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Identification of active retinaldehyde dehydrogenase isoforms in the postnatal human eye.

Harper AR, Wiechmann AF, Moiseyev G, Ma JX, Summers JA - PLoS ONE (2015)

Bottom Line: RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE.In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT

Background/objectives: Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Methodology: Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.

Principal findings: In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.

Conclusions/significance: Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

No MeSH data available.


Related in: MedlinePlus

Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.(A) Low power magnification of ocular tissues demonstrating RALDH1 labeling (red) in the retina and choroid. (B) Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. (C, D) Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (blue). BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.
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pone.0122008.g006: Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.(A) Low power magnification of ocular tissues demonstrating RALDH1 labeling (red) in the retina and choroid. (B) Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. (C, D) Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (blue). BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.

Mentions: In order to determine the cellular localization of RALDH1, immunohistochemistry was performed on sections of human posterior ocular tissues (retina, RPE, choroid) (Fig. 6). When sections of human fundus were probed with anti-human RALDH1 followed by anti-rabbit IgG conjugated to AlexFlour 568 (red), RALDH1 could be detected in the retina and choroid (Fig. 6A). Based on morphology and placement in the retina, RALDH1 immunopositive labeling was observed in rod outer segments and a subpopulation of cone outer segments. Additionally, punctate labelling could be detected throughout the inner plexiform layer and inner limiting membrane, possibly associated with Müller cells. No labeling of the retina or choroid was found in negative controls in which non-immune IgG was used as the primary antibody (Fig. 6B). Non-specific labelling was detected in the RPE and is most likely due to autofluorescence of RPE-associated lipofuscin (discussed above) [24].


Identification of active retinaldehyde dehydrogenase isoforms in the postnatal human eye.

Harper AR, Wiechmann AF, Moiseyev G, Ma JX, Summers JA - PLoS ONE (2015)

Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.(A) Low power magnification of ocular tissues demonstrating RALDH1 labeling (red) in the retina and choroid. (B) Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. (C, D) Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (blue). BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4368790&req=5

pone.0122008.g006: Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.(A) Low power magnification of ocular tissues demonstrating RALDH1 labeling (red) in the retina and choroid. (B) Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. (C, D) Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (blue). BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.
Mentions: In order to determine the cellular localization of RALDH1, immunohistochemistry was performed on sections of human posterior ocular tissues (retina, RPE, choroid) (Fig. 6). When sections of human fundus were probed with anti-human RALDH1 followed by anti-rabbit IgG conjugated to AlexFlour 568 (red), RALDH1 could be detected in the retina and choroid (Fig. 6A). Based on morphology and placement in the retina, RALDH1 immunopositive labeling was observed in rod outer segments and a subpopulation of cone outer segments. Additionally, punctate labelling could be detected throughout the inner plexiform layer and inner limiting membrane, possibly associated with Müller cells. No labeling of the retina or choroid was found in negative controls in which non-immune IgG was used as the primary antibody (Fig. 6B). Non-specific labelling was detected in the RPE and is most likely due to autofluorescence of RPE-associated lipofuscin (discussed above) [24].

Bottom Line: RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE.In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT

Background/objectives: Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Methodology: Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.

Principal findings: In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.

Conclusions/significance: Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

No MeSH data available.


Related in: MedlinePlus