(A) hMEC-1 and TIME MECs treated with recMFAP5 had significantly higher motility rates than did MECs treated with the control buffer, and the stimulatory effect of MFAP5 on cell motility was abrogated in cells preloaded with the cell-permeant calcium chelator BAPTA-AM (mean ± SEM of 3 independent experiments; P < 0.01, by 2-tailed Student’s t test). (B) Fluorescence micrographs show that MFAP5-induced stress fiber formation was abrogated in MECs that had been preloaded with BAPTA-AM, suggesting that calcium signaling is involved in modulating MFAP5 function. Red: F-actin; blue: nuclei. Scale bars: 5 μm. (C–E) Mean normalized time courses of calcium mobilization induced by treating hMEC-1 cells with recMFAP5 in the absence and presence of calcium channel blockers. Calcium influx was monitored with confocal fluorescence microscopy. recMFAP5 was added to the imaging chamber at t0. Blue lines indicate the mean; red lines indicate the SEM. The inositol 1,4,5-triphosphate receptor inhibitor xestospongin C abrogated calcium mobilization, while inhibition of ryanodine receptor with ryanodine did not prevent calcium mobilization. (F) Mean normalized time courses of store-operated calcium entry. Thapsigargin was used to empty intracellular Ca²⁺ stores in the absence of extracellular Ca²⁺. Addition of Ca²⁺ to the medium at t0 resulted in rapid extracellular Ca²⁺ entry, which was inhibited by preincubation with SKF96365. Solid lines indicate the mean. Dotted lines indicate the SEM. (G) Western blot analyses showing that hMEC-1 and TIME endothelial cells treated with recMFAP5 had higher expression of p-FAK (Y861), p–PLC-γ1 (Y783), p-PKCθ (T538), p-ERK1/2 (T202/Y204), p-MLC2 (T18/S19), p-CREB (S133), c-Jun, and p–c-Jun (S73) compared with control cells. Relative normalized protein expression levels with respect to the corresponding controls are shown. Note: The blot groupings for p-CREB in hMEC-1 and TIME MECs and p-PKCθ in TIME MECs were generated from multiple gels that were run in parallel. (H) MFAP5-induced microvascular endothelial cell motility was suppressed in MECs treated with anti–α_Vβ₃ integrin antibodies. hMEC-1 and TIME MECs were treated with 50 ng/ml recMFAP5 in the presence of an anti-α₅ antibody, an anti-α_vβ₃ antibody, or the control IgG, and the effect on cell motility was determined by a Boyden chamber cell motility assay (mean ± SEM of 3 independent experiments; P < 0.01, by 2-tailed Student’s t test).