PEDF and VEGF-A Output from Human Retinal Pigment Epithelial Cells Grown on Novel Microcarriers

Human retinal pigment epithelial (hRPE) cells have been tested as a cell-based therapy for Parkinson's disease but will require additional study before further clinical trials can be planned. We now show that the long-term survival and neurotrophic potential of hRPE cells can be enhanced by the use of FDA-approved plastic-based microcarriers compared to a gelatin-based microcarrier as used in failed clinical trials. The hRPE cells grown on these plastic-based microcarriers display several important characteristics of hRPE found in vivo: (1) characteristic morphological features, (2) accumulation of melanin pigment, and (3) high levels of production of the neurotrophic factors pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A). Growth of hRPE cells on plastic-based microcarriers led to sustained levels (>1 ng/ml) of PEDF and VEGF-A in conditioned media for two months. We also show that the expression of VEGF-A and PEDF is reciprocally regulated by activation of the GPR143 pathway. GPR143 is activated by L-DOPA (1 μM) which decreased VEGF-A secretion as opposed to the previously reported increase in PEDF secretion. The hRPE microcarriers are therefore novel candidate delivery systems for achieving long-term delivery of the neuroprotective factors PEDF and VEGF-A, which could have a value in neurodegenerative conditions such as Parkinson's disease

A role for myocilin in receptor-mediated endocytosis.

Myocilin is a broadly expressed protein that when mutated uniquely causes glaucoma. While no function has been ascribed to explain focal disease, some properties of myocilin are known. Myocilin is a cytoplasmic protein that also localizes to vesicles specifically as part of a large membrane-associated complex with properties similar to the SNARE machinery that function in vesicle fusion. Its role in vesicle dynamics has not been detailed, however myocilin intersects with the endocytic compartment at the level of the multivesicular body. Since internalized GPCRs are sorted in the multivesicular body, we investigated whether myocilin functions in ligand-dependent GPR143 endocytosis. Using recombinant systems we found that the kinetics of myocilin recruitment to biotinylated membrane proteins was similar to that of arrestin-3. We also co-localized myocilin with GPR143 and Arrestin-2 by confocal microscopy. However, wild-type myocilin differed significantly in its association kinetics and co-localization with internalized proteins from mutant myocilin (P370L or T377M). Moreover, we found that myocilin bound to the cytoplasmic tail of GPR143, an interaction mediated by its amino terminal helix-turn-helix domain. Hydrodynamic analyses show that the myocilin-GPR143 protein complex is >158 kD and stable in 500 mM KCl, but not 0.1% SDS. Collectively, data indicate that myocilin is recruited to the membrane compartment, interacting with GPCR proteins during ligand-mediated endocytosis and that GPCR signaling underlies pathology in myocilin glaucoma

Ligand-dependent co-localization of myocilin and GPR143.

<p>Confocal micrographs of GPR143 linked to GFP and myocilin identified by immunofluorescence. Cells were harvested at 0, 5, 20, 40, 60 after exposure to 1 µM l-DOPA. Overlaid images illustrate co-localization of GPR143 and myocilin starting at 5 minutes and continuing through 20 minutes, with little co-localization at 40 minutes and no co-localization at time 0 and 60 minutes. Insets (shown for 5, 20 and 40 min) are 4x computer magnification of a region denoted with an arrow illustrating co-localization. Bar equals 25 µM.</p

Co-localization of myocilin and arrestin 2.

<p>Confocal micrograph of arrestin 2 linked to GFP and myocilin observed by immunofluorescence at time 0, 10, and 25 minutes after l-DOPA treatment. The images are overlaid to illustrate co-localization at 10 and lack of co-localization at 0 and 25 minutes. Bar equalsµM.</p

Glaucoma-associated mutations alter ligand-stimulated recruitment of MYOC to cell surface membranes containing GPR-143.

<p>Cell surface proteins of CHO and MCF7 cells stably expressing GPR-143 and MYOC (WT, P370L or T377M) were first biotinylated then cells were treated with L-DOPA for the times indicated. (A) Biotinylated proteins were captured (bound, b) using streptavidin chromatography and distribution of myocilin (MYOC), and actin compared to unbound (u) using western blot analyses. Panel B compares densitometric analysis of band intensities of myocilin protein isoforms in bound fraction over time. Shown is a representative experiment, repeated a total of eight times (4 times each in CHO and MCF7 cells). In panel C the mean (± SD) amount of MYOC in the bound fraction are compared at 2 time points, 20 and 60 minutes from 8 experiments (* p<0.05).</p

Myocilin is part of a large complex of proteins bound to the cytoplasmic domain of GPR143.

<p>Elution fractions 3-6, containing WT MYOC from GPR143:MBP column were pooled and maintained in PBS prior to sedimentation, or adjusted to 0.1% SDS or 500 mM KCl to determine the stability of the protein complex. Samples were then subjected to glycerol gradient sedimentation, SDS-PAGE and analyzed by western blot (panel A). Densitometric analysis of the relative MYOC band intensities in the various fractions is displayed in panel B. Parallel gradients were run with proteins of known molecular weights, which were visualized by silver stain (not shown). The peaks of elution profiles for the standards are indicated with arrows and corresponding molecular weight. Results shown are from a single experiment that is representative of 3 experiments in total.</p

Interaction between myocilin and the cytoplasmic domain of GPR143.

<p>Recombinant GPR143-maltose binding protein (MBP) fusion protein or MBP alone were bound to immobilized amylose and lysates from COS cells expressing full-length myocilin tagged with GFP (MYOC-GFP) or the HTH domain (N-terminal 58 amino acids, HTH-GFP) of myocilin were passed over the columns. Columns were washed and bound proteins were eluted by competition with excess maltose. Shown are western blot analysis of elution fractions from columns containing immobilized GPR143:MBP or MBP. Results are representative of 3 experiments with each construct.</p

Time-course of ligand-dependent myocilin recruitment to cell-surface membranes containing GPR-143.

<p>After labeling cell surface proteins with biotin, CHO and MCF7 cells stably expressing MYOC and GPR143 were stimulated with l-DOPA, then proteins were harvested at the times illustrated. (A). Shown are representative western blots of cell lysates subjected to strepavidin chromatography. Proteins in bound (b) and unbound (u) fractions were probed for myocilin (MYOC) and actin abundance. (B). Specific bands from blots were analyzed using ImageJ to quantify band intensity and kinetic results of bound fraction for all three proteins analyzed for an individual experiment is shown. Results are representative of 4 experiments for each cell type tested. The mean values of the bound MYOC at time zero and at 20 min are compared for the experiments in aggregate, and found to be significantly different (* p<0.05).</p