Aims/hypothesis. Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. Methods. Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. Results. Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HGcultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. Conclusions/interpretation. In diabetes, the expression and activity of eNOS is regulated through glucosemediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1. Abbreviations: AP-1, activator protein-1 · EC, endothelial cell · eNOS, endothelial nitric oxide synthase · FBS, fetal bovine serum · HG, high glucose · MnSOD, manganese superoxide dismutase · MOI, multiplicity of infection · NF-κB, nuclear factor-κB · NG, normal glucose · ROS, reactive oxygen species · UCP-1, uncoupling protein-1

Giardia lamblia infections are nearly universal among children in low-income countries and are syndemic with the triumvirate of malnutrition, diarrhea, and developmental growth delays. Amidst the morass of early childhood enteropathogen exposures in these populations, G. lamblia-specific associations with persistent diarrhea, cognitive deficits, stunting, and nutrient deficiencies have demonstrated conflicting results, placing endemic pediatric giardiasis in a state of equipoise. Many infections in endemic settings appear to be asymptomatic/ subclinical, further contributing to uncertainty regarding a causal link between G. lamblia infection and developmental delay. We used G. lamblia H3 cyst infection in a weaned mouse model of malnutrition to demonstrate that persistent giardiasis leads to epithelial cell apoptosis and crypt hyperplasia. Infection was associated with a Th2-biased inflammatory response and impaired growth. Malnutrition accentuated the severity of these growth decrements. Faltering malnourished mice exhibited impaired compensatory responses following infection and demonstrated an absence of crypt hyperplasia and subsequently blunted villus architecture. Concomitantly, severe malnutrition prevented increases in B220 + cells in the lamina propria as well as mucosal Il4 and Il5 mRNA in response to infection. These findings add insight into the potential role of G. lamblia as a "stunting" pathogen and suggest that, similarly, malnourished children may be at increased risk of G. lambliapotentiated growth decrements.

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...

Background: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota.Objective: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls.Design: With the use of a 1H nuclear magnetic resonance spectroscopy–based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model.Results: We showed considerable shifts within the intestinal microbiota 14–24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both well-nourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed.Conclusions: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction.

Objective-Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte-EC interactions in vivo. Methods and Results-We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-␣ in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-␣-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-␣ plus SEW2871-treated mice had few monocytes bound (PϽ0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte-EC interactions in response to TNF-␣. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun. Conclusions-Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-␣-mediated monocyte-EC interactions in vivo. Key Words: endothelium Ⅲ sphingosine-1-phosphate Ⅲ inflammation Ⅲ Endothelium differentiation gene (Edg) receptors I nflammation is a hallmark of atherosclerosis and diabetes. 1 Monocyte-endothelial interactions are key initiating events of inflammation. 2 Activated monocytes release tumor necrosis factor (TNF)-␣ that mediates a variety of pathological vascular responses. 3 Monoclonal antibody therapies to reduce TNF-␣ have reduced inflammation in several chronic diseases, 4 but these therapies do not prevent the initiation of inflammation.Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid in circulation that evokes a variety of cellular responses, including cell migration, vascular maturation, and lymphocyte homing. 5 However, the role of S1P in vascular inflammation is unknown. S1P binds to 5 G protein-coupled receptors (Edg receptors) on endothelium and activates these receptors in the low nanomolar range. 6,7 In the vasculature, S1P receptors are found on both endothelial cells (ECs) and monocytes. 8,9 S1P1 is involved in endothelial migration and blood vessel formation. 10 S1P3 regulates heart rate in mice 11 and vascular tone through activation of endothelial nitric oxide synthase (eNOS). 12,13 In the current study, we report that S1P prevents TNF-␣-mediated monocyte adhesion to intact aorta and to cultured primary isolates of aortic endothelial cells. We found that the protective, antiinflammatory action of S1P is primarily mediated through binding to the endothelial S1P1 receptor. MethodsDetailed Methods for all experiments can be found online. Please see http://atvb.ahajournals.org. Results S1P and an S1P1 Receptor Agonist Prevent TNF-Mediated Monocyte-Endothelial InteractionsWe injected C57BL/6J (B6) mice with 0.5 g of recombinant murine TNF-␣ intravenously, and either 5 mg/kg of the selective S1P1 receptor agonist, SEW2871 (5...

Background-12/15-lipoxygenase (12/15-LO) activity leads to the production of the proinflammatory eicosanoids 12-S-hydroxyeicosatetraenoic acid (12SHETE) and 13-S-hydroxyoctadecadienoic acid. We have previously shown a 3.5-fold increase in endothelial intercellular adhesion molecule (ICAM)-1 expression in mice overexpressing the 12/15-LO gene. We examined whether 12/15-LO activity regulated endothelial ICAM-1 expression. Methods and Results-Freshly isolated aortic endothelial cells (EC) from 12/15-LO transgenic mice had significantly greater nuclear factor-B (NF-B) activation and ICAM mRNA expression compared with C57BL/6J control. 12/15-LO transgenic EC showed elevated RhoA activity, and inhibition of RhoA using either C3 toxin or the Rho-kinase inhibitor Y-27632 blocked NF-B activation, ICAM-1 induction, and monocyte adhesion. Furthermore, we show that 12SHETE activates protein kinase C␣, which forms a complex with active RhoA and is required for NF-B-dependent ICAM expression in response to 12SHETE. A key early event in vascular inflammation is the interaction of monocytes and endothelial cells (EC) in the vessel wall. 1 Activated monocytes interact with intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on the EC surface where the monocyte adheres firmly to the endothelium and transmigrates through the EC monolayer. [2][3][4] Monocytes are the primary inflammatory cells localized to human atherosclerotic plaques and play a major role in atherosclerotic plaque progression. 5 12/15-lipoxygenase (12/15-LO) incorporates molecular oxygen in a stereospecific manner into arachidonic and linoleic acids to generate 12-and 15-S-hydroxyeicosatetraenoic acids (12SHETE/15SHETE) and 13-S-hydroxyoctadecaenoic acid (13SHODE), respectively. 6 -8 Several vascular cell types produce 12/15-LO eicosanoids, including EC, smooth muscle cells, monocytes, and platelets. Several studies, including ours, have indicated that the 12/15-LO pathway is proinflammatory and increases atherosclerosis development in mice. 9 -13 12SHETE, a major product of 12/15-LO, is present in the vasculature in the nanomolar range and activates EC to stimulate monocyte adhesion. 14, 15 We have previously reported a significant increase in expression of ICAM-1 on aortic endothelium of 12/15-LO transgenic mice (12/15-LOTG), suggesting 12SHETE stimulates monocyte adhesion through enhancing endothelial ICAM-1 expression. 13 ICAM-1 and VCAM-1 are both believed to be important for early atherogenesis. 16,17 Several studies have indicated that deletion of ICAM-1 in apoE-deficient mice attenuates atherosclerosis progression, 18 -20 although Cybulsky and colleagues reported that VCAM-1, rather than ICAM-1, was the primary regulator of atherosclerosis. 21 Kevil et al reported that ICAM-1 was critical for mediating monocyte adhesion to murine EC, 22 although Huo and colleagues reported that VCAM-1 played a critical role in mediating this process. 23 We have previously reported that both VCAM-1 and ICAM-1 mediate monocyte:endothelial...

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 ϳ16 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-␣-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 M cinnamyl-3,4-dihydroxy-␣-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 ␣ 4 ␤ 1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of ␤ 2 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.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.