We have shown that the 12/15-lipoxygenase (12/15-LO) product 12S-hydroxyeicosatetraenoic acid increases monocyte adhesion to human endothelial cells (EC) in vitro. Recent studies have implicated 12/15-LO in mediating atherosclerosis in mice. We generated transgenic mice on a C57BL/6J (B6) background that modestly overexpressed the murine 12/15-LO gene (designated LOTG). LOTG mice had 2.5-fold elevations in levels of 12S-hydroxyeicosatetraenoic acid and a 2-fold increase in expression of 12/15-LO protein in vivo. These mice developed spontaneous aortic fatty streak lesions on a chow diet. Thus, we examined effects of 12/15-LO expression on early events leading to atherosclerosis in these mice. We found that, under basal unstimulated conditions, LOTG EC bound more monocytes than B6 control EC (18 ؎ 2 versus 7 ؎ 1 monocytes/field, respectively; p < 0.0001). Inhibition of 12/15-LO activity in LOTG EC using a 12/15-LO ribozyme completely blocked monocyte adhesion in LOTG mice. Thus, 12/15-LO activity is required for monocyte/EC adhesion in the vessel wall. Expression of ICAM-1 in aortic endothelia of LOTG mice was increased severalfold. VCAM-1 expression was not changed. In a series of blocking studies, antibodies to ␣ 4 and ␤ 2 integrins in WEHI monocytes blocked monocyte adhesion to both LOTG and B6 control EC. Inhibition of ICAM-1, VCAM-1, and connecting segment-1 fibronectin in EC significantly reduced adhesion of WEHI monocytes to LOTG EC. In summary, these data indicate that EC from LOTG mice are "pre-activated" to bind monocytes. Monocyte adhesion in LOTG mice is mediated through ␤ 2 integrin and ICAM-1 interactions as well as through VLA-4 and connecting segment-1 fibronectin/ VCAM-1 interactions. Thus, 12/15-LO mediates monocyte/EC interactions in the vessel wall in atherogenesis at least in part through molecular regulation of expression of endothelial adhesion molecules.Murine 12/15-lipoxygenase (12/15-LO) 1 incorporates molecular oxygen in a stereospecific manner into arachidonic and linoleic acids to generate 12S-and 15S-hydroxyeicosatetraenoic acids (12S-HETE) and 13S-hydroxyoctadecadienoic acid (13S-HODE) (1-3). Murine 12/15-LO is similar biochemically and structurally to the porcine leukocyte-type 12-LO and human 15-LO enzymes (2-4). There also exists a murine platelet 12-LO, which utilizes arachidonic acid solely as a substrate to generate 12S-HETE (4, 5).The exact biologic functions of 12/15-LO are unknown. However, considerable evidence exists to support a role for 12/ 15-LO in promoting both diabetes and atherosclerosis (6 -9). Nadler and co-workers (6) have shown that mice deficient in 12/15-LO are protected from development of low dose streptozotocin-induced diabetes. We have recently shown that diabetic db/db mice produce significant quantities of 12S-HETE in vivo (10). Importantly, using a catalytic ribozyme to inactivate 12/ 15-LO mRNA, we have shown that disruption of 12/15-LO mRNA in diabetic db/db mice blocks monocyte adhesion (10). Striking evidence for the role of 12/15-LO in atherogenes...

Abstract-The development of atherosclerosis is accelerated in individuals with type 2 diabetes. Adhesion of monocytes to the vascular endothelium is a key initial step in atherogenesis. We have previously shown that monocyte adhesion to human aortic endothelial cells (HAECs) cultured long-term in high-glucose medium (25 mmol/L, 2 passages) is increased compared with cells grown in normal glucose (5 mmol/L). One potential mechanism for increased monocyte adhesion to HAECs under hyperglycemic conditions is via the 12-lipoxygenase (12-LO) pathway. In this study, we demonstrated in HAECs that the major LO metabolite of arachidonic acid was the 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which was increased severalfold in HAECs cultured under high-glucose conditions. Furthermore, treatment of HAECs with 12(S)-HETE induced monocyte, but not neutrophil, adhesion an average of 3-fold (range of 1.5-to 5-fold) compared with untreated cells (75Ϯ5 versus 26Ϯ1 monocytes per field, respectively, PϽ0.001). Expression of the adhesion molecules vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 was not significantly increased. However, both glucose and 12(S)-HETE induced a 60% increase in HAEC surface expression of connecting segment-1 (ie, CS-1) fibronectin, a ligand for very late-acting antigen-4 (VLA-4). The antibodies used to block monocyte integrin VLA-4 and leukocyte function-related antigen-1, a monocytic counterreceptor for intercellular adhesion molecule-1, inhibited the ability of both 12-LO products and high glucose to induce monocyte adhesion. These results definitively demonstrate for the first time in HAECs that the 12-LO pathway can induce monocyte-endothelial cell interaction and that the effects of glucose may be mediated, at least in part, through this pathway. Thus, these results suggest that the 12-LO pathway may play a role in the increased susceptibility of diabetics to atherosclerosis.

Abstract-The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose-and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reversephase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. Key Words: 12-lipoxygenase Ⅲ ribozyme Ⅲ adenovirus Ⅲ endothelium Ⅲ vascular smooth muscle cells L ipoxygenase (LO) activation has been implicated in numerous diseases including atherosclerosis and diabetes. Certain pathological changes that occur in diabetic vascular disease, such as adhesion of leukocytes to the endothelium and chemotaxis of smooth muscle cells (SMCs), may share a common pathway through LO. Upregulation of LO activity and enhanced formation of LO products have been reported in vitro and in vivo. We and others have reported increased LO activity and elevated levels of LO products, but not cyclooxgenase products, in porcine aortic endothelial cells (PAECs) 1 and porcine SMCs (PSMCs) 2 when cultured in high glucose (HG) conditions. Also, enhanced production of HETEs has been observed in patients with diabetic renal disease 3 and in vessels from infants of diabetic mothers. 4 Exposure to extracellular stimulants such as angiotensin II, platelet-derived growth factor (PDGF), interleukin-4, and interleukin-1 has also led to increased synthesis of LO products in vascular and mononuclear cells. 2,[5][6][7][8] These results suggest that many of the biological effects of the agents involved in stimulating inflammatory processes may be mediated through LO and its products.The LOs responsible for regulating specific vascular functions have not been determined. The number of mammalian LO sequences published includes at least 18 different sequences representing 7 isoforms in 7 ...