ICAM-1 Antibodies Research Articles

Optimization of ultrasound contrast agents with computational models to improve selection of ligands and binding strength

Diagnosis of cardiovascular disease is currently limited by the testing modality. Serum tests for biomarkers can provide quantification of severity but lack the ability to localize the source of the cardiovascular disease, while imaging technology such as angiography and ultrasound can only determine areas of reduced flow but not the severity of tissue ischemia. Targeted imaging with ultrasound contrast agents offers the ability to locally image as well as determine the degree of ischemia by utilizing agents that will cause the contrast agent to home to the affected tissue. Ultrasound molecular imaging via targeted microbubbles (MB) is currently limited by its sensitivity to molecular markers of disease relative to other techniques (e.g., radiolabeling). We hypothesize that computational modeling may provide a useful first approach to maximize microbubble binding by defining key parameters governing adhesion. Adhesive dynamics (AD) was used to simulate the fluid dynamic and stochastic molecular binding of microbubbles to inflamed endothelial cells. Sialyl Lewis(X) (sLe(x)), P-selectin aptamer (PSA), and ICAM-1 antibody (abICAM) were modeled as the targeting receptors on the microbubble surface in both single- and dual-targeted arrangements. Microbubble properties (radius [R(c)], kinetics [k(f), k(r)], and densities of targeting receptors) and the physical environment (shear rate and target ligand densities) were modeled. The kinetics for sLe(x) and PSA were measured with surface plasmon resonance. R(c), shear rate, and densities of sLe(x), PSA, or abICAM were varied independently to assess model sensitivity. Firm adhesion was defined as MB velocity <2% of the free stream velocity. AD simulations revealed an optimal microbubble radius of 1-2 µm and thresholds for kf(in) ( >10(2) s(-1)) and kr(o) (<10(-3) s(-1)) for firm adhesion in a multi-targeted system. State diagrams for multi-targeted microbubbles suggest sLe(x) and abICAM microbubbles may require 10-fold more ligand to achieve firm adhesion at higher shear rates than sLe(x) and PSA microbubbles. The AD model gives useful insight into the key parameters for stable microbubble binding, and may allow flexible, prospective design, and optimization of microbubbles to enhance clinical translation of ultrasound molecular imaging.

Intranasal pretreatment with toll like receptor 9 ligand CpG oligodeoxynucleotides prevents the development of allergic rhinitis in juvenile guinea pigs

To evaluate the therapeutic effect of intranasal oligodeoxynucleotides with CpG motifs (CpG ODN) in prevention of allergic rhinitis in juvenile guinea pigs. Juvenile guinea pigs aged from 7 to 10 weeks were administrated with CpG ODN alone or combined with OVA at single dose concentration intranasally (on day 0, 5, 10, 15 in sequence) while control and blank group were administrated with saline. Both experimental and control animals were again sensitized by OVA (on day 18, 25), and 14 days after second sensitization animals were challenged by OVA intranasally (on days 39 and 46). Two hours after challenge, the animals were sacrificed. Then Hemotoxin and Eosin stain were carried out to analyze local eosinophilic reactions and nasal lesions. Local and systemic cytokines interleukin IL-5 and IFN-γ levels were examined by ELISA. Immunofluorescence was carried out with ICAM-1 antibody. Statistical analysis was performed using a SPSS 11.0 software. In CpG ODN-administration or CpG ODN with OVA-administration group allergic rhinitis symptoms were not as severe as model control group (P < 0.05). Compared with the model control group, CpG ODN-administration did not increase production of OVA-specific Th1 cytokine IFN-γ but decreased productions of ovalbumin-specific Th2 cytokines IL-5 both in serum and nasal specimen (q value were 3.890 and 4.019, P < 0.05). Moreover, nasal lesions with infiltration of mean (x ± s) eosinophils (20.0 ± 9.6) in CpG group animal were prominently reduced by the CpG ODN-treatment compared with the control animals (53.5 ± 19.8) and CpG+OVA group (9.5 ± 5.7) were lower than CpG-M+OVA group (49.2 ± 18.9), the differences were significant (q value were 3.785 and 4.576, P < 0.05). Immunofluorescence results showed lower ICAM-1 expression in nasal specimen of CpG group compared with model group and CpG plus OVA group animal to CpG mimics plus OVA group (Z value were 3.697 and 3.765, P < 0.05). Intranasal administration of CpG oligodeoxynucleotides with or without allergen may be an effective way to prevent the development of allergic rhinitis.

