Inhibition Of VCAM-1 Research Articles

Knockdown of mouse VCAM-1 by vector-based siRNA

Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.

Various adhesion molecules impair microvascular leukocyte kinetics in ventilator-induced lung injury

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.

VLA-4 antagonist WAY160103 (WAY) inhibits VCAM-1 activated eosinophil (EOS) respiratory burst but promotes eosinophil derived neurotoxin (EDN) degranulation

Rationale EOS are selectively recruited to the lung during asthma exacerbations and play an important role in airway inflammation. EOS participation is initiated by adhesion of circulating cells via VLA-4 (a4β1) to VCAM-1 expressed on pulmonary endothelium. Inhibition of this process has been studied with a variety of VLA-4 antagonists as possible therapeutic agents to block the participation of EOS in allergic airway inflammation. Although integrin (b1, b2) ligation has been closely related to functional activation of EOS, studies on the effects of these antagonists on other EOS functions have been limited. We hypothesized that the small molecule VLA-4 inhibitor, WAY160103, affects not only EOS adhesion but also other b-integrin-dependent functions. Methods Human peripheral blood EOS were isolated and assayed for transendothelial migration (TNF-a activated human pulmonary microvascular endothelial cells [HPMEC]), respiratory burst (superoxide anion generation) and EDN degranulation (RIA). Results Although WAY inhibited VCAM-1 adhesion, it had no effect on EOS transmigration across VCAM-1 upregulated HPMEC monolayers. WAY did, however, give a concentration-dependent inhibition of VCAM-1 activated superoxide anion generation similar to its effects on EOS adhesion. Finally, WAY promoted EDN degranulation possibly due to a shift from VLA-4 to b2-dependent adhesion necessary for EOS degranulation. Conclusion Although inhibition of EOS adhesion to VCAM-1 may suppress an early step in cell infiltration, other pathways (selectins) can initiate EOS extravascular migration. The effects of VLA-4 inhibitors on other inflammatory EOS functions must be taken into account when testing these compounds as possible therapeutic agents in allergic disease and asthma.

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions — (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-α, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-α and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-α. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Griseofulvin has a potential to modulate the expression of cell adhesion molecules on leukocytes and vascular endothelial cells

Griseofulvin has been used as an antifungal drug for many years, but it has recently been shown effective for several inflammatory skin diseases. We therefore investigated its putative immunomodulatory roles by flow cytometric analysis of cell adhesion molecules on human leukocytes and human vascular endothelial cells. Griseofulvin downregulated L-selectin expression on neutrophils, but not on lymphocytes, in a dose-dependent manner. Griseofulvin did not affect CD11b/CD18 expression on neutrophils. On human dermal microvascular endothelial cells (HDMEC), griseofulvin inhibited the expression of TNFα-induced VCAM-1 dose-dependently, and this inhibition was fully reversible. Similarly, griseofulvin inhibited the induction of VCAM-1 expression on both TNFα- and IL-1α-stimulated human umbilical vein endothelial cells (HUVEC). In addition, it partially inhibited the induction of E-selectin expression, whereas it had a marginal effect on ICAM-1 expression. Reverse transcription-polymerase chain reaction of TNFα-stimulated HDMEC showed inhibition of VCAM-1, but not ICAM-1 gene transcription. These results indicate potent immunomodulatory properties of griseofulvin, which may be associated with its feature as a microtubule antagonist.

Inhibition of VCAM-1 expression in the arterial wall is shared by structurally different antioxidants that reduce early atherosclerosis in NZW rabbits

We previously established that probucol decreases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expression that normally accompanies atherogenesis. To determine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expression and whether the degree of VCAM-1 inhibition correlates with the reduction of atherosclerosis or the antioxidant protection of LDL. Atherogenesis and VCAM-1 mRNA and protein were determined in four groups of NZW rabbits (n = 6;-8) fed 0.5% cholesterol alone or supplemented with 0.1% vitamin E, a low dose (0.04;-0.075%) of probucol yielding the same degree of antioxidant protection of plasma LDL as vitamin E, or a high dose (0.5%) of probucol, and in normocholesterolemic rabbits. After 81 days, extensive atherosclerosis and a greater than 4-fold up-regulation of VCAM-1 mRNA was seen in rabbits on high cholesterol diet, mostly in the intima. Treatment with vitamin E, high-dose probucol, and low-dose probucol significantly decreased VCAM-1 mRNA by 49.0, 74.9, and 57. 5%, respectively, and reduced atherosclerosis in adjacent segments of the thoracic aorta to a similar degree as reported by previous studies. Immunocytochemistry confirmed that lesions of antioxidant-treated animals also contained less VCAM-1 protein. Neither the degree of VCAM-1 inhibition nor the extent of atherosclerosis correlated with the degree of antioxidant protection of plasma LDL.In summary, treatment with structurally unrelated antioxidants conveyed different degrees of antioxidant protection to plasma LDL but significantly reduced VCAM-1 expression in vivo and inhibited atherogenesis. This is consistent with the assumption that antiatherogenic effects of antioxidants may in part be mediated by interference with oxidation-dependent intracellular signaling.

