The expression of heparanases is modulated by proinflammatory cytokines, and the enzymes can convert the bFGF binding and proliferation suppressive effects of ephrinB2. (A) Western blot for Hpa-1 and -2 in HUAECs and U937 cells. After stimulation of the above-mentioned substance for 24 hours, the cell lysates were obtained and underwent immunoblot assay for Hpa-1 and -2. β-Actin was used as an internal control. Two bands, about 35 kDa and about 175 kDa, could be detected in HUAECs stimulated with 20 ng/mL IL-1β or TNF-α on the immunoblot of Hpa-2 while 1 band of about 70 kDa was detected in HUAECs treated with IL-1β or TNF-α as well as U937 cells treated with ionomycin and PMA. (B) HUAEC cultures were either pretreated with 200 ng/mL preclustered ephrinB2 or clustering Ab alone in M199 media with 1% FCS for 12 hours. Then each group was divided into 2 subgroups. One subgroup received heparitinase (heparinase III; Flavobacterium heparinum [EC 4.2.2.8]) in a final concentration of 5 mIU/mL and was incubated at 37° C for 45 minutes. The other subgroup received no treatment of heparitinase. The media were then collected and centrifuged to discard the pellets and then heated to 50° C for 10 minutes to eliminate residual enzymatic activity. After returning to room temperature, the media were further divided into 2 different regimens: they either did or did not receive 10 ng/mL bFGF and then were added to 24-well HUAEC culture plates (5 × 10³ cells per well per 500 μL). The cultures were incubated for 3 days and then assessed by MTT assays. The samples of each tested group were in sextuplicate, and the experiment was performed in triplicate. Each data set was normalized with the average value of the negative control group. Bars represent the mean ± SD from 6 data sets of 1 representative experiment. eB2 indicates ephrinB2. *P < .05, significantly greater than the bFGF alone group (fourth bar from the left). #P < .05, significantly smaller than the bFGF alone group. (C) HUAECs were seeded on 12-well culture dishes in a density of 1 × 10⁵ cells per well 24 hours before the binding assay. After 2 washes with binding buffer, 1 mL conditioned media with or without pretreatment of ephrinB2 and heparitinase as in a previous experiment, combined with 20 ng bFGF-FITC, was added to each well and then incubated at 4° C for 1 hour. Each well was washed twice with binding buffer, and then 1 mL of 2 M NaCl in 20 mM sodium acetate (pH 4.0) was used to dissociate the bound bFGF-FITC. To determine the fluorescence level, 150 μL of the supernatant in each well was transferred to an ELISA plate. The samples of each tested group were in sextuplicate, and the experiment was repeated twice. Each data set was normalized with the average value of the negative control group. Bars represent the mean ± SD from 6 data sets of 1 representative experiment. eB2 indicates ephrinB2; Hpa, heparitinase. *P < .05, significantly greater than the negative control group. #P < .05, significantly smaller than the negative control group.