Effect of hyperketonemia on chemokine secretion, adhesion molecule expression, and monocyte adhesion to endothelial cells

Frequent episodes of hyperketonemia are associated with higher incidences of vascular disease. We examined the hypothesis that hyperketonemia increases monocyte adhesion to endothelial cells (EC). Human U937 monocytes or THP‐1 monocytes and HUVECs were cultured with AA (0–10 mM) or BHB (0–10 mM) for 24 hours. Results ( ± SE) show that there was significant increase in IL‐8 and MCP‐1 secretion in AA treated THP‐1 and U937 monocytes and HUVECs, however, BHB had no effect. There was a 10.5% ± 0.04 (ns) increase in protein levels of LFA‐1 in 4 mM AA treated U937 monocytes compared to control and a 13% ± 4.3 (p&lt;0.04) increase in surface expression. There was a 21% ± 3.5 (p&lt;0.01) increase in CD14 surface expression on U937 and a 3.9% ± 1.8 (ns) in THP‐1 treated with 4 mM AA compared to control whereas BHB had no effect. Surface ICAM‐1 increased by 23.8% ± 0.02 (p&lt;0.05) and total protein expression by 40% in HUVECs treated with 4 mM AA compared to the control. Results showed a 33.7% ± 2.3 (p&lt;0.01) increase in U937 adhesion and a 23.5% ± 3.8 (p&lt;0.05) increase in THP‐1 adhesion to HUVEC monolayer's treated with 4 mM AA compared to the control. Monocyte adhesion could be blocked by 14% ± 3.6 (p&lt;0.04) when HUVEC's were pre‐incubated with ICAM‐1 antibody. Thus, hyperketonemia increases monocyte adhesion to EC's by upregulation of LFA‐1, CD‐14 and ICAM‐1, which may contribute to the excess vascular disease in diabetes.

The Epitope Targeted by Apoptosis-Inducing ICAM-1 Antibody B11 Is Highly Expressed in Multiple Myeloma.

Abstract 4897Complex adhesive and non-cognate interactions participate in multiple myeloma disease progression, resistance to apoptosis, and development of drug resistance. In spite of significant recent attempts to develop new drug classes targeting both myeloma and its microenvironment, multiple myeloma remains an incurable disease warranting development of more effective therapies. Applying novel combined target and drug discovery methodology we isolated a human tumor cell apoptosis-inducing antibody (IgG B11) targeting ICAM-1, as previously described. ICAM-1 is a cell adhesion molecule strongly implicated in myeloma pathophysiology, both regarding bone marrow stromal cell mediated disease progression and cell adhesion mediated drug resistance. We here characterize B11 epitope expression by multicolor FACS analysis in 25 patients investigated for multiple myeloma referred to Department of Hematology, Lund University Hospital, Lund, Sweden and 5 healthy controls. The B11 epitope was highly expressed in plasma cells in 5 of 5 patients with myeloma and in 1 of 1 patient with AL (light chain) amyloidosis. A comprehensive preclinical program assessing IgG B11 anti-myeloma activity and evaluating IgG B11 safety has been conducted. Based on these studies, and having received an investigational new drug (IND) approval from the U.S. Food and Drug Administration (FDA), clinical phase I trials with IgG B11 will soon commence. DisclosuresVeitonmäki:BioInvent International: Employment. Lindblom:BioInvent International: Employment, Equity Ownership. Frendéus:BioInvent International AB: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