The inhibition of endothelial activation by unsaturated fatty acids

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-kappaB (NF-kappaB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-kappaB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.

Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4.

1. Expression of cell adhesion molecules (CAM) on the lung microvascular endothelium is believed to play a key role in the recruitment of leukocytes in pulmonary inflammation. Moreover, regulation of CAM expression may be an important mechanism through which this inflammation may be controlled. Experimental evidence has suggested that combined phosphodiesterase (PDE) 3 and 4 inhibitors increase cyclic AMP levels within cells greater than inhibition of either isoenzyme alone. In the present study we assessed the effect of combinations of rolipram (PDE4 inhibitor), ORG 9935 (PDE3 inhibitor) and salbutamol (beta-agonist) on CAM expression and neutrophil or eosinophil adhesion to human lung microvascular endothelial cells (HLMVEC). 2. Tumour necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin expression were measured on HLMVEC monolayers at 6 h by a specific ELISA technique in the presence of different combinations of medium, rolipram, ORG 9935 and salbutamol. 3. Rolipram in combination with salbutamol, but neither agent alone, inhibited TNF-alpha-induced E-selectin expression, whilst ICAM-1 and VCAM-1 expression were not affected. ORG 9935 had no significant effect on CAM expression alone. However, in combination with rolipram a syngergistic inhibition of VCAM-1 and E-selectin, but not ICAM-1, expression was observed. No further inhibition was seen in the additional presence of salbutamol. 4. Neutrophil adhesion to TNF-alpha-stimulated (6 h) HLMVEC was mainly E-selectin dependent in this model, as ENA2 an anti-E-selectin monoclonal antibody (mAb) abrogated neutrophil adhesion. Eosinophil adhesion was E-selectin-, ICAM-1- and VCAM-1-dependent, as assessed by the inhibitory activity of ENA2 and the ability of a mAb to the ICAM-1 ligand, CD18, and a mAb to the VCAM-1 ligand, VLA4, to attenuate adhesion. 5. Rolipram in the presence of salbutamol or ORG 9935 significantly inhibited neutrophil adherence to TNF-alpha-stimulated HLMVEC. Eosinophil adherence to monolayers was inhibited only when HLMVEC were activated in the presence of rolipram and ORG 9935. 6. Collectively, the findings presented in this manuscript suggest that inhibition of PDE4 with appropriate activation of adenylate cyclase is sufficient to inhibit induction of E-selectin expression on HLMVEC to a level that has functional consequences for neutrophil adhesion. In contrast, combined inhibition of PDE3 and 4 isoenzymes is necessary to inhibit VCAM-1 and to have inhibitory effects on eosinophil adhesion to activated HLMVEC. Upregulation of ICAM-1 expression on HLMVEC does not appear to be modulated by PDE3 and 4 inhibition. These data may have implications for the use of selective PDE4 inhibitors in lung inflammation.

VCAM-1/α4-integrin adhesion pathway: Therapeutic target for allergic inflammatory disorders

Lymphocyte recirculation and leukocyte extravasation involve a multistep process that is central to immune surveillance and the rapid response of white blood cells to sites of injury or infection. Interaction of vascular adhesion molecules (VCAM-1, ICAM-1, and selectins) with ligands on the leukocyte surface (integrins, carbohydrates, and mucin-like molecules) regulate diapedesis. The nature of an inflammatory stimulus ultimately determines the pattern of endothelial adhesion molecule expression and the avidity state of their counterreceptors, thus dictating to a large extent whether a subclass of leukocytes will play a dominant role in the immune response. Immunoglobulin superfamily member VCAM-1 recognizes α4β1 integrin, expressed on all leukocytes except neutrophils. Blockade or inhibition of VCAM-1/α4β1 interaction is expected to have therapeutic potential in treating various inflammatory disorders and autoimmune diseases since this adhesion pathway has a major influence on eosinophil, lymphocyte, and monocyte trafficking. This review summarizes some of the strategies that are currently used to selectively inhibit the VCAM-1/α4 integrin pathway, including soluble VCAM-Ig fusion protein, peptide antagonists, antisense oligonucleotides, natural products, and neutralizing antibodies to VCAM-1 or α4 integrin. (J Allergy Clin Immunol 1996;98:270-7.)