Research ArticleLuteolin protects against vascular inflammation in mice and TNF-alpha-induced monocyte adhesion to endothelial cells via suppressing IΚBα/NF-κB signaling pathway
Introduction
Atherosclerosis is one of the major chronic diseases in human. The formation of atherosclerotic vascular disease involves complex pathological processes. Recent basic, clinical and epidemiological studies have demonstrated that chronic inflammation plays a key role in the initiation and progression of atherosclerosis [1]. Indeed, one of the key early events in the pathogenesis of atherosclerosis is inflammation-triggered endothelial activation that leads to the adhesion of monocytes to the endothelium followed by their transmigration into the subendothelial space [2], [3], [4]. This process is primarily mediated by several intracellular signaling events that lead to the elevated expression of a number of proinflammatory chemokines, such as interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1), and several endothelial adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin. These chemokines and adhesion molecules play key roles in the firm adhesion of monocytes to the activated endothelial cells (ECs) [2], [3], [4].
Accumulating evidence suggests that tumor necrosis factor (TNF)-α, a key cytokine in the inflammatory cascade, is a proatherosclerotic factor that triggers vascular inflammation and the subsequent development of atherosclerosis [5]. TNF-α has been found to mediate interaction of invading monocytes with vascular ECs, thereby triggering extracellular matrix deposition in aortic vessels [5]. Consistently, human studies have demonstrated that TNF-α is remarkably elevated in the plasma and arteries of subjects with vascular complications [6] and that anti-TNF-α therapy improved aortic stiffness and carotid intima media thickness in patients with inflammatory arthropathies [7]. These results indicate that TNF-α is critically involved in the pathogenesis of atherosclerosis. TNF-α can trigger several intracellular signaling events that ultimately up-regulate the expression of chemokines IL-8 and MCP-1 and adhesion molecules VCAM-1, ICAM-1 and E-selectin. It is well established that activation of nuclear factor (NF)-κB is essential for the transcriptional regulation of TNF-α-induced IL-8 and MCP-1, as well as adhesion molecules [8], [9]. The p65 heterodimer, which is expressed in vascular cells, is one of the most abundant forms of the NF-κB family members. The increased nuclear translocation of the p65 subunit is detected in the intimal thickening of ECs of human atherosclerotic lesions [10]. Since inflammation-induced endothelial dysfunction is important in the development of atherosclerosis, search for agents that can attenuate TNF-α-induced NF-κB activation in ECs could be an effective strategy to prevent vascular endothelial dysfunction.
In recent years, flavonoids have drawn wide scientific attention because of their diverse health benefits, and accumulating epidemiological studies show a positive relationship between flavonoid intake and reduced risk of cardiovascular disease (CVD) [11], [12], [13], [14]. Luteolin is a bioflavonoid present in many medicinal plants as well as in some commonly consumed fruits and vegetables including green leafy spices such as parsley, sweet peppers and celery [11], [12], [13]. Previous studies showed that luteolin possess numerous beneficial medicinal properties including antioxidant, anti-inflammatory and antiallergic activity [15], [16], [17], [18], [19]. Data from in vitro studies also suggest a protective role of luteolin in the vascular system and the beneficial effect of luteolin on inflammatory process and inflammatory-associated CVD [15], [16], [17], [18], [19]. In this context, luteolin at higher doses (≥25 μM) inhibited oxidized low-density lipoprotein and TNF-α-induced VCAM-1 expression [15], [16], [17], [18], [19]. Luteolin at pharmacological concentrations showed lowering plasma lipids [20], inhibiting cholesterol biosynthesis [21] and increasing eNOS gene expression [22]. Luteolin also protected against Fe(2+)-induced lipid peroxidation and dose dependently showed potent radical scavenging ability and Fe(2+)-chelating ability [15], but those effects require higher doses that are unachievable by dietary intake of this compound.
While those previous studies provide evidence for a protective effect of luteolin against vascular dysfunction, most of the reported results reflected a pharmacological, rather than physiological, effect of luteolin because the effective concentrations used in most of the studies are well above achievable plasma luteolin levels (≤2 μM) in both rodents and humans following consumption of various luteolin-containing bioflavonoid products [14], [15], [16], [17], [18], [19]. Indeed, the average plasma concentrations of luteolin can reach 0.99 μM after the consumption of a complex meal rich in flavonoids (equivalent to average dietary intake of luteolin 8.08 mg/day per person) in 92 students with a range of 20–28 years [14]. Although the levels of conjugated luteolin can temporarily reach about 10 μM in plasma just after oral ingestion of a milligram of luteolin [15], [16], [17], [18], [19], under the healthy condition, the majority of the conjugates cannot be absorbed by the cells because they are excreted via the kidney within 25 h after ingestion [15], [16], [17], [18], [19]. Previous studies reported that the intracellular physiological concentrations of luteolin were found to be less than 2 μM [15], [16], [17], [18], [19]. As mentioned previously, the concentrations (>10 μM) used in most of the previous studies are far greater than the physiologically relevant concentrations of luteolin (<2 μM). Therefore, the biological relevance of previous findings is largely unclear, and the cellular or molecular action of luteolin at physiologically relevant concentrations needs to be further defined. In addition, the effect of luteolin on proinflammatory mediator-induced vascular inflammation in vivo is largely unknown. We thus investigated whether luteolin at physiologically achievable concentrations (≤2 μM) prevents TNF-α-induced endothelial inflammation in ECs by examining monocyte–ECs interaction, the production of chemokines and adhesion molecules, as well as the NF-κB pathway in ECs. We hypothesize that luteolin protects against vascular inflammation. We further examined the effect of dietary intake of luteolin on TNF-α-induced vascular inflammation in mice.
