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Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia.

Dharmarajan S, Gurel Z, Wang S, Sorenson CM, Sheibani N, Belecky-Adams TL - Mol. Vis. (2014)

Bottom Line: However, as expected, the profiles including the oxidative agent and growth factor were not identical.Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment.BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University-Purdue University Indianapolis,, Indianapolis, IN ; Center for Regenerative Biology and Medicine, Indiana University- Purdue University Indianapolis, Indianapolis, IN.

ABSTRACT

Purpose: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells.

Methods: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry.

Results: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection.

Conclusions: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

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Bone morphogenetic protein (BMP) type I receptors in the mature mouse retina. A: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize Müller glial and retinal astrocyte marker glutamine synthetase (GS) and BMP receptor 1A (BMPR1A). Thin plane confocal microscopy with y,z (strips to right of panels), and x,z planes (strips at bottom of panels) are shown in the last panels in the row. BMPR1A was localized to the Müller glial processes in the outer nuclear layer (ONL) and in Müller glial cell or retinal astrocyte processes in the ganglion cell layer (GCL, arrows A-C). BMPR1A was also detectable in the photoreceptor outer segments, the inner plexiform layer (IPL), cell bodies within the ganglion cell layer, and the nerve fiber layer (NFL). B: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and BMPR1B. BMPR1B appeared to be localized to the outer segments (OS), outer plexiform layer (OPL) and IPL and, Müller glial cell processes as well as Müller glial/retinal astrocyte processes in the GCL and NFL (arrows). C: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and activin receptor like kinase 2 (ALK2). The distribution of ALK2 receptors was similar to that of BMPR1A, with the majority of signal being localized to the end feet within the GCL and NFL. D: Müller glia isolated from P30 retinas were co-labeled for GS and BMPR1A, 1B and ALK2. The isolated cells showed the distribution of receptors to be primarily in the processes and the end feet of the Müller glia, as seen in the P30 tissues. n=3 different eyes for each label. Scale bar A=50 μm applies to A, B, C.
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f1: Bone morphogenetic protein (BMP) type I receptors in the mature mouse retina. A: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize Müller glial and retinal astrocyte marker glutamine synthetase (GS) and BMP receptor 1A (BMPR1A). Thin plane confocal microscopy with y,z (strips to right of panels), and x,z planes (strips at bottom of panels) are shown in the last panels in the row. BMPR1A was localized to the Müller glial processes in the outer nuclear layer (ONL) and in Müller glial cell or retinal astrocyte processes in the ganglion cell layer (GCL, arrows A-C). BMPR1A was also detectable in the photoreceptor outer segments, the inner plexiform layer (IPL), cell bodies within the ganglion cell layer, and the nerve fiber layer (NFL). B: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and BMPR1B. BMPR1B appeared to be localized to the outer segments (OS), outer plexiform layer (OPL) and IPL and, Müller glial cell processes as well as Müller glial/retinal astrocyte processes in the GCL and NFL (arrows). C: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and activin receptor like kinase 2 (ALK2). The distribution of ALK2 receptors was similar to that of BMPR1A, with the majority of signal being localized to the end feet within the GCL and NFL. D: Müller glia isolated from P30 retinas were co-labeled for GS and BMPR1A, 1B and ALK2. The isolated cells showed the distribution of receptors to be primarily in the processes and the end feet of the Müller glia, as seen in the P30 tissues. n=3 different eyes for each label. Scale bar A=50 μm applies to A, B, C.

Mentions: BMPs bind preferentially to three type I receptors; receptor IA, IB, or ALK2. Double-label immunofluorescence was used to determine whether any of the type I BMP receptors was expressed by Müller cells or retinal astrocytes. Sections through adult murine retina were colabeled with antibodies specific for GS, which labels Müller glial cells and retinal astrocytes, and BMPR1A, BMPR1B, or ALK2 (Figure 1). All three receptors showed similar labeling patterns in the retina with Müller glial cell processes labeled in the outer nuclear layer (ONL), outer plexiform layer (OPL), and GCL (Figure 1A-C). Photoreceptor outer segments (OS) and ganglion cell bodies were also clearly labeled for all three receptors. To confirm the label of Müller glial cells and their processes by antibodies to type I receptors, enzymatically dissociated cells were colabeled with antibodies to GS and BMPRIA, BMPR1B, or ALK2 (Figure 1D). Immunolabeled Müller glia were readily apparent in dissociated samples, as they had retained their shape throughout the processing. All three receptors appeared to colabel GS (+) cells, indicating that the cells are responsive to BMPs in vivo.


Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia.

Dharmarajan S, Gurel Z, Wang S, Sorenson CM, Sheibani N, Belecky-Adams TL - Mol. Vis. (2014)

Bone morphogenetic protein (BMP) type I receptors in the mature mouse retina. A: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize Müller glial and retinal astrocyte marker glutamine synthetase (GS) and BMP receptor 1A (BMPR1A). Thin plane confocal microscopy with y,z (strips to right of panels), and x,z planes (strips at bottom of panels) are shown in the last panels in the row. BMPR1A was localized to the Müller glial processes in the outer nuclear layer (ONL) and in Müller glial cell or retinal astrocyte processes in the ganglion cell layer (GCL, arrows A-C). BMPR1A was also detectable in the photoreceptor outer segments, the inner plexiform layer (IPL), cell bodies within the ganglion cell layer, and the nerve fiber layer (NFL). B: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and BMPR1B. BMPR1B appeared to be localized to the outer segments (OS), outer plexiform layer (OPL) and IPL and, Müller glial cell processes as well as Müller glial/retinal astrocyte processes in the GCL and NFL (arrows). C: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and activin receptor like kinase 2 (ALK2). The distribution of ALK2 receptors was similar to that of BMPR1A, with the majority of signal being localized to the end feet within the GCL and NFL. D: Müller glia isolated from P30 retinas were co-labeled for GS and BMPR1A, 1B and ALK2. The isolated cells showed the distribution of receptors to be primarily in the processes and the end feet of the Müller glia, as seen in the P30 tissues. n=3 different eyes for each label. Scale bar A=50 μm applies to A, B, C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4119236&req=5

f1: Bone morphogenetic protein (BMP) type I receptors in the mature mouse retina. A: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize Müller glial and retinal astrocyte marker glutamine synthetase (GS) and BMP receptor 1A (BMPR1A). Thin plane confocal microscopy with y,z (strips to right of panels), and x,z planes (strips at bottom of panels) are shown in the last panels in the row. BMPR1A was localized to the Müller glial processes in the outer nuclear layer (ONL) and in Müller glial cell or retinal astrocyte processes in the ganglion cell layer (GCL, arrows A-C). BMPR1A was also detectable in the photoreceptor outer segments, the inner plexiform layer (IPL), cell bodies within the ganglion cell layer, and the nerve fiber layer (NFL). B: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and BMPR1B. BMPR1B appeared to be localized to the outer segments (OS), outer plexiform layer (OPL) and IPL and, Müller glial cell processes as well as Müller glial/retinal astrocyte processes in the GCL and NFL (arrows). C: Retinal sections of 4 week-old retina double-labeled using antibodies that recognize GS and activin receptor like kinase 2 (ALK2). The distribution of ALK2 receptors was similar to that of BMPR1A, with the majority of signal being localized to the end feet within the GCL and NFL. D: Müller glia isolated from P30 retinas were co-labeled for GS and BMPR1A, 1B and ALK2. The isolated cells showed the distribution of receptors to be primarily in the processes and the end feet of the Müller glia, as seen in the P30 tissues. n=3 different eyes for each label. Scale bar A=50 μm applies to A, B, C.
Mentions: BMPs bind preferentially to three type I receptors; receptor IA, IB, or ALK2. Double-label immunofluorescence was used to determine whether any of the type I BMP receptors was expressed by Müller cells or retinal astrocytes. Sections through adult murine retina were colabeled with antibodies specific for GS, which labels Müller glial cells and retinal astrocytes, and BMPR1A, BMPR1B, or ALK2 (Figure 1). All three receptors showed similar labeling patterns in the retina with Müller glial cell processes labeled in the outer nuclear layer (ONL), outer plexiform layer (OPL), and GCL (Figure 1A-C). Photoreceptor outer segments (OS) and ganglion cell bodies were also clearly labeled for all three receptors. To confirm the label of Müller glial cells and their processes by antibodies to type I receptors, enzymatically dissociated cells were colabeled with antibodies to GS and BMPRIA, BMPR1B, or ALK2 (Figure 1D). Immunolabeled Müller glia were readily apparent in dissociated samples, as they had retained their shape throughout the processing. All three receptors appeared to colabel GS (+) cells, indicating that the cells are responsive to BMPs in vivo.

Bottom Line: However, as expected, the profiles including the oxidative agent and growth factor were not identical.Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment.BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University-Purdue University Indianapolis,, Indianapolis, IN ; Center for Regenerative Biology and Medicine, Indiana University- Purdue University Indianapolis, Indianapolis, IN.

ABSTRACT

Purpose: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells.

Methods: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry.

Results: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection.

Conclusions: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

Show MeSH
Related in: MedlinePlus