Soluble ICAM-1 and VCAM-1 as markers of endothelial activation

Activated endothelium releases the soluble adhesion molecules vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1). Measurement of fluid-phase adhesion molecules is therefore used to quantify endothelial activation, but it is unclear which is the better marker. The aims of the study were to compare the relationships between mRNA, surface and total expression and released VCAM-1 and ICAM-1 in endothelial cell cultures during activation, and to compare human umbilical vein endothelial cells (HUVEC) with the microvascular cell line HMEC-1. sVCAM-1 better represented mRNA and surface expression changes in HUVEC undergoing endotoxin stimulation than did sICAM-1. Very little VCAM-1 was released from endotoxin-stimulated HMEC-1, and sICAM-1 seemed a better activation marker for these cells. During incubation of HUVEC in media with glucose concentrations of 5.6, 10.6 or 20.6 m m , VCAM-1 was released to the media in a dose-dependent way without changes in surface expression. ICAM-1 was not influenced by the glucose concentration. There are situations when VCAM-1 concentrations in the media do not mirror the surface expression on HUVEC in culture, indicating that measurements of soluble adhesion molecules may not necessarily be representative of the conditions on the cell surface. Endothelium from different locations showed varying responses with respect to VCAM-1 and ICAM-1 liberation upon endotoxin stimulation. Thus, both sVCAM-1 and sICAM-1 should be quantified in clinical studies of endothelial activation until their characteristics are better clarified.     

Maternal serum levels of VCAM-1, ICAM-1 and E-selectin in preeclampsia

Endothelial dysfunction is thought to be a central pathogenic feature in preeclampsia on the basis of elevated adhesion molecules. The aim of the present study was to compare the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin) in sera of normal and preeclamptic pregnancies. We studied the serum levels of sVCAM-1, sICAM-1 and sE-selectin in normal pregnant women (n=63), mild preeclampsia (n=33) and severe preeclampsia (n=82). Concentrations of soluble adhesion molecules were determined with enzyme-linked immunoassay (ELISA). Serum concentrations of sVCAM-1 were significantly higher in both mild (p=0.004) and severe preeclampsia (p=0.000) than normal pregnancy. There were also significant differences in sVCAM- 1 levels between mild and severe preeclampsia (p=0.002). sICAM-1 levels of severe preeclampsia were statistically different from those of normal pregnancy (p=0.038). Levels of sE-selectin were elevated in both mild (p=0.011) and severe preeclampsia (p=0.000) compared to normal pregnancy, but no statistical difference between the mild and severe preeclampsia (p=0.345). These results suggest that all three soluble adhesion molecules are increased in severe preeclampsia, and sVCAM-1 among them may be useful in predicting the severity of preeclampsia.     

Expression of ICAM-1, VCAM-1, E-selectin and TNF-alpha on the endothelium of femoral and iliac arteries in thromboangiitis obliterans

Immunohistochemical light and electron microscopical analysis of surgical biopsies obtained from femoral and iliac arteries of patients with thromboangiitis obliterans (TAO) were performed to investigate the presence of tumour necrosis factor-alpha (TNF-alpha) and expression of the endothelial cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Expression of ICAM-1, VCAM-1 and E-selectin was increased on endothelium and some inflammatory cells in the thickened intima in all TAO patients. Ultrastructural immunohistochemistry revealed contacts between mononuclear blood cells and ICAM-1-, and E-selectin-positive endothelial cells. These endothelial cells showed morphological signs of activation. The present data indicate that endothelial cells are activated in TAO and that vascular lesions are associated with TNF-alpha secretion by tissue-infiltrating inflammatory cells, ICAM-1-, VCAM-1- and E-selectin expression on endothelial cells and leukocyte adhesion via their ligands. The preferential expression of inducible adhesion molecules in microvessels and mononuclear inflammatory cells suggests that angiogenesis contributes to the persistence of the inflammatory process in TAO.     

