with control subjects (135±41 ng/mL). The highest sP-selectin values were measured in a stroke patient with fever of unknown origin, who died of cerebral herniation due to swelling of the infarcted hemisphere (414.80 ng/mL), and in a patient requiring hemodialysis (490.30 ng/mL). Omitting these values from the analysis still resulted in a value of P<.0001.

View this table: [in this window] [in a new window]   Table 1. Baseline Characteristics

View this table: [in this window] [in a new window]   Table 2. Concentrations of Adhesion Molecules

View larger version (21K): [in this window] [in a new window]   Figure 1. Concentrations of soluble adhesion molecules in acute ischemic stroke (), in previously symptomatic carotid atherosclerosis (), and in control patients ().

sE-selectin concentrations were also significantly elevated in acute ischemic stroke (53±42 ng/mL; P=.001) as well as in carotid atherosclerosis (49±25 ng/mL; P=.0007) compared with control subjects (35±15 ng/mL), although with considerable overlap. The highest values occurred in the patient with a fatal stroke who also had an excessively high P-selectin value (254.70 ng/mL) and in a patient without a known concomitant disease, who had a stenosis of the right ICA of more than 70% (155.30 ng/mL). Omitting these values from the analysis resulted in values of P=.0017 and P=.0011, respectively. The Spearman correlation coefficient of sP-selectin and sE-selectin was .36 (P=.0004).

No differences were found with respect to sICAM-1 and sVCAM-1 concentrations, except for a trend toward higher sVCAM-1 levels in the patients with carotid atherosclerosis (P=.07). Excluding the two patients with hypertension from the control group led to similar probability values for all comparisons except sVCAM-1, which became significantly elevated in carotid atherosclerosis (P=.04).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This is the first study to demonstrate an increase of soluble endothelial adhesion molecules in patients with moderate to severe stenosis of the ICA revealed by a previous TIA or minor ischemic neurological deficit. We also confirmed earlier reports of a significant increase of sP-selectin and sE-selectin concentrations in acute ischemic stroke.^29,30 We could not show any differences between patients with an acute ischemic stroke and with previously symptomatic ICA stenosis. Since vascular risk factors such as hypertension were more common in the vascular patients than in the control subjects, it cannot be excluded that part of the elevation of sP- and sE-selectin can be attributed to the presence of these risk factors. To assess their influence on circulating adhesion molecules, a controlled study matching for all known vascular risk factors is necessary. However, in a previous study comparing circulating adhesion molecule concentrations in patients with acute ischemic stroke and in patients with and without vascular risk factors, sE-selectin was elevated in the stroke patients, whereas no difference in sE-selectin levels was found between the two other groups with normal levels (sP-selectin was not tested).³⁰ Contrary to our findings, a more recent article reported no difference in sE-selectin levels between acute ischemic stroke patients, age- and risk factor–matched control subjects, and healthy young control subjects, whereas sICAM-1 was increased in the stroke patients.³¹ At present, because of largely overlapping ranges, elevated levels of sP-selectin and sE-selectin cannot be used to distinguish between patients and asymptomatic control subjects. However, they may prove to be valuable as intraindividual markers of severity of atherosclerotic disease in the follow-up of selected patients.

Wu et al²⁹ also reported elevation of sP-selectin in patients with acute ischemic stroke. They concluded that elevated sP-selectin results from platelet activation and endothelial damage in the acute phase. Indeed, the correlation we found between sP-selectin and sE-selectin suggests that part of sP-selectin originates from endothelial cells and the remainder from platelets. Platelet activation in focal cerebral ischemia has been shown in MCA occlusion and reperfusion experiments in animals. Okada et al¹⁶ demonstrated elevated P-selectin in infarcted tissue at 4 and 24 hours of reperfusion in conjunction with an increase of glycoprotein IIb/IIIa receptor antigen, indicating the presence of activated platelets. In acute ischemic stroke, generation of thrombin, oxygen radicals, and other stimulants of endothelial cells and platelets leads to an increase of both endothelial and platelet P-selectin expression, which in turn leads to further adhesion of leukocytes and subsequent release of cytokines and oxygen radicals, increase of tissue damage, exposure of tissue factor, activation of coagulation, and stimulation of endothelial cells and platelets, in an ongoing cycle. Acting through these processes, various systemic concomitant disorders such as atherosclerotic vascular disease and infectious diseases may contribute to increase of soluble adhesion molecules.