B599 Apoptosis-Inducing ICAM-1 Antibody BI-505 Is a Potent Inhibitor of Multiple Myeloma

B599 Apoptosis-Inducing ICAM-1 Antibody BI-505 Is a Potent Inhibitor of Multiple Myeloma

In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

ICAM-1 and p38 MAPK mediate fibrinogen-induced migration of human vascular smooth muscle cells

Fibrinogen deposition in the vessel wall represents an independent atherogenic risk factor. In Boyden-chamber assays, fibrinogen concentration-dependently (1–100 μM) induced migration of human vascular smooth muscle cells (SMC). This was inhibited by antibodies to intercellular adhesion molecule-1 (ICAM-1, 10 μg/ml), and by inhibitors of PI3-kinase (LY294002, 10 μM) and MAPK (mitogen-activated protein kinase) p38 (SB203580, 10 μM). The MEK (MAP kinase kinase) inhibitor PD98059 (10 μM) and the GPIIb/IIIa antagonist abciximab (10 μg/ml) had no effect. ICAM-1 antibodies inhibited fibrinogen-induced Akt and p38 phosphorylation. Thus fibrinogen stimulates human SMC migration through binding to ICAM-1 and activation of Akt and p38.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH) 2D 3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-κB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10 − 10 M 1,25-(OH) 2D 3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH) 2D 3. ECF-L-Fc by itself only modestly increased NF-κB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1α and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10 − 10 M 1,25-(OH) 2D 3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH) 2D 3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase

Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

Phosphatidylinositol 3-Kinase γ Signaling through Protein Kinase Cζ Induces NADPH Oxidase-mediated Oxidant Generation and NF-κB Activation in Endothelial Cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

Spatial regulation of shear stress in cultured human aortic endothelial cell functions.

Early lesions of atherosclerosis tend to occur in regions of a vessel that experience low shear stress and reversing flow patterns. The recruitment of monocytes into endothelium is a key step in vascular inflammation that contributes to the development of atherosclerosis. Vascular adhesion molecules (VAM) that mediate leukocyte arrest are regulated on endothelium by cytokine stimulus and fluid shear stress. However, the variations in protein expression (VCAM-1, ICAM-1, and E-selectin) under different shear stresses on human aortic endothelial cells (HAEC), and the effects on monocyte recruitment, are ill-defined. In order to observe changes in cell function over a broad shear range in a flow channel, a 20mm long microfluidic chamber with continuous increase in shear field was created using soft lithography. Shear stress in the range of 0–15 dyne/cm2 was applied to HAECs for 4 hours during stimulation with tumor necrosis factor-α (30U). Immunofluorescence microscopy showed that the mean pixel intensity (MPI) of images acquired from the high shear region (14 dyne/cm2, 3 mm from the inlet) was elevated by 32% when compared with those from low shear region (1.5 dyne/cm2, 18mm downstream) when labeled with ICAM-1 antibody. In contrast, MPIs were suppressed by 27% and 26%, respectively, using VCAM-1 and E-selectin antibodies. We also found that more monocyte-endothelium interactions occurred at regions exposed to low shear stress than those at high shear filed. The results suggest shear stress affects characteristic changes specific to each VAM, which in turn influences efficiency of rolling-arrest.

