中国科技论文在线 http://www.paper.edu.cn Original article 87 Telmisartan ameliorates lipopolysaccharide-induced innate immune response through peroxisome proliferator-activated receptor-g activation in human monocytes Tao Pang, Julius Benicky, Juan Wang, Martina Orecna, Enrique Sanchez-Lemus and Juan M. Saavedra Objective Angiotensin II type 1 receptor (AT1) blockers anti-inflammatory effects of telmisartan were prevented by (ARBs) reduce the bacterial endotoxin lipopolysaccharide both PPARg antagonism and PPARg gene silencing. Anti- (LPS)-induced innate immune response in human inflammatory effects of ARBs correlated with their PPARg circulating monocytes expressing few AT1. To clarify the agonist potency. mechanisms of anti-inflammatory effects of ARBs with Conclusion Our observations demonstrate that in human different peroxisome proliferator-activated receptor-g monocytes, ARBs inhibit the LPS-induced pro-inflammatory (PPARg)-activating potencies, we focused our study on response to a major extent through the PPARg activation telmisartan, an ARB with the highest PPARg-stimulating pathway and may be beneficial for the treatment of activity. cardiovascular and metabolic disorders in which Methods Human circulating monocytes and monocytic inflammation plays a major role. J Hypertens 30:87–96 Q THP-1 (human acute monocytic leukemia cell line) cells 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins. were exposed to 50 ng/ml LPS with or without pre- incubation with telmisartan. AT1 mRNA and protein Journal of Hypertension 2012, 30:87–96 expressions were determined by real-time PCR and membrane receptor binding assay, respectively. The Keywords: angiotensin receptor blockers, cytokines, inflammation, expression of pro-inflammatory factors was determined by peroxisome proliferator-activated receptor-g, transcription factors real-time PCR, western blot analysis and ELISA. PPARg Abbreviations: ABCG1, ATP-binding cassette subfamily G member 1; Ang activation was measured by electrophoretic mobility shift II, angiotensin II; ARB, Ang II receptor blocker; AT1, Ang II type 1 receptor; CD36, cluster of differentiation 36; COX-2, cyclooxygenase-2; EMSA, assay and its role was determined by pharmacological electrophoretic mobility shift assay; ERK1/2, extracellular signal-regulated inhibition and PPARg gene silencing. kinases 1/2; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; IkB- a, inhibitor of kB-a; LPS, lipopolysaccharide; MAPK, mitogen-activated Results In human monocytes, telmisartan significantly protein kinase; MCP-1, monocyte chemotactic protein-1; NF-kB, nuclear factor-kB; PGE2, prostaglandin E2; PPARg, peroxisome proliferator- attenuated the LPS-induced expression of pro- activated receptor-g; ROS, reactive oxygen species; THP-1, human acute inflammatory factors, the release of pro-inflammatory monocytic leukemia cell line; TNFa, tumor necrosis factor-a cytokines and prostaglandin E2, nuclear factor-kB Section on Pharmacology, Division of Intramural Research Programs, Department activation and reactive oxygen species formation. In THP-1 of Health and Human Services, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, USA cells, telmisartan significantly reduced LPS-induced tumor necrosis factor-a, inhibitor of kB-a, monocyte chemotactic Correspondence to Tao Pang, PhD, Section on Pharmacology, Department of Health and Human Services, National Institute of Mental Health, National protein-1 (MCP-1) and lectin-like oxidized low-density Institutes of Health, 10 Center Drive, Building 10, Room #2D-57, Bethesda, MD lipoprotein receptor-1 gene expression and MCP-1-directed 20892, USA Tel: +1 301 451 8379; fax: +1 301 402 0337; e-mail: [email protected] migration. Telmisartan also stimulated the expression of the PPARg target genes cluster of differentiation 36 and ATP- Received 7 July 2011 Revised 15 September 2011 binding cassette subfamily G member 1 in monocytes. The Accepted 5 October 2011 Introduction Most of the ARBs are biphenyl-tetrazole derivatives with Excessive angiotensin II (Ang II) type 1 receptor (AT1) similar but not identical pharmacological profiles [5]. The stimulation is associated with hypertension, and AT1 biphenyl-nontetrazole telmisartan is structurally unique blockers (ARBs) were developed for the treatment of and, in addition to its AT1 blocking properties, functions as high blood pressure to antagonize increased Ang II- a partial agonist of the peroxisome proliferator-activated dependent vasoconstriction [1]. ARBs protect end organs receptor-g (PPARg) [6], an intracellular nuclear hormone not only because they ameliorate hypertension, but also receptor regulating multiple pathways involved in carbo- as a consequence of beneficial effects on associated hydrate and lipid metabolism [7] and the control of expres- inflammatory and metabolic alterations beyond their sion of pro-inflammatory genes [8,9]. Full PPARg agonists effect on blood pressure control [2,3]. The protective reduce inflammation and metabolic alterations associated effect of ARBs is enhanced with increasing doses beyond with cardiovascular disease [10]. However, their associated full AT1 blockade [4], suggesting beneficial mechanisms toxicity limits their therapeutic value [11], and the identi- additional to AT1 blockade. fication of telmisartan as a well tolerated bifunctional 0263-6352 ß 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI:10.1097/HJH.0b013e32834dde5f 转载 中国科技论文在线 http://www.paper.edu.cn 88 Journal of Hypertension 2012, Vol 30 No 1 molecule to treat both hemodynamic and metabolic altera- All experiments were performed on nondifferentiated tions generated a high degree of enthusiasm [6]. It was later THP-1 cells. recognized that other ARBs, such as candesartan, activate PPARg in vitro and in vivo [12,13]. Angiotensin II receptor binding assay Untreated THP-1 cells (10 million) or human monocytes Anti-inflammatory effects of ARBs, first established in the (150 million) were harvested and homogenized in ice-cold peripheral vasculature [14] were later demonstrated in the buffer containing 50 mmol/l Tris-HCl pH 7.5, 1 mmol/l cerebral vasculature [15] and in stress-induced gastric ethylene glycol tetraacetic acid and 5 mmol/l MgCl2.A ulcerations [16]. These observations suggest that ARBs piece of fresh rat kidney cortex (100 mg) to be used as a may exert general anti-inflammatory effects beyond those positive control was homogenized in the same buffer. associated with cardiovascular and metabolic disease. Evi- Crude membrane fraction was pelleted by centrifugation dence in support of this hypothesis was obtained in at 20 000g for 20 min at 48C and the pellets were resus- normotensive rodents in which candesartan reduced the pended in 200 ml of the same buffer. Protein content was peripheral and brain acute inflammation following assessed by the Bradford reagent. The binding assay was systemic administration of the bacterial endotoxin lipopo- performed as previously described [23], with 0.25 nmol/l lysaccharide (LPS) [17–19]. Anti-inflammatory effects of [125I]Sar1-Ile8-Ang II as a receptor ligand. candesartan were also demonstrated in cultured circulating human monocytes expressing few AT1 [20]. Real-time PCR We hypothesized that, in human circulating monocytes Human monocytes or THP-1 cells were pre-incubated for 2 h with ARBs or vehicle (dimethylsulfoxide) followed with poor AT1 expression, the anti-inflammatory effects of ARBs may be associated with PPARg activation. We by exposure to 50 ng/ml LPS or physiological saline, compared ARBs with different PPARg-activating poten- composed of 0.9% w/v sodium chloride, for additional cies and focused on the effect of telmisartan, an ARB with 2 h, and reverse transcriptase-PCR measurements were the highest PPARg-stimulating activity. We studied LPS- performed as described [20] (see Supplemental Materials induced inflammation in monocytes as a relevant model, and Methods, http://links.lww.com/HJH/A136). because these cells are critical in the development of Western blot analysis insulin resistance, diabetes and arteriosclerosis; plasma Human monocytes were pre-incubated for 2 h with tel- LPS is elevated in obesity and diabetes [21]; and low- misartan or vehicle followed by addition of 50 ng/ml LPS grade chronic inflammation with increased expression of or physiological saline, composed of 0.9% w/v sodium monocyte-derived inflammatory factors is an established chloride, for different times, as indicated in the figures. component of vascular inflammatory disease in hyperten- Nuclear and whole-cell protein extracts were subjected to sion [14]. the separation by SDS-PAGE and analyzed as described Materials and methods in Supplemental Materials and Methods (http:// links.lww.com/HJH/A136). Detailed methods can be found in Supplemental Materials and Methods sections (http://links.lww.com/ Measurement of prostaglandin E2 production HJH/A136). Human monocytes were incubated for 24 h with 50 ng/ml LPS alone or in a combination with different concen- Human peripheral blood monocytes trations of telmisartan. The cell supernatants were col- Human peripheral blood mononuclear cells were isolated lected at the end of incubation for prostaglandin E2 by density gradient centrifugation of heparinized blood, (PGE2) measurement by PGE2 enzyme immunoassay collected by leukaphoresis of healthy volunteers at the (EIA) kit (Cayman Chemical, Ann Arbor, Michigan, Department of Transfusion Medicine, National Insti- USA) according to the manufacturer’s instructions. tutes of Health