It could be argued that elevated sP-selectin is a consequence of platelet activation induced by retrieval of blood. However, in a study of different ways of plasma preparation, no influence on sP-selectin levels was found either by drawing of blood with different techniques or by addition of various protease inhibitors, irrespective of in vitro platelet activation, as demonstrated by elevated levels of ß-thromboglobulin and platelet factor 4.³² In another study we also did not find an influence of addition of hirudin and prostaglandin E₁, nor of prolonged centrifugation, on sP-selectin in our plasma samples.⁴

ICAM-1 and VCAM-1 are upregulated by cytokines and other mediators of inflammation. Hence, an increased expression is expected in such disorders as sepsis and other infectious diseases, inflammatory disorders, autoimmune diseases, atherosclerosis and other vasculopathies, and acute ischemic events such as myocardial infarction and ischemic stroke.^2,3 Indeed, the expression of ICAM-1 and VCAM-1 is found on human atherosclerotic plaques.^5,6 Also, in view of the relevance of vascular risk factors, increased expression of ICAM-1 was found in carotid artery sections of spontaneously hypertensive rats.³³ However, we could not demonstrate an increase of sICAM-1 and sVCAM-1 in our two vascular patient groups, in contrast to an earlier report in which increased levels of sICAM-1 and sVCAM-1 were found in 22 acute stroke patients compared with 22 control subjects without vascular risk factors.³⁰ In another study sICAM-1 levels were significantly lower in 14 acute stroke patients compared with 12 healthy control subjects.³⁴ The question of an explanation of these differences arises.

Administration of heparin to stroke patients in the study of Fassbender et al,³⁰ as opposed to low-dose aspirin in most of our patients, may be an explanation. Recently, several studies have focused on the influence of anti-inflammatory agents on nuclear factor-B, a transcription factor that is critical for the expression of several genes, including E-selectin, ICAM-1, and VCAM-1. In vitro experiments showed aspirin to inhibit activation of nuclear factor-B in a dose-dependent way.^35,36 Subsequently, tumor necrosis factor- or interleukin-1–induced expression of adhesion molecules may be inhibited. This was demonstrated for E-selectin and VCAM-1, but not for ICAM-1, in one study.³⁷ However, aspirin concentrations in these experiments were rather high. Comparable concentrations in vivo can only be attained by toxic dosages. It is still unclear whether these in vitro effects play a role in vivo, and how different oral doses of aspirin affect expression of endothelial adhesion molecules.

Another more plausible explanation for some of these contradictory results has been presented in a recent study on expression of ICAM-1, VCAM-1, and E-selectin in the human carotid bifurcation.³⁸ Expression of VCAM and E-selectin was limited to specimens with plaques and was absent in normal arteries. However, strong focal expression of ICAM-1, limited to the lateral outer wall of the ICA, was also present in vessels with no or low-grade stenosis. The authors conclude that the expression of ICAM-1 in the carotid artery precedes the first signs of atherosclerosis. If we assume that carotid plaques are indicative of more widespread atherosclerosis and consider that we excluded patients with symptomatic vascular diseases, but not with vascular risk factors, from the control group, this may explain why no difference was found for sICAM-1, whereas sE-selectin was significantly elevated and sVCAM-1 showed a trend toward increased concentrations in patients with carotid stenosis.

In conclusion, elevated plasma concentrations of sE-selectin indicate activation of endothelial cells, whereas increased levels of sP-selectin reflect activation of both endothelial cells and platelets in acute ischemic stroke and in carotid atherosclerosis associated with previous ischemic neurological deficits.

	Selected Abbreviations and Acronyms

 

ICA = internal carotid artery MCA = middle cerebral artery MoAb = monoclonal antibody sE-selectin = soluble E-selectin (s)ICAM-1 = (soluble) intercellular adhesion molecule-1 sP-selectin = soluble P-selectin (s)VCAM-1 = (soluble) vascular cell adhesion molecule-1 TIA = transient ischemic attack

	Acknowledgments

 
We kindly thank A. Algra, MD, for his helpful comments.

Received June 3, 1997; revision received July 2, 1997; accepted August 14, 1997.

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