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Pigment epithelium-derived factor released by Müller glial cells exerts neuroprotective effects on retinal ganglion cells.

Unterlauft JD, Eichler W, Kuhne K, Yang XM, Yafai Y, Wiedemann P, Reichenbach A, Claudepierre T - Neurochem. Res. (2012)

Bottom Line: Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss.Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition.Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Liebigstrasse 10-14, 04103, Leipzig, Germany.

ABSTRACT
Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.

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PEDF expression in rodent retina a relative expression of PEDF mRNA in rat Müller glia cells related to β-actin compare to rat RPE cells in vitro (in thousands). There was no statistical differences (n = 4) and therefore expression levels are comparable in both cell types in vitro suggesting that Müller glia cell is a source of PEDF within retina. b–d Immunohistological staining of mouse adult retina with antibodies against cralbp (b), and PEDF (c) and the resulting merged image d together with nucleus marker DAPI. Cralbp staining reveales the typical Müller glial cells morphology running radially through the entire retina thickness from the outer limiting membrane (OLM) to the inner limiting membrane (ILM) where the glia cells extend large end feet (long arrow in b–d) embedding the retinal ganglion cell (RGC) bodies. Retinal pigmented epithelium (RPE) cells are heavily stained with both cralbp and PEDF antibody, suggesting a high synthesis of this growth factor. Note the clear exclusion zone of PEDF staining above the RPE layer delimiting the Bruch’s membrane (BM, black asterisk in d). Within the neural retina, PEDF staining co-localizes mainly with cralbp in Müller glial cells. The signal is particularly strong at their end feet (long arrow in b, c, d) in close vicinity to RGC. In addition, in the outer plexiform layer corresponding to the photoreceptor synapses area, PEDF is seen in structures devoid of cralbp staining probably accounting for perivascular structure and/or extracellular deposit (small arrow in c, d). ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; scale bar in b: 20 μm
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Fig1: PEDF expression in rodent retina a relative expression of PEDF mRNA in rat Müller glia cells related to β-actin compare to rat RPE cells in vitro (in thousands). There was no statistical differences (n = 4) and therefore expression levels are comparable in both cell types in vitro suggesting that Müller glia cell is a source of PEDF within retina. b–d Immunohistological staining of mouse adult retina with antibodies against cralbp (b), and PEDF (c) and the resulting merged image d together with nucleus marker DAPI. Cralbp staining reveales the typical Müller glial cells morphology running radially through the entire retina thickness from the outer limiting membrane (OLM) to the inner limiting membrane (ILM) where the glia cells extend large end feet (long arrow in b–d) embedding the retinal ganglion cell (RGC) bodies. Retinal pigmented epithelium (RPE) cells are heavily stained with both cralbp and PEDF antibody, suggesting a high synthesis of this growth factor. Note the clear exclusion zone of PEDF staining above the RPE layer delimiting the Bruch’s membrane (BM, black asterisk in d). Within the neural retina, PEDF staining co-localizes mainly with cralbp in Müller glial cells. The signal is particularly strong at their end feet (long arrow in b, c, d) in close vicinity to RGC. In addition, in the outer plexiform layer corresponding to the photoreceptor synapses area, PEDF is seen in structures devoid of cralbp staining probably accounting for perivascular structure and/or extracellular deposit (small arrow in c, d). ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; scale bar in b: 20 μm

