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Attainment of polarity promotes growth factor secretion by retinal pigment epithelial cells: relevance to age-related macular degeneration.

Sonoda S, Sreekumar PG, Kase S, Spee C, Ryan SJ, Kannan R, Hinton DR - Aging (Albany NY) (2009)

Bottom Line: PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures.Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures.Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Keck School of Medicine of University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
The antiangiogenic and neurotrophic growth factor, pigment epithelial derived factor (PEDF), and the proangiogenic growth factor, vascular endothelial growth factor-A (VEGF), are released from retinal pigment epithelial (RPE) cells where they play a critical role in the pathogenesis of age-related macular degeneration (AMD). Since RPE polarity may be altered in advanced AMD, we studied the effect of polarization of differentiated, human RPE monolayer cultures on expression and secretion of PEDF and VEGF. Polarized RPE demonstrated apical microvilli, expression of tight junction proteins, apical localization of Na/K- ATPase, and high transepithelial resistance (490 +/- 17 Omega x cm(2)). PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures. Polarization of the RPE monolayer increased PEDF secretion, which was predominantly apical, by 34 fold (p<0.02) and VEGF secretion, which was predominantly basolateral, by 5.7 fold (p<0.02). Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures. Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

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Cell cycle analysis of polarized RPE monolayers. (A, B, C)                                            Expression of p27 (green) and its localization to nuclei (blue). (D, E,                                                    F). Polarized RPE cultures show lack of expression of Ki-67 (green) in                                            the nuclei. Nuclei counterstained blue with DAPI.
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Figure 5: Cell cycle analysis of polarized RPE monolayers. (A, B, C) Expression of p27 (green) and its localization to nuclei (blue). (D, E, F). Polarized RPE cultures show lack of expression of Ki-67 (green) in the nuclei. Nuclei counterstained blue with DAPI.

Mentions: It has been reported previously that cellular proliferation/cell cycle can influence the amount of PEDF secretion by human fibroblast-like cells [33,34]. We determined whether cells were in cycle vs cellular quiescence by evaluating the nuclear expression of Ki-67 (cells in cycle) and p27 (cellular quiescence) under three conditions, viz. confluent RPE (condition 1), confluent-quiescent non-polarized RPE (condition 2), and confluent polarized RPE (condition 3). Polarized RPE monolayers showed almost 100% positivity for p27 and barely any cells (<0.1%) positive for Ki-67 indicating that these cells were in a quiescent stage (Figure 5; Table 1). On the other hand, the just confluent non-polarized RPE cells showed an opposite staining pattern with almost 90% of cells positive for Ki-67 and <1% of cells positive for p27 indicating that these cells were in cell cycle (Table 1). To determine whether the differences in growth factor secretion between polarized and non-polarized confluent cells were due to differences in cell cycle, we also evaluated confluent-quiescent cultures (condition 2; cells cultured an additional 7 days in 1% FBS) for their expression of Ki-67 and p27, and their levels of growth factor secretion. Confluent-quiescent, non-polarized cultures were predominantly quiescent with <5% Ki-67 positivity and almost 60% p27 positivity (Table 1); a pattern that was close to that of polarized RPE monolayers (Table 1). While confluent polarized RPE showed 33.6 fold increased PEDF secretion compared to confluent non-polarized RPE, the confluent-quiescent RPE showed only a two fold increase (2.20 ± 0.21, mean ± SEM) compared to confluent non-polarized RPE cells. These results provide strong support for the contention that polarization, rather than quiescence, largely contributes to increased PEDF secretion found in confluent polarized monolayers.


Attainment of polarity promotes growth factor secretion by retinal pigment epithelial cells: relevance to age-related macular degeneration.

Sonoda S, Sreekumar PG, Kase S, Spee C, Ryan SJ, Kannan R, Hinton DR - Aging (Albany NY) (2009)

Cell cycle analysis of polarized RPE monolayers. (A, B, C)                                            Expression of p27 (green) and its localization to nuclei (blue). (D, E,                                                    F). Polarized RPE cultures show lack of expression of Ki-67 (green) in                                            the nuclei. Nuclei counterstained blue with DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2837203&req=5

Figure 5: Cell cycle analysis of polarized RPE monolayers. (A, B, C) Expression of p27 (green) and its localization to nuclei (blue). (D, E, F). Polarized RPE cultures show lack of expression of Ki-67 (green) in the nuclei. Nuclei counterstained blue with DAPI.
Mentions: It has been reported previously that cellular proliferation/cell cycle can influence the amount of PEDF secretion by human fibroblast-like cells [33,34]. We determined whether cells were in cycle vs cellular quiescence by evaluating the nuclear expression of Ki-67 (cells in cycle) and p27 (cellular quiescence) under three conditions, viz. confluent RPE (condition 1), confluent-quiescent non-polarized RPE (condition 2), and confluent polarized RPE (condition 3). Polarized RPE monolayers showed almost 100% positivity for p27 and barely any cells (<0.1%) positive for Ki-67 indicating that these cells were in a quiescent stage (Figure 5; Table 1). On the other hand, the just confluent non-polarized RPE cells showed an opposite staining pattern with almost 90% of cells positive for Ki-67 and <1% of cells positive for p27 indicating that these cells were in cell cycle (Table 1). To determine whether the differences in growth factor secretion between polarized and non-polarized confluent cells were due to differences in cell cycle, we also evaluated confluent-quiescent cultures (condition 2; cells cultured an additional 7 days in 1% FBS) for their expression of Ki-67 and p27, and their levels of growth factor secretion. Confluent-quiescent, non-polarized cultures were predominantly quiescent with <5% Ki-67 positivity and almost 60% p27 positivity (Table 1); a pattern that was close to that of polarized RPE monolayers (Table 1). While confluent polarized RPE showed 33.6 fold increased PEDF secretion compared to confluent non-polarized RPE, the confluent-quiescent RPE showed only a two fold increase (2.20 ± 0.21, mean ± SEM) compared to confluent non-polarized RPE cells. These results provide strong support for the contention that polarization, rather than quiescence, largely contributes to increased PEDF secretion found in confluent polarized monolayers.

Bottom Line: PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures.Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures.Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Keck School of Medicine of University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
The antiangiogenic and neurotrophic growth factor, pigment epithelial derived factor (PEDF), and the proangiogenic growth factor, vascular endothelial growth factor-A (VEGF), are released from retinal pigment epithelial (RPE) cells where they play a critical role in the pathogenesis of age-related macular degeneration (AMD). Since RPE polarity may be altered in advanced AMD, we studied the effect of polarization of differentiated, human RPE monolayer cultures on expression and secretion of PEDF and VEGF. Polarized RPE demonstrated apical microvilli, expression of tight junction proteins, apical localization of Na/K- ATPase, and high transepithelial resistance (490 +/- 17 Omega x cm(2)). PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures. Polarization of the RPE monolayer increased PEDF secretion, which was predominantly apical, by 34 fold (p<0.02) and VEGF secretion, which was predominantly basolateral, by 5.7 fold (p<0.02). Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures. Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

Show MeSH
Related in: MedlinePlus