Expression and secretion of MMPs by purified MKs.

(A) RT-PCR analysis of purified MKs revealed the expression of MMP-9 and TIMP-1, but not of MMP-2. (B) Day 14 ex vivo–expanded MKs (double pure) were cultured with or without rhSDF-1 (200 ng/mL per day) and thrombin (1 U/mL) for 48 hours, and the culture supernatants were analyzed by gelatinolytic zymography. Supernatants from MK cultures have gelatinolytic activity corresponding to human pro–MMP-9 (92 kd) and active MMP-9 (83-88 kd). (C) Quantification of the proteolytic activity detected in MK culture supernatants by densitometry. Data are shown as relative densitometry units. SDF-1 and thrombin stimulation of MK resulted in 95% and 60% increases in pro–MMP-9 and 200% and 65% increases in active MMP-9, respectively. (D) Western blot analysis of MK culture supernatants confirmed the increased expression of pro–MMP-9 on stimulation with SDF-1 and thrombin. Results are representative of 3 separate experiments.