title: Angiogenesis related gene expression profiles of EA.hy926 cells induced by irbesartan: a possible novel therapeutic approach.
abstract: BACKGROUND
  Angiogenesis occurs commonly in various physiological and pathological processes. Improving blood supply through promoting angiogenesis is a novel approach for treating ischemic diseases. Angiotensin II type 1 receptor blockers (ARBs) dominate the management of hypertension, but evidence of their role in angiogenesis is contradictory. Here we explored the angiogenic effects of ARBs through characterizing gene expression of the human umbilical vein endothelial cell line EA.hy926 exposed to irbesartan.
  
  
  METHODS
  The human umbilical vein endothelial cell line EA.hy926 was grown for 72 hours after treatment with different concentrations of irbesartan. The cell proliferative capacity was assessed by CCK8 assay at 24, 48 and 72 hours. Gene expression levels in EA.hy926 cells responding to irbesartan were measured under optimal proliferation conditions by microarray analysis using Affymetrix U133 plus 2.0. The differential expression of genes involved in angiogenesis was identified through cluster analysis of the resulting microarray data. Quantitative RT-PCR and Western blotting analyses were used to validate differential gene expression related to the angiogenesis process.
  
  
  RESULTS
  In the 10(-4), 10(-5), 10(-6) mol/L treatment groups, cell proliferation studies revealed significantly increased proliferation in EA.hy926 cells after 24 hours of irbesartan treatment. However, after 48 and 72 hours of treatment with different concentrations of irbesartan, there was no significant difference in cell proliferation observed in any treatment group. We selected the group stimulated with irbersartan at a concentration of 10(-6) mol/L for microarray experiments. Statistical analysis of the microarray data resulted in the identification of 56 gene transcripts whose expression patterns were significantly correlated, negatively or positively, with irbesartan treatment. Cluster analysis showed that these genes were involved in angiogenesis, extracellular stimulus, binding reactions and skeletal system morphogenesis. Of these 56 genes we identified seven genes (VEGF, KDR, PTGS2, PLXND1, ROBO4, LMO2, and COL5A1) involved in the angiogenesis process. qRT-PCR analysis of these genes confirmed the microarray results. Protein expression of three VEGF pathway genes (VEGF, KDR, and PTGS2) was further confirmed by Western blotting.
  
  
  CONCLUSIONS
  Our study showed that irbesartan may induce angiogenic effects in vascular endothelial cells. It suggested that the mechanism of angiogenic effects of ARBs might be attributed to the signaling cascade from angiotensin receptors in the VEGF pathway. It also provided evidence indicating that ARBs could be used as a novel therapeutic approach to treat chronic ischemic heart disease as well as anti-hypertensive agents.
authors.affiliation: Department of Geriatric Cardiology, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Ma
authors.forename: Xue-chun
authors.initials: XC
authors.lastname: Lu
authors.forename: Yun
authors.initials: Y
authors.lastname: Luo
authors.forename: Jian
authors.initials: J
authors.lastname: Cao
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Yan
authors.initials: Y
authors.lastname: Gao
authors.forename: Xian-feng
authors.initials: XF
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Fan
mesh_terms: D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology*; D000959:Antihypertensive Agents / Q000494:pharmacology*; D001713:Biphenyl Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D000077405:Irbesartan; D017202:Myocardial Ischemia / Q000188:drug therapy; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D013777:Tetrazoles / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D047228:Angiotensin II Type 1 Receptor Blockers; D000959:Antihypertensive Agents; D001713:Biphenyl Compounds; D013777:Tetrazoles; D000077405:Irbesartan