Structural and functional changes in heparan sulfate proteoglycan expression associated with the myofibroblastic phenotype

Abstract

The principal cells implicated as the source of the extracellular matrix in areas of progressive fibrosis are fibroblasts with the phenotypic appearance of myofibroblasts. This report describes differences in heparan sulfate proteoglycan expression between myofibroblasts and normal fibroblasts, associated with impaired responses to fibroblast growth factor-2 (FGF-2). Although both cell types responded to platelet-derived growth factor, myofibroblasts, unlike fibroblasts, did not proliferate to FGF-2. A response was acquired, however, when myofibroblasts were incubated with FGF-2 in the presence of heparan sulfate (HS) and heparin. Selective digestion with pronase, NaOH/NaBH(4), heparinase I, or low pH nitrous acid showed that each HS-glycosaminoglycan region comprised a pronase-resistant peptide separating two HS chains. The HS-glycosaminoglycan chains from myofibroblasts were larger (K(av), 0.32; molecular weight, 50 kd) than those from fibroblasts (K(av), 0.4; molecular weight, 33 kd), although their disaccharide composition was identical. The chains from myofibroblasts, however, contained three, compared to two, heparinase 1-resistant sequences separated by larger contiguous areas of low sulfation. Furthermore, although there was no difference in FGF-2-binding affinity between the two cell types, the chains secreted by myofibroblasts had twice the binding capacity of those from fibroblasts. Thus, it is likely that the difference in response to FGF-2 is because of a difference in FGF-2 sequestration and receptor interaction with FGF-2-HS complexes. A comparative investigation into HS fine structure is being undertaken to examine these findings in more detail.

Figure 1.

Renal fibroblasts (A, C, and E) and myofibroblasts (B, D, and F) were grown to subconfluence and examined after fixation by immunochemistry for vimentin (A and B), α-SMA (C and D), or desmin (E and F). Original magnifications: ×250 (A–C, E, F); ×400 (D).

Figure 2.

The effect of growth factors on cell proliferation. Fibroblasts (□) and myofibroblasts (▪) were growth-arrested in serum-free medium for 48 hours and then incubated with either PDGF (A) or FGF-2 (B) for 48 hours. Proliferation of the cells was measured by MTT assay and the results are expressed as mean percentage (±1 SD, n = 3) of the number of cells present in the absence of growth factors.

Figure 3.

The effect of co-incubation of FGF-2 with heparin or HS chains on myofibroblast proliferation. Myofibroblasts were growth-arrested in serum-free medium for 48 hours then incubated with FGF-2 in the absence (▒) or presence (▪) of 10 U/ml of heparin (A) or HS chains (B) for 48 hours. Proliferation was measured by MTT assay and the results expressed as mean percentage (±1 SD, n = 3) of the number of cells present in the absence of the growth factor. The proliferative effect of FGF-2 on fibroblasts (□) is also shown as control.

Figure 4.

Sepharose CL-4B chromatography of ³⁵S HSPGs. Aliquots of ³⁵S-labeled proteoglycans (normalized to cell number) were incubated with chondroitin ABC lyase overnight. The remaining HSPGs were recovered by alcohol precipitation and chromatographed on a Sepharose CL-4B column. Fibroblasts (○) and myofibroblasts (•): conditioned medium (A), cell surface (B), and cell layer extract (C).

Figure 5.

Sepharose CL-6B chromatography of cell-surface HS GAG chains. Fibroblasts (○) and myofibroblasts (•) were metabolically labeled with [³H]-glucosamine for 24 hours. The trypsin extract was then obtained as described. After digestion with chondroitin ABC lyase, the remaining HS GAGs were isolated using a mini DEAE column. Aliquots were then chromatographed on a Sepharose CL-6B column after no further treatment (A), incubation with NaOH-NaBH₄ (B), and incubation with heparinase 1 (C). Results shown are representative of three identical experiments.

Figure 6.

BioGel P-10 chromatography of nitrous acid-treated HS GAGs. Aliquots of the ³H-labeled HS GAG chains from fibroblasts (○) and myofibroblasts (•) were alcohol precipitated and incubated with ice-cold nitrous acid. The products were then separated on a BioGel P-10 column equilibrated with 0.2 mol/L of NH₄HCO₃ (see Materials and Methods). A: Culture medium; B: cell surface; and C: cell layer extract. The number of disaccharides making up each oligosaccharide peak is shown. Results are representative of three identical experiments.

Figure 7.

BioGel P-10 chromatography of heparinase 3-digested HS GAGs. Aliquots of the ³H-labeled HS GAGs obtained from fibroblasts (○) and myofibroblasts (•) were alcohol precipitated and incubated with heparinase 3. The products were then separated on a BioGel P-10 column equilibrated with 0.2 mol/L of NH₄HCO₃. A: Culture medium; B: cell surface; and C: cell layer extract. Results shown are representative of three identical experiments.

Figure 8.

The binding of HS GAGs to FGF-2. ³H-labeled HS GAGs were incubated with 100 ng of _rhuman FGF-2 overnight and then passed over a 0.22-μm pore nitrocellulose membrane. Nonbound material was removed and bound material eluted with a stepwise gradient of NaCl. The relative binding affinity of the HS GAGs from fibroblasts (□) and myofibroblasts (▪) was determined as a percentage of the total eluted radioactivity (mean ± 1 SD, n = 3).