39.1.3 VEGF Expression was Determined by ELISA

The sandwich ELISA method was used for the measurement of the VEGF expression. The VEGF monoclonal antibody was used for coating and the VEGF polyclonal antibody (Santa Cruz, CA USA) was used for detection. By this method, the detection sensitivity of VEGF was 9 pg/ul and reception rate was 102%. The assay was performed according to the instructions of the kit (Quantikineo, CA USA). The OD was analyzed to calculate the VEGF concentration using regression analysis according to the standard curve.

39.1.4 Statistical Analysis

The data was further analyzed using SPSS software (Windows 13.0). Then Dunnett comparison was used for comparisons of different groups.

39.2 Results

39.2.1The Maximum Inhibition of VEGF Expression by Protamine Sulfate

We found that the VEGF expression was inhibited by protamine sulfate with a dosedependent pattern from 10 to 80 ug/ml of protamine. The maximum inhibition was observed when 80 ug/ml of protamine sulfate was used. We observed that VEGF expression was reduced 47% from 810 to 383 pg/ml when 80 ug/ml of protamine sulfate was used for inhibition of VEGF expression. However, we also found that the VEGF expression could not be inhibited when 160 ug/ml of protamine sulfate was used; the reason remains unclear. Most cells died when 640 ug/ml of protamine sulfate was used for VEGF inhibition. Taken together, 80 ug/ml of protamine sulfate causes the inhibition of maximum VEGF expression (Fig. 39.1).

39.2.2Protamine Sulfate Inhibits the RF/6A Cell VEGF Expression at the Hypoxic Condition

The cells were divided into four groups including normal control group, hypoxia group, and normal with protamine sulfate treatment group and hypoxia with protamine sulfate treatment group. The final concentration of protamine sulfate is 80 ug/ml. The cells were cultured for 5 days, then 50 ul of culture medium from each group was used for VEGF measurement and 50 ul of fresh medium was added to the culture plate to keep the total volume constant. In order to have enough nutrition for the cells, we kept 20 ml medium in the plates. VEGF was detected at 0, 24, 48, 72 and 96 h after the addition of protamine sulfate. We found that (1) the

Fig. 39.1 The inhibition of VEGF expression by protamine sulfate of different concentrations. VEGF expression was inhibited by protamine sulfate with a dose-dependent pattern. The maximum inhibition of VEGF expression was observed with 80 ug/ml of protamine sulfate

VEGF expression in hypoxic condition was always higher than that of normal condition, and the VEGF expression increased significantly at 72 h, 96 h and 120 days (p <0.05). (2) The VEGF expression in hypoxia with protamine sulfate treatment group was significantly higher than that of the hypoxia group at 72 and 96 h (p <0.05, p <0.01, respectively). Accordingly, protamine sulfate could inhibit VEGF expression at hypoxic condition (Fig. 39.2).

39.2.3Protamine Sulfate Inhibits the Binding of VEGF to Its Receptor

The four groups of cells were smeared on slides for immunocytochemistry studies after 96 h of culture (Fig. 39.3b). The semi-quantity results were as follows: A = 2.70 ± 0.06, B = 5.02 ± 0.20, C = 1.03 ± 0.16, D = 3.75 ± 0.63, X2 analysis with single factor showed significant difference between different groups(F = 43.43, P = 1.02E-06; between group A and group C and between group B and group D(t = 7.10, P = 0.00086; t = 2.79; P = 0.038) (Fig. 39.3a). The staining color in protamine sulfate groups was obviously slighter than those in their control groups in normal or hypoxic condition. These results can be explained by two possibilities:

(1) protamine sulfate inhibited the binding of VEGF to its receptors; (2)protamine sulfate inhibited VEGF expression, so the VEGF-VEGFR complex was reduced. In any event, protamine sulfate can inhibit the binding of VEGF and its receptor in hypoxic condition.