Fig 1: Elevated pressure alters PEDF production and localization in RGCs and Müller cells in vitro. A. Graphical representation of PEDF concentration (pg/ml; y-axis) in media from primary cultures of purified RGCs exposed to ambient or elevated pressure for 48 hours, as measured by multiplex ELISA. Asterisk denotes p<0.05. B. Fluorescent micrographs of PEDF (green) and γ-synuclein (red) immunolabeling in purified, primary cultures of RGCs exposed to either ambient (top panels) or elevated (bottom panels) pressure for 48 hours reveals a reduction in localization of PEDF to neurites following exposure to elevated pressure (black arrowheads) versus ambient pressure (white arrowheads). C. Graphical representation of PEDF concentration (pg/ml; y-axis) in media from primary cultures of purified Müller cells exposed to ambient or elevated pressure for 48 hours, as measured by multiplex ELISA. D. Fluorescent micrographs of PEDF (green) and glutamine synthetase (red) immunolabeling in purified, primary cultures of Müller cells exposed to either ambient (top panels) or elevated (bottom panels) pressure for 48 hours reveals retraction in cellular processes that is associated with a reduction in PEDF staining following exposure to elevated pressure (black arrowheads), as compared to ambient pressure (white arrowheads).
Fig 2: PEDF signaling protects RGCs from pressure-induced apoptosis. A. Graphical representation of the percentage TUNEL+RGCs treated with vehicle or the PEDF-R specific inhibitor aglistatin (50 µM, 100 µM, 200 µM, 400 µM) and exposed to ambient or elevated pressure for 48 hours. Asterisks denote statistical significance (p<0.05) between vehicle and drug treatments within each pressure condition. B. Graphical representation of the same data in (A) depicted as the percentage change in TUNEL+RGCs between ambient and elevated pressure within each drug treatment. Asterisks denote statistical significance between ambient and elevated pressure within each drug treatment (p<0.05). C. Graphical representation of the percentage of TUNEL+RGCs treated with vehicle, 50ng/ml recombinant PEDF (rPEDF) or 100 ng/ml rPEDF and exposed to ambient or elevated pressure for 48 hours. Asterisks denote statistical significance (p<0.05).
Fig 3: PEDF is constitutively expressed in healthy retina. A. Graphical representation of pedf total mRNA levels in naïve C57 retina as measured by qRT-PCR. Y-axis represents the average delta of threshold cycle (Δ-CT) values for pedf normalized to the control gene gapdh. B. Representative micrograph of longitudinal sections of retina from naïve mice immunolabeled with antibody against PEDF with (right panel) and without (left) pre-absorption with recombinant PEDF (rPEDF). Morphology consistent with localization to RGCs (white arrowheads) and Müller cells (black arrowheads). C. Retinal layer-specific quantification of PEDF labeling, expressed in intensity (arbitrary units) per area (mm2). All asterisks denote p<0.05. ONL: Outer Nuclear Layer; OPL: Outer Plexiform Layer; INL: Inner Nuclear Layer; IPL: Inner Plexiform Layer; GCL: Ganglion Cell Layer; NFL: Nerve Fiber Layer.
Fig 4: PEDF is associated with RGCs and Müller cell endfeet in the ganglion cell and nerve fiber layers. A. Representative confocal micrographs with orthogonal view (bottom panel) of wholemount retina from naïve mice co-immunolabeled with antibodies against PEDF (green) and the RGC marker γ-synuclein (red). PEDF localizes to the area surrounding PEDF γ-synuclein+ RGCs in the ganglion cell layer (GCL; arrowheads). B. Representative confocal micrographs with orthogonal view (bottom panel) of wholemount retina from naïve mice co-immunolabeled with antibodies against PEDF (green) and the Müller cell marker glutamine synthetase (GluSyn; blue). PEDF localizes to the area surrounding Müller cell endfeet in the nerve fiber layer (NFL).
Fig 5: Elevated IOP increases PEDF expression in murine retina. A. Graphical representation of changes in pedf total mRNA in retina from saline- and microbead-injected mice, as measured by qPCR. Y-axis represents the fold-change in Δ-CT values for pedf (normalized to the control gene gapdh) microbead-injected, as compared to saline-injected. B. Representative micrographs of immunolabeling for PEDF in longitudinal sections of retina from saline- (right panel) and microbead-injected (left panel) mice demonstrate increased intensity of labeling in all layers of retina from microbead-injected eyes, as compared to saline-injected eyes. The pattern of PEDF immunolabeling is consistent with RGC soma in the GCL (white arrowheads) and Müller cell processes and endfeet in the ONL and NFL (black arrowheads), respectively. C. Retinal layer-specific quantification of PEDF labeling, expressed in intensity (arbitrary units) per area (mm2). All asterisks denote p<0.05. ONL: Outer Nuclear Layer; OPL: Outer Plexiform Layer; INL: Inner Nuclear Layer; IPL: Inner Plexiform Layer; GCL: Ganglion Cell Layer; NFL: Nerve Fiber Layer
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human Serpin F1/PEDF Protein, CF