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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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Confocal and electron microscopic characterization of polarized RPE cells.                                                Evidence for tight junction proteins and polarity in fetal RPE cells                                                cultured on Transwell filters for 6 weeks.  (A, B)                                                Immunofluorescence staining of tight junction proteins ZO-1 and occludin. (C)                                                Localization of Na/K- ATPase to the apical plasma membrane as shown in the                                                confocal vertical (X-Z) section (white arrow). (D) Well                                                differentiated apical microvilli observed by scanning electron microscopy                                                (SEM). (E) Well developed microvilli (mv), localization of pigment                                                on the apical side (asterisks), nuclei on basal side (N), and presence of                                                tight-junctional complexes (arrows) by transmission electron microscopy                                                (TEM).
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Figure 1: Confocal and electron microscopic characterization of polarized RPE cells. Evidence for tight junction proteins and polarity in fetal RPE cells cultured on Transwell filters for 6 weeks. (A, B) Immunofluorescence staining of tight junction proteins ZO-1 and occludin. (C) Localization of Na/K- ATPase to the apical plasma membrane as shown in the confocal vertical (X-Z) section (white arrow). (D) Well differentiated apical microvilli observed by scanning electron microscopy (SEM). (E) Well developed microvilli (mv), localization of pigment on the apical side (asterisks), nuclei on basal side (N), and presence of tight-junctional complexes (arrows) by transmission electron microscopy (TEM).

Mentions: As in native tissue, human RPE cells on Transwell filters formed a monolayer, were well pigmented, and were arranged in a regular hexagonal array. Confocal immunofluorescent studies of cultures grown in 1% FBS for one month showed that the intercellular assemblage outlining each cell was positively stained for tight junction protein is ZO-1 and occludin (Figure 1A, B). To establish that the cultured RPE cells exhibit polarity, we stained for the apical marker enzyme Na/K- ATPase. As expected, Na/K- ATPase was localized to the apical plasma membrane of the RPE cells as shown in the confocal vertical (X-Z) section (Figure 1C, arrow). Figure1D shows a scanning electron micrograph of the apical surface of the RPE monolayer with well-developed apicalmicrovilli. Furthermore, transmission electron micrographs show that RPE have basally located nuclei, contain melanin pigment granules that congregate on the apical side of the cytoplasm, and exhibit well-developed tight-junctional complexes and apical microvilli (Figure 1E).


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)

Confocal and electron microscopic characterization of polarized RPE cells.                                                Evidence for tight junction proteins and polarity in fetal RPE cells                                                cultured on Transwell filters for 6 weeks.  (A, B)                                                Immunofluorescence staining of tight junction proteins ZO-1 and occludin. (C)                                                Localization of Na/K- ATPase to the apical plasma membrane as shown in the                                                confocal vertical (X-Z) section (white arrow). (D) Well                                                differentiated apical microvilli observed by scanning electron microscopy                                                (SEM). (E) Well developed microvilli (mv), localization of pigment                                                on the apical side (asterisks), nuclei on basal side (N), and presence of                                                tight-junctional complexes (arrows) by transmission electron microscopy                                                (TEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Confocal and electron microscopic characterization of polarized RPE cells. Evidence for tight junction proteins and polarity in fetal RPE cells cultured on Transwell filters for 6 weeks. (A, B) Immunofluorescence staining of tight junction proteins ZO-1 and occludin. (C) Localization of Na/K- ATPase to the apical plasma membrane as shown in the confocal vertical (X-Z) section (white arrow). (D) Well differentiated apical microvilli observed by scanning electron microscopy (SEM). (E) Well developed microvilli (mv), localization of pigment on the apical side (asterisks), nuclei on basal side (N), and presence of tight-junctional complexes (arrows) by transmission electron microscopy (TEM).
Mentions: As in native tissue, human RPE cells on Transwell filters formed a monolayer, were well pigmented, and were arranged in a regular hexagonal array. Confocal immunofluorescent studies of cultures grown in 1% FBS for one month showed that the intercellular assemblage outlining each cell was positively stained for tight junction protein is ZO-1 and occludin (Figure 1A, B). To establish that the cultured RPE cells exhibit polarity, we stained for the apical marker enzyme Na/K- ATPase. As expected, Na/K- ATPase was localized to the apical plasma membrane of the RPE cells as shown in the confocal vertical (X-Z) section (Figure 1C, arrow). Figure1D shows a scanning electron micrograph of the apical surface of the RPE monolayer with well-developed apicalmicrovilli. Furthermore, transmission electron micrographs show that RPE have basally located nuclei, contain melanin pigment granules that congregate on the apical side of the cytoplasm, and exhibit well-developed tight-junctional complexes and apical microvilli (Figure 1E).

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