Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in culture.

Proteoglycans (PGs) were extracted from the [35S]-sulfate labelled medium and cell layer of proliferating human gingival epithelial cells and analyzed by ion exchange and molecular sieve chromatography, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sepharose CL-6B contained two species--a quantitatively major peak at K(av) = 0.30 (M(r) congruent to 235,000 on SDS-PAGE) and a quantitatively minor peak at K(av) = 0.39. The major peak was sensitive to alkaline borohydride, shifting to K(av) = 0.45, and nitrous acid degradation, indicating the presence of heparan sulfate PG with glycosaminoglycan chains with M(r) congruent to 26,000. The minor peak is chondroitin/dermatan sulfate PG with glycosaminoglycan chains of M(r) = 22,200 as indicated by sensitivity to alkaline borohydride (shifting to K(av) = 0.48) and chondroitin ABC lyase digestion. The [35S]-sulfate labelled material from the cell layer eluted in a broad peak between 0-0.50 M NaCl from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K(av) = 0.20, 0.31, and 0.75. The largest peak (K(av) = 0.20 and M(r) congruent to 245,000 on SDS-PAGE) shifted elution position to K(av) = 0.50 after alkaline borohydride treatment and was completely sensitive to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M(r) congruent to 20,000. Two peaks containing [35S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K(av)S = 0.42 (M(r) congruent to 30,500) and 0.75 (M(r) congruent to 5300). The larger peak was predominantly chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase while the chains at K(av) = 0.75 were predominantly heparan sulfate with 83% susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.

Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment.

Failure of arteriovenous communications used for chronic hemodialysis was studied during sequential 5-year periods after placement of either endogenous Brescia-Cimino (B-C) fistulas (50 patients) or polytetrafluoroethylene (PTFE, Gore-Tex) grafts (66 patients). Venous stenosis near the anastomosis was the reason for failure in 45% of PTFE grafts compared with 16% of B-C fistulas (p less than 0.001). Failure occurred, on average, 16 months after PTFE graft placement compared with 22 for B-C fistulas (p = NS). Proximal vein segments removed from five failed and two functioning PTFE graft communications were studied using light and electron microscopy and immunocytochemical techniques. All venous segments removed during surgical shunt repair exhibited a marked intimal hyperplasia. The intimal cellular component was almost exclusively smooth muscle. Accumulation of intracellular lipid droplets was not seen. Foam cells as well as extracellular lipid deposits were absent; macrophages and lymphocytes were absent from the zone of proliferation. Ultrastructural examination revealed a large proportion of extracellular matrix surrounding smooth muscle cells in the neointima. Collagen and elastin were present in the extracellular matrix, in greatest concentration deeper in the intima. Closer to the lumen, most of the extracellular volume consisted of proteoglycan. Hemosiderin was absent from the lesions as were consistent signs of luminal and intimal fibrin. Uniform intimal gradients of actin, collagen, and proteoglycan suggest that this is a steadily progressive, rather than episodic, proliferative response. These clinical and histologic observations and an analysis of hemodynamic stresses support the postulate that upstream release of platelet-derived growth factor, and possibly, shear-induced intimal injury stimulate this response. This myointimal proliferative process provides a readily accessible model of fibromuscular hyperplasia in humans; its understanding may lead to effective methods for its prevention and may provide clues to the pathogenesis of arteriosclerosis.