Lynn L. Stoll

Ghrelin Inhibits Proinflammatory Responses and Nuclear Factor-κB Activation in Human Endothelial Cells

Background—Ghrelin is a novel growth hormone–releasing peptide that has been shown to improve cachexia in heart failure and cancer and to ameliorate the hemodynamic and metabolic disturbances in septic shock. Because cytokine-induced inflammation is critical in these pathological states and because the growth hormone secretagogue receptor has been identified in blood vessels, we examined whether ghrelin inhibits proinflammatory responses in human endothelial cells in vitro and after administration of endotoxin to rats in vivo. Methods and Results—Human umbilical vein endothelial cells (HUVECs) were treated with or without tumor necrosis factor-&agr;(TNF-&agr;), and induction of proinflammatory cytokines and mononuclear cell adhesion were determined. Ghrelin (0.1 to 1000 ng/mL) inhibited both basal and TNF-&agr;–induced cytokine release and mononuclear cell binding. Intravenous administration of ghrelin also inhibited endotoxin-induced proinflammatory cytokine production in rats in vivo. Ghrelin inhibited H2O2-induced cytokine release in HUVECs, suggesting that the peptide blocks redox-mediated cellular signaling. Moreover, ghrelin inhibited basal and TNF-&agr;–induced activation of nuclear factor-κB. Des-acyl ghrelin had no effect on TNF-&agr;–induced cytokine production in HUVECs, suggesting that the antiinflammatory effects of ghrelin require interaction with endothelial growth hormone secretagogue receptors. Conclusions—Ghrelin inhibits proinflammatory cytokine production, mononuclear cell binding, and nuclear factor-κB activation in human endothelial cells in vitro and endotoxin-induced cytokine production in vivo. These novel antiinflammatory actions of ghrelin suggest that the peptide could play a modulatory role in atherosclerosis, especially in obese patients, in whom ghrelin levels are reduced.

Proinflammatory phenotype of perivascular adipocytes: influence of high-fat feeding.

Adipose tissue depots originate from distinct precursor cells, are functionally diverse, and modulate disease processes in a depot-specific manner. However, the functional properties of perivascular adipocytes, and their influence on disease of the blood vessel wall, remain to be determined. We show that human coronary perivascular adipocytes exhibit a reduced state of adipocytic differentiation as compared with adipocytes derived from subcutaneous and visceral (perirenal) adipose depots. Secretion of antiinflammatory adiponectin is markedly reduced, whereas that of proinflammatory cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1, is markedly increased in perivascular adipocytes. These depot-specific differences in adipocyte function are demonstrable in both freshly isolated adipose tissues and in vitro–differentiated adipocytes. Murine aortic arch perivascular adipose tissues likewise express lower levels of adipocyte-associated genes as compared with subcutaneous and visceral adipose tissues. Moreover, 2 weeks of high-fat feeding caused further reductions in adipocyte-associated gene expression, while upregulating proinflammatory gene expression, in perivascular adipose tissues. These changes were observed in the absence of macrophage recruitment to the perivascular adipose depot. We conclude that perivascular adipocytes exhibit reduced differentiation and a heightened proinflammatory state, properties that are intrinsic to the adipocytes residing in this depot. Dysfunction of perivascular adipose tissue induced by fat feeding suggests that this unique adipose depot is capable of linking metabolic signals to inflammation in the blood vessel wall.

Crosstalk between perivascular adipose tissue and blood vessels.

Crosstalk between cells in the blood vessel wall is vital to normal vascular function and is perturbed in diseases such as atherosclerosis and hypertension. Perivascular adipocytes reside at the adventitial border of blood vessels but until recently were virtually ignored in studies of vascular function. However, perivascular adipocytes have been demonstrated to be powerful endocrine cells capable of responding to metabolic cues and transducing signals to adjacent blood vessels. Accordingly, crosstalk between perivascular adipose tissue (PVAT) and blood vessels is now being intensely examined. Emerging evidence suggests that PVAT regulates vascular function through numerous mechanisms, but evidence to date suggests modulation of three key aspects that are the focus of this review: inflammation, vasoreactivity, and smooth muscle cell proliferation.