Schistosoma mansoni schistosomula reduce E-selectin and VCAM-1 expression in TNF-alpha-stimulated lung microvascular endothelial cells by interfering with the NF-kappaB pathway

The recruitment of immune cells into the lungs is a key step in protection against murine schistosomiasis. In this phenomenon, pulmonary (micro)vascular endothelial cells (EC) probably play a central role, by expressing specific adhesion molecules on their surface. Recently, we have shown that Schistosoma mansoni schistosomula, the parasitic stage which resides in the lungs, could activate microvascular EC to acquire an anti-inflammatory phenotype. In the present study, we tested the hypothesis that schistosomula could also regulate the expression of adhesion molecules in vitro by human lung microvascular EC (HMVEC-l) in the present of the pro-inflammatory cytokine TNF-alpha. We found that lipophilic substance(s) present in the excretory/secretory products from schistosomula selectively reduce the TNF-alpha-induced synthesis of E-selectin and VCAM-1 mRNA and proteins without affecting ICAM-1. This inhibitory effect appears to be mediated by a cyclic AMP/protein kinase A (cAMP/PKA) pathway that probably interferes with the NF-kappaB pathway induced by TNF-alpha at the level of the E-selectin promoter, whereas a cAMP-independent pathway appears to operate in VCAM-1 down-modulation. Finally, schistosomula also significantly reduce the VLA-4/VCAM-1-dependent adherence of leukocytes to TNF-alpha-stimulated HMVEC-l. We speculate that this mechanism could represent a new stratagem that parasites may use to escape the immune system by controlling leukocyte recruitment to the lungs.     

Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Expression of allograft endothelial VCAM-1 can be manipulated with antagonist of IFN-alpha or IL-4 and is not required for allograft rejection.

This report provides evidence to support the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and IL-4 promote the expression of new endothelial surface molecules in rejecting murine heterotopic cardiac allografts. The microvascular endothelia of these cardiac allografts all develop strong reactivity with the monoclonal antibodies (mAbs) YN1.1/74 (anti-ICAM-1), M/K-2 (anti-VCAM-1) and MECA-32 (undefined molecule) within 3 to 5 days of graft implantation. Daily treatment of the allograft recipients with pentoxifylline (PTX), soluble TNF receptor (TNFR:Fc), anti-interleukin-4 (IL-4) mAb (11B11), or soluble IL-4 receptor, each abrogate the expression of endothelial VCAM-1 and reduce the endothelial reactivity with the mAbs YN1.1/74 and MECA-32 to levels found in cardiac isografts. This is accompanied by a reduction, but not an elimination, of interstitial leukocytic infiltration. Despite this, cardiac allograft recipients treated with PTX or the mAb 11B11 rejected allografts at the same rate as untreated allograft recipients, ie, within 10 to 12 days after graft implantation. These rejected grafts contained mRNAs for TNF-alpha and IL-4, as well as for all other cytokines that have been associated with rejecting allografts. This suggests that endothelial VCAM-1 expression, which is characteristic of rejection, is not an essential element of the rejection process. Interestingly, the grafts from the PTX-treated recipients continued to display rare, isolated VCAM-1 positive cells in the interstitium, which may be dendritic cells. In general, these studies demonstrate a role for IL-4 and TNF-alpha in the alterations of vascular endothelial phenotype observed in rejecting cardiac allografts. They also demonstrate that endothelial VCAM-1 expression is not essential for the allograft rejection process, and that the role of VCAM-1 in this process may be more subtle than was initially suspected.     

Soluble ICAM-1, VCAM-1 and E-selectin in children from families with high risk of atherosclerosis

The genetic predisposition for cardiovascular disease seems to play an important role in atherogenesis. Atherosclerosis, which can be clinically asymptomatic for many years, begins early in life. Therefore finding markers of early atherosclerotic process would be of great importance for screening and early treatment of these children. As the result of endothelial dysfunction, the adhesion molecules (VCAM-1, ICAM-1, ELAM) are overexpressed. These molecules are shed from the surface and can be measured, as soluble forms in serum. Therefore they can be regarded as early markers of atherosclerosis. The aim of the study was to measure the serum levels of soluble adhesion molecules ELAM, ICAM-1, VCAM-1 and plasma lipid profile--total (TC), LDL (LDL-C) and HDL-cholesterol (HDL-C) and triglycerides (TG) in children from families of high risk for cardiovascular diseases. Forty-eight children were studied, 24 children from high risk families, according to NCEP definition: one or two parents had clinical manifestation of cardiovascular disease before the age of 65 years (mother) or 55 years (father). Twenty-four healthy children without familial history of cardiovascular disease were used as the control. Children of either group did not have any metabolic diseases. The concentration of sELAM, sICAM-1 and sVCAM-1 were assessed using ELISA kits. Soluble ICAM-1 level was significantly higher in high risk group in comparison to control (p<0.02). The soluble VCAM-1 and ELAM levels did not differ significantly between the groups. There were no changes in total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides between the groups. In normolipidemic children from families with high risk for atherosclerosis the soluble ICAM-1 levels are significantly higher as compared to control.     

Mast cell-mediated induction of ICAM-1, VCAM-1 and E-selectin in endothelial cells in vitro: constitutive release of inducing mediators but no effect of degranulation

 Mast cell (MC)-mediated induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and of E-selectin was studied in cultures of rat heart endothelial cells (EC) and human umbilical vein EC (HUVEC) respectively. MC induced VCAM-1 and E-selectin, but hardly any ICAM-1 in EC. Induction was not dependent on MC degranulation, but seemed to be provoked by constitutively released substances, other than histamine, from MC. Co-incubation of MC and EC, allowing for direct contact between the two cell types, was more potent in induction than MC co-incubated separately from EC using a permeable membrane. MC were less potent in induction than exogenous added cytokines or LPS. Induction of cell adhesion molecules in rat heart EC was MC-specific, since EC incubations with either rat cardiomyocytes or heart fibroblasts had no effect. The data show that rat MC, independent of degranulation, secrete mediators relevant for the induction of a specific set of EC adhesion molecules in vitro. This suggests a (supportive) role for MC in cell-adhesion molecule induction in the endothelium in settings of early or mild inflammation. The results are discussed in the context of inflammatory processes in the heart in vivo. Received: 29 November 1996 / Received after revision: 10 June 1997 / Accepted: 22 July 1997     

Resting and activated T cells induce expression of E-selectin and VCAM-1 by vascular endothelial cells through a contact-dependent but CD40 ligand-independent mechanism

This study explored the effect on endothelial cell (EC) activation of contact with T lymphocytes, which occurs during lymphocyte emigration into inflamed tissues. Addition of T cells to umbilical vein or dermal microvascular EC monolayers stimulated expression of EC E-selectin and VCAM-1. This response required direct cell:cell contact, but not T-cell activation. The capacity of resting CD4+ T cells to activate EC was restricted to the CD45RO+ subset and could be enhanced by 6 h prestimulation of T cells with PMA and ionomycin. The EC-stimulating capacity of resting or activated T cells was independent of CD40 ligand. Furthermore, inhibition of TNF-alpha/beta and IL-1alpha/beta, together with CD40 ligand, failed to inhibit EC activation by resting T cells and only inhibited the response to PMA- and ionomycin-activated T cells by 40 +/- 18%. Our data suggest that T-cell-EC interactions can lead to EC activation through a novel contact-dependent, but CD40 ligand-independent, mechanism.     

Expression of CD34 in pulmonary endothelial cells in vivo.

OBJECTIVES: The morphological phenotype of endothelial cells (EC) is specific for individual types of vessels. There are, however, no studies on the phenotypical diversity of EC in human lung tissue. The influences exerted by physiological factors (e.g. age, sex) or pathological factors (e.g. pulmonary hypertension) on EC have not been ascertained up to now. METHODS: In order to determine the influence of pulmonary hypertension (PH), age and sex on EC, we localized the expression of the endothelial marker CD34 immunohistologically by light microscopy and laser scanning microscopy in lung tissue specimens from children and adults. RESULTS: Capillary EC showed a stronger staining reaction than EC in arteries, veins, arterioles or venules. The staining intensity increased with age in veins and arteries and decreased with age in venules, arterioles and capillaries. Sex exerted no statistically significant influence. CD34 was generally more strongly expressed in specimens with PH than in those without. CONCLUSION: The study demonstrates for the first time that (1) CD34 is heterogeneously expressed by human pulmonary EC, and (2) the physiological/pathophysiological factors age/PH influence CD34 expression. Hence a correlation between CD34 expression and its role as adhesion molecule and a link between CD34 expression and maturation are subject of discussion.     

The relationship of soluble ICAM-1, VCAM-1, P-selectin and E-selectin to cardiovascular disease risk factors in healthy men and women.

BACKGROUND: Clinical studies have shown that elevated serum concentrations of cell adhesion molecules such as inter-cellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-selectin (ESEL) and P-selectin (PSEL) may be independent risk factors for atherosclerosis and cardiovascular disease (CVD). Less is known of the relationship of these inflammatory markers with established CVD risk factors in healthy individuals, particularly women. OBJECTIVE: The aim of this study was to examine cross-sectional relationships between the concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1, sPSEL and sESEL) and smoking behaviour, body composition, blood pressure, serum lipids and physical activity in a large sample of healthy men and women, with special emphasis on interactions between smoking and other CVD risk factors. SUBJECTS: The analysis included 592 healthy white adults aged 18-82 years. RESULTS: There were no sex differences in the concentrations of sICAM-1, sVCAM-1 and sPSEL, but men had higher sESEL levels than women (p < 0.0001). Male and female smokers had higher sICAM-1 and sESEL levels than non-smokers and soluble cell adhesion molecules (CAMs) were correlated with the pack-years of cigarette smoking (r = 0.3-0.4, p < 0.0001, significant in women only). Significant independent associations were found between soluble CAMs and smoking, waist-hip ratio (WHR), blood pressure, high density lipoprotein cholesterol and total cholesterol. Furthermore, significant interaction effects were found in women, such that the relationship between CAMs and lipid concentrations and WHR were stronger in smokers than non-smokers. In conclusion, the concentration of soluble CAMs, particularly sICAM-1 and sESEL, reflect the level of established CVD risk factors in apparently healthy men and women, adding to the evidence that these factors contribute to CVD through their inflammatory effects on the vascular endothelium.     

Circulating ICAM-1, VCAM-1, E-selectin, P-selectin, and TNFRII in patients with coronary artery disease

OBJECTIVE: To evaluate the relationship between the serum concentration of tumor necrosis factor receptor 2 (TNFRII) and some adhesion molecules [including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-Selectin, and E-Selectin] and coronary artery stenosis. DESIGN AND SETTING: Observational (cross-sectional) study in a University Heart Hospital in Tehran, Iran. PATIENTS: 75 patients with angiographically proven coronary artery disease were compared with 81 individuals who had undergone coronary angiography with no significant evidence of stenosis (control subjects). METHODS: Soluble adhesion molecules and TNFRII were determined by enzyme-linked immunosorbent assay technique. sICAM-1 and sP-selectin values were significantly higher in patients with coronary artery disease than in control subjects [146(38) vs. 132(48) p < 0.04 and 275(107) vs. 241(104) ng/ml p < 0.04 respectively]. Multiple logistic regression analysis showed sICAM-1 an independent discriminating risk factor for coronary artery disease (p < 0.03). Prediction models that incorporated sICAM-1 in addition to other established coronary risk factors were significantly better at predicting risk than the models based on the other risk factors alone. Multiple regression analysis indicated that sP-selectin levels were greater in patients with single-vessel disease than in the respective normal (p < 0.01). CONCLUSIONS: Our findings suggest that sICAM-1 has an association with s1 coronary artery disease as such; the evaluation of this marker may improve the coronary risk assessment in Iranian patients.     

Circulating ICAM-1, VCAM-1, E-selectin, P-selectin, and TNFalphaRII in patients with coronary artery disease

OBJECTIVE: To evaluate the relationship between the serum concentration of TNFalphaRII and some adhesion molecules (including ICAM-1, VCAM-1, P-selectin and E-selectin) and coronary artery stenosis. DESIGN AND SETTING: Observational (cross-sectional) study in a university heart hospital in Tehran, Iran. PATIENTS: 81 patients with angiographically proven coronary artery disease were compared with 75 individuals who had undergone coronary angiography with no significant evidence of stenosis (control subjects). METHODS: Soluble adhesion molecules and TNFalphaRII were determined by enzyme-linked immunosorbent assay technique. sICAM-1 and sP-selectin values were significantly higher in patients with coronary artery disease than in control subjects (146 +/- 38 vs. 132 +/- 48 p < 0.04 and 275 +/- 107 vs. 241 +/- 104 ng/ml p < 0.04 respectively). Multiple logistic regression analysis showed sICAM-1 as an independent discriminating risk factor for coronary artery disease (p < 0.03). Prediction models that incorporated sICAM-1 in addition to other established coronary risk factors were significantly better at predicting risk than the models based on the other risk factors alone. Multiple regression analysis indicated that sP-selectin levels were greater in patients with single-vessel disease than in the respective normal (p < 0.01). CONCLUSIONS: Our findings suggest that sICAM-1 has an association with stable coronary artery disease and the evaluation of this marker may improve the coronary risk assessment in Iranian patients.     

Expression of endothelial adhesion molecules in vivo. Increased endothelial ICAM-2 expression in lymphoid malignancies.

The authors have compared the reactivity of monoclonal antibodies (MAb) directed against endothelial adhesion molecules (ICAM-1, ICAM-2, VCAM-1, ELAM-1) in hyperplastic, nonmalignant, and malignant lymph nodes. The authors demonstrate that the reactivity with ICAM-1 MAb is stronger in the high endothelial venules (HEV) and other smaller vessels (SV) in lymphomas compared with hyperplastic lymph nodes. Similarly, the reactivity of an ICAM-2 MAb (6D5) was shown to be higher in malignant lymph nodes compared with nonmalignant ones. ICAM-2 MAb stained both the HEV and SV. VCAM-1 MAb reacted strongly with germinal centers and its endothelial reactivity was higher in the lymphoma nodes. ELAM-1 MAb stained only faintly some endothelial cells in malignant tissue. These data suggest that besides the known regulatable endothelial adhesion molecules ICAM-1 and VCAM-1, the expression of ICAM-2 can be modified.     

Evidence for endocytosis of E-selectin in human endothelial cells.

E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.     

Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Roles for TNF and IL4.

The in vivo mechanisms of vascular endothelial activation and VCAM-1 expression were studied in murine heterotopic cardiac grafts. Preliminary studies demonstrated that cardiac allograft endothelia develop reactivity with MECA-32 monoclonal antibody (MAb) and M/K-2 (anti-VCAM-1) MAb within 3 days of transplantation, whereas cardiac isografts develop MECA-32 reactivity but no M/K-2 reactivity. Additional studies demonstrated that a single treatment of cardiac isograft recipients with the anti-CD3 MAb 145-2C11 induces VCAM-1 expression on isograft microvascular endothelia within 24 hours. We have used this experimental system to identify the cytokines responsible for expression of VCAM-1 and MECA-32 MAb reactivity on graft vascular endothelia. We report that the expression of VCAM-1 on isograft endothelia that was induced with anti-CD3 MAb was blocked by simultaneous treatment with either pentoxifylline, soluble tumor necrosis factor (TNF) receptor (TNFR-Fc), anti-IL4 MAb, or soluble IL4R, but not by anti-IFN-gamma MAb. Alternatively, a similar pattern of isograft endothelial VCAM-1 expression was stimulated in the absence of anti-CD3 MAbs with a single injection of human recombinant TNF-alpha, or with recombinant murine IL4 provided as IL4/anti-IL4 MAb complexes. In addition, the IL4-induced VCAM-1 expression was completely blocked by a single intravenous treatment of the isograft recipients with TNFR:Fc. This suggests that high concentrations of TNF-alpha can stimulate endothelial VCAM-1 expression, but these concentrations are apparently not achieved in cardiac isografts. In the absence of an inducing agent such as anti-CD3 MAb, the stimulation of VCAM-1 expression with exogenous IL4 may reflect functional interaction between endogenous TNF and exogenous IL4, as suggested by the blocking experiments with TNFR:Fc. Although cardiac isograft endothelia normally develop reactivity with MECA-32 MAb within 3 days of transplantation, treatment of cardiac isograft recipients with anti-CD3 MAb accelerated MECA-32 reactivity to within 24 hours of transplantation. This accelerated expression can be experimentally manipulated in the same way as M/K-2 reactivity, suggesting that similar mechanisms may control the development of these two inflammatory endothelial phenotypical markers, despite their differential expression in cardiac isografts and allografts.     

E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

Endothelial cells respond to several cytokines by a rapid increase in expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1), followed by a gradual decline. The fate of these molecules, which was so far unknown, was studied. Specific sandwich ELISA for the detection of soluble (s)E-selectin and sICAM-1 were developed. In supernatant, centrifuged 3 hr at 100,000 g to remove microparticles, from human umbilical vein endothelial cells (HUVEC) activated with tumour necrosis factor (TNF), interleukin-1 (IL-1) or lipopolysaccharide (LPS), E-selectin and ICAM-1 molecules could be detected. Biochemical analysis revealed that sE-selectin migrated as a band of approximately 94,000 MW. The amount of soluble adhesion molecules released was directly correlated with cell surface expression. Maximal release of E-selectin was observed 6-12 hr after activation of HUVEC and decreased to below detection limit 24 hr after activation. After activation, release of ICAM-1 gradually increased with ICAM-1 cell surface expression, and reached a plateau after 24 hr, which was constant for 3 days. Since E-selectin and ICAM-1 are highly expressed at inflammatory sites, the resulting high concentrations of released E-selectin and ICAM-1 may affect interactions of leucocytes with endothelial cells. The physiological role, however, of the release of E-selectin and ICAM-1 remains to be elucidated.