Induction of human monocyte migration by ECM.
(A) Correlation between monocyte migration and VEGF-A and PlGF levels contained in ECM. Two 105 monocytes obtained from peripheral blood were placed in the upper compartment of a modified Boyden chamber. ECM was collected at day 6 of culture and added to the lower compartment of the chamber. ECM a to ECM f corresponds to increasing ECM quantities. Different ECM volumes (a = 0.6 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 6 mL, f = 9 mL) were concentrated and used under a final volume of 600 μL so that concentration factors were 1 for a, 3.3 for b, 5 for c, 6.6 for d, 10 for e, and 15 for f. PlGF and VEGF-A contents were determined by ELISA. PlGF picograms per well, VEGF-A picograms per well: levels of PlGF × 10−1(■) and VEGF-A (▴) in the different ECMs. Number of migrated cells (♦) represents the mean number of migrated cells during 3 independent experiments ± SD. (B) Monocyte chemotactic activities of ECM and recombinant factors. Culture medium (control), chemotactic tripeptide fMLP (6 μmoles per well), VEGF-165 (10 ng per well), PlGF-1 (10 ng per well), and both VEGF-165 (10 ng per well) and PlGF-1 (10 ng per well) or ECM were added to the lower compartment. ECM a and f are described in Figure 3A. Anti-PlGF blocking antibodies (ECMf Ab PlGF), anti-VEGF blocking antibodies (ECMf Ab VEGF), or both (ECMf Ab PlGF Ab VEGF) were added to ECMf. Number of migrated cells represents the mean number of migrated cells during 3 independent experiments ± SD.
