Cold Pressor Test Raises Serum Concentrations of ICAM-1, VCAM-1, and E-Selectin in Normotensive and Hypertensive Patients
Abstract In patients with essential hypertension, elevated soluble E-selectin (sE-selectin) levels may indicate endothelial cell injury or activation. We therefore sought to ascertain whether arterial blood pressure increased by the cold pressor test can modify serum concentrations of sE-selectin and other soluble forms of adhesion molecules, such as soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1), or the expression of any adhesion molecules in circulating monocytes and lymphocytes. Our findings show that levels of sE-selectin, sVCAM-1, and sICAM-1 are higher in patients with essential hypertension than in normotensive subjects (sICAM-1, 380±52 versus 262±96 ng/mL, P<.05; sVCAM-1, 720±52 versus 625±38 ng/mL, P<.05; and sE-selectin, 75±21 versus 61±22 ng/mL, P<.05). Furthermore, in normotensive and hypertensive patients, the cold pressor test caused an increase in serum concentrations of sICAM-1, sVCAM-1, and sE-selectin, but it did not cause changes in the expression of adhesion molecules in circulating monocytes and lymphocytes. High arterial blood pressure may therefore increase the production of serum adhesion molecules, probably through endothelial activation.
Transfer of leukocytes across the vascular endothelium depends on the interaction between leukocytes and endothelial cells and is mediated by cell adhesion molecules, including ICAM-1, E-selectin, and VCAM-1.1 2 Increases in the soluble form of these molecules have been found in association with endothelial cell activation and inflammation.
Blann et al3 found increased sE-selectin and sICAM-1 values in inflammatory vasculitis, as well as increased sE-selectin, sICAM-1, and sVCAM-1 levels in patients with ischemic heart disease, whereas sICAM-1 and sVCAM-1 levels were raised in patients with peripheral vascular disease of the carotid, iliac, or femoral arteries. High sE-selectin levels have also been reported in patients with essential hypertension.4 Our aim was therefore to ascertain whether the increase in arterial blood pressure induced by the CPT can modify serum concentrations of adhesion molecules or their expression in monocytes and lymphocytes.
The study was carried out on 71 subjects subdivided into two groups. Group 1 included 36 patients with essential hypertension (20 males, 16 females; mean age 48±2 years; body mass index, 25.1±3.9 kg/m2) and group 2 included 35 normotensive subjects (20 males, 15 females; mean age 47±2 years; body mass index, 25.5±5.1 kg/m2). Patients considered hypertensive had blood pressure higher than 160/90 mm Hg in at least four sets of readings taken at 1-week intervals.
Hypertension had been recently diagnosed in all subjects, and none had ever been treated with drugs. These subjects all led sedentary lives and were not involved in athletic training. Exclusion criteria included alcohol or drug abuse; suspected or proven secondary hypertension; kidney, heart, or hepatic disease; thyroid disease; postural hypotension; diabetes mellitus; and/or treatment with nonsteroidal anti-inflammatory drugs, laxatives, lithium salts, or any drugs that might influence blood pressure. Control subjects had a supine systolic blood pressure lower than 140 mm Hg and a diastolic blood pressure lower than 90 mm Hg.
Written informed consent was obtained from all subjects, and the approval of our ethics committee was obtained for all the procedures used.
At 7 am, after an overnight fast, an indwelling catheter was inserted into the antecubital vein for blood sampling, the first samples being taken at 8 am. After subjects had rested supine for 60 minutes in a room maintained at 25°C to 30°C, resting blood pressure was measured. Using an automated measurement and recording device (Sentry Device Inc), systolic and diastolic blood pressures were monitored throughout the experimental period.
Five minutes after basal measurements had been obtained, the CPT was performed (t 0). The patients’ hands were immersed to just above the wrists in cold water (3°C to 5°C) for 4 minutes. Serum samples were taken at −5, 0, and 4 minutes (immediately before hands were removed from the water) and at 15 minutes to measure sICAM-1 (ICAM-1 Predicta kit, Genzyme) and sE-selectin and sVCAM-1 (Bender Med System, Genzyme Corporation); hematocrits were also evaluated at the same times.
Plasma norepinephrine and epinephrine also were measured at the same times, and dihydroxybenzylamine was added to each sample aliquot as an internal standard. The mixture was buffered to pH 7.8 to 8.2 with Tris-HCl buffer (pH 8.6), and acid-washed alumina was added. The solution was mixed for 15 minutes, the supernatant was removed, and the alumina was washed with ice-cold distilled water. After 200 μL of 0.1 mol/L perchloric acid was added to extract the catecholamine, the sample was injected into a high-performance liquid chromatograph (Beckman Instruments) with an electrochemical detector (model 5000 ESA, coulometric method for highly sensitive cells).
Direct immunofluorescence was carried out to measure antigen expression in monocytes and lymphocytes. One hundred microliters of venous blood was incubated for 30 minutes at 4°C with a panel of selected monoclonal antibodies at saturating concentrations in a double-labeling procedure; the monoclonal FITC-conjugated antibodies used were anti-CD11a (LFA-1α), anti-CD11b (C3b), anti-CD15s (sialyl- Lewis-x), and anti-CD49d (VLA-α4) (Becton Dickinson and T Cell Sciences, Inc).