Low-Level Endotoxin Induces Potent Inflammatory Activation of Human Blood Vessels: Inhibition by Statins

Background—Low-level endotoxemia (ie, ≥50 pg/mL) in apparently healthy subjects was recently identified as a powerful, independent risk factor for atherosclerosis. Methods and Results—We treated human saphenous veins (HSVs) with low levels of endotoxin. Release of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA. Superoxide was determined by using the fluorescent probe dihydroethidium (HE), and monocyte binding was assessed with calcein-labeled U-937 cells. Three- to 4-fold increases in MCP-1 and IL-8 release were observed at endotoxin concentrations of 100 pg/mL; these increases were inhibited by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin. Studies in cultured endothelial cells suggest that the mechanism is related to inhibition of isoprenylation (ie, geranylgeranylation) rather than cholesterol formation. Endotoxin produced dose-dependent increases in HE fluorescence that were inhibited by the superoxide dismutase mimics Tiron and MnTBAP. Endotoxin potently induced U-937 cell binding to HSV; binding was inhibited by both Tiron and atorvastatin. Toll-like receptor-4 expression was detected in cultured HSV endothelial and smooth muscle cells and in intact HSV. Conclusions—Clinically relevant levels of endotoxin, as reported in ambulatory populations, have profound inflammatory effects on intact HSV. Inhibition of endotoxin-induced vascular inflammation might contribute to the beneficial effects of statins in treating atherosclerosis.

Endotoxin, TLR4 signaling and vascular inflammation : Potential therapeutic targets in cardiovascular disease

Cardiovascular disease ranks among the leading causes of morbidity and mortality in adult populations in the Western world. Significant progress in understanding the etiology of cardiovascular disease has come from recent recognition that chronic inflammation plays a key role in its development. The principal mediators of this inflammatory response, and the mechanisms by which they work, however, are incompletely understood. Moreover, the complex nature of the inflammatory response poses significant challenges to the development of effective and targeted treatments. Potentially promising targets to reduce inflammation in atherosclerosis include Toll-like receptor (TLR) pathways and anti-inflammatory factors that modulate TLR signaling. In this review, we outline studies that provide insight into the links between cardiovascular disease and inflammation, focusing on innate immunity and endotoxin/TLR4 signaling. We also discuss the contribution of specific host immune/inflammatory responses to atherogenesis, and describe cellular signaling pathways (lipopolysaccharide-binding protein [LBP], CD14, MD-2, TLR4, MyD88, and NF-kappaB, among others) that play key roles in innate immune signaling. Finally, we discuss the therapeutic potential of modulating these cellular signaling pathways as future strategies for the prevention and treatment of cardiovascular disease, including such approaches as specific targeting of the TLR4 signaling pathway, antibiotic therapy, drug classes with broad anti-inflammatory activity (statins, thiazolidinediones), and the potential of vaccine development. Because of the complexity of the links between low-level chronic infections, inflammation, and atherosclerosis, treatment and prevention of cardiovascular disease will likely require an integrated approach that utilizes a combination of these strategies to target the underlying inflammatory processes.

CHANGES IN SERUM INFLUENCE THE FATTY ACID COMPOSITION OF ESTABLISHED CELL LINES

SummaryThe fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines.

Prostaglandin production by 3T3-L1 cells in culture

Rapidly growing cultures of 3T3-L1 preadipocytes produce large quantities of prostaglandins when they are either stimulated with the calcium ionophore A23187 or incubated with arachidonic acid. The main prostaglandin produced under all conditions was prostaglandin E2. Prostaglandin production in response to ionophore stimulation or incubation with arachidonic acid decreased markedly, however, as the cultures approached confluence, were maintained in the confluent state, or were stimulated to differentiate. Enrichment of confluent, differentiated cultures with arachidonic acid did not enhance prostaglandin production. Recovery of prostaglandin production occurred when logarithmic growth was reinstituted by reseeding confluent cultures at low cell densities, but sparse cultures maintained in a low-growth phase did not recover the ability to produce large amounts of prostaglandin E2. Therefore, the decline in prostaglandin synthetic capacity appears to be associated with the decrease in growth rate as the cells approach confluence. Media conditioned by confluent cells reduced prostaglandin E2 production when added to rapidly growing cells, suggesting that an inhibitor of prostaglandin synthesis may be formed by the confluent cultures. Nondifferentiating 3T3 fibroblasts, which similarly release mainly prostaglandin E2, also exhibited a decrease in prostaglandin production as the cultures became confluent. The amounts of prostaglandins produced by 3T3 cells in the confluent state were much greater, however, than those produced by confluent or differentiated 3T3-L1 cultures. These findings suggest that the low capacity to produce prostaglandins may be involved in either the induction or maintenance of differentiation in 3T3-L1 cells.

Regulation of Endotoxin-Induced Proinflammatory Activation in Human Coronary Artery Cells: Expression of Functional Membrane-Bound CD14 by Human Coronary Artery Smooth Muscle Cells

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.

Epoxyeicosatrienoic Acids Increase Intracellular Calcium Concentration in Vascular Smooth Muscle Cells

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.

Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues

Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.