PMC

VEGF increases the expression of P-Rex1 and CXCR4 in human microvascular endothelial cells. A, total RNA was isolated from control of VEGF-stimulated HMEC cells stimulated with 100 ng/ml VEGF for the indicated times. RT-PCR assays were done to detect the expression of P-Rex1, CXCR4, and TIAM. A representative experiment is shown. B, -fold increase in mRNA expression was determined by densitometric analysis after normalization using β-actin as control. The mean and S.E.M values of seven experiments are shown. *, p < 0.05 difference with respective unstimulated controls.

P-Rex1 is an essential element in the angiogenic response elicited by SDF-1. A, P-Rex1 knockdown in endothelial cells caused a marked inhibition of the angiogenic effect elicited by SDF-1 but not by VEGF. Serum-starved P-Rex1 knockdown (shP-Rex1) or control endothelial cells (sh control) were added onto Matrigel and incubated without (negative control) or with 100 ng/ml SDF-1, 100 ng/ml VEGF, or 5% FBS at 37°C in a5%CO₂ atmosphere for 9 to 12 h. Representative photographs show the angiogenic effect of the indicated stimuli assessed as the acquisition of a cord-like interconnected structure of endothelial cells. B, bars represent the quantitative analysis of the angiogenic effect of SDF-1 or VEGF assessed as the capillary tube length normalized to the effect of FBS. Data are shown as mean + S.E.M of six experiments for SDF-1 and FBS and three experiments for VEGF. *, p < 0.001 difference with the corresponding effect in the control group.

SDF-1 induces microvascular endothelial cell migration via PRex1. P-Rex1 knockdown or control HMEC cells were subjected to chemotaxis assays in Boyden chambers. Cells were starved for 12 h in serum-free media and stimulated with different concentrations of SDF-1 as indicated (from 50 to 200 ng/ml) or VEGF (100 ng/ml) for6hat 37°C, ina5%CO₂ atmosphere. FBS (10%) was used as positive control, basal migration was assessed in media lacking chemoattractants. Migrating cells were stained, and relative cell migration was determined by densitometry of scanned filters. Relative migration was obtained by adjusting to the effect observed in cells migrating toward FBS, which was adjusted to 100%. Three independent experiments were averaged and plotted. Error bars indicate S.E.M., *, p < 0.001 difference with control group. A representative filter of a migration assay is shown.

SDF-1 promotes Rac1 activation via P-Rex1 in microvascular endothelial cells. A, P-Rex1 knockdown in endothelial cells. HMEC cells were infected with lentiviral vectors carrying the sequence to produce a P-Rex1-specific shRNA (shPRex-1) or a scrambled sequence for a control shRNA (sh control). Cell lysates were resolved on SDS-PAGE gels and analyzed by immunoblotting with P-Rex1 and GAPDH antibodies. B, P-Rex1 knockdown or control HMEC cells, infected with the corresponding lentiviral vectors, were stimulated with 50 ng/ml SDF-1 or 10 ng/ml VEGF for 5 and 1 min, respectively. The active form of Rac (Rac-GTP) was isolated with GST-PAK-CRIB beads. Total cell lysates and pull-downs were resolved on SDS-PAGE gels and analyzed by immunoblotting for Rac1, phospho-ERK (pERK), ERK-2, and GAPDH. A representative blot detecting the indicated proteins is shown. C, relative -fold increase of Rac activation and ERK phosphorylation was quantified by densitometric analysis of six independent experiments and adjusted to the effect of VEGF in control cells which was set to 100%. Error bars indicate S.E.M.; *, p < 0.05 difference with the corresponding effect in the control group.

Effect of VEGF-pretreatment on SDF-1-induced migration and angiogenic response in human microvascular endothelial cells. A, VEGF-pretreatment increases SDF-1-induced endothelial cell migration. Serum-starved HMEC cells, incubated with or without VEGF (100 ng/ml) for 12 h, were subjected to chemotaxis assays. Cells were stimulated with different concentrations of SDF-1 (5 or 50 ng/ml), VEGF (100 ng/ml), SDF-1 + VEGF (S+V, 50 and 100 ng/ml, respectively), or 10% FBS for6hat 37°C in a 5% CO₂ atmosphere. Relative cell migration (percentage of FBS) was determined by comparing control cells with cells preincubated with VEGF. Three independent experiments were averaged and plotted. A representative filter of a migration assay is shown. Graph represents the average results of three independent experiments. Error bars indicate S.E.M., *, p < 0.05 versus the same concentration of ligand for control group (no VEGF pretreatment). B, HMEC control and VEGF-pretreated cells were subjected to in vitro angiogenesis assays on Matrigel in the presence of 100 ng/ml SDF-1, 100 ng/ml VEGF, or 5% FBS at 37°C, 5% CO₂ for 9 to 12 h. As a negative control, cells were incubated in serum-free media (basal). Length of cord-like structures was quantified using ImageJ program and normalized to the effect of FBS, which was considered 100%. Graph represents the average results of three independent experiments. Error bars indicate S.E.M., **, p < 0.001 versus the same concentration of ligand for control group (no VEGF pretreatment).

SDF-1 induces Rac1 activation, endothelial cell migration, and in vitro angiogenesis. A, expression of CXCR4 and CXCR7 in HMEC and MDA-MB-231 cells. Extraction of total RNA was performed as described under Materials and Methods, and RT-PCR assays of MDA-MB-231 and HMEC cells were performed. As shown, CXCR4 was the only receptor for SDF1 detected in HMEC cells. B, HMEC cells, starved in serum-free media, were stimulated with 50 ng/ml SDF-1 or 10 ng/ml VEGF for the indicated times. The active form of Rac (Rac-GTP) was isolated using GST-PAK-CRIB beads. Total cell lysates and pull-downs were resolved on SDS-polyacrylamide gels and analyzed by immunoblotting for Rac1, phospho-ERK (pERK), ERK-2, and actin. A representative experiment is shown. C, relative -fold increase of Rac activation and ERK phosphorylation was quantified by densitometric analysis of three independent experiments and adjusted to the effect of VEGF, which was set to 100%. D, serum-starved HMEC cells were subjected to chemotaxis assays in serum-free media containing the indicated concentrations of SDF-1 (from 25 to 200 ng/ml) or 10% FBS used as positive control. Migration assays were done in Boyden chambers for6hat 37°C in a 5% CO₂ atmosphere. Migrating cells were stained, and the relative cell migration was quantified by densitometry of scanned filters; a representative filter is shown. Graphs represent the average results of three independent experiments, error bars indicate S.E.M. E, serum-starved HMEC cells were subjected to in vitro angiogenesis assays on Matrigel in the presence of 100 ng/ml SDF-1 or 5% FBS at 37°C, 5% CO₂ for 9 to 12 h. As a negative control, cells were incubated in the absence of SDF-1 or FBS. Length of cord-like structures was quantified using ImageJ program and normalized to the effect of FBS, which was considered 100%. Bars represent the average results of three independent experiments. Error bars indicate S.E.M.; *, p < 0.05 difference with the corresponding effect in the control group.