HMWHA and LMWHA treated with hyaluronidase inhibit fibrocyte differentiation.

<p>(<b>A</b>) HMWHA and LMWHA were treated with 10 U/ml of hyaluronidase for 1 and 4 hours at 37°C. Samples were analyzed by agarose gel electrophoresis. M, 1 Kb ladder; Lane 1, HMWHA; Lane 2, HMWHA incubated with hyaluronidase for 1 hour; Lane 3, HMWHA incubated with hyaluronidase for 4 hours; Lane 4, LMWHA; Lane 5, LMWHA incubated with hyaluronidase for 1 hour; Lane 6, LMWHA incubated with hyaluronidase for 4 hours; Lane 7, PBS; Lane 8, PBS incubated with hyaluronidase for 1 hour; and Lane 9, PBS incubated with hyaluronidase for 4 hours. (<b>B</b>) Human PBMC were cultured in 300 µg/ml of 4 hours-hyaluronidase-treated or untreated HMWHA and LMWHA. After 5 days, the cells were air dried, fixed, stained, and the number of fibrocytes was counted. The results are mean ± SEM of SFM percent control (n = 6 separate experiments). The number of fibrocytes in SFM controls from the 6 donors was 883, 1029, 808, 288, 400, and 288. * indicates p<0.05, ** p<0.01, *** p<0.001 as determined by t-test. Using the non-parametric Mann Whitney two-tailed t-test, 300 µg/ml HMWHA significantly increases the number of fibrocytes when compared to control with p<0.001, but 300 µg/ml hyaluronidase incubated HMWHA is not significantly different from control. Also, 300 µg/ml LMWHA significantly decreases the number of fibrocytes when compared to control with p<0.001, and 300 µg/ml hyaluronidase incubated LMWHA also significantly decreases the number of fibrocytes when compared to control with p<0.001. Using the non-parametric Mann Whitney one-tailed t-test, 300 µg/ml hyaluronidase incubated HMWHA significantly decreases the number of fibrocytes when compared to control with p<0.05.