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. 2017 Dec 18;128(2):589–606. doi: 10.1172/JCI95200

Figure 5. LPP mediates the effect of MFAP5 on endothelial cell motility and monolayer permeability.

Figure 5

(A) hMEC-1 and TIME endothelial cells treated with MFAP5 had markedly increased motility potential compared with control cells. This increase in motility induction was abrogated in cells transfected with LPP-targeting siRNA but not in cells transfected with control scrambled siRNA, which suggests that LPP mediates the effect of MFAP5 on endothelial cell motility (mean ± SEM of 3 independent experiments; *P < 0.01, by 2-tailed Student’s t test). (B) A significantly greater number of hMEC-1 and TIME cells invaded through porous Matrigel-coated cell culture inserts in the presence of recMFAP5 than in the absence of recMFAP5. The effect of MFAP5 on promoting invasive potential was abrogated in endothelial cells transfected with LPP-targeting siRNAs (mean ± SEM of 3 independent experiments; P values were determined by 2-tailed Student’s t test). (C) Micrographs show that recMFAP5 enhanced the tubular network formation of hMEC-1 and TIME cells seeded on Matrigel in a dose-dependent manner. Scale bars: 50 μm. (D) Image analyses showed dose-dependent increases in tube length, tube area, number of segments, and number of branch points for tubes formed from hMEC-1 and TIME cells seeded onto MFAP5-containing Matrigel compared with those formed from cells seeded onto control Matrigel (mean ± SEM of 3 independent experiments; P values were determined by 2-tailed Student’s t test). (E) Monolayer permeability analyses using the xCELLigence system show that MFAP5-treated, confluent endothelial cell monolayer hMEC-1 and TIME cultures had a marked decrease in impedance compared with PBS-treated cells (mean ± SEM of 4 independent experiments). (F) Effect of MFAP5 on the permeability of endothelial cell monolayers. hMEC-1 and TIME monolayers treated with recMFAP5 had a significantly greater amount of fluorescence-labeled dextran in the bottom wells of Transwells than did those treated with PBS (mean ± SEM of 3 independent experiments; P values were determined by 2-tailed Student’s t test). (G) Effect of LPP silencing on MFAP5-enhanced endothelial cell permeability. hMEC-1 and TIME monolayers treated with recMFAP5 had a significantly greater amount of fluorescence-labeled dextran in the bottom wells of Transwells than did those treated with PBS, and the effect was abrogated when hMEC-1 and TIME were transfected with LPP-targeting siRNA (mean ± SEM of 3 independent experiments; *P < 0.01, by 2-tailed Student’s t test).