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Leukocyte recruitment to inflamed tissues is the cornerstone of inflammatory responses and the driving force behind the establishment of inflammatory bowel disease, consisting of Crohn's disease and ulcerative colitis. It has been reported that angiogenic cytokines contribute to this inflammatory response that facilitates the chronic nature of disease. We have previously reported (Goebel S, Huang M, Davis WC, Jennings M, Siahaan TJ, Alexander JS, Kevil CG. Am J Physiol Gastrointest Liver Physiol 290: G648-G654, 2006) that vascular endothelial growth factor (VEGF)-A can stimulate neutrophil adhesion to colon microvascular endothelial cells in a [[beta].sub.2]-integrin (Itgb2)-dependent manner. However, it is not known which of the specific leukocyte integrins are critical for VEGF-A-dependent neutrophil and T cell recruitment. Here we examine the differential importance of either [alpha]-integrin (Itga)L or ItgaM in governing neutrophil and T cell adhesion to VEGF-A-activated colonic endothelium. Using an in vitro parallel-plate flow chamber model, we found that genetic deficiency of ItgaM completely blunted neutrophil adhesion to VEGF-A-stimulated endothelium, whereas ItgaL deficiency only partly blocked neutrophil adhesion. Deficiency of ItgaM did significantly decrease neutrophil rolling, whereas deficiency of ItgaL did not. We found that genetic deficiency of either ItgaL or ItgaM did significantly blunt T cell adhesion to VEGF-A-stimulated colon endothelium. We also found that genetic deficiency of these Itgas significantly attenuated T cell rolling behavior. Lastly, we examined whether VEGF-A-mediated leukocyte recruitment occurred through different VEGF receptor (VEGFR) pathways and found that VEGFR2 activation regulates neutrophil recruitment, whereas both VEGFR1 and VEGFR2 modulate T cell recruitment. Together, these data identify differential molecular mechanisms of VEGF-A-mediated leukocyte recruitment. colitis; leukocyte; angiogenesis; vascular endothelial growth factor receptor; adhesion doi: 10.1152/ajpgi.00202.2010.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by increased leukocyte recruitment and subsequent tissue damage. An increase in the density of the microvasculature of the colon during IBD has been suggested, leading to the concept that angiogenesis may play a pathological role in IBD. Increased tissue and serum levels of the angiogenic cytokine VEGF-A have been reported in cases of active IBD. In this study, we examined the hypothesis that VEGF-A exerts a proinflammatory effect on colon microvascular endothelium that contributes to colonic inflammation. Leukocyte adhesion to VEGF-A-stimulated colon microvascular endothelial cells was examined using a parallel-plate hydrodynamic flow chamber. ICAM-1 adhesion molecule expression on colonic microvascular endothelium also was determined in response to VEGF-A stimulation, along with characterization of leukocyte adhesion molecule expression. High-dose VEGF-A (50 ng/ml) stimulation increased neutrophil and T cell adhesion to and decreased rolling velocities on activated endothelium, whereas low-dose VEGF-A (10 ng/ml) was without effect. Colonic endothelium constitutively expressed ICAM-1, which was significantly increased by treatment with 50 ng/ml VEGF-A or 10 ng/ml TNF-[alpha] but not 10 ng/ml VEGF-A. T cells expressed CD18 and CD1 la with no expression of CD11b, whereas neutrophils expressed CD18, CD11a, and CD11b. Finally, VEGF-A-dependent leukocyte adhesion was found to occur in a CD18-dependent manner. These results demonstrate that VEGF-A levels found in IBD exert a proinflammatory effect similar to other inflammatory agents and suggest that this cytokine may serve as an intermediary between angiogenic stimulation and cell-mediated immune responses. angiogenesis; inflammation; CD18; integrin: colitis; vascular endothelial growth factor

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity. tert-butyl hydroperoxide; diamide; dehydroisoandrosterone; glucose6-phosphate dehydrogenase; antisense

Sasaki, Makoto, John W. Elrod, Paul Jordan, Makoto Itoh, Takashi Joh, Alireza Minagar, and J. Steven Alexander. CYP450 dietary inhibitors attenuate TNF-[alpha]-stimulated endothelial molecule expression and leukocyte adhesion. Am J Physiol Cell Physiol 286: C931-C939, 2004; 10.1152/ajpcell.00351.2003.--Enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and other endothelial cell adhesion molecules (ECAMs) are associated with the onset and progression of inflammatory bowel disease (IBD). We show in this study that two cytochrome P-450 (CYP450) inhibitors from Citrus paradis (grapefruit), bergamottin, and 6',7'dihydroxybergamottin (DHB) block tumor necrosis factor (TNF)-[alpha]stimulated expression of MAdCAM-1 in cultured endothelial cells and also reduce [[alpha].sub.4][[beta].sub.7]-dependent lymphocyte adhesion. Bergamottin (20-50 [micro]M) or DHB (10-30 [micro]M) pretreatment dose-dependently reduced TNF-[alpha]-mediated expression of MAdCAM-1 and lymphocyte adhesion. Bergamottin and DHB also prevented expression of two other ECAMs, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (but not E-selectin). SKF-525a, a specific CYP450 inhibitor, also blocked the expression of MAdCAM-1 mediated by TNF-[alpha]. Similar to SKF-525a (20 [micro]M), bergamottin (20 [micro]M) and DHB (20 [micro]M) directly inhibited the activity of CYP450 3A4. These results suggest that natural CYP450 inhibitors may be effective in reducing ECAM expression and leukocyte adhesion and therefore be useful in the clinical treatment of inflammatory states like IBD. cytochrome P-450; inflammatory bowel disease; lymphocytes; mucosal adhesion cell adhesion molecule-1