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Abstract: Objective: In whole genome and single gene analyses, genetic variation at the vascular cell adhesion molecule-1 (VCAM-1) locus has been associated with inflammatory disease and stroke in sickle cell anaemia. In the current work, we investigated the functional impact of VCAM-1 missense variants and their effect on cell–cell adhesion. Methods and results: To determine the functional in vitro relevance of five missense VCAM-1 variants (S318F; T384A; G413A; L555V; I716L), we generated wild type and single variant VCAM-1 forms [318F, 384A, 413A, 555V, 716L] in EA.hy926 endothelial cells. Real-time PCR, western blot and ELISA analyses revealed significant differences in mRNA and protein levels for VCAM-1 variants. Monocytic cell lines THP-1 and U937 showed significantly increased adhesion to endothelial cells overexpressing VCAM-1 forms 318F, 555V and 716L compared to those overexpressing wild type VCAM-1 (p < 0.05). Conclusions: VCAM-1-dependent cell adhesion to endothelial cells in vitro is significantly increased when expressing VCAM-1 missense mutations 318F, 555V and 716L. The underlying mechanism involves altered VCAM-1 protein levels and function. This observation may be of particular relevance for chronic inflammatory pathophysiologic conditions involving cell–cell adhesion such as atherosclerosis and other proinflammatory conditions. [Copyright &y& Elsevier]

Abstract: Objectives: Vascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli. Methods: Endothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence. Results: siRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective. Conclusion: VEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions. [Copyright &y& Elsevier]

Abstract: Objective: : The scavenger receptor SR-PSOX/CXCL16, which is identical to the chemokine CXCL16, is thought to be involved in atherogenesis. However, the presence and function of SR-PSOX/CXCL16 in the endothelium of atherosclerotic arteries has not been substantiated. Methods and results: : In rabbit aorta immunocytochemistry revealed SR-PSOX/CXCL16 primarily in the endothelium at sites predisposed to lesion formation, in the endothelium of early atherosclerotic lesions, and mainly in intimal macrophages of more developed lesions, indicating that SR-PSOX/CXCL16-expression shifts during atherogenesis. In addition to its function as scavenger receptor and chemokine, SR-PSOX mediated the adhesion of THP-1 monocytes to endothelial cells in vitro. Both THP-1 monocytes and endothelial cells express SR-PSOX/CXCL16, and THP-1 monocytes express CXCR6, the specific receptor for SR-PSOX/CXCL16. Anti-SR-PSOX/CXCL16 and anti-CXCR6 antibody block monocyte adhesion, showing that SR-PSOX/CXCL16–CXCR6 interaction mediates monocyte-endothelial cell adhesion. SR-PSOX/CXCL16 expression of endothelial cells is upregulated by pro-inflammatory cytokines, and is reversed by incubation with ciglitazone and lovastatin. Conclusions: : We suggest that SR-PSOX/CXCL16 may promote the adhesion of monocytes to the endothelium during early atherogenesis and that accumulating cytokines enhance SR-PSOX/CXCL16-mediated adhesion by upregulating SR-PSOX/CXCL16 expression. Manipulation of SR-PSOX/CXCL16 expression with anti-inflammatory agents may be of therapeutic value. [Copyright &y& Elsevier]

Abstract: Objective: Uniform laminar shear stress (LS) and disturbed turbulent shear stress (DS) are thought to play opposite roles in preventing or inducing atherosclerosis. Endothelial cell (EC) growth and monocyte adhesion to ECs, an early event in atherosclerosis, are also oppositely regulated by LS and DS. However, how atherogenesis is affected by the regulation of growth by blood flow is unknown. Here we examined the role of p21 Sdi/Cip/Waf1 (p21), a growth inhibitor induced by LS, in monocyte adhesion to ECs. Methods: p21 was overexpressed by transfecting a p21-expressing adenoviral vector into ECs. Factors linking EC growth, monocyte adhesion, and p21 were examined by microarray analysis, PCR and Western blotting. Results: Compared with DS, in the presence or absence of TNFα, LS significantly inhibited EC growth and monocyte adhesion to ECs. Both EC proliferation and monocyte adhesion induced by DS were inhibited by p21-overexpression. LS suppressed the expression of thioredoxin-interacting protein (TXNIP). Thioredoxin (TRX) activity, which is suppressed by TXNIP, was therefore higher under LS than DS, as reported previously. p21-overexpression significantly suppressed the DS-induced TXNIP expression, and inhibited the expression of vascular cell adhesion molecule 1 and chemokine (C–C motif) ligand 5 (CCL5/RANTES), which stimulates leukocyte recruitment and is downregulated by ROS scavenging. Conclusion: p21 may function to prevent atherogenesis by regulating the redox balance, which leads to the inhibition of adhesion molecule and chemokine expression in ECs under LS. [Copyright &y& Elsevier]

