Endothelial Nuclear Factor-κB Translocation and Vascular Cell Adhesion Molecule-1 Induction by Complement

Abstract

Abstract-We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-B (NF-B) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-B translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O 2 ) of hypoxic (12 hours, 1% O 2 ) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-B translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-B translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-B translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) Key Words: adhesion molecules Ⅲ hypoxia Ⅲ inflammation Ⅲ nitric oxide Ⅲ immunotherapy I ncreasing evidence suggests that the terminal complement complex (C5b-9) plays an integral role in the pathogenesis of atherosclerosis 1-5 and vascular injury after ischemiareperfusion, cardiopulmonary bypass, and acute myocardial infarction. 6 -11 In addition to amplifying the local inflammatory response by inducing endothelial interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion, 12 C5b-9 also influences endothelial vascular tone [13][14][15] 16 ). Yet the mechanisms by which C5b-9 influences endothelium-dependent relaxation and leukocyte adhesion molecule expression have not been fully elucidated.Vascular tone and leukocyte adhesion molecule expression can be regulated by NO. 18,19 NO synthesized by the vascular endothelium induces vasodilation by activation of soluble guanylate cyclase and the subsequent increase of cGMP. 20 In addition to being a potent smooth muscle relaxant, NO inhibits platelet aggregation 21 and suppresses endothelial neutrophil adhesion molecule expression. 19 Adhesion molecules regulated by NO include P-selectin, 22 VCAM-1, 23 and ICAM-1. 24 Specifically, increased levels of NO are associated with decreased leukocyte adhesion molecule expression. [23][24][25][26] The mechanisms by which NO modulates expression of these adherence molecules is unclear but have been specul...

Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression

“…It has been shown that iNOS mRNA can be regulated transcriptionally via NF-B in the rat intestine (37). We have previously shown that the terminal complement cascade can directly regulate the translocation of NF-B in an in vitro model (7). Our present findings extend our in vitro findings to an animal model and demonstrate that NF-B activation (e.g., increased p-I B-␣ expression) can be inhibited by anti-C5 treatment.…”

Complement Plays an Important Role in Spinal Cord Injury and Represents a Therapeutic Target for Improving Recovery following Trauma

“…C5a can also upregulate expression of the adhesion molecules P-and E-selectin, intercellular adhesion molecule-1 and vascular adhesion molecule-1 on endothelial cells. [22][23][24][25] Of note, C5a is implicated in brain cryoinjury, because injury and neutrophil extravasation is reduced in C5-deficient mice and C5a receptor antagonist-treated mice. 26 However, we cannot exclude an important role for the terminal product of the complement cascade, the membrane attack complex (MAC).…”

A Keratin Peptide Inhibits Mannose-Binding Lectin

“…C3 was detected using a peroxidase-conjugated polyclonal goat anti-human C3 Ab (Cappel, West Chester, PA) and developed with ABTS. VCAM-1 expression was measured using a mAb to human VCAM-1 (mAb 6G10) (24) as previously described (25). All ELISA data represent the mean Ϯ SE.…”