Glucose regulates interleukin-8 production in aortic endothelial cells through activation of the p38 mitogen-activated protein kinase pathway in diabetes.

We have shown that chronic elevated glucose (25 mm) increases monocyte adhesion to human aortic endothelial cells (EC). This increased adhesion is mediated primarily through induction of interleukin (IL)-8 via activation of the transcription factor AP-1 (Srinivasan, S., Yeh, M., Danziger, E. C., Hatley, M. E., Riggan, A. E., Leitinger, N., Berliner, J. A., and Hedrick, C. C. (2003) Circ. Res. 92, 371-377). In the current study, we identified the elements in the AP-1 transcriptional complex that are activated by glucose. These elements include c-Jun, c-Fos, and Fra-1. AP-1 is activated by cellular oxidative stress, and we have reported significant production of ROS by high glucose-cultured cells. We examined signaling pathways upstream of AP-1 in EC that lead to AP-1 activation by HG. EC cultured in 25 mm glucose had a 2-fold increase in p38 phosphorylation compared with control normal glucose-cultured EC. Inhibition of the p38 pathway using 5 microm SB203580 significantly reduced glucose-mediated IL-8 mRNA production by 60%. Furthermore, blocking p38 pathway activation using a dominant-negative p38 construct significantly reduced glucose-mediated monocyte adhesion by 50%. Thus, glucose-stimulated monocyte adhesion is primarily regulated through phosphorylation of p38 with subsequent activation of AP-1, leading to IL-8 production. To study this pathway in the setting of diabetes, we used the db/db mouse. P38 phosphorylation was increased in diabetic db/db mice compared with control mice. We found a dramatic elevation in plasma levels of KC, the mouse ortholog of IL-8 in diabetic db/db mice (1800 +/- 100 pg/ml KC in db/db versus 300 +/- 75 pg/ml in C57BL/6J control mice, p < 0.0001). Inhibition of the p38 pathway in diabetic db/db mice significantly reduced monocyte adhesion by 50%. Taken together, these data indicate that chronic elevated glucose in diabetes activates the p38 MAP kinase pathway to increase inflammatory IL-8 gene induction and monocyte/endothelial adhesion.

Modulation of PPARalpha expression and inflammatory interleukin-6 production by chronic glucose increases monocyte/endothelial adhesion.

OBJECTIVE: We have previously reported increased monocyte adhesion to human aortic endothelial cells (HAECs) cultured in 25 mmol/L glucose (HG) compared with normal glucose (NG) (5.5 mmol/L). In this study, we explored mechanisms that contribute to increased monocyte adhesion by elevated glucose. METHODS AND RESULTS: We found that HAECs cultured in HG have increased production of the chemokine interleukin-6 (IL-6). We examined whether IL-6 directly modulated monocyte adhesion to EC. Inhibition of IL-6 using a neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50%, and addition of IL-6 directly to human EC stimulated monocyte adhesion. PPARalpha has been reported to negatively regulate expression of IL-6 in vascular cells, so we examined PPARalpha-associated signaling in EC. A known PPARalpha agonist, Wy14,643, prevented glucose-mediated IL-6 production by EC and reduced glucose-mediated monocyte adhesion by 40%. HG-cultured HAEC had a 50% reduction in expression of PPARalpha compared with control EC. Primary aortic EC isolated from PPARalpha knockout (KO) mice showed increased monocyte adhesion compared with EC isolated from control mice. PPARalpha KO EC also had increased production of IL-6. Finally, we measured IL-6 levels in diabetic db/db mice and found significant 6-fold elevations in IL-6 levels in db/db EC. CONCLUSIONS: These data indicate that IL-6 production is increased in diabetes and contributes to early vascular inflammatory changes. PPARalpha protects EC from glucose-mediated monocyte adhesion, in part through regulation of IL-6 production.

12/15-Lipoxygenase activity mediates inflammatory monocyte/endothelial interactions and atherosclerosis in vivo.

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 alpha(4) and beta(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 beta(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.

Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice.

Atherosclerosis is a major complication of diabetes. Up to 16 weeks of age, the db/db mouse is insulin-resistant and hyperglycemic and is a good model of Type 2 diabetes. After approximately 16 weeks of age, the mice develop pancreatic beta cell failure that can progress to a Type 1 diabetes phenotype. We have previously shown that glucose increases production of endothelial 12/15 lipoxygenase (12/15LO) products in vitro. In young 10-week-old Type 2 diabetic db/db mice, we found significant elevations in levels of urinary 12/15LO products, 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 13S-hydroxyoctadecaenoic acid (13S-HODE) in vivo compared with C57BLKS/J mice. Using isolated primary aortic endothelial cells (ECs) from db/db mice and WEHI78/24 mouse monocyte cells in static adhesion assays, we found increased WEHI monocyte adhesion to db/db ECs (14 +/- 2 monocytes/field for db/db ECs versus 4 +/- 1 monocytes/field for C57BLKS/J ECs, p < 0.002). Thus, ECs from db/db mice appear to be "pre-activated" to bind monocytes. Analysis of db/db ECs revealed a 2-fold elevation in 12/15LO protein compared with C57BLKS/J EC. To determine that 12/15LO products were responsible for the increased monocyte adhesion observed with db/db ECs, we inhibited expression of murine 12/15LO using either an adenovirus expressing a ribozyme to 12/15LO (AdRZ) or with the 12/15LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 microm cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate significantly reduced monocyte adhesion to db/db endothelium (p < 0.009). Thus, inhibition of the murine 12/15LO in db/db mice significantly reduced monocyte/endothelial interactions. We also found that adhesion of monocytes to diabetic db/db ECs was mediated by interactions of alpha4beta1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of beta2 integrins with endothelial intercellular adhesion molecule 1. In summary, regulation of the 12/15LO pathway is important for mediating early vascular changes in diabetes. Modulation of the 12/15LO pathway in the vessel wall may provide therapeutic benefit for early vascular inflammatory events in diabetes.