Mentions: To evaluate the significance of Müller-cell related PEDF production in the retina we first compared their PEDF mRNA level to that of those cells thought to be main producers of PEDF in the eye, viz. RPE cells. Semi-quantitative real-time PCR analysis revealed that in vitro, level of PEDF mRNA in rat Müller and RPE cells were comparable (Fig. 1a). Relative PEDF mRNA levels (in thousands) related to β-actin were found to be 27.3 ± 2.0 in rat Müller cells and 17.6 ± 2.7 in rat RPE cells (n = 4). As we were testing in this study the effect of PEDF on mouse RGC we therefore analyzed the localisation of PEDF in mouse retina. We used the cralbp antibody to specifically label Müller glial cell within the retina. In addition to typical radial morphology of Müller cell in the retinal compartment, cralpb also label RPE and choroid (Fig. 1b). PEDF antibody revealed a highly similar localization. PEDF was found in cell structures running radially through the retinal layers from the outer to the inner limiting membranes (Fig. 1c). Merged image confirmed that, within the retina, PEDF was present in Müller glial cell (Fig. 1d). Signal was found especially strong at their large end feet (long arrow in Fig. 1c, d) in close contact with the nearby RGC, visualized using DAPI nuclear staining (Fig. 1d). PEDF staining was also found in the RPE but no signal was detected in the choroid as the limit of the double staining marked the Bruch’s membrane (asterisk in Fig. 1d). Based on those observations we then aimed to determine whether PEDF was able to exert a direct neuroprotective activity on pure RGC culture. We observed that hypoxia (0.2% O2) causes a compromised survival of RGC when stained by calcein-AM, which diffuses through cell membranes and is cleaved in the living cell by non-specific esterases to generate a fluorescent product, calcein. (Fig. 2a) When counting the number of calcein positive RGC versus the total number of cells in the field we found a ratio significantly reduced (°°P < 0.01) in hypoxic condition versus normoxia (Fig. 2b). Under normoxia, RGC survival in absence of growth factor was 50.3 ± 0.1% and dropped to 33.0 ± 4.3%. However, addition of PEDF (range 0.02–0.5 nM [1–25 ng/ml]) to RGC cultures resulted in a dose-dependent increase of RGC survival, under normoxia (53.6 ± 2.5%; 60.4 ± 2.3%; and 69.1 ± 4.5% with 1, 5 and 25 ng/ml of PEDF respectively) that reached significance for the two higher doses of PEDF (**P < 0.01). This neuroprotective effect was also found under hypoxia (0.2% O2) as RGC survival increased along with PEDF concentration (33.3 ± 3.9%; 38.8 ± 5.4%; 49.0 ± 6.0%); however, survival rate was found significantly improved only with 25 ng/ml PEDF (**P < 0.01). CNTF was previously reported as a strong survival factor for RGC [35] and was used here to compare the efficiency of PEDF treatments. We found a survival of 80.7 ± 5.7% under normoxia and only 54.1 ± 1.9% under hypoxia that was significantly higher than control culture in normoxia and hypoxia (**P < 0.01). With the 25 ng/ml treatment we significantly increased the RGC survival in both normoxia and hypoxia. The survival rate did not reach a plateau in normoxia, however under hypoxia, 25 ng/ml PEDF had a nearly similar neuroprotective effect as CNTF treatment.Fig. 1


Pigment epithelium-derived factor released by Müller glial cells exerts neuroprotective effects on retinal ganglion cells.

Unterlauft JD, Eichler W, Kuhne K, Yang XM, Yafai Y, Wiedemann P, Reichenbach A, Claudepierre T - Neurochem. Res. (2012)

PEDF expression in rodent retina a relative expression of PEDF mRNA in rat Müller glia cells related to β-actin compare to rat RPE cells in vitro (in thousands). There was no statistical differences (n = 4) and therefore expression levels are comparable in both cell types in vitro suggesting that Müller glia cell is a source of PEDF within retina. b–d Immunohistological staining of mouse adult retina with antibodies against cralbp (b), and PEDF (c) and the resulting merged image d together with nucleus marker DAPI. Cralbp staining reveales the typical Müller glial cells morphology running radially through the entire retina thickness from the outer limiting membrane (OLM) to the inner limiting membrane (ILM) where the glia cells extend large end feet (long arrow in b–d) embedding the retinal ganglion cell (RGC) bodies. Retinal pigmented epithelium (RPE) cells are heavily stained with both cralbp and PEDF antibody, suggesting a high synthesis of this growth factor. Note the clear exclusion zone of PEDF staining above the RPE layer delimiting the Bruch’s membrane (BM, black asterisk in d). Within the neural retina, PEDF staining co-localizes mainly with cralbp in Müller glial cells. The signal is particularly strong at their end feet (long arrow in b, c, d) in close vicinity to RGC. In addition, in the outer plexiform layer corresponding to the photoreceptor synapses area, PEDF is seen in structures devoid of cralbp staining probably accounting for perivascular structure and/or extracellular deposit (small arrow in c, d). ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; scale bar in b: 20 μm
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Related In: Results  -  Collection

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Fig1: PEDF expression in rodent retina a relative expression of PEDF mRNA in rat Müller glia cells related to β-actin compare to rat RPE cells in vitro (in thousands). There was no statistical differences (n = 4) and therefore expression levels are comparable in both cell types in vitro suggesting that Müller glia cell is a source of PEDF within retina. b–d Immunohistological staining of mouse adult retina with antibodies against cralbp (b), and PEDF (c) and the resulting merged image d together with nucleus marker DAPI. Cralbp staining reveales the typical Müller glial cells morphology running radially through the entire retina thickness from the outer limiting membrane (OLM) to the inner limiting membrane (ILM) where the glia cells extend large end feet (long arrow in b–d) embedding the retinal ganglion cell (RGC) bodies. Retinal pigmented epithelium (RPE) cells are heavily stained with both cralbp and PEDF antibody, suggesting a high synthesis of this growth factor. Note the clear exclusion zone of PEDF staining above the RPE layer delimiting the Bruch’s membrane (BM, black asterisk in d). Within the neural retina, PEDF staining co-localizes mainly with cralbp in Müller glial cells. The signal is particularly strong at their end feet (long arrow in b, c, d) in close vicinity to RGC. In addition, in the outer plexiform layer corresponding to the photoreceptor synapses area, PEDF is seen in structures devoid of cralbp staining probably accounting for perivascular structure and/or extracellular deposit (small arrow in c, d). ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; scale bar in b: 20 μm
Mentions: To evaluate the significance of Müller-cell related PEDF production in the retina we first compared their PEDF mRNA level to that of those cells thought to be main producers of PEDF in the eye, viz. RPE cells. Semi-quantitative real-time PCR analysis revealed that in vitro, level of PEDF mRNA in rat Müller and RPE cells were comparable (Fig. 1a). Relative PEDF mRNA levels (in thousands) related to β-actin were found to be 27.3 ± 2.0 in rat Müller cells and 17.6 ± 2.7 in rat RPE cells (n = 4). As we were testing in this study the effect of PEDF on mouse RGC we therefore analyzed the localisation of PEDF in mouse retina. We used the cralbp antibody to specifically label Müller glial cell within the retina. In addition to typical radial morphology of Müller cell in the retinal compartment, cralpb also label RPE and choroid (Fig. 1b). PEDF antibody revealed a highly similar localization. PEDF was found in cell structures running radially through the retinal layers from the outer to the inner limiting membranes (Fig. 1c). Merged image confirmed that, within the retina, PEDF was present in Müller glial cell (Fig. 1d). Signal was found especially strong at their large end feet (long arrow in Fig. 1c, d) in close contact with the nearby RGC, visualized using DAPI nuclear staining (Fig. 1d). PEDF staining was also found in the RPE but no signal was detected in the choroid as the limit of the double staining marked the Bruch’s membrane (asterisk in Fig. 1d). Based on those observations we then aimed to determine whether PEDF was able to exert a direct neuroprotective activity on pure RGC culture. We observed that hypoxia (0.2% O2) causes a compromised survival of RGC when stained by calcein-AM, which diffuses through cell membranes and is cleaved in the living cell by non-specific esterases to generate a fluorescent product, calcein. (Fig. 2a) When counting the number of calcein positive RGC versus the total number of cells in the field we found a ratio significantly reduced (°°P < 0.01) in hypoxic condition versus normoxia (Fig. 2b). Under normoxia, RGC survival in absence of growth factor was 50.3 ± 0.1% and dropped to 33.0 ± 4.3%. However, addition of PEDF (range 0.02–0.5 nM [1–25 ng/ml]) to RGC cultures resulted in a dose-dependent increase of RGC survival, under normoxia (53.6 ± 2.5%; 60.4 ± 2.3%; and 69.1 ± 4.5% with 1, 5 and 25 ng/ml of PEDF respectively) that reached significance for the two higher doses of PEDF (**P < 0.01). This neuroprotective effect was also found under hypoxia (0.2% O2) as RGC survival increased along with PEDF concentration (33.3 ± 3.9%; 38.8 ± 5.4%; 49.0 ± 6.0%); however, survival rate was found significantly improved only with 25 ng/ml PEDF (**P < 0.01). CNTF was previously reported as a strong survival factor for RGC [35] and was used here to compare the efficiency of PEDF treatments. We found a survival of 80.7 ± 5.7% under normoxia and only 54.1 ± 1.9% under hypoxia that was significantly higher than control culture in normoxia and hypoxia (**P < 0.01). With the 25 ng/ml treatment we significantly increased the RGC survival in both normoxia and hypoxia. The survival rate did not reach a plateau in normoxia, however under hypoxia, 25 ng/ml PEDF had a nearly similar neuroprotective effect as CNTF treatment.Fig. 1

Bottom Line: Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss.Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition.Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Liebigstrasse 10-14, 04103, Leipzig, Germany.

ABSTRACT
Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.

Show MeSH
Related in: MedlinePlus