Figure 4
Figure 4. NF-κB pathway up-regulates VEGFR-3 expression and activates lymphatic endothelial cells. (A) VEGFR-3 promoter activity in RLECs and H-LLY cells cotransfected with VEGFR-3−849/+55 and pCMV-Flag-p50, pCMV-Flag-p65, or empty control plasmids. Promoter activity is normalized per milligram of protein. Data presented for each cell line as the mean promoter activity ± SEM of 3 independent experiments performed in duplicate ± SEM (total n = 6 per experimental condition). ***P < .001 versus control as determined by Student unpaired t test. (B) ChIP was performed using RLECs and anti-p65, -p50, and -acetylated histone H3 antibodies (positive control), or nonspecific rabbit IgG (negative control). Immunoprecipitated chromatin was visualized by PCR using primers either flanking (−403/−238 bp) or upstream of putative NF-κB binding sites (−813/−403 bp). Data are representative of 4 independent ChIP experiments with similar results. (C-F) qRT-PCR analysis of NF-κB p50 and p65 (C), E-selectin (D), VEGFR-3 (E), and LYVE-1 (F) mRNA expression in HDLECshtert treated with IL-3 (10 ng/mL) or LPS (100 ng/mL) for 6 or 24 hours. The relative expression of each target was normalized to β-actin. Data are presented as the mean values of 3 independent experiments ± SEM. *P < .05, **P < .01, and ***P < .001 versus control as determined by Student unpaired t test. (G-I) RLEC proliferation induced by 72-hour exposure to VEGF-C152S (25-200 ng/mL; G), IL-3 (5-100 ng/mL; H), and LPS (50-1000 ng/mL; I). (J) Additive proliferative effects of RLECs treated with VEGF-C152S (100 ng/mL), IL-3 (10 ng/mL), or LPS (500 ng/mL) alone compared with pretreatment with IL-3 (10 ng/mL) or LPS (500 ng/mL) followed by stimulation with VEGF-C152S (100 ng/mL). (G-J) Data are presented as the average cell number of 3 independent experiments ± SEM (total n = 6 per condition). (K) Migration of RLECs induced by treatment with VEGF-C152S (200 ng/mL), IL-3 (10 ng/mL), or LPS (500 ng/mL) and combined treatment with IL-3 (10 ng/mL) and VEGF-C152S (200 ng/mL) or LPS (500 ng/mL) and VEGF-C152S (200 ng/mL). RLEC migration toward 0.25% FBS was used as a negative control. Data presented as average fold increase in RLEC migration ± SEM of 3 independent experiments. (J-K) *P < .05, **P < .01, and ***P < .001 versus control. ##P < .01 and ###P < .001 versus cytokine treatment alone. +P < .05, ++P < .01, and +++P < .001 versus VEGF-C152S treatment alone. All statistical tests were done by Student unpaired t test.

NF-κB pathway up-regulates VEGFR-3 expression and activates lymphatic endothelial cells. (A) VEGFR-3 promoter activity in RLECs and H-LLY cells cotransfected with VEGFR-3−849/+55 and pCMV-Flag-p50, pCMV-Flag-p65, or empty control plasmids. Promoter activity is normalized per milligram of protein. Data presented for each cell line as the mean promoter activity ± SEM of 3 independent experiments performed in duplicate ± SEM (total n = 6 per experimental condition). ***P < .001 versus control as determined by Student unpaired t test. (B) ChIP was performed using RLECs and anti-p65, -p50, and -acetylated histone H3 antibodies (positive control), or nonspecific rabbit IgG (negative control). Immunoprecipitated chromatin was visualized by PCR using primers either flanking (−403/−238 bp) or upstream of putative NF-κB binding sites (−813/−403 bp). Data are representative of 4 independent ChIP experiments with similar results. (C-F) qRT-PCR analysis of NF-κB p50 and p65 (C), E-selectin (D), VEGFR-3 (E), and LYVE-1 (F) mRNA expression in HDLECshtert treated with IL-3 (10 ng/mL) or LPS (100 ng/mL) for 6 or 24 hours. The relative expression of each target was normalized to β-actin. Data are presented as the mean values of 3 independent experiments ± SEM. *P < .05, **P < .01, and ***P < .001 versus control as determined by Student unpaired t test. (G-I) RLEC proliferation induced by 72-hour exposure to VEGF-C152S (25-200 ng/mL; G), IL-3 (5-100 ng/mL; H), and LPS (50-1000 ng/mL; I). (J) Additive proliferative effects of RLECs treated with VEGF-C152S (100 ng/mL), IL-3 (10 ng/mL), or LPS (500 ng/mL) alone compared with pretreatment with IL-3 (10 ng/mL) or LPS (500 ng/mL) followed by stimulation with VEGF-C152S (100 ng/mL). (G-J) Data are presented as the average cell number of 3 independent experiments ± SEM (total n = 6 per condition). (K) Migration of RLECs induced by treatment with VEGF-C152S (200 ng/mL), IL-3 (10 ng/mL), or LPS (500 ng/mL) and combined treatment with IL-3 (10 ng/mL) and VEGF-C152S (200 ng/mL) or LPS (500 ng/mL) and VEGF-C152S (200 ng/mL). RLEC migration toward 0.25% FBS was used as a negative control. Data presented as average fold increase in RLEC migration ± SEM of 3 independent experiments. (J-K) *P < .05, **P < .01, and ***P < .001 versus control. ##P < .01 and ###P < .001 versus cytokine treatment alone. +P < .05, ++P < .01, and +++P < .001 versus VEGF-C152S treatment alone. All statistical tests were done by Student unpaired t test.

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