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Dr. May J. Reed MD
Associate Professor of Medicine
University of Washington School of Medicine
Fibroblast Function In Aging:
Wound repair consists of several stages overlapping stages: inflammation, tissue deposition, and remodeling. It is generally accepted that aging is associated with delays in each of these phases, but the degree of the deficit is highly variable among aged subjects. We wished to compare the ability of dermal fibroblasts from young and aged donors to attach, spread, and migrate on collagen matrices, and then relate these functions to biochemical mediators of cellular movement: the major collagen integrin, a2b1; the matrix metalloproteases, MMP1, MMP2, MMP9, and MMP13; and the primary natural inhibitor of MMPs, TIMP1. Dermal fibroblasts from healthy young and aged male donors enrolled in the Baltimore Longitudinal Study on Aging were obtained from the NIA Aging Cell Culture Repository of the Coriell Institute for Medical Research. Cells from donors 22-30 years of age (n=5) "young fibroblasts" and 81-92 years of age (n=5) "aged fibroblasts" were analyzed. All experiments were performed on cells at less than 60% of their in vitro life span to exclude confounding by in vitro aging. Radial migration studies were performed on 100mg/ml of collagen I. Studies of attachment and adhesion were performed on plastic, 1, and 100mg/ml of collagen I. Function of the a2b1 integrin was measured by attachment to an immobilized antibody to the intact heterodimer. For biochemical analyses, all cells were plated overnight in DMEM with 5% fetal bovine serum, rinsed twice in serum-free media, then incubated for 48 hours under serum free conditions. Media were collected, cells were trypsinized, counted, and lysed in integrin lysis buffer. Media and lysates from equivalent numbers of cells were analyzed by zymography and western lotting. As expected, young fibroblasts proliferated twice as fast as aged cells. However, there was significant variability in migratory ability among aged fibroblasts, with 2 of the aged fibroblast lines demonstrating radial movement similar to that of young fibroblasts. In assays of attachment and spreading, aged and young fibroblasts showed similar adhesion on high concentrations of collagen (100mg/ml), but aged cells demonstrated impaired attachment on low concentrations of collagen (1mg/ml) and on plastic. Although biochemical analyses demonstrated similar levels of a2 integrin protein in aged and young cells, the aged fibroblasts that migrated in a manner similar to young cells demonstrated enhanced a2b1 function relative to aged cells with delayed migration. Synthesis of MMP1 and 9 were increased to a greater extent in aged fibroblasts who demonstrated a deficit in migration relative to young fibroblasts, but levels of active MMP2 were higher in all the aged donors irrespective of their motility. There was no effect of age or migratory ability on MMP13 or TIMP1. We conclude that dermal fibroblasts from healthy aged donors are variable in their migratory ability on collagen I. Relative to young fibroblasts and aged fibroblasts without a migratory deficit, aged fibroblasts with slowed migration have decreased function of a2b1 and higher levels of MMP1 and MMP9. In contrast, aged fibroblasts show increased active MMP2 compared to young cells irrespective of their migratory ability. Fibroblasts from young and aged donors had similar synthesis of MMP13 and TIMP1. These data demonstrate that functional deficits in fibroblasts with aging are variable and that certain biochemical features point to mechanisms that are operative in fibroblasts with impaired migration. Therapeutic interventions that reverse these biochemical changes may enhance wound repair in aged tissues.
Coauthor(s): Nancy Ferara