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  • Title: Proteoglycans in the substratum adhesion sites of human papillary or reticular dermal fibroblasts. Aging in vivo or in vitro.
    Author: Kent WM, Funderburg FM, Culp LA.
    Journal: Mech Ageing Dev; 1986 Jan; 33(2):115-37. PubMed ID: 3959599.
    Abstract:
    Sulfate-radiolabeled proteoglycans (PG) have been characterized from the tissue culture substratum adhesion sites of two human dermal fibroblast subpopulations, papillary (PAP) or reticular (RET), to determine the consequences of aging of these cells either in vivo or in vitro upon properties of these cells: matrix interfacing molecules. Cells were isolated from a 1-day-old male infant (patient 5) and a 78-year-old male (patient 8) and, after longterm radiolabeling in culture, detached from the substratum by EGTA treatment. The substratum adhesion sites were then extracted with a mixture of 1% octylglucoside, 1 M NaCl, and 0.5 M guanidine hydrochloride (GdnHCl) in acetate buffer with various protease inhibitors; these reagents quantitatively solubilize PG from adhesion sites and can be readily removed to test biological activities. PAP adhesion sites contained significantly more free chains of glycosaminoglycan than the sites of RET cells. Fractionation on DEAE-Sepharose columns under two different sets of gradient elution conditions [DEAE-I in which only acetate buffer was used; or DEAE-II in which acetate buffers were supplemented with 8 M urea] identified two major classes of PG in both PAP and RET cells - heparan sulfate proteoglycan (HS-PG) and chondroitin, dermatan sulfate proteoglycans (CS, DS-PG) - with an increased proportion of HS-PG in cells which had aged in vivo or in vitro (late-passage cells also generate a low molecular weight component that resolves on these columns). On DEAE-I columns, 70-80% of the PG forms high molecular weight aggregates that require high concentrations of urea or GdnHCl for further fractionation (DEAE-II conditions). Subsequent fractionation of the two PG classes was performed using three affinity chromatography systems. On platelet factor-4 (PF4) Sepharose columns, the HS-PGs from all cells studied bound completely and eluted with considerable heterodispersity. The CS, DS-PGs from middle-passage cells bound completely to PF4 as well but gave a more homodisperse pattern of elution; in contrast, late-passage (in vitro-aged) adhesion sites contained CS, DS-PGs that were more heterodisperse and that contained a high-avidity class. On plasma fibronectin (pFN)-Sepharose columns, the HS-PGs of middle or late-passage cells bound completely and eluted with a homodisperse pattern; in contrast, the HS-PGs from in vivo-aged cells contained 15-20% of their molecules which failed to bind to the column and a small subset which bound with greater avidity.(ABSTRACT TRUNCATED AT 400 WORDS)
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