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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 7 (BMP7) treatment of MIO-M1 Müller glial cell line increases glial fibrillary acidic protein (GFAP) expression. A: Expression patterns for a panel of markers associated with reactive gliosis were compared in human MIO-M1 Müller glial cells treated with sodium peroxynitrite, or BMP7 for 24 or 36 h. For each experimental treatment, cells were treated with vehicle, sodium peroxynitrite, or BMP7 and real-time quantitative PCR (RT-qPCR) was undertaken. Levels of mRNA were normalized to internal control β2 Microglobulin, and values were plotted relative to control levels. Each sample was run in triplicate and experiment repeated 3 times for each gene. Values represented are means±SEM. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Any change above or below 1.0 indicates a change relative to control values. Peroxynitrite-treated MIO-M1 cells showed a statistically significant increase above the 1.5-fold level in paired-homeobox 2 (Pax2), acid sensing ion channel 1 (Asic1), lipocalin2 (Lcn2), potassium inwardly rectifying channel 2.1 (Kir2.1), Gfap, and phosphacan (Pcan) in comparison to vehicle-treated cells. Twenty-four hours following BMP7 addition, the MIO-M1 cells only showed a statistically significant increase above the 1.5-fold level in Gfap and Pax2, and a decrease in Txnip, galectin 3 (Gal3), Lcn2, and matrix metalloproteinase 9 (Mmp9). At the 36 h time point Gfap, and toll-like receptor 4 (Tlr4) levels of mRNA were increased above the 1.5-fold level in comparison to vehicle-treated cells and Spp1 was decreased. B: western blot analysis was performed for GFAP, S100β, TXNIP, and PAX2, with β-TUBULIN used as a loading control. Densitometric data shown are means+/−SEM of 3 trials. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Densitometric analysis of the blots showed a statistically significant increase in protein levels of GFAP and TXNIP in the peroxynitrite treatment. A significant increase in levels of GFAP, S100β, and TXNIP was observed in the 24 h BMP7 treatment, while a significant decrease was observed in PAX2 levels in the 36 h bmp7 treatment.
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f4: Bone morphogenetic protein 7 (BMP7) treatment of MIO-M1 Müller glial cell line increases glial fibrillary acidic protein (GFAP) expression. A: Expression patterns for a panel of markers associated with reactive gliosis were compared in human MIO-M1 Müller glial cells treated with sodium peroxynitrite, or BMP7 for 24 or 36 h. For each experimental treatment, cells were treated with vehicle, sodium peroxynitrite, or BMP7 and real-time quantitative PCR (RT-qPCR) was undertaken. Levels of mRNA were normalized to internal control β2 Microglobulin, and values were plotted relative to control levels. Each sample was run in triplicate and experiment repeated 3 times for each gene. Values represented are means±SEM. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Any change above or below 1.0 indicates a change relative to control values. Peroxynitrite-treated MIO-M1 cells showed a statistically significant increase above the 1.5-fold level in paired-homeobox 2 (Pax2), acid sensing ion channel 1 (Asic1), lipocalin2 (Lcn2), potassium inwardly rectifying channel 2.1 (Kir2.1), Gfap, and phosphacan (Pcan) in comparison to vehicle-treated cells. Twenty-four hours following BMP7 addition, the MIO-M1 cells only showed a statistically significant increase above the 1.5-fold level in Gfap and Pax2, and a decrease in Txnip, galectin 3 (Gal3), Lcn2, and matrix metalloproteinase 9 (Mmp9). At the 36 h time point Gfap, and toll-like receptor 4 (Tlr4) levels of mRNA were increased above the 1.5-fold level in comparison to vehicle-treated cells and Spp1 was decreased. B: western blot analysis was performed for GFAP, S100β, TXNIP, and PAX2, with β-TUBULIN used as a loading control. Densitometric data shown are means+/−SEM of 3 trials. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Densitometric analysis of the blots showed a statistically significant increase in protein levels of GFAP and TXNIP in the peroxynitrite treatment. A significant increase in levels of GFAP, S100β, and TXNIP was observed in the 24 h BMP7 treatment, while a significant decrease was observed in PAX2 levels in the 36 h bmp7 treatment.

Mentions: Similar to the retinal astrocyte cultures, treatment of the Müller glial cell line MIO-M1 with peroxynitrite also led to increases in some of the markers in the reactive gliosis panel (Figure 4A). Western blots showed a modest increase in protein levels of GFAP and S100β; however, no change was noted in PAX2 expression (Figure 4B). Treatment of MIO-M1 cells with BMP7 yielded similar, but slightly different changes in mRNA levels following treatment for 24 or 36 h. At 24 h, Gfap and Pax2 mRNA levels were increased in comparison to control vehicle-treated cells above the 1.5-fold threshold, while there was a decrease in Txnip, Gal3, Lcn2, and Mmp9 levels below the 0.5-fold threshold (Figure 4A). Western blotting showed statistically significant changes in GFAP, S100β, and TXNIP protein levels in comparison to controls; however, increases in PAX2 were not statistically significant (Figure 4B). Treatment of cells with BMP7 for 36 h yielded increases in Gfap and Tlr4 mRNA levels in comparison to control cultures and a decrease in secreted phosphoprotein 1 (Spp1; Figure 4A). By western blot, densitometric analysis showed a statistically significant decrease in PAX2 levels and no significant changes in GFAP, S100β, or TXNIP levels in comparison to vehicle-treated cells (Figure 4B).


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 7 (BMP7) treatment of MIO-M1 Müller glial cell line increases glial fibrillary acidic protein (GFAP) expression. A: Expression patterns for a panel of markers associated with reactive gliosis were compared in human MIO-M1 Müller glial cells treated with sodium peroxynitrite, or BMP7 for 24 or 36 h. For each experimental treatment, cells were treated with vehicle, sodium peroxynitrite, or BMP7 and real-time quantitative PCR (RT-qPCR) was undertaken. Levels of mRNA were normalized to internal control β2 Microglobulin, and values were plotted relative to control levels. Each sample was run in triplicate and experiment repeated 3 times for each gene. Values represented are means±SEM. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Any change above or below 1.0 indicates a change relative to control values. Peroxynitrite-treated MIO-M1 cells showed a statistically significant increase above the 1.5-fold level in paired-homeobox 2 (Pax2), acid sensing ion channel 1 (Asic1), lipocalin2 (Lcn2), potassium inwardly rectifying channel 2.1 (Kir2.1), Gfap, and phosphacan (Pcan) in comparison to vehicle-treated cells. Twenty-four hours following BMP7 addition, the MIO-M1 cells only showed a statistically significant increase above the 1.5-fold level in Gfap and Pax2, and a decrease in Txnip, galectin 3 (Gal3), Lcn2, and matrix metalloproteinase 9 (Mmp9). At the 36 h time point Gfap, and toll-like receptor 4 (Tlr4) levels of mRNA were increased above the 1.5-fold level in comparison to vehicle-treated cells and Spp1 was decreased. B: western blot analysis was performed for GFAP, S100β, TXNIP, and PAX2, with β-TUBULIN used as a loading control. Densitometric data shown are means+/−SEM of 3 trials. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Densitometric analysis of the blots showed a statistically significant increase in protein levels of GFAP and TXNIP in the peroxynitrite treatment. A significant increase in levels of GFAP, S100β, and TXNIP was observed in the 24 h BMP7 treatment, while a significant decrease was observed in PAX2 levels in the 36 h bmp7 treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Bone morphogenetic protein 7 (BMP7) treatment of MIO-M1 Müller glial cell line increases glial fibrillary acidic protein (GFAP) expression. A: Expression patterns for a panel of markers associated with reactive gliosis were compared in human MIO-M1 Müller glial cells treated with sodium peroxynitrite, or BMP7 for 24 or 36 h. For each experimental treatment, cells were treated with vehicle, sodium peroxynitrite, or BMP7 and real-time quantitative PCR (RT-qPCR) was undertaken. Levels of mRNA were normalized to internal control β2 Microglobulin, and values were plotted relative to control levels. Each sample was run in triplicate and experiment repeated 3 times for each gene. Values represented are means±SEM. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Any change above or below 1.0 indicates a change relative to control values. Peroxynitrite-treated MIO-M1 cells showed a statistically significant increase above the 1.5-fold level in paired-homeobox 2 (Pax2), acid sensing ion channel 1 (Asic1), lipocalin2 (Lcn2), potassium inwardly rectifying channel 2.1 (Kir2.1), Gfap, and phosphacan (Pcan) in comparison to vehicle-treated cells. Twenty-four hours following BMP7 addition, the MIO-M1 cells only showed a statistically significant increase above the 1.5-fold level in Gfap and Pax2, and a decrease in Txnip, galectin 3 (Gal3), Lcn2, and matrix metalloproteinase 9 (Mmp9). At the 36 h time point Gfap, and toll-like receptor 4 (Tlr4) levels of mRNA were increased above the 1.5-fold level in comparison to vehicle-treated cells and Spp1 was decreased. B: western blot analysis was performed for GFAP, S100β, TXNIP, and PAX2, with β-TUBULIN used as a loading control. Densitometric data shown are means+/−SEM of 3 trials. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Densitometric analysis of the blots showed a statistically significant increase in protein levels of GFAP and TXNIP in the peroxynitrite treatment. A significant increase in levels of GFAP, S100β, and TXNIP was observed in the 24 h BMP7 treatment, while a significant decrease was observed in PAX2 levels in the 36 h bmp7 treatment.
Mentions: Similar to the retinal astrocyte cultures, treatment of the Müller glial cell line MIO-M1 with peroxynitrite also led to increases in some of the markers in the reactive gliosis panel (Figure 4A). Western blots showed a modest increase in protein levels of GFAP and S100β; however, no change was noted in PAX2 expression (Figure 4B). Treatment of MIO-M1 cells with BMP7 yielded similar, but slightly different changes in mRNA levels following treatment for 24 or 36 h. At 24 h, Gfap and Pax2 mRNA levels were increased in comparison to control vehicle-treated cells above the 1.5-fold threshold, while there was a decrease in Txnip, Gal3, Lcn2, and Mmp9 levels below the 0.5-fold threshold (Figure 4A). Western blotting showed statistically significant changes in GFAP, S100β, and TXNIP protein levels in comparison to controls; however, increases in PAX2 were not statistically significant (Figure 4B). Treatment of cells with BMP7 for 36 h yielded increases in Gfap and Tlr4 mRNA levels in comparison to control cultures and a decrease in secreted phosphoprotein 1 (Spp1; Figure 4A). By western blot, densitometric analysis showed a statistically significant decrease in PAX2 levels and no significant changes in GFAP, S100β, or TXNIP levels in comparison to vehicle-treated cells (Figure 4B).

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