Selective regulation of MAP kinases and Chemokine expression after ligation of ICAM-1 on human airway epithelial cells

BackgroundIntercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like cell adhesion molecule expressed on the surface of multiple cell types, including airway epithelial cells. It has been documented that cross-linking ICAM-1 on the surface of leukocytes results in changes in cellular function through outside-inside signaling; however, the effect of cross-linking ICAM-1 on the surface of airway epithelial cells is currently unknown. The objective of this study was to investigate whether or not cross-linking ICAM-1 on the surface of airway epithelial cells phosphorylated MAP kinases or stimulated chemokine expression and secretion.MethodsThe human lung adenocarcinoma (A549) cells and primary cultures of normal human bronchial epithelial (NHBE) cells were used in these studies. To increase ICAM-1 surface expression, cultures were stimulated with TNFα to enhance ICAM-1 surface expression. Following ICAM-1 upregulation, ICAM-1 was ligated with a murine anti-human ICAM-1 antibody and subsequently cross-linked with a secondary antibody (anti-mouse IgG(ab')2) in the presence or absence of the MAP kinase inhibitors. Following treatments, cultures were assessed for MAPK activation and chemokine gene expression and secretion. Control cultures were treated with murine IgG1 antibody or murine IgG1 antibody and anti-mouse IgG(ab')2 to illustrate specificity. Data were analyzed for significance using a one-way analysis of variance (ANOVA) with Bonferroni post-test correction for multiple comparisons, and relative gene expression was analyzed using the 2-ΔΔCT method.ResultsICAM-1 cross-linking selectively phosphorylated both ERK and JNK MAP kinases as detected by western blot analysis. In addition, cross-linking resulted in differential regulation of chemokine expression. Specifically, IL-8 mRNA and protein secretion was not altered by ICAM-1 cross-linking, in contrast, RANTES mRNA and protein secretion was induced in both epithelial cultures. These events were specifically inhibited by the ERK inhibitor PD98059. Data indicates that ICAM-1 cross-linking stimulates a synergistic increase in TNFα-mediated RANTES production involving activation of ERK in airway epithelial cells.ConclusionResults demonstrate that cytokine induced ICAM-1 on the surface of airway epithelial cells induce outside-inside signaling through cross-linking ICAM-1, selectively altering intracellular pathways and cytokine production. These results suggest that ICAM-1 cross-linking can contribute to inflammation in the lung via production of the chemokine RANTES.

Enhanced monocyte binding to human cytomegalovirus-infected syncytiotrophoblast results in increased apoptosis via the release of tumour necrosis factor alpha

We have shown that monocytes bound to intercellular adhesion molecules (ICAM-1) on syncytialized placental trophoblasts (ST) induce trophoblast apoptosis, and that ST infection by human cytomegalovirus (HCMV) up-regulates ICAM-1. We hypothesize that the focal loss of trophoblast seen in HCMV-infected placenta is mediated by increased adherence of monocytes at sites of infection. We find that ST cultures (differentiated from primary cytotrophoblasts) increase monocyte binding when infected with HCMV. Monocyte adhesion was inhibited by antibodies to ICAM-1 and its ligand leukocyte function-associated molecule (LFA-1) on monocytes. When co-cultured with adhering monocytes, infected ST cultures had higher levels of apoptosis than infected cultures alone. Although trophoblast apoptosis clustered around adhering monocytes, it occurred only in non-infected cells. Blocking monocyte binding with ICAM-1 and LFA-1 antibodies reduced the rate of apoptosis to that of the infected culture. Co-cultures incubated with TNFalpha antibody and EGF inhibited both monocyte- and HCMV-induced apoptosis but did not block binding. We conclude that HCMV stimulates ST culture expression of ICAM-1, which binds to LFA-1 on monocytes that release TNFalpha, thereby inducing apoptosis of neighbouring uninfected trophoblasts. The above data indicates that trophoblast loss associated with HCMV infection can be caused by increased monocyte adhesion to ST.

Use of ICAM-1 antibodies and antisense oligonucleotides to inhibit transmigration of neutrophils.

