Purification and Characterization of Proteoglycan from Bovine Aortic Endothelial Cells Conditioned Media, and its Interaction with Basic Fibroblast Growth Factor (bFGF)

Cultured bovine aortic endothelial (BAE) cells were found to synthesize and secrete heparan sulfate proteoglycans (HSPG), which bound basic fibrobalst growth factor (bFGF). bFGF is a known mitogen for vascular smooth muscle cells, and is indicated to have a role in some proliferative vascular disorders. In the present study, we have purified proteoglycans from BAE cells conditioned media (BAE PG), and further separated the PG into two fractions, PG-I and PG-II, by ion exchange chromatography on a Q-Sepharose column using a linear salt gradient (0.15 M to 1.2 M). PG-I and PG-II elute at 0.85M salt and 0.1M salt respectively. BAE PG is primarily composed of heparan sulfate, which is accessible to the digestion of Heparinase I/III and nitrous acid treatment; and a small amount of chondroitin sulfate, which can be digested by Chondroitinase ABC. Gel filtration chromatography (Sepharose CL-2B and CL-4B columns) showed that BAE PG consisted of two different sized peaks, and had an average molecular weight of approximately 5 x 10⁵ Da. SDS-PAGE with silver staining indicated that BAE PG had two core proteins with estimated sizes of 300kDa and 320kDa, which corresponded to the core protein of PG-I and PG-II respectively. Western blotting with anti-perlecan primary antibody recognized the core proteins of BAE PG. Size exclusion chromatography (Sepharose CL-6B column) following β-elimination showed that BAE PG had GAG chains with an estimated size less than 2 x 10⁵ Da.

A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, K_D, of (1.6 ± 0.8) x 10⁻¹⁰ M; the dissociation rate constant, k_r, of 0.01 min⁻¹; the association rate constant, k_f, of 6.2 x 10⁷ M⁻¹min⁻¹ and the total binding sites of the proteoglycan, R_T, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds.