title: Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.
abstract: BACKGROUND
  Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear.
  
  
  OBJECTIVE
  To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process.
  
  
  METHODS
  TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi.
  
  
  RESULTS
  Clopidogrel at 20 μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form.
  
  
  CONCLUSIONS
  Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process.
authors.affiliation: Centro de Excelencia en Medicina Traslacional, CEMT-BIOREN, Universidad de La Frontera, Temuco, Chile.|Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Alvaro
authors.identifier: http://orcid.org/0000-0003-3428-8332
authors.initials: A
authors.lastname: Cerda
authors.affiliation: Centro de Excelencia en Medicina Traslacional, CEMT-BIOREN, Universidad de La Frontera, Temuco, Chile.
authors.forename: Monica
authors.initials: M
authors.lastname: Pavez
authors.affiliation: Centro de Excelencia en Medicina Traslacional, CEMT-BIOREN, Universidad de La Frontera, Temuco, Chile.
authors.forename: Victor
authors.initials: V
authors.lastname: Manriquez
authors.affiliation: Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte, Natal, RN, Brazil.
authors.forename: Andre Ducati
authors.initials: AD
authors.lastname: Luchessi
authors.affiliation: Centro de Excelencia en Medicina Traslacional, CEMT-BIOREN, Universidad de La Frontera, Temuco, Chile.
authors.forename: Pamela
authors.initials: P
authors.lastname: Leal
authors.affiliation: Centro de Excelencia en Medicina Traslacional, CEMT-BIOREN, Universidad de La Frontera, Temuco, Chile.
authors.forename: Felipe
authors.initials: F
authors.lastname: Benavente
authors.affiliation: Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Cristina Moreno
authors.initials: CM
authors.lastname: Fajardo
authors.affiliation: Centro de Biología Molecular y Farmacogenética, CBMF-BIOREN, Universidad de La Frontera, Temuco, Chile.
authors.forename: Luis
authors.initials: L
authors.lastname: Salazar
authors.affiliation: Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Mario Hiroyuki
authors.initials: MH
authors.lastname: Hirata
authors.affiliation: Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Rosario Dominguez Crespo
authors.initials: RDC
authors.lastname: Hirata
mesh_terms: D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics; D000077144:Clopidogrel; D016207:Cytokines / Q000096:biosynthesis*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D009569:Nitric Oxide / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis / Q000235:genetics; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C111835:CCL2 protein, human; D015815:Cell Adhesion Molecules; D018932:Chemokine CCL2; D016207:Cytokines; D010975:Platelet Aggregation Inhibitors; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D000077144:Clopidogrel; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D013988:Ticlopidine
keywords: Adhesion molecules; Clopidogrel; Inflammation; Nitric oxide; Pleiotropic effects