Section snippets
Reagents
Calcein O, where O=−diacetate tetrakis (acetoxymethyl) ester (calcein-AM), RPMI-1640 and Dulbecco’s modified Eagle’s Medium (DMEM), recombinant human TNF-α and Lipofectamine transfection reagent were purchased from Life Technologies (Grand Island, NY, USA). Recombinant murine TNF-α was from PeproTech Inc. (Rocky Hill, NJ, USA). Endothelial growth supplements EGM2 and M199 media were purchased from Lonza (Walkersville, MD, USA). Enzyme-linked immunosorbent assay (ELISA) kits for human and mouse
Luteolin inhibits TNF-α-induced binding of monocytes to ECs
Since inflammation-induced mononuclear cell adhesion to ECs is an important step in the development of atherosclerosis, we determined if luteolin can block inflammation-induced adhesion of monocytes to EA.Hy 926 cells, a permanent HUVEC line which is often used as a model of endothelium for studies of various physiological and pathological processes, especially in vascular inflammation research. Exposure of ECs to 10 ng/ml TNF-α for 24 h significantly increased adhesion of monocytes to ECs (Fig. 1
Discussion
Chronic inflammation of ECs and subsequent recruitment of monocytes into the arterial wall play an important role in initiating atherogenesis. Monocytes in the subendothelial space then develop into macrophages, which ultimately become lipid-rich foam cells, a hallmark of the atherosclerotic plaque [36]. Thus, protection of monocytes from adhesion to the endothelium could be a useful strategy for the prevention of atherosclerosis. Indeed, atherosclerosis fails to develop in animal models where
Disclosure statements
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from National Center for Complementary and Alternative Medicine in the National Institutes of Health (1R15AT005372 to Z. Jia and 1R01AT007077-01 to D. Liu).
References (60)
- et al.
17-epiestriol, an estrogen metabolite, is more potent than estradiol in inhibiting vascular cell adhesion molecule 1 (VCAM-1) mRNA expression
J Biol Chem
(2003) - et al.
Polyphenols and disease risk in epidemiologic studies
Am J Clin Nutr
(2005) - et al.
Consumption of foods rich in flavonoids is related to a decreased cardiovascular risk in apparently healthy French women
J Nutr
(2004) - et al.
Protective effect of luteolin on an oxidative-stress model induced by microinjection of sodium nitroprusside in mice
J Pharmacol Sci
(2013) - et al.
Bioavailable flavonoids to suppress the formation of 8-OHdG in HepG2 cells
Arch Biochem Biophys
(2006) - et al.
Luteolin inhibits microglia and alters hippocampal-dependent spatial working memory in aged mice
J Nutr
(2010) - et al.
Intestinal absorption of luteolin and luteolin 7-O-beta-glucoside in rats and humans
FEBS Lett
(1998) - et al.
Genistein prevents hyperglycemia-induced monocyte adhesion to human aortic endothelial cells through preservation of the cAMP signaling pathway and ameliorates vascular inflammation in obese diabetic mice
J Nutr
(2012) - et al.
Genistein inhibits TNF-alpha-induced endothelial inflammation through the protein kinase pathway A and improves vascular inflammation in C57BL/6 mice
Int J Cardiol
(2013) - et al.
Sulforaphane reduces vascular inflammation in mice and prevents TNF-alpha-induced monocyte adhesion to primary endothelial cells through interfering with the NF-kappaB pathway
J Nutr Biochem
(2014)
Glucose regulates interleukin-8 production in aortic endothelial cells through activation of the p38 mitogen-activated protein kinase pathway in diabetes
J Biol Chem
Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2-mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis
Am J Pathol
Arterial colony stimulating factor-1 influences atherosclerotic lesions by regulating monocyte migration and apoptosis
J Lipid Res
The androgen derivative 5alpha-androstane-3beta,17beta-diol inhibits tumor necrosis factor alpha and lipopolysaccharide induced inflammatory response in human endothelial cells and in mice aorta
Atherosclerosis
Hematein inhibits tumor necrotic factor-alpha-induced vascular cell adhesion molecule-1 and NF-kappaB-dependent gene expression in human vascular endothelial cells
Biochem Biophys Res Commun
Role of the histone H3 lysine 4 methyltransferase, SET7/9, in the regulation of NF-kappaB-dependent inflammatory genes. Relevance to diabetes and inflammation
J Biol Chem
Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells
J Biol Chem
Age-related changes in the elastic tissue of the human thoracic aorta
Atherosclerosis
Resveratrol prevents the development of abdominal aortic aneurysm through attenuation of inflammation, oxidative stress, and neovascularization
Atherosclerosis
Raloxifene inhibits matrix metalloproteinases expression and activity in macrophages and smooth muscle cells
Pharmacol Res
Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association
Circulation
Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation
Am J Physiol Heart Circ Physiol
Ginkgo biloba extract inhibits tumor necrosis factor-alpha-induced reactive oxygen species generation, transcription factor activation, and cell adhesion molecule expression in human aortic endothelial cells
Arterioscler Thromb Vasc Biol
Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals
Arterioscler Thromb Vasc Biol
Mechanisms of vascular smooth muscle cell migration
Circ Res
C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women
N Engl J Med
Effect of 1-year anti-TNF-alpha therapy on aortic stiffness, carotid atherosclerosis, and calprotectin in inflammatory arthropathies: a controlled study
Am J Hypertens
Inhibition of nuclear factor-kappa B nuclear localization reduces human E-selectin expression and the systemic inflammatory response
Circulation
Hyperglycemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells
Diabetes
Flavonoids for reduction of atherosclerotic risk
Curr Atheroscler Rep
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