After staining, red blood cells were lysed for 10 minutes using a commercial reagent. The samples were then centrifuged for 5 minutes at 500g, washed twice in PBS containing 0.1% sodium azide, and resuspended in 300 μL of PBS for flow-cytometric analysis. Immunofluorescence analysis was performed using a FACScan flow cytometer (Becton Dickinson).
All data were collected using a logarithmic amplification of fluorescence signals. Results are expressed as a relative mean fluorescence intensity channel. The statistical analysis was performed using Student’s t test. The Pearson correlation coefficient was used to analyze any correlations, a value of P<.05 being considered significant. Results are given as mean±SD.
Patients with essential hypertension had serum concentrations of ICAM-1, VCAM-1, and E-selectin that were significantly higher than those in normotensive subjects (Table⇓).
After 4 minutes, the CPT had increased arterial blood pressure, plasma catecholamines, and serum concentrations of sICAM-1, sVCAM-1, and sE-selectin in both normotensive and hypertensive patients. At 15 minutes, all the values had returned to basal levels (Table⇑). No significant changes were observed in hematocrit values. After the CPT, no change was found in the expression of adhesion molecules in circulating monocytes and lymphocytes (data not shown). No statistically significant correlation was found between soluble molecules and plasma concentrations of epinephrine and norepinephrine either at rest or after CPT, nor was any correlation found between arterial pressure and adhesion molecules.
Our findings confirm the observation made by Blann et al4 that sE-selectin levels are higher in patients with essential hypertension than in normotensive subjects and demonstrate that a similar increase occurs for sICAM-1 and sVCAM-1 levels. Our most important finding, however, was that the pressure increase induced by CPT was in itself enough to increase serum concentrations of the adhesion molecules studied, not only in hypertensive but also in normotensive subjects.
The data obtained from the measurement of soluble adhesion molecules in hypertensive patients may in fact be interpreted in various ways. These molecules may be a marker of activation, and in the case of sE-selectin, for example, increased serum levels would indicate endothelial activation. Yet hypertension is characterized by endothelial cell alteration, shown by the increase in levels of von Willebrand factor, which is a marker of endothelial cell damage.5
Blann et al,4 however, found no correlation between sE-selectin levels and von Willebrand factor concentrations in hypertensive patients and suggest that raised levels of sE-selectin in hypertension indicate endothelial activation rather than endothelial cell injury.
In our experimental model, the CPT produced a rapid, transient, and slight vasopressor stimulus, which was unlikely to have caused true endothelial damage, especially in normotensive subjects. This finding suggests that adhesion molecules might be useful in monitoring endothelial cell activation rather than endothelial damage.
The mechanisms by which increased blood pressure causes an increase in adhesion molecules are not yet fully understood. Increased vascular distending and transmural pressures occurring with increased arterial pressure may be enough to cause endothelial cell activation, with a consequent change in the serum levels of adhesion molecules.
The increase in serum adhesion molecules after the CPT may also be due to sympathetic stimulation induced by the CPT itself.6 It is well known that in experimental hypertension, morphological changes occur in arterial endothelial cells in response to norepinephrine infusion.7 8 Nevertheless, Kugelmass et al9 found that intravenous administration of norepinephrine does not cause a significant increase in selectin (specifically P-selectin) expression. In our model, no statistically significant correlation was found between soluble molecules and plasma concentrations of epinephrine and norepinephrine either at rest or after CPT.
The lack of significant alterations in the expression of the adhesion molecules studied on the surface of monocytes and lymphocytes could further show the exclusive endothelial interest of the response to the pressure increase caused by the CPT. Alternatively, this finding might have been due to the different chronology of the events themselves, and a modified expression of adhesion molecules on blood cells may occur after changes in serum levels of sICAM-1, E-selectin, and sVCAM-1.
In conclusion, our findings and those of others suggest that the alterations in the serum concentrations of some adhesion molecules in hypertensive patients may be a sign of simple endothelial activation secondary to stress to vessels caused by blood pressure increase. Further studies could contribute to a clearer understanding of the inner physiopathological mechanism underlying this phenomenon.
Selected Abbreviations and Acronyms
|CPT||=||cold pressor test|
|ICAM-1||=||intercellular adhesion molecule-1|
|VCAM-1||=||vascular cell adhesion molecule-1|
- Received November 7, 1996.
- Revision received December 4, 1996.
- Accepted March 11, 1997.
Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood. 1994;84:2068-2101.
Robertson D, Johnson GA, Robertson RM, Nies A, Shand DG, Gates JA. Comparative assessment of stimuli that release neuronal and adrenomedullary catecholamines in man. Circulation. 1979;59:637-643.
Luscher T, Raij L, Vanhoutte P. Endothelium-dependent vascular responses in normotensive and hypertensive Dahl rats. Hypertension. 1987;9:157-163.
Kugelmass AD, Shannon RP, Yeo EC, Ware JA. Intravenous cocaine induces platelet activation in the conscious dog. Circulation. 1995;1:1336-1340.