Fig 1.
Hypoxia induced migration of V-SMC and A-SMC.
Scratch wound assays were used to determine relative migration of cells under normoxia or hypoxia. A. Representative phase contrast images of V-SMC and A-SMC immediately after the scratch (0 hour) are shown. Cells were fixed and stained with DAPI after 24-hour treatment with normoxia or hypoxia. Representative images are shown. B. Relative migration and wound closure was determined in three independent experiments. Error bars indicate S.D. Data are the mean ± SD of at least three independent experiments. * represents P<0.05.
Fig 2.
ECIS was used to determine TER. A. Flow chart of the experimental design. B. Real-time tracings of V-SMC and A-SMC, normalized to the time of wounding, representing average cellular migration over a period of 15 hours are shown. The tracings represent average TER (quadruplicates) of V-SMC and A-SMC. C. Histogram shows average rate of SMC migration from quadruplicate cultures over 5 hours. * indicates statistical significance (P <0.05).
Fig 3.
Hypoxic endothelial cell conditioned medium augments SMC migration.
A. and B. Trans cellular electrical resistance (TER) tracing indicates real-time cell migration. Endothelial cell-derived growth factors in HECM stimulated SMC to migrate faster when compared to NECM. C. Effect of endothelial cell conditioned media (NECM and HECM) on SMC migration in scratch wound assays. Cell migration was normalized to EBM-2 control (100%). Error bars indicate S.D. Data are the mean ± SD from three independent experiments. * Represents P<0.05. D and E. Effect of endothelial cell conditioned media (NECM and HECM) on SMC migration in Boyden chamber assays. Values are normalized to EBM-2 control (100%). Error bars indicate S.D. Data are the mean ± SD of three independent experiments.
Fig 4.
VEGFR-1 and PDGF-BB play a role in SMC migration.
A. and B. Real-time tracings of A-SMC and V-SMC, normalized to the time of wounding, representing average cellular migration over a period of 5 hours, with or without pre-incubation with VEGFR-1 and PDGF-BB neutralizing antibody, in the presence of HECM are shown.
Fig 5.
Hypoxia differentially up regulates VEGFR-1 in SMC.
A. RT-PCR was used to determine hypoxia-induced changes in VEGFR-1 transcript levels. Representative gel showing VEGFR-1 mRNA levels in V-SMC and A-SMC under normoxia and hypoxia. B. Representative western blot showing hypoxia induced changes in VEGFR-1 protein levels in V-SMC and A-SMC. C. RT-PCR amplified fragments were resolved in agarose gels and scanned. Values from three independent experiments are shown. Transcript levels in A-SMC under normoxic conditions are considered as 1 to calculate relative transcript levels in V-SMC in normoxia and hypoxia. Error bars indicate S.D. * Represents P <0.05. D. The figure represents relative levels of VEGFR-1 protein, normalized to A-SMC levels under normoxia. Error bars indicate S.D. Data are the mean ± SD of at least three independent experiments. * represents P <0.05.
Fig 6.
p38 MAPK pathway is involved in the paracrine-stimulation of SMC by endothelial cells.
A. Upper panel: Representative western blot shows the levels of phosphorylated p38 MAPK and total p38 MAPK in A-SMC and V-SMC, when incubated with EBM-2, NECM and HECM under hypoxia for 3 hours. Lower panel: The figure represents relative levels of phosphorylated p38 MAPK normalized to total p38 MAPK levels. Error bars indicate S.D. Data are the mean ± SD of at least three independent experiments. B. and C. Real-time tracings of V-SMC and A-SMC, normalized to the time of wounding, representing average cellular migration with or without SB203580 (p38MAPK inhibitor), in the presence of HECM, over a period of 8 hours. The tracings represent average TER (quadruplicates) of V-SMC and A-SMC in the presence or absence of SB203580.
Fig 7.
Schematic diagram showing paracrine and autocrine stimulation of SMC migration.
The diagram shows a differential increase in VEGFR-1 in V-SMC and A-SMC under hypoxia, which results in phosphorylation of p38 MAPK, leading to increased migration in V-SMC compared to A-SMC.