The increase in adhesion molecule expression during the initial phase of inflammation leads to transmigration of neutrophils. Inhibition of this transmigration could decrease inflammatory response and tissue damage. Uptake of fluorescein-labelled oligonucleotides, expression of ICAM-1 and neutrophil migration were studied using the bilayer of epithelial (H292) and endothelial (ECV-304) cell lines and neutrophils from peripheral blood of healthy volunteers. The inhibition of ICAM-1 expression was time dependent for both cell lines, with inhibition of more than 50% after 48 h. Antisense oligos inhibited transmigration already after 4 h of incubation (6.6 +/- 2.5% versus 18.6 +/- 2.5% inhibition, p < 0.01). Antisense was more effective in preventing fMLP-induced neutrophil migration than ICAM-1 antibodies (88 +/- 3.8% versus 67 +/- 7% inhibition, p = 0.02). Antisense oligos cause a decrease in ICAM-1 expression and inhibit transmigration of neutrophils. However the effectiveness of antisense is higher than monoclonal antibodies, neither of them is able to block the migration completely. This suggests the existence of ICAM-1 independent mechanisms that are responsible for migration.

Lymphocyte homing in xenotransplanted human thyroid tissue can be inhibited by LFA-1 and ICAM-1 antibodies.

Homing of lymphocytes is an important factor with respect to the initiation of the autoimmune process in Graves' disease (GD). As previously shown, human lymphocytes, particularly of intrathyroidal origin, derived from patients with GD, are able to migrate into normal xenotransplanted thyroid tissue and induce functional and histological changes. The aim of this study was to investigate the effect of LFA-1 and ICAM-1 antibodies on the homing of lymphocytes of different origin into xenografted human thyroid tissue. Eighty-five nude mice bearing 8-week-old xenografts of normal human thyroid tissue were treated twice with anti-CD 54 (anti-ICAM-1), anti-CD 11a (anti-LFA-1), a combination of both, or, serving as controls, iso-antibodies without specific binding capacity or saline. Thereafter, intrathyroidal (ITL) or peripheral blood lymphocytes (PBL) obtained from 4 patients with GD or saline were injected into the animals (i.v., 0.2 mL, 10(6) cells). After 48 hours the mice were sacrificed and transplants as well as mice thyroids were examined by immunohistochemical staining with Ki67, CD3, HLA-II (DAKO, Hamburg), IgG, CD44, ICAM-1, and VCAM-1 (Immunotech, Hamburg). Pretreatment with anti-ICAM-1 and anti-LFA-1 decreased lymphocyte homing (CD3-staining), and expression of HLA-II, IgG, CD44, and VCAM-1 in the transplants. Our data show that [ICAM-1/LFA-1 stimulated (induced)] lymphocyte homing and subsequently thyrocyte proliferation are inhibited by ICAM-1 and LFA-1 antibodies in xenotransplanted thyroid tissue. This suggests that ICAM1 and LFA-1 play an important role in the early steps of autoimmune thyroid disease. The inhibition/suppression of ICAM-1 and LFA-1 interaction by respective antibodies, as demonstrated in the present study, may provide a new concept for prophylaxis and therapy.

Upregulation of P-selectin and intercellular adhesion molecule-1 after retinal ischemia-reperfusion injury.

Vascular endothelial cells and leukocytes express several inflammatory adhesion receptors, such as selectins and cell adhesion molecules, after retinal ischemia. They mediate the transmigration process of leukocytes. For further understanding of the role of leukocytes after retinal ischemia-reperfusion injury, the responses of the leukocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule (ICAM)-1 during retinal ischemia and reperfusion were examined. Male pigmented rats were subjected to retinal ischemia by a 1-hour ligation of the optic nerve followed by reperfusion. Gene and protein expression of P-selectin and ICAM-1 were studied at 0.5, 1, 6, 12, and 24 hours after the onset of reperfusion with semiquantitative polymerase chain reaction and Western blot analysis. Immunohistochemical methods were used to detect specific lesions expressing ICAM-1. Significant upregulation of P-selectin and ICAM-1 mRNA (at 6, 12, and 24 hours of reperfusion) were observed, with the expression peaks of both P-selectin and ICAM-1 mRNA occurring at 12 hours after reperfusion. P-selectin protein gradually increased and reached a maximum 6 hours after reperfusion, whereas ICAM-1 protein increased until 24 hours after reperfusion. Immunostaining with ICAM-1 antibodies was positive in endothelial cells after ischemia-reperfusion injury. Retinal ischemia-reperfusion stimulates P-selectin and ICAM-1 expression. Endothelial ICAM-1 expression in retinal vessels was observed. Upregulation of P-selectin and ICAM-1, which contribute to leukocyte rolling and adhesion, were observed after retinal ischemia.

Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress.

Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.

Characterization of ischemia/reperfusion injury after pancreas transplantation and reduction by application of monoclonal antibodies against ICAM-1 in the rat

Background. Reperfusion injury contributes to early organ malfunction after transplantation. The aim of this study was to characterize the ischemia-reperfusion injury after pancreas transplantation and to evaluate the effect of monoclonal antibody therapy against ICAM-1. Methods. Twenty-five heterotopic pancreas transplantations were performed in syngenic rats in a no-touch technique. Microcirculation and leukocyte-endothelial interaction in the grafts were evaluated by intravital microscopy at 1 hour (I), 6 hours (II), 12 hours (III), and 24 hours (IV) after reperfusion, and 12 hours after reperfusion (V) in the therapeutic group. In (V) antibodies against ICAM-1 were applied as bolus at reperfusion and continuously for 6 hours. Next to intravital microscopy, histologic scores, myeloperoxidase levels, and ICAM-1 expression were determined. Results. Microcirculation was significantly reduced in capillaries and postcapillary venules in the time course after reperfusion (capillary: 0.96 ± 0.08 mm/s [I] to 0.45 ± 0.07 mm/s [IV] [P <.01]) and was improved significantly by therapy with ICAM-1 antibodies (0.98 ± 0.06 mm/s, (P <.01). Leukocyte-endothelial interaction significantly increased 6 hours after reperfusion (II) (P <.01). These changes were paralleled by an increased endothelial expression of ICAM-1 in immunohistochemistry. Histologic evaluation showed increased inflammation at 12 hours after reperfusion (P <.01), which could be diminished by the administration of ICAM-1 antibodies (P <.05). Conclusion. Increased endothelial expression of ICAM-1 after pancreas transplantation is positively correlated with microcirculatory impairment. Important steps of pancreatic inflammation take place approximately 6 hours after reperfusion. Prophylactic application of monoclonal antibodies against ICAM-1 reduces reperfusion injury and successfully prevents the occurrence of graft pancreatitis. (Surgery 2003;134:63-71.)

The hyaluronan receptor for endocytosis (HARE) is not CD44 or CD54 (ICAM-1)

Mammalian liver contains an endocytic, recycling receptor that mediates the clearance of hyaluronan (HA) and chondroitin sulfate from the circulation. McCourt et al. [J. Biol. Chem. 269 (1994) 30081] previously reported that this endocytic liver HA receptor was ICAM-1. In contrast, we purified this HA receptor for endocytosis (HARE) from rat liver sinusoidal endothelial cells (LECs) and obtained two novel large proteins [Zhou et al., J. Biol. Chem. 274 (1999) 33831]. The goal of the present study was to clarify this inconsistency and determine whether CD44, which is also an HA receptor, or ICAM-1 (CD54) is identical to, or is part of, HARE. Although isolated liver LECs contain HARE, CD44, and ICAM-1, confocal fluorescence microscopy showed that the two latter proteins have cellular distributions that are distinct from and essentially nonoverlapping with HARE. HA accumulation by cultured LECs was inhibited >98% by an antibody against HARE and unaffected by antibodies to ICAM-1 or CD44, indicating that virtually all specific HA uptake is mediated by HARE and not by ICAM-1 or CD44. Finally, no reactivity was observed against purified HARE in an ELISA-based assay using CD44 or ICAM-1 antibodies. The results confirm that the mammalian endocytic HA receptor is HARE and is not ICAM-1 or CD44.