title: Deletion of FHL2 gene impaired ischemia-induced blood flow recovery by modulating circulating proangiogenic cells.
abstract: OBJECTIVE
  The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in inhibiting inflammatory angiogenesis. Here, we tested the hypothesis that impaired ischemia-induced neovascularization in mice lacking FHL2 is related to a defect in proangiogenic cell mobilization and functions in vasculogenesis.
  
  
  RESULTS
  Unilateral hindlimb ischemia surgery was conducted in FHL2(-/-) mice and wild-type (FHL2(+/+)) mice. After hindlimb ischemia surgery, expression of FHL2 protein was noted in ischemic tissues of wild-type mice. All FHL2-null mice (100%) suffered from spontaneous foot amputation, but only 20% of wild-type mice had ischemia-induced foot amputation after ischemic surgery. Blood flow recovery was significantly impaired in FHL2(-/-) mice when compared with that in wild-type mice as determined by laser Doppler imaging. Histological analysis revealed that the capillary density in the ischemic limb was increased in wild-type mice, whereas no such increase was noted in FHL2(-/-) mice. Flow cytometry demonstrated that the number of CD34(+) or CD34(+)/Sca-1(+)/Flk-1(+) in peripheral blood after ischemic surgery significantly decreased in FHL2-null mice than those in wild-type mice after hindlimb ischemia surgery. FHL2 deficiency impaired ex vivo angiogenesis in mouse aortic-ring culture assay, which revealed that the mean density of the microvessels was significantly higher in the wild-type aorta than in the FHL2(-/-) aorta. Western blot analysis showed that vascular endothelial growth factor (VEGF), interleukin-6, matrix metalloproteinase-2, matrix metalloproteinase-9, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 levels were significantly downregulated in ischemic muscles in FHL2-null mice compared with wild-type mice. Deletion of FHL2 protein by FHL2 small interfering RNA impaired VEGF production under hypoxia conditions, and also suppressed endothelial progenitor cell angiogenic functions, but these effects could be recovered by administration of VEGF.
  
  
  CONCLUSIONS
  Deficiency of FHL2 impairs ischemia-induced neovascularization, and these suppressive effects may occur through a reduction in proangiogenic cell mobilization, migration, and vasculogenesis functions.
authors: Division of Cardiology, Taipei Veterans General Hospital, Taipei, Taiwan.
authors: Po-Hsun
authors: PH
authors: Huang
authors: Chi-Yu
authors: CY
authors: Chen
authors: Chih-Pei
authors: CP
authors: Lin
authors: Chao-Hung
authors: CH
authors: Wang
authors: Hsiao-Ya
authors: HY
authors: Tsai
authors: Wei-Yuh
authors: WY
authors: Lo
authors: Hsin-Bang
authors: HB
authors: Leu
authors: Jaw-Wen
authors: JW
authors: Chen
authors: Shing-Jong
authors: SJ
authors: Lin
authors: Pao-Hsien
authors: PH
authors: Chu
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000097:blood; D000950:Antigens, Ly / Q000097:blood; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western; D016026:Bone Marrow Transplantation; D002196:Capillaries / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D006614:Hindlimb; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D007511:Ischemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D060850:LIM-Homeodomain Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D017078:Laser-Doppler Flowmetry; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008565:Membrane Proteins / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism* / Q000473:pathology; D018919:Neovascularization, Physiologic; D034622:RNA Interference; D020127:Recovery of Function; D012039:Regional Blood Flow; D013234:Stem Cells / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors; D046509:Tissue Culture Techniques; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000097:blood
chemical_list: D018952:Antigens, CD34; D000950:Antigens, Ly; D015415:Biomarkers; C105987:FHL2 protein, human; C491382:Fhl2 protein, mouse; D015850:Interleukin-6; D060850:LIM-Homeodomain Proteins; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D009124:Muscle Proteins; D014157:Transcription Factors; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse