High performance size exclusion chromatography of liver heparan sulfates. A Dionex HPLC system was used to equilibrate a prepacked Superdex 75 HR column (10 × 300 mm; GE Healthcare) at 0.5 ml/min in 10 mm HEPES buffer, 150 mm NaCl (pH 7.2). The anion exchange chromatography-purified liver HS samples were fractionated to check the intact full chains. Then the high molecular weight ³H-labeled male liver HS chains (A) and low molecular weight ³H-labeled female liver HS chains (B) were separated and run again on the same column to confirm the homogeneity. The male liver HS (C) and female liver HS (D) were run on the Superdex 200 HR column (10 × 300 mm) to check the size of the HS chain. The Superdex 200 column was calibrated using gel filtration high and low molecular weight protein calibration marker proteins and even-numbered heparin oligosaccharides derived from heparin. The column void (V₀) and total (V_t) volumes were determined using blue dextran 2000 and sodium dichromate, respectively. The elution volumes (V_e) of protein standards were converted into a calibration chart of K_av against molecular mass (K_av = (V_e − V_o)/(V_t − V_o). A line of best fit was fitted to the calibration data using Microsoft Excel, and the equation of this line was used to estimate mass according to observed K_av. The total number of disaccharide repeats was calculated based on the elution position of the known heparin oligosaccharides (dp2–dp26). The completed arrows indicate the elution positions of high and low molecular weight protein standards (1, ferritin; 2, aldalose; 3, conalbumin; 4, ovalbumin; 5, carbonic anhydrase; 6, ribonuclease A; 7, aprotinin), and the dotted lines indicate the elution positions of heparin oligosaccharide standards (1, dp26; 2, dp20; 3, dp16; 4, dp12; 5, dp8; 6, dp6).

Strong anion exchange high performance liquid chromatography profiles of male and female liver HS after cleavage with heparin lyases. Disaccharides were prepared by exhaustive digestion with a combination of heparinases I, II, and III; disaccharides were purified by Bio-Gel P2 gel filtration chromatography and then resolved on a Pro-Pac PA1-SAX-HPLC column eluted with a gradient of NaCl as described under “Experimental Procedures.” The elution was monitored using a UV detector at 232 nm. A, male liver HS after digestion with heparinase I, II, and III; B, female liver HS after digestion with heparinase I, II, and III. The column was calibrated using 12 unsaturated heparin disaccaride standards from Iduron (1, ΔHexUA-GlcN (Δdi-nonS); 2, ΔHexUA-GlcNAc (aΔdi-nonS); 3, ΔHexUA-GlcN,6S (Δdi-mono6S); 4, ΔHexUA,2S-GlcN (Δdi-mono2S); 5, ΔHexUA-GlcNSO₃ (Δdi-monoNS); 6, ΔHexUA-GlcNAc,6S (aΔdi-mono6S); 7, ΔHexUA,2S-GlcNAc (aΔdi-mono2S); 8, ΔHexUA,2S-GlcN,6S (Δdi-di(2,6)S); 9, ΔHexUA-GlcNSO₃,6S (Δdi-di(6,N)S); 10, ΔHexUA,2S-GlcNSO₃ (Δdi-di(2,N)S); 11, ΔHexUA,2S-GlcNAc,6S (aΔdi-di(2,6)S); 12, ΔHexUA,2S-GlcNSO₃,6S (Δdi-tri(2,6,N)S)). The numbers on the peaks correspond to the elution positions of known disaccharide standards.

Effect of male and female liver HS on the growth of hMSC. Cells were cultured for 7 days in normal maintenance medium in the presence of different concentrations (12.5, 1.25, 0.625, 0.312, or 0.156 μg/ml) of male liver HS (A) and female liver HS (B). Viable cell counts were determined at days 1, 3, 5, and 7. C, the importance of FGFR signaling for HS activity was investigated by supplementing serum-starved subconfluence cultures with male or female HS (2.5 μg/ml) or FGF2 (2.5 ng/ml) in the presence or absence of an FGFR1 inhibitor (SU5402 at 20 μm) for 60 min. Total protein was extracted for Western blotting and first probed for activated ERK1/2 expression (pERK1/2). Membranes were then striped and reprobed with anti-ERK1/2 antibody. D, the amount of pERK1/2 protein was determined by densitometry. E, cells were cultured for 5 days with male HS (2.5 μg/ml) or FGF2 (2.5 ng/ml) in the presence or absence of an FGFR1 inhibitor (SU5402 at 20 μm). Error bars, ± S.E.

The expression profile of osteogenic marker genes after 7, 14, and 21 days of HS treatment. hMSCs were seeded at 3000 cells/cm² and grown in osteogenic medium with or without liver HS for 21 days with medium changes every 3 days. Total RNA was extracted at days 7, 14, and 21, and quantitative real-time polymerase chain reaction was performed using sequence-specific primers (Table 1) and probes as markers of osteoblast differentiation. The expression levels of osteogenic markers alkaline phosphatase (ALP), BSP2, osteopontin (OPN), and Runx2 were normalized to universal 18 S ribosomal RNA. The data represent mean relative expression units (REU) ± S.E. (error bars) (n = 3).

Bio-Gel P10 size exclusion chromatography profiles of male and female liver HS after cleavage with heparinase I, heparinase II, or heparinase III. A, male liver HS after digestion with heparinase I; B, female liver HS after digestion with heparinase I; C, male liver HS after digestion with heparinase II; D, female liver HS after digestion with heparinase II; E, male liver HS after digestion with heparinase III; F, female liver HS after digestion with heparinase III. The columns void (V₀) and total (V_t) volumes were determined using hemoglobin and sodium dichromate, respectively. The column was calibrated using even-numbered heparin oligosaccharides derived from heparin. The enzyme-digested peaks were identified by the elution positions of the known heparin oligosaccharides. The arrows indicate the elution positions of heparin oligosaccharide standards (1, dp10; 2, dp6; 3, dp4; 4, dp2).

The percentage composition of sulfated and N-unsubstituted disaccharides of male and female liver tissues analyzed by SAX-HPLC. A, the percentage of total, O-, N-, 6-O-, and 2-O-sulfated disaccharides among total disaccharides of male and female liver HS. B, the percentage of N-unsubstituted disaccharides among total disaccharides of male and female liver HS. C and D, growth factors binding to male and female mouse liver HS. Shown is growth factor binding ability of male (C) and female (D) mouse liver HS coated on Iduron heparin/GAG binding plates. Error bars, ± S.E.

Osteogenic differentiation of hMSCs 21 days after induction with osteogenic medium. A, phase-contrast morphology of hMSCs grown in osteogenic medium with or without (0.3 or 1 μg/ml) liver HS after a 21-day culture period. All pictures are shown in ×10 magnification. B, mineralized nodules formed by controls and HS-treated hMSCs were stained with Alizarin red S and Von Kossa. Representative dishes of different concentrations of HS on hMSC at 21 days of culture were stored. C, quantitative analysis of Alizarin red S and Von Kossa staining density was measured with BioQuant software. Representative examples of triplicate wells are shown. Scale bar, 100 μm.

Proposed structural model of male and female mouse liver intact HS chains. The structure of male and female mouse liver HS chains has been predicted with combined data obtained from chain length (Fig. 1), heparin lyase digestion patterns (Fig. 2), SAX-HPLC disaccharide profile (Fig. 3), percentage of disaccharide composition (Table 1), and percentage of sulfated and N-unsubstituted disaccharides (Fig. 4). Note that there are three repeats of duplicated GlcA-GlcNAc, four repeats of triplicated GlcA-GlcNH₃⁺, and two repeats of triplicated GlcA-GlcNH₃⁺(6S).