Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Adhesion of human monocytic THP-1 cells to endothelial cell adhesion molecules or extracellular matrix proteins via beta1 integrins regulates heparin binding epidermal growth factor-like growth factor (HB-EGF) expression

Adherence to endothelium and then to the extracellular matrix is a prerequisite for extravasation of monocytes into injured tissues. There, monocytes differentiate into macrophages and express heparin binding epidermal growth factor-like growth factor (HB-EGF), a key growth factor involved in normal wound healing. We investigated whether the interaction of human monocytic THP-1 cells with the endothelial cell adhesion molecules (vascular CAM-1, VCAM-1; intercellular adhesion molecule-1 ICAM-1 and endothelial-selectin, E-selectin), or the extracellular matrix (ECM) proteins (fibronectin, FN; laminin, LN and fibrinogen, FG) regulate HB-EGF expression. We have shown that adherence of THP-1 cells via VCAM-1, E-selectin or FN, which are all overexpressed at sites of inflammation, potentiates HB-EGF mRNA expression. In contrast, adhesion of THP-1 cells via ICAM-1 or FG, has no significant effect. Since THP-1 cells interact with ICAM-1 and FG through beta2 integrins, and with VCAM-1 and FN via beta1 integrins, regulation of HB-EGF expression appears to be specific to beta1 integrin ligation. In addition, we demonstrate that THP-1 binding to LN, through the beta1 integrin VLA-6, down regulates HB-EGF expression. Thus physiologically, transient destruction of LN and expression of VCAM-1, E-selectin and fibronectin at sites of inflammation, may locally induce HB-EGF overexpression.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs). We examined whether caveolae are involved in monocyte adhesion to ECs responding to a synergy of hypercholesterolemia and inflammation. Treating human umbilical vein ECs with cholesterol enhanced endotoxin lipopolysaccharide (LPS)-induced monocyte adhesion. Use of isolated caveolae-enriched membranes revealed that cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), co-localized with Cav-1 in caveolae. LPS upregulated CAMs expression and increased the co-localization. Cholesterol exposure decreased the level of CAMs in the caveolae. Co-immunoprecipitation and confocal microscopy revealed that ICAM-1 interacted with Cav-1. Electron microscopy showed that ICAM-1 was mainly located in caveolae. Cholesterol exposure decreased this interaction and drove ICAM-1 out of caveolae. Knockdown of Cav-1 reduced the synergistic effects of cholesterol and inflammation. In vivo, ICAM-1 and Cav-1 co-localization was lower in the aortic endothelium of ApoE^−/− mice than in that of wild-type controls. Cav-1 negatively regulates monocyte adhesion by the co-localization of CAMs in caveolae, which is disturbed by cholesterol. Thus, our study suggests a molecular basis underlying the synergistic effects of hypercholesterolemia and inflammation in atherogenesis.     

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.     

Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions

Chemokines such as the monocyte chemol attractant protein-1 (MCP-1) convert monocyte rolling to firm arrest under physiological flow conditions via integrin activation and simultaneously activate phosphoinositide 3-kinase (PI3K). Here we used adenoviral gene transfer and biochemical inhibitors to manipulate PI3K-dependent pathways in human monocytes. In in vitro lipid kinase assays from purified human monocytes, we showed that MCP-1 activates the "classical" PI3Kalpha pathway and not PI3Kgamma, a PI3K isoform thought to be activated only by the betagamma complex of heterotrimeric G proteins. The activity of PI3Kalpha in purified human monocytes was evident within 30 s. MCP-1-induced monocyte arrest was significantly inhibited both by wortmannin (n = 4; p < 0.01) and LY294002 (n = 4; p < 0.01) with restoration of the rolling phenotype (p < 0.05 for both inhibitors, compared with rolling of control monocytes after MCP-1 treatment). To test the hypothesis that activation of PI3K is sufficient to induce monocyte adhesion, we transduced the monocytic THP-1 cell line with a recombinant adenovirus (Ad) carrying a constitutively active mutant of PI3K (Ad.BD110). We examined the ability of these cells to adhere to human vascular endothelium (HUVEC) transduced with adenoviruses carrying E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1. Under flow conditions, ICAM-1- and VCAM-1-dependent firm adhesion of Ad.BD110-transduced THP-1 cells was enhanced compared with THP-1 cells infected with control Ad (n = 4; p < 0.01 for both). Adhesion augmented by constitutive PI3K activation was entirely abrogated by pretreatment with wortmannin (n = 3; p < 0.01). In contrast, a constitutively active Akt construct had no effect on THP-1 adhesion (n = 3; p = NS). We conclude that PI3K activation is necessary and sufficient to enhance monocytic adhesion under physiological flow conditions. BD110-expressing THP-1 cells should provide a useful tool for identifying the signaling pathways downstream of PI3K that are necessary for monocyte recruitment relevant to a variety of human vascular pathologies.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Cryptotanshinone inhibits oxidized LDL-induced adhesion molecule expression via ROS dependent NF-κB pathways

Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, play important roles in the initial stage of atherosclerosis. Cryptotanshinone (CPT), a natural compound isolated from Salvia miltiorrhiza Bunge, exhibits anti-atherosclerotic activity although the underlying mechanisms remain elusive. In this study, the protective effect of CPT against oxidized low-density lipoprotein (ox-LDL)-induced adhesion molecule expression was investigated in human umbilical vein endothelial cells. Ox-LDL significantly induced ICAM-1, VCAM-1, and E-selectin expression at the mRNA and protein levels but reduced eNOS phosphorylation and NO generation, which were reversed by CPT pretreatment. Sodium nitroprusside, a NO donor, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF-κB inhibitor, inhibited ox-LDL-induced ICAM-1, VCAM-1, and E-selectin expression. Ox-LDL-induced ROS production was significantly inhibited by CPT and NAC. Furthermore, ox-LDL activated the NF-κB signaling pathway by inducing phosphorylation of IKKβ and IκBα, promoting the interaction of IKKβ and IκBα, and increasing p65 nuclear translocation, which were significantly inhibited by CPT. In addition, CPT, NAC, and BAY117082 inhibited ox-LDL-induced membrane expression of ICAM-1, VCAM-1, E-selectin, and endothelial–monocyte adhesion and restored eNOS phosphorylation and NO generation. Results suggested that CPT inhibited ox-LDL-induced adhesion molecule expression by decreasing ROS and inhibiting the NF-κB pathways, which provides new insight into the anti-atherosclerotic mechanism of CPT.     

Effect of high glucose concentrations on expression of ELAM-1, VCAM-1 and ICAM-1 in HUVEC with and without cytokine activation

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.     

Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells

Chlamydophila pneumoniae is a common respiratory pathogen that has been shown to be associated with coronary artery disease. Recent studies have shown that one of the possible mechanisms of the atherogenicity of C. pneumoniae is overexpression of cell adhesion molecules (CAMs) in infected endothelial cells. We investigated whether exposure of C. pneumoniae-infected endothelial cells to oxidized LDL (oxLDL) leads to further upregulation of CAMs. Flow cytometry and immunoblot analysis of human aortic endothelial cells (HAECs) was performed for intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ICAM-1 was expressed in 78.7% of C. pneumoniae-infected HAECs. The addition of oxLDL (100 microg/ml) to infected HAECs increased the proportion of ICAM-1-positive cells to 92%. VCAM-1 was only observed in 9.3% of infected HAECs, and the addition of oxLDL had no further effect on the surface expression of VCAM-1. C. pneumoniae also upregulated the surface expression of E-selectin on 52.2% of the cells, and incubation with oxLDL further increased the proportion of positive cells to 63.64%. In conclusion, C. pneumoniae upregulated the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin on HAECs. The addition of oxLDL to the infected cells further enhanced the surface expression of ICAM-1 and E-selectin.     

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.     

The role of the cytoskeleton in cellular adhesion molecule expression in tumor necrosis factor-stimulated endothelial cells

Leukocyte infiltration is a hallmark of the atherosclerotic lesion. These cells are captured by cellular adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule (PECAM), and E-selectin, on endothelial cells (EC). We examined the role of the actin cytoskeleton in tumor necrosis factor-alpha (TNF-alpha)-induced translocation of CAMs to the cell surface. Human aortic EC were grown on 96-well plates and an ELISA was used to assess surface expression of the CAMs. TNF-alpha increased VCAM-1, ICAM-1, and E-selectin by 4 h but had no affect on the expression of PECAM. A functioning actin cytoskeleton was important for VCAM-1 and ICAM-1 expression as both cytochalasin D, an actin filament disruptor, and jasplakinolide, an actin filament stabilizer, attenuated the expression of these CAMs. These compounds were ineffective in altering E-selectin surface expression. Myosin light chains are phosphorylated in response to TNF-alpha and this appears to be regulated by Rho kinase instead of myosin light chain kinase. However, the Rho kinase inhibitor, Y27632, had no affect on TNF-alpha-induced CAM expression. ML-7, a myosin light chain kinase inhibitor, had a modest inhibitory effect on the translocation of VCAM-1 but not on ICAM-1 or E-selectin. These data suggest that the surface expression of VCAM-1 and ICAM-1 is dependent on cycling of the actin cytoskeleton. Nevertheless, modulation of actin filaments via myosin light chain phosphorylation is not necessary. The regulation of E-selectin surface expression differs from that of the other CAMs.     

Attenuation of leukocyte-endothelium interaction by antioxidant enzymes

This report assessed the effect of overexpressing Cu,Zn superoxide dismutase (SOD) and/or catalase on the interaction of mononuclear cells (MNCs) and endothelial cells (ECs). ECs were obtained from the aorta of wild-type mice and transgenic mice overexpressing Cu,ZnSOD and/or catalase. MNCs were obtained from wild-type mice. Treatment of wild-type ECs with CuSO4-oxidized low-density lipoprotein (oxLDL) significantly elevated the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and increased the adherence of MNCs. Overexpression of Cu,ZnSOD and/or catalase in ECs attenuated the adherence of MNCs and the expression of cell adhesion molecules induced by oxLDL. For example, ECs overexpressing Cu,ZnSOD and/or catalase showed significantly less expression of VCAM-1 and ICAM-1 and less number of adherent MNCs than wild-type ECs. Moreover, ECs overexpressing Cu,ZnSOD and catalase in combination showed significantly less expression of VCAM-1 and ICAM-1 and less number of adherent MNCs than those overexpressing either Cu,ZnSOD or catalase alone. These results suggest that combinational overexpression of Cu,ZnSOD and catalase can reduce the expression of cell adhesion molecules and inhibit the adherence of leukocyte to ECs more efficiently than overexpression of Cu,ZnSOD or catalase alone.     

Expression of Cell Adhesion Molecules, their Ligands and Tumour Necrosis Factor α in the Liver of Patients with Metastatic Gastrointestinal Carcinomas

The expression of the following cell adhesion molecules, their β1 and β2 integrin ligands and the cytokine tumour necrosis factor-α (TNF-α) was investigated by light and electron microscope immunohistochemistry in the liver tissue in 20 patients with colorectal and gastric cancer also presenting with liver metastases: intercellular adhesion molecule-1 (ICAM-1), vascular endothelial adhesion molecule-1 (VCAM-1), E-selectin, leucocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We have found a parallel enhancement of the adhesion molecules and of TNF-α in liver sinusoids surrounding metastases. The expression of ICAM-1 was enhanced on sinusoidal cells in all zones of the acinus. VCAM-1 immune reactivity was diffuse but less intensive in the lobule. E-selectin expression was observed in sinusoidal cells attached to metastases. In tumour metastases the expression of ICAM-1, VCAM-1, and E-selectin was visible on the tumour vascular endothelium. Tumour infiltrating host cells sowing positive immunoreactivity for ICAM-1, VCAM-1, LFA-1, Mac-1, and VLA-4 were located mainly at the boundary between liver parenchyma and the metastasis. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the cellular membrane and in some transport vesicles of gastric metastatic cells. Further, the expression of all adhesion molecules was confirmed to sinusoidal endothelial cells and tumour vessels. It is concluded that the enhanced expression of adhesion molecules in liver sinusoids could be a marker for the assessment of the ability of sinusoidal endothelial cells to control the recruitment of leukocytes and monocytes to the metastatic site. They could also direct the adhesion of new circulating tumour cells to sinusoidal endothelium.     

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.