Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (≤1 ng/ml) caused production of chemotactic cytokines in HCAEC. Endotoxin-induced cytokine production was maximal at LPS-binding protein:soluble CD14 ratios <1, typically observed in individuals with subclinical infection; higher LPS-binding protein:soluble CD14 ratios were inhibitory. Endotoxin potently activated HCASMC, with cytokine release >10-fold higher in magnitude at >10-fold lower threshold concentrations (10–30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.

Pseudomonas aeruginosa secretes numerous factors that alter host cell function and may contribute to disease pathogenesis. Among recognized virulence factors is the redox-active phenazine pyocyanin. We have recently demonstrated that the precursor for pyocyanin, phenazine-1-carboxylic acid (PCA), increases oxidant formation and alters gene expression in human airway epithelial cells. We report in this work that PCA and pyocyanin increase expression of ICAM-1 both in vivo and in vitro. Moreover, phenazines enhanced cytokine-dependent increases in IL-8 and ICAM-1. Antioxidant intervention studies indicated both similarities and differences between PCA and pyocyanin. The thiol antioxidant N-acetyl cysteine, extracellular catalase, and inducible NO synthase inhibitors inhibited ICAM-1 and IL-8 increases in response to both phenazines. However, pyocyanin was significantly more sensitive to N-acetylcysteine inhibition. Interestingly, hydroxyl radical scavengers inhibited the response to pyocyanin, but not to PCA. These studies suggest that P. aeruginosa phenazines coordinately up-regulate chemokines (IL-8) and adhesion molecules (ICAM-1) by mechanisms that are, at least in part, oxidant dependent. However, results indicate that the mechanisms by which PCA and pyocyanin exert their effects are not identical, and not all antioxidant interventions are equally effective in inhibiting phenazine-mediated proinflammatory effects.

Our goal is to harness the protective effects of mammalian hibernation to prevent and to treat cardiovascular disease (CVD). CVD is among the leading causes of morbidity and mortality in the United States. The specific objective of this study was to determine the effect of hibernation on the cardiac delta opioid system, and to determine the mechanisms by which plasma from hibernating woodchucks (HWP) is cardioprotective. Hearts were collected from summer woodchucks and squirrels, and from animals at defined times in the hibernation cycle. Tissues were analyzed by real‐time PCR for gene expression and by Western blot analysis for protein levels. Immunofluorescence microscopy was used to determine the effects of HWP on cellular localization of the delta opioid receptor (DOR). Hibernation induced dynamic changes in cardiac expression of opioid genes, and altered protein levels of the delta opioid precursor proenkephalin and of DOR. HWP increased localization of DOR to the plasma membrane of primary human vascular cells, suggesting that mobilization of DOR to cell surfaces may contribute to cardioprotection. These data support a role for delta opioids and their receptor in the hibernation phenotype, and provide insights that will further the development of therapies based on the natural protective processes of hibernation. Funded by the Department of Emergency Medicine, The University of Iowa.

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