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.
Highlights
The retinal pigment epithelium (RPE), strategically located between the light sensitive photoreceptors and the choroid, is a monolayer of highly specialized cells that serves as the outer blood-retinal barrier, selectively transporting biomolecules between the neural retina and choriocapillaris, and secreting factors that protect their health and integrity [1,2]
We have studied the expression and secretion of the two key growth factors linked to age‐related macular degeneration (AMD) viz pigment epithelial derived factor (PEDF) and vascular endothelial growth factor‐A (VEGF) in confluent human RPE and in highly polarized human RPE monolayers
Our data show that both PEDF and VEGF are secreted from RPE, with levels of PEDF secretion three orders of magnitude greater than that for VEGF
Summary
The retinal pigment epithelium (RPE), strategically located between the light sensitive photoreceptors and the choroid, is a monolayer of highly specialized cells that serves as the outer blood-retinal barrier, selectively transporting biomolecules between the neural retina and choriocapillaris, and secreting factors that protect their health and integrity [1,2]. It is common for differentiated cells to lose their specialized properties after multiple passages; ARPE-19 cells showed relatively low transepithelial resistance (TER) and depend on highly specific culture conditions for the development of functional tight junctions [11,12,13]. In a report comparing the barrier properties of ARPE-19, D-407, primary RPE cells from C57Bl/6 mouse, and primary human fetal RPE, only those culture systems with well differentiated monolayers showing high TER (>500 Ω·cm2) were found to be suitable for studying growth factor regulation [14]. There has been relatively little specific focus on differences between nonpolarized and highly polarized human RPE cells from individual donors with respect to the level of growth factor expression and secretion
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