Abstract: Acute, short-term hyperglycemia is becoming recognized as an important risk factor for several diseases. In the present study, using human aortic endothelial cells (HAECs), we investigated whether short-term high glucose exposure, either on the scale of hours, could enhance the monocyte adhesion and migration to the subendothelium via increasing expression of adhesion molecules and release of chemotactic factors. HAECs stimulated with 25mM d(+)glucose (HG) for not more than 12h, exhibited rapid up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein. Although intercellular adhesion molecule-1 (ICAM-1) is considered as a marker of the activation of the atherogenic process, early up-regulation was not observed, and VCAM-1 and MCP-1 protein enhance was sufficient to increase the adhesiveness of human monocytes U-937 to HAECs and their transmigration into the subendothelial space after 4h HG stimulation; both effects were prevented by interfering with monoclonal antibodies against VCAM-1, CD11b, and MCP-1. An increased intracellular oxidative stress, a translocation of NF-κB to the nucleus and a prevention of adhesion and transmigration of U-937 by interfering with NF-κB inhibitors was also observed after a short HG treatment. Taken together, these results suggest that either acute hyperglycemic spikes could exert an influence on the onset of diabetic complications and on the development of the atherogenic profile on diabetic and non-diabetic subjects. [Copyright &y& Elsevier]

Abstract: Leukocyte recruitment and adhesion to the endothelium are critical steps in the early phase of atherosclerosis. Synthetic ligands of peroxisome proliferator-activated receptors (PPARs) were shown to reduce cytokine-stimulated leukocyte–endothelial cell interactions by inhibiting the NF-κB mediated inflammatory response. Conjugated linoleic acids (CLA), which are natural ligands of PPARs, were demonstrated to have anti-inflammatory and anti-atherogenic properties in vivo. With a view to elucidating the anti-atherogenic mechanisms of CLA, the present study aimed to explore the effects of cis-9, trans-11 CLA and trans-10, cis-12 CLA on cytokine-induced chemokine release, surface expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and U937 monocyte adhesion in human aortic endothelial cells (HAEC). Treatment of HAECs with 2ng/mL of TNFα markedly increased expression of adhesion molecules, U937 monocyte adhesion, and release of the monocyte chemoattractant protein (MCP)-1. However, treatment of HAECs with either CLA isomer or linoleic acid did not modulate the cytokine-induced expression of ICAM-1, VCAM-1, and E-selectin, U937 cell adhesion and MCP-1 release. In addition, both CLA isomers and linoleic acid slightly increased PPARγ DNA-binding activity, but did not alter DNA-binding activity of NF-κB. In conclusion, CLA isomers showed no effect on cytokine-induced monocyte–endothelial cell interactions and on the molecular mechanisms regulating these processes in HAEC. This study suggests that anti-atherogenic effects of CLA observed in vivo are probably not mediated by reduced monocyte–endothelial cell interactions. [Copyright &y& Elsevier]

Abstract: The magnitude of postprandial lipemia has been identified as independent risk factor for the development of coronary artery disease. To test the effect of postprandial versus postabsorptive low-density lipoproteins (LDL) on the expression of adhesion molecules, LDL were isolated from healthy subjects before and 4h after ingestion of a standardized fatty test meal. We used flow cytometry and Northern blotting to quantify cell adhesion molecules in human aortic endothelial cells (HAEC). The adherence of leukocytes to HAEC was analyzed using a monocyte adhesion assay. Incubation of HAEC with postprandial, but not postabsorptive LDL induced a two-fold increase in the surface expression of intercellular adhesion molecule-1 (ICAM-1), but not of E-selectin or vascular cell adhesion molecule-1. In addition, increased amounts of ICAM-1 transcripts were found in HAEC treated with postprandial LDL. The adhesion of monocytes to HAEC was enhanced after pretreatment with postprandial, but not with postabsorptive LDL. We conclude that postprandial, but not postabsorptive LDL increase the surface expression of ICAM-1 in HAEC apparently by de novo protein synthesis leading to increased adhesion of monocytes. The upregulation of ICAM-1 by postprandial LDL may explain part of the proatherogenic effect of high postprandial lipemia. [Copyright &y& Elsevier]