title: Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence.
abstract: OBJECTIVE
  Venous reflux precedes the development of venous ulcers. Our earlier work showed that the fibroblasts that are cultured from these wounds display more characteristics of senescence. We evaluated fibroblast senescence in patients with venous reflux but without ulcers to further investigate the role of venous reflux in the predisposition to venous ulcers.
  
  
  METHODS
  Fibroblasts that were isolated from skin biopsy specimens of the "gaiter" area (distal) and of the ipsilateral thigh of the same patient (proximal) were compared. Twelve patients with venous reflux (9 patients in clinical, etiologic, anatomic, and pathologic classification 4; 3 patients in classification 5) with an average venous filling index of 5.45 mL/s and 4 patients without venous reflux were enrolled in the study. The growth rates, the response to basic fibroblast growth factor (b-FGF), and the senescence markers (beta-galactosidase activity at a pH level of 6, unstimulated fibroblasts fibronectin protein, and messenger RNA levels) were determined for each cell population.
  
  
  RESULTS
  The number of senescence-associated beta-galactosidase positive cells (8.3% +/- 1.9% vs 2.2% +/- 0.8%; P =.008) and the level of cellular fibronectin protein (455.7 +/- 80 vs 210 +/- 51; P =.04) and messenger RNA (16.8 +/- 6.8 vs 13.5 +/- 5.7; P =.042) were significantly higher in the distal fibroblasts as compared with the proximal fibroblast cultures. The growth rates of the distal fibroblasts were lower when compared with the proximal fibroblasts (15,746 +/- 4287 cells/day vs 29,550 +/- 5035 cells/day; P <.002) but were not different in the presence of b-FGF (41,717 +/- 9542 cells/day vs 47,030 +/- 6133 cells/day; P =.53). In the patients without venous reflux, no site differences were noted in the growth rates or the senescence markers between the proximal and distal fibroblasts.
  
  
  CONCLUSIONS
  Distal fibroblasts that are isolated from patients with venous reflux display more senescence characteristics than do proximal fibroblasts and have significantly lower growth rates. Despite senescence, b-FGF restored the distal-fibroblasts growth rate to that of the stimulated proximal fibroblasts, which proposes a therapeutic role for b-FGF. These changes precede ulcer formation and suggest a mechanism that is focal and intrinsically related to venous reflux.
authors.affiliation: Section of Vascular Surgery, Boston University Medical Center, Boston, MA 02118, USA.
authors.forename: M V
authors.initials: MV
authors.lastname: Mendez
authors.forename: A
authors.initials: A
authors.lastname: Stanley
authors.forename: T
authors.initials: T
authors.lastname: Phillips
authors.forename: M
authors.initials: M
authors.lastname: Murphy
authors.forename: J O
authors.initials: JO
authors.lastname: Menzoian
authors.forename: H Y
authors.initials: HY
authors.lastname: Park
mesh_terms: D000328:Adult; D000368:Aged; D002455:Cell Division; D002478:Cells, Cultured; D016922:Cellular Senescence; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000473:pathology*; D005353:Fibronectins / Q000032:analysis / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007866:Leg; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D012867:Skin / Q000473:pathology; D014689:Venous Insufficiency / Q000473:pathology*; D001616:beta-Galactosidase / Q000032:analysis
chemical_list: D005353:Fibronectins; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D001616:beta-Galactosidase