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Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE.

Klettner A, Kaya L, Flach J, Lassen J, Treumer F, Roider J - Mol. Vis. (2015)

Bottom Line: In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side.In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side.In contrast, an apical effect of VEGFR-2 inhibition was not found.

View Article: PubMed Central - PubMed

Affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.

ABSTRACT

Purpose: Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells.

Methods: RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed.

Results: In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the western blot experiments exhibited a significant decrease in the VEGF-A protein under SU1498 treatment. In the RPE/choroid organ cultures, PlGF was initially found mainly on the basal site with only minute amounts of PlGF found apically. NF-κB and SP-1 were strongly involved in PlGF regulation apically and basally, while VEGFR2 and to a lesser degree p38 displayed some regulation at the basal site. In the primary RPE cell culture, PlGF was not found on the apical or basal side.

Conclusions: VEGF-A and PlGF were constitutively secreted and regulated by the RPE/choroid complex, with PlGF secreted mainly by the choroid. Although the transcription factors NF-κB and SP-1 were involved in apical and basal regulation of both growth factors, VEGFR-2 displayed a strong polarity, with regulation of apical VEGF-A and basal PlGF secretion.

No MeSH data available.


Related in: MedlinePlus

Influence of p38 on apical and basal VEGF-A secretion. A: In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B: On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side (C), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h (D). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p<0.01, ++ p<0.01 +++=p<0.001. n=4 (A, B), n=6 (C, D). Bars depict the mean and the standard deviation.
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f5: Influence of p38 on apical and basal VEGF-A secretion. A: In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B: On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side (C), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h (D). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p<0.01, ++ p<0.01 +++=p<0.001. n=4 (A, B), n=6 (C, D). Bars depict the mean and the standard deviation.

Mentions: In the RPE/choroid organ culture, the inhibition of p38 displayed a less pronounced effect on the apical side, with no significant effect found at 24 h (control: 466.7±337.5 pg/ml, inhibitor: 213.3±144.5 pg/ml), and a significant decrease in VEGF-A secretion was observed at 48 h (control: 283.5±100.0 pg/ml, inhibitor: 158.5±90.1 pg/ml, p<0.05; Figure 5A). At the basal side, an effect was observed, with VEGF-A secretion significantly reduced at 24 h (control: 654.6±252.5 pg/ml, inhibitor: 283.4±213.3 pg/ml, p<0.05) and at 48 h (control: 632.4±228.1 pg/ml, inhibitor: 238.1±158.2 pg/ml, p<0.01; Figure 5B). In the cell culture, no significance influence of the inhibition of p38 on VEGF-A secretion was found on the apical side (0 h: 221.4±103.0 pg/ml; 24 h: 184.2±56.6 pg/ml, 48 h: 191.3±56.0 pg/ml; Figure 5C). In contrast, similar to the findings in the RPE/choroid, the inhibition of p38 significantly reduced the secretion of p38 on the basal side at 24 h (0 h: 680.1±175.8 pg/ml, 24 h: 534.2±164.8 pg/ml, p<0.001) and at 48 h (48 h: 518.4±176.9 pg/ml, p<0.001; Figure 5D).


Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE.

Klettner A, Kaya L, Flach J, Lassen J, Treumer F, Roider J - Mol. Vis. (2015)

Influence of p38 on apical and basal VEGF-A secretion. A: In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B: On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side (C), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h (D). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p<0.01, ++ p<0.01 +++=p<0.001. n=4 (A, B), n=6 (C, D). Bars depict the mean and the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499472&req=5

f5: Influence of p38 on apical and basal VEGF-A secretion. A: In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B: On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side (C), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h (D). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p<0.01, ++ p<0.01 +++=p<0.001. n=4 (A, B), n=6 (C, D). Bars depict the mean and the standard deviation.
Mentions: In the RPE/choroid organ culture, the inhibition of p38 displayed a less pronounced effect on the apical side, with no significant effect found at 24 h (control: 466.7±337.5 pg/ml, inhibitor: 213.3±144.5 pg/ml), and a significant decrease in VEGF-A secretion was observed at 48 h (control: 283.5±100.0 pg/ml, inhibitor: 158.5±90.1 pg/ml, p<0.05; Figure 5A). At the basal side, an effect was observed, with VEGF-A secretion significantly reduced at 24 h (control: 654.6±252.5 pg/ml, inhibitor: 283.4±213.3 pg/ml, p<0.05) and at 48 h (control: 632.4±228.1 pg/ml, inhibitor: 238.1±158.2 pg/ml, p<0.01; Figure 5B). In the cell culture, no significance influence of the inhibition of p38 on VEGF-A secretion was found on the apical side (0 h: 221.4±103.0 pg/ml; 24 h: 184.2±56.6 pg/ml, 48 h: 191.3±56.0 pg/ml; Figure 5C). In contrast, similar to the findings in the RPE/choroid, the inhibition of p38 significantly reduced the secretion of p38 on the basal side at 24 h (0 h: 680.1±175.8 pg/ml, 24 h: 534.2±164.8 pg/ml, p<0.001) and at 48 h (48 h: 518.4±176.9 pg/ml, p<0.001; Figure 5D).

Bottom Line: In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side.In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side.In contrast, an apical effect of VEGFR-2 inhibition was not found.

View Article: PubMed Central - PubMed

Affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.

ABSTRACT

Purpose: Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells.

Methods: RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed.

Results: In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the western blot experiments exhibited a significant decrease in the VEGF-A protein under SU1498 treatment. In the RPE/choroid organ cultures, PlGF was initially found mainly on the basal site with only minute amounts of PlGF found apically. NF-κB and SP-1 were strongly involved in PlGF regulation apically and basally, while VEGFR2 and to a lesser degree p38 displayed some regulation at the basal site. In the primary RPE cell culture, PlGF was not found on the apical or basal side.

Conclusions: VEGF-A and PlGF were constitutively secreted and regulated by the RPE/choroid complex, with PlGF secreted mainly by the choroid. Although the transcription factors NF-κB and SP-1 were involved in apical and basal regulation of both growth factors, VEGFR-2 displayed a strong polarity, with regulation of apical VEGF-A and basal PlGF secretion.

No MeSH data available.


Related in